Journal of the Indian Institute of Science Bangalore 560 ...journal.iisc.ernet.in/archives/archives/v9-79/Vol.69(1-6)1989/Vol.69(4...Volume 69 Journal of the Indian Institute of Science

Volume 69

Journal of the Indian Institute of Science Bangalore 560 012, India

July-August 1989 Number 4

CONTENTS

Endocrinology

R. Ravindra and C. E. Grosvenor Role of microtubles and microfilaments in 249 polypeptide and steroid hormone secretion: A review

Algology

S. G. Sarwar and D. P. Zutshi Periphytic algal flora of Phragmites 275 communis Trin.

Short Communications

Aquatic Ecology

B. Jyothi, G. Sudhakar and V. Venkateswarlu

Phytohormones

N. Krishnan and S. Chandrasekharan

11% Theses Abstracts

Ecological studies on a desmid bloom 285

Effect of a commercial growth promoter 291 (Trade name Vipul) on fenugreek and its root nodule bacteria

Book Reviews

J . Indian Inst. Sci., July-Aug., 1989,69,249-274 "Ind~an Institute of Science.

and steroid hormone secretion: a review

R. RAVINDRA* Departmen1 ot" Pharmacology & Tox~cology. Med~cal College of Gcorgm, Augusta. CA, USA, 30912

AND

C. E GROSVENOK Department of Molecular and Cell Blology, Pennsylvania Stale University, Universrty Park, PA, USA, 16802.

Received on August 25. 1988; Revised on February 20, 1989.

Abstract

The important part played by the cytoskeleton In the dynamic process of polypeptide and steroid hormone secretion has been examined. Certain specilic endocrine tissues have been chosen to analyse the information avadable regarding hormone secretion. For example, for polypeptide hormones the anterior lobe of the pituitary and the islets of Langerhans have been discussed whereas for the steroid hormones, the ovaries and the adrenals have been chosen for discussion.

Evidence suggesting the involvement of exocytosis in the secretion of the two types of hormones has heen d~scussed. Most ofthe data reeardine the role of the cytoskcleton in hormone secretion has so far been obtained . - by employing drugs known to affect either microlilaments or microtubules-thereby studying the effects of oharmacolonical doses ofthe druns on hormonal secretion. At these high doses. the s~ecrficilv of the diue effects . . isquestlonable. On theotherhand,however,afew investigators haveexamined the biochemistry of thecytoskeleton durmg hormone secretion. Sensitive methods have been standardised to study the equilibrium between soluble and polymer~sed tubulin or actin pools, the assembly of tubuhn into microtubules and the GTPase activ~ty of tubulin.

Key words: Secretion, tubulm, actin, hormones.

1. Introduction

Although on the role of the cytoskeleton in hormone action were published not too long ago, discoveries made since then have prompted us to attempt this treatise. Although hormone secretion involves components besides cytoskeleton, such as CAMP, CaZ+, calmodulin and phosphoinositides, we have decided to restrict the scope of this paper to the role of microtubules and microIilaments in hormone secretion. We will examine the present understanding of polypeptide and steroid hormone secretion and the studies

'Reprint requests

250 R. RAVINDRA AND C E. GROSVENOR

on the role of cytoskeleton therein. We will present evidence to support the theory that secretion of protein as well as steriod hormones foliows a similar pattern in that these two types of hormones are packaged into secretory granules and are transportcd to the plasma membrane by the microtubular system for subsequent exocytosis. We will also examine the reports supporting the hypothesis that steroid hormones interact with the cytoskeleton prior to activating the genome.

2. Cytoskeleton

For brevity, we will includc only the important aspects of the three components of the cytoskeleton. For more information regarding these cytoskeletal elements, the reader is refcrred to detailed woiks on mi~ro tubules*~~, acting-" and intermediate l i l ament~ '~ .

? 2.1: Microtubules

Tubulin, the monomeric unit of microtubules, has a molecular weight of about 110,000 daltons and is composed of two non- den tical subunits, a and fl. Tubulins have remained very stable in evolution, histones apparently being the only class of proteins which have undergone less change since the origin of eukaryotes. Common antigenic determinants in microtubules from mammals, birds, reptiles, teleosts and diptera have been reportedL3. Tubulin is a glycoprotein with approximately 1.2mole neutral sugar per dimerI4. The possibility of tubulin being associated with phospholipids was s ~ g g e s t e d ' ~ by experiments wherein the addition of phospholipase A to bmin extract having the capacity to form microtubules, prevented the assembly at 37°C.

Tubulin has two guanosine nucleotide-binding sites per dimer. One is exchangeable (E), and the other nonexchangeable (N). Although at both sites GTP is bound non- covalently, the N-site can only be removed by denaturing the protein, whereas the E-site GTP is exchangeable with free GTP. The E-site GTP is hydrolysed during tubulin polymerisation8~'b. Tubulin is accompanied on the electrophoresis columns by several associated proteins ofhigher molecular weight. The presence of thesemicrotubule-associated proteins (MAPs), often in stoichiometric relation to tubulin, suggests a regulatory role of MAPs in the structure of microtubules".

Tubulin from rat brain could be assembled in vitro, to form microt~bules '~ with an exterior diameter of 24nm, provided the following conditions are met: 1) concentration of tubulin is sufficiently high, 2) GTP i s added, 3) Ca2+ is removed using EGTA, and 4) the temperature is 37°C with a pH of6.8. The assembly reaction isinhibited at cold temperatures. Microtubules generally grow in definite directions from initiation centres such as basal bodies or centrioles. Studies conducted in vitro on the directionality of microtubule growth from such initiation sites revealed that microtubules mainly increase their length in one directior~. Tubulin assembly appears to be a sequential process, consisti~~g of nucleation, elongation, ~ o ~ ~ m e r - p o l y m e r equilibrinm and length redi~tribution"~~-"~.

There are quite a few reliable methods3' to monitor the in vitro assembly of tuhulin into microtubules: turbidity measurements at 350nm, electron microscopy, viscosity measure-

CYTOSKELETON AND HORMONE SECRETION 251

ments and sedimentation of the polymer, estimation of the monomers remaining in the supernatant, flow birefringence, dark-field light microscopy, x-ray diffraction, laser light scattering, filtration, calorimetry and immunochemistry. Microtubule function is generally investigated using c ~ l c h i c i n e ~ ~ ~ ~ ' , v inb la~t ine~~, D,043,44 and T ~ X O P - ~ ~ (see refs 40,42, 43 for reviews). Colchicine is considered to be a prototype of the class of chemical inhibitors that act on microlubules. It is known that colchicine inhibits the assembly of tubulin monomers into microtubule polymers, resulting finally in the accumulation of tubulin in soluble pools. Addition of vinblastine to cells results in precipitation of tubulin. On the other hand, D,O and Taxol are known to alter the equilibrium between soluble and polymerised form of tubulin by overstabilising them in their polymerised form.

2.2. Microfilaments

Actin is ubiquitous in eukaryotes and has been highly conserved during e v o l ~ t i o n ~ ~ ~ ~ ~ . Actin comprises 15-20Gf the total cell protein, and approximately 50% of total actin in non-muscle cells exists as a soluble pool, i.e., not polymerised into F-actin. G-actin is a 42,000 dalton protein with one mol ATP bound per monomer and one mol Ca2+ bound to a specific divalent cation-binding site, different from that of the ATP-binding ~ i t e ~ ' . ~ ~ . There are many proteins that form tight complexes with G-actin, thus facilitating an increase in the pool of non-polymerised actin in the cell; there are also proteins in the cell that are known to accelerate the rate of actin depolymerisation". G-actin, under defined in uitro conditions, polymerises into F-actinlo. The microfilament polymer is a two-start, double- stranded, right-handed helix. The filament diameter is approximately 5 to 7nm. The polymerisation process is similar to that of tubulin, consisting of nucleation, elongation, and monomer-polymer equilibrium. Actin has an intrinsic ATPase activity. G-actin as well as F-actin are both capable of ATP h y d r o l y s i ~ ~ ~ , ~ ~ . A continued hydrolysis of ATP by F-actin takes place during the interconversion of G-actin to F-actin. This does not mean that ATP hydrolysis is a prerequisite for polymerisation. The rate of polymerisation and the critical concentration of actin are the same in the presence of the non-hydrolysable nucleotide AMPPNP as in the presence of ATPS5. Cytochalasins, a group of low-molecular weight fungal metabolites, stimulate the ATPase activity of G-actin by 30fold. Cytochalasin B preferentially blocks elongation of F-actin, and cytochalasins, in general, inhibit the rate of actin p o l y m e r i s a t i ~ n ~ ~ . ~ ~ . Methods to monitor the assembly of G-actin to F-actin are similar to those mentioned above for tubulinS8.

2.3. Intermediatefilaments

In contrast to microtubules and microfilaments, the intermediate filaments are composed of heterogeneous subunit protein^'^-^^. The biochemical and immunological differences in these subunit profeins have resulted in identification of five major subclasses of intermediate fdaments: 1) tonofilaments consisting of cyto- or prekeratins (40-68 kDa), 2) neurofilaments composed of triplet proteins (68,160,200 kDa), 3) glial filaments with glial iibrillary acidic proteins (51 kDa), 4) desmin filaments made up of desmin (53 kDa) and 5) vimentin filaments with vimentin (53 kDa). Intermediate filaments are highly insoluble in physiological ionic solutions. The filanients can be soluhilised into their constituent subunit

252 R. RAVINDRA AND C. t. GROSVENOR

proteins only under denaturing conditions or at very low ionic strength". The denatured subunit proteins assemble into intermediate filaments (inner diameter 7 -1 1 nm); th: 7s process is independent of accessory protcins or other cofactors, suggesting that the information to assemble inlo the polymer is present in the secondary structure of the subunit proteins themselves. A pool of unpolymerised subunit proteins does not seem to exist in the cell. The intermediate filament polymers are extraordinarily stable, and, to date, there is no known specific agcnt that causes depolymerisation of the intermediate filament polymer^'^ The recent finding that the Caz+-activated proteolytic enzymes are able to degrade the intermediate filaments in uitro led to the hypothesis that the changes in intracellular Ca2+ concentration could be regulating the assembly and disassembly in vivo'2,66-68.

There is a considerable body of evidencc that intermediate filaments interact with microtubules and micr~fi laments~~- '~; the disassembly of microtubules and microfilaments by drugs such as colchicine or cytochalasin is also accompanied by a rea'rrdngement df intermediate filament^^"^^-^^. Microinjection of antibodies against intermediate filament proteins into cells results in a rapid and reversible collapse of all the intermediate filaments into a tight perinuclear cap. However, this dramatic rearrangement of intermediate filaments did not change Lhe cytoplasmic distribution of microtubules and microfilaments, and the microinjected cells retained normal mitotic and cytokinetic p r ~ p e r t i e s ' ~ - ~ ~ .

Intermediate filaments as they normally occur in thecell are considered to play a structural role in reinforcing the cyto~keleton'~. Recent reportsN'-", however, suggest that the subunit protcins of the intermediate filaments could act as transmitters of signals from thc cell periphery to the nucleus. The subunit proteins are known to bind to nucleic acids9'. Purified vimentm binds to different fractions of avian erythrocyte membranes through two distinct d o r n a i n ~ ~ ~ . ~ ~ . Sites located at the carhoxy-terminal end or the vimentin moleculc hind specifically to nuclear envelopes in a cooperative fashion; the plasma membrane fraction interacts in a saturable manner with the amino-terminal head of the vimentin molecule.

3. Mechanism of hormone secretion

The general pathway of polypeptide hormone secretion in vertebrates is well d o c ~ m e n t e d ~ ~ - ' ~ ~ . From their site of synthesis in the rough endoplasrnic reticulum, the proteins are transported Lo the Golgi apparatus, where glycosylation and packaging into secretory granules take place. From the Golgi apparatus the secretory granules are transported to the cell membrane where exocytosis occurs. Exocytosis is a process by which cells release materials into the extracellular milieu. Upon appropriate stimulus, the secretory granules migrate.to the luminal plasma membrane, fuse with the membrane, and the hormones are then secreted.

On the other hand, it was believed that steroid hormones, after being synthesised in the steroidogenic tissue, are released into the extracellular milieu by simple d i f f u s i ~ n ' ~ ' ~ ' ~ ~ . However, recent report^'^^-'^^ suggest that steroid hormone secretion by the corpus luteum as well as the adrenals could he an active process related to or identical with exocytosis. A correlation between the presence and concentration of densely staining granules in the corpus luteum and progesterone secretion has been notedlo3. Quirk et a1105


reported that progesterone is localised within electron-dense granules and suggested that these granules are released into extracellular medium by exocytosis. Sawyer et allo4 proposed that a progesterone-carrier protein (without progesterone) was packaged into secretory granules at the level of the Golgi apparatus; these granules pick up progesterone from the tubular elements of the agranular endoplasmic reticulum as they move towards the plasma membrane; finally the granules are exocytosed.

Although it is generally accepted that cytoskeleton plays a major role in the transport of secretory granules from the Golgi apparatus to the plasma membrane, the exact mechanism of this transport is yet to be delineated. Most of the work regarding the involvement of cytoskeleton in hormone secretion was performed using drugs such as colchicine, yinblastine or cytochalasin. These agents are known to perturb either the microtubules or microfilaments, and the effects these compounds exert on hormone secretion have been related to the cytoskeleton. However, more often than not, these agents were employed at high doses and at these concentrations the drugs are known to affect other cellular functions as we114,110-115, besides disrupting the cytoskeleton. Therefore, the results obtained with the drugs should be viewed with caution.

The very heterogeneous nature of intermediate filaments-there being at least five protein subunits-and the unavailability of a drug known to affect their function, have made it difficult for the endocrinologist to study the role of the intermediate filaments during hormone secretion. However, the three components of the cytoskeleton are known to interact with each other, and any stimulus affecting one of the three could also affect the other two.

3.1. Anterior pituitary lobe

Anti-cytoskeletal drugs were employed to demonstrate the involvement of microtubules and microfilaments in the secretion of gonadotropins by the anterior pituitary lobe in response to GnRHH6-''l. Colchicine was observed to: inhibit the secretion of ACTH'22 in the rat in vitro; accumulate secretory granules and cause disappearance of microtubules in somatotrophic cells1z3; inhibit PRL and GH s e ~ r e t i o n ' ~ ~ - ' ~ ~ . In only one case colchicine was shown to stimulate"' the release of LH, FSH, and TSH. Vincristine, another drug which interacts with the microtubules, was shown to inhibit PRL and GH relea~e'~'. All these s t u d i e ~ " ~ ~ ~ ~ ~ employed high concentr&ions of the drugs, and the specificity of their effects on the microtubules was not evaluated with the use of lumicolchicine, an inactive isomer of colchicine which does not affect the microtubule function.

Shino et a1 employed electron microscopy to correlate PRL secretion to the microtubules in the anterior pituitary Although they visualised the microtubules, they could not observe changes in the tuhulin during increased PRL secretion. Sheterline etal, using morphometric techniques, demonstrated that stimulation of GH secretion from bovine somatotrophs by non-physiological secretagogues, was accompanied by a decrease in the polymerised m i c r ~ t u b u l e s ~ ~ ~ . Holck et al, employing immunofluorescent methods, observed more intense tuhulin staining of gonadotrophs in castrated rats1j4. This finding was later confirmed by Valenti et a1 by direct measurements of total tubulin by the colchicine binding a ~ s a y ' ~ ~ , ' ~ ~ . Using morphological techniques, it was demonstrated that in human FSH-

254 R. RAVlNDRA AND C E. GROSVENOR

producing adenomas, microtubules were often conspicuous and might increase in number in the cytoplasm of the adenoma the gonadotrophic cells of the non-tumorous pituitary contained only a few micro tubule^'^^. Niwa eta1 demonstrated that in uitro treatment of rat prolactinoma cells with bromocriptine resulted in changing the fine reticular networks of microtubules into coarse aggregates, thus implicating microtubules in PRL secretionL3'.

In our attempts to examine the anterlor pituitary lobe microtubules, we have chosen the lactating rat as our model and stimulated PRL secretion by suckling the mothers with the pups. Instead of using drugs which are known to disrupt the microtubule system and then examining hormone secretion, we have applied a physiological stimulus to release PRL from the anterior pituitary lobe and then studied the pituitary tubulin status. Using the 3H-colchicine-binding assay, we have initially observed that suckling resulted in a significant elevation in tubulin levels in the anterior pituitary lobe'40. Next we have standardised the buffer conditions to estimate the soluble and polymerised tubulin pools in the anterior pituitary lobe and reported that suckling resulted in a shift in the equilibrium between soluble and polymerised tubulin pools in the anterior pituitary14'. The total amount of the two tubulin pools did not change appreciably during suckling (fig. 1). In another study we have assayed the GTPase activity of the soluble and polymerised tubulin pools and observed (fig.2) that the GTPase activities of the two tubulin pools during suckling also appeared to be in a dynamic flux'4'. We followed these observations with the standardiza- tion of a method to monitor the in uitro assembly of anterior pituitary lobe tubulin into

0 30 60 90 mln Suckling

FIG- 1. Efiect of suckllng on the soluble (SQ and polymerised (PQ tubulin pools Each point represents the mean i s e m from six to eight rats. One-half microgram of protein from each antenor pituitary lobe was assayed in triplicate for colchidne binding. For PT: 0 us 30 min, P < 0.05; 30 us 60min. P=NS; 60 us 90 min,P<0.05. For ST0 us30min, pc0.05;30us 60min, P=NS; 60 us 90 min, Ps0.05 (Reproduced with permission from ref. 141).

0 3 0 60 90

mln SUCKLING

FIG. 2. Erect of suckling on GTPase activity in the soluble and polymerised tubulin fractions. Each point represents mean _t sem obtamed from slx to etght rats. The GTPase activity in soluble and polymerised fractions from individual pituitary eland was assayed . . - In lrlpllwle uslng?S$g prurein From each fracuon. For ST lSol~blcrubulmr O rr IS mm P<OOS: IS us 60 mln. P<0.05; 60 us 90' min=NS. For PT' (polymerised tubulin). 0 us 15 min P<O.OS; 15 us 30, MI, 90 min P<O.O5 (Ravindra and Grosvenor, unpublished data).

CVTOSKELFTON AND HORMONE SECRETION

FIG. 3. EIlecr of su~kling on tubulin assembly. The antenor pilultary giands from iix suckled (fur 30 mm) and SIX nonsuckled rats were ~nd~v~dua l ly processed to obtain the 'tubulm fracoon' Protein ob tamd from each pilu~tary gland was incubated in dupl~carc at a cmcentratmn o f 0 6 rng/ml In MES buffer, pH 6.8. 37°C. GTP 2 mM for one mln. Thc results are expressed as mean _isern and the suckled group (hatched bar) is sigm- ficanrly dlllercnt (P<O.05) from the nansuckled group (open bar) (Figure reproducd wlth permusion from reference 143)

microtubules. This technique was found to he sensitive and capable of measuring the assembly in approximately 2pg of tubulin present in the 100,000 x g supernatants. After extensively validating this protocol we have observed (fig. 3) that suckling affected the extent to which anterior pituitary tubulin of the rat assembled into micr~tubules '~~. These observations are consistent with the hypothesis that microtubules are being recruited to transport PRL granules from the Golgi apparatus to the plasma membrane. This process could conceivably involve many cycles of polymerisation/depolymerisation. Although our studies suggest that microtubules could be involved in PRL secrelion by the anterior pituitary lobe, it was pointed out that the changes we have observed in the microtubules during PRL secretion may not be specifically occurring in the lactotrophs, as we were dealing with the total cell population of the anterior pituitary lobe. In the rat, suckling slimulates the release of TSI-1144.'45, GH14' and ACTH/fl-end~rphin'~' in addition to PRL. However, electron microscopy has revealed that in most species examined, PRL secretory cells increase in number and cytoplasmic volume and become the dominant cell type during la~ta t ion '~? We have attempted to address this question by employing drugs that are known to affect PKL secretion. We have demonstrated that domperidone-elevated anterior pituitary lobe polymerised tubulin levels and plasma PRL concentration comparable to those increases caused by suckling. Rromocriptine blocked the suckling-induced rise in polymerised tubulin and the rise in plasma PRL levels as well'4'. Domperidone was also demonstratcd to increase the soluble tubulin GTPase activity comparable with that

256 R. RAVINDRA AND C. E. CROSVENOR

at 15min suckling, and bromocriptine blocked the suckling-induced rise in the polymerised tubulin GTPase activity (Ravindra & Grosvenor, manuscript in preparation). The in vitro assembly or anterior pituitary lobe tubulin inro microtubules was also affected by domperidone, bromocriptine, and oPRLi4!'. Treatment of lactating rats with oPRL prior to suckling was shown to inhibit the relcase of PRL into plasma150; the same dose of oPRL also resulted in a shift in the equilibrium in the two tubulin pools and their GTPase activities (Ravindra & Grosvenor, unpublished results). Furthermore, we have observed that the equilibrium between the two tubulin pools was also affected in the lactotrophs of ovariectomised rats primed with estradiol (Ravindra, Hymer & Grosvenor, preliminary observations). Thus, these results support our contention that the changes seen in the anterior pit~itary microtubules in response to a physiological stimulus could be correlated to those occurring in the lactotrophs during PRL secretion.

Sherline eta1 reported that porcine pituitary secretory granules, enriched in G H and PRL, bound to brain microtubules in vitro, but not to depolymerised microtubules (i.e., tubulin), suggesting that microtubules might facilitate the cell interior to cell surface movement of PRL secretory granules by providing tracks along which granules could move'51. Bloom eta1 published a good method to purify tubulin from the bovine anterior pituitary tissue and GH, cells which secrete G H and PRL15'. These methods should stimulate more work regarding the biochemical interaction between the secretory granules and tubulin system in the anterior pituitary lobe.

The role of actin in the pituitary hormone secretion bas not been as vigorously pursued as that of tubulin. Benzonana c ta l adapted the DNAse method to the anterior pituitary lobe for estimating the G-actin ~onten t"~ . Ostlund et demonstrated that anterior pituitary gland secretory granules bound to G-actin in vitro.

3.2. Islets ofLangerhans

The secretion of insulin has been well studied, using either the whole organ or isolated ce{]s155-~160 . Although the bulk of the work was done using pharmacological doses of drugs known to disrupt the cytoskeleton, some studies combined this approach with morphological examination of the cytoskeleton, while others investigated the biochemistry of the cytoskeleton in these cells. Taken together, these results suggest that insulin secretion is a two-step process, involving both microtubules and microfilaments. The first step involves the transport of the insulin secretory granules from the Golgi apparatus to the periphery of the cell; microtubules appear to be involved in this process. In the second step, the insulin-containing secretory granules which are at the periphery of the cell membrane fuse with the plasma membrane to release insulin into the extracellular milieu; microfilaments are implicated in this step.

The secretion of glucagon has not received much attention, and the reports are rather a r n b i g u o u ~ ~ " " ~ ~ .

3.3. Adrenals

Information regarding adrenal steroid secretion was obtained mainly with the use of drugs


that arc known to interact with the cytoskeleton. Vinblastine caused the appearance of tubulin crystals in the ratIb3 and stimulated the secretion of adrenal steroid hormones in the mouseLb4. Stimulation of tissue cultures by ACTH and CAMP appeared to increase the number of visible microtubules as seen by immunofluorescence, indicating that the hormone might ~nduce tubulin polymerisalion. The nncrotubules thus formed could help in the translocation of steroid containing granules'65. Payet era1 reported that administration of colchicine to rats stimulated aldosterone and corticosterone ~ e c r e t i o n ' ~ ~ . Colchicine and other antimicrotubular drugs increased steroid secretion by cultures of Y-1 adrenal tumor cells"j7, and in normal rat adrenocortical ~ e l l s ' " ~ ' ~ ~ . O'Hare, however, found that steroid secretion in the normal rat adrenal cells was inhibited by these drugs170.

Cytochalasins stimulated Y-1 cell steroidogenesis within an hour of incubation in uitro; this effect of the drug was reversible by washing17'. On the other hand, other studies found that in the Y-1 cells, cytochalasin and anti-actin antibodies inhibited the ACTH- or CAMP-stimulated steroid secretion2,"' 12. Cytochalasin also inhibited the ACTH-stimulated steroid secretion by normal adrenal cell^'^^^"^. Employing dispersed bovine adrenal cells, it was reported that actin fibre bundles were distributed transversely in the cytoplasm; after the addition of ACTH or cAMP the microfilaments became inconspicuous with dot-like appearance and their distribution pattern was altered from circular to It was also observed'76 that after ACTH treatment, bovine adrenocortical cells became rounded with the breakdown of microtubules. It is doubtful that there is any correlation between steroidogenesis and cell shape. Using immunoelectron microscopy, Loesser and Malamed'" showed that in freshly isolated rat adrenocortical cells, ACTH had no effect in actin content in cytoplasm, mitochondria or lipid droplets; ACTH increased the actin concentration in the peripheral cytoplasmic band. These findings are in contradiction to those reported by Cheitlin and R a m a ~ h a n d r a n ' ~ ~ , " ~ . These conflicting results suggest that more work needs to be done to clarify the role of cytoskeleton in the secretion of adrenal steroids.

3.4. Ouary

Soto et aPaO reported that the stimulation of progestins by hCG and hLH in human granulosa cells in uilro was accompanied by an alteration in the cell shape, and the changes in cell shape brought about by the hormones were mimicked by treating the cultures with cytochalasin B or D. However, the response of the cells to these agents was much more rapid than the hormones, occurring within 20min as compared to 4 h with the hormones. Using rat granulosa cell cultures, it was o h ~ e r v e d ' ~ ~ - ' ~ ~ that colchicine, cytochalasin and Ca2+ ionophore-stimulated steroid secretion and was accompanied by a change in cell morphology. Zor dcmonstrated that the presence of anti-actin antibodies or cytochalasin B in the culture medium prevented the rat Graafian follicles from responding to LII or FSH; they also reported that colchicine did not impair the response to LH but prevented the stimulatory effect of FSH and PGE, on follicular cAMP production.

In bovine luteal cells colcemid, vinblastine and cytochalasin B inhibited the CAMP- or LH-induced morphological changes, suggesting that LH and cAMP could be promoting

258 R. R A V l N D R A A N D C E. G R O S V E N O R

the formation of cytoskeleton1s6. Cytochalasin inhibited hCG-induced progesterone production by rat187 luteal cells in uitro: cytochalasin inhibited basal as well as LH- stimulated progesterone production by the bovinelS8 luteal tissue. Cytochalasin and colchicine inhibited progesterone production by o ~ i n e ' ~ ~ . ' ~ ~ luteal cells in vitro. In viuo treatment of ewes191 and rats'92 with pharmacological doses of colchicine resulted in a significant reduction in plasma progesterone. However, anti-microtubule drugs had no effect on either the basal or LH-stimulated steroid production in uitro by collagenase- dispersed ratIn7 luteal cells and bovineLa8 luteal tissue. Using ovine luteal tissue, Sawyer etalro4 reported that LH-stimulated progesterone secretion was significantly reduced in the presence of colchicine. It was observed that in viuo colchicine treatment inhibited the in uitro progesterone production (cells + medium) by collagenase-dispersed rat luteal cells, but did not alter themicrotubule content as assessed by quantitative electron m i c r ~ s c o p y ~ ' ~ . We know from experience that sensitive biochemical methods are required to monitor the subtle changes in the equilibrium between the soluble and polymerised tubulin poolsL40-143.149

The use of collagenase or trypsin to prepare cells from the luteal tissue could affect their function, and this might complicate matters when the cells are immediately incubated with cytoskeletal inhibitors. Carnegie et a1 have, in fact, demonstratedln4 that when rat granulosa cells were grown on collagen gels, they secreted almost three fold more progesterone than cells cultured in minimal essential medium alone. Also, it is a general practice to keep the luteal tissue on ice until the required amount of tissue is dissected out of the animals. It is knowna that in vitro microtubules (polymerised from purified brain tubulin) depolymerise when exposed to cold temperatures and can be readily repolymerised by incubating at 37°C. However, we do not have extensive knowledge of the behaviour of microtubules present in a tissue. In one study using the hamster corpora lutea the tissue was not exposed to either enzymes or cold tempe~atures''~. The tissue was kept in minimal essential medium at room temperature for not more than 15min and then was incubated at 37°C. Vinblastine and colchicine inhibited progesterone secretion in vitro by the corpora lutea; the effect of colchicine was observed at about 30min and was significant at 60 min, indicating that these drugs act rapidly. In an attempt to demonstrate the specificity of colchicine binding and relate its eMect to the disassembly of microtubules, experiments were conducted with lumicolchicine, an isomer of colchicine that has no effect on microtubule assembly. Lumicolchicine did not inhibit progesterone secretion by the hamster corpora lutea. Moreover, colchicine inhibition of progesterone secretion could be overcome by preincubating the corpora lutea with D,O. D 2 0 is known to alter the equilibrium between soluble and polymerised form of t u b ~ l i n ~ ~ , ~ ~ . Preincubation of corpora lutea with anti-tubulin antiodies prevented the inhibitory effect of colchicine on progesterone secretion; anti-tubulin antibodies did not affect basal or LH-stimulated progesterone secretion. This observation suggested that the antibody bound to cell surface of the luteal tissue and prevented the effects of colchicine. The presence of a plasma membrane-associated tubulin has been demonstrated in the brain of a few specie^'^^.'^^. The presence of plasma membrane- associated tubulin in the hamster corpus luteum might explain these effects of anti-tubulin antibodies. Zor eta1 also observed that the presence of antibodies to tubulin in the medium did not inhibit the stimulatory effect of LH on the rat Graafian follicles'85,

CYTOSKELETON A N 3 HORMONE SECRETION 259

It was observed that estradiol inhibited the progesterone secretion by the hamster corpus luteum by interfering with microtubule function of the lateal ce13'94,'97-199 . Sub-optimal concentrations of estradiol and colchicine added together resulted in a maximal inhibition of steroid secretion. Preincubating the corpora lutea with either anti-tubulin antibodies or D 2 0 prevented the inhibitory effect of estradiol on steroid secretion, suggesting that the hormone could be initially binding to a membrane component'94. The concept that steroid hormones interact with membrane components is relatively new200-z09. It was assumed until r e c ~ n t l y ~ ' ~ that steroid hormonespassively diffuse to bind to the cytoplasmicreceptors. The hormone-receptor complexcs thus formed provoke increased transcription of specific genes, Leading to the accumulation of specific ~ R N A S ~ " - " ~ . The initial localisation of thc receptor in the absence of hormone is not known. It is suggested that the receptor may be attached to, or a component of, the plasma membrane from which it is quite easily detached during homogenisation and hence obtained in the ~ y t o p l a s m ~ ' ~ ~ ~ ' ~ . A marked saturability and temperature dependence of steroid hormone entry, which cannot be attributed to the funclion olcyloplasmic r e ~ e p t o r s ~ ' ~ - ~ ' ~ , has been noted. Rat endometrial cells exposed to estradiol for a brief period of time exhibited pronounced altered membrane function^^^'^^^^ and micropinocytot~c vacuolation of p l a ~ m a l e m m a ~ ' ~ .

R ~ p o r t s ~ ' ~ - ~ ~ ' from the Szego laboratory suggest that estradiol interacts with components of biological membranes and may enter cells by a membrane-mediated process. Szego and coworkers demonstrated specific, saturable and temperature-dependent cell surface- binding sites on endometrial cclls ro estradiol-BSA conjugatc immobilised to nylon fibreszo3. Employing carefully controlled homogenisation and isolation procedures that are different from the methods generally usedzz", Pietras and Szego2I3 demonstrated that approximately 27% ofreceptor component with high affinity and ligand specificity for binding estradiol-178 is concentrated in plasma membranes purified from isolated uterine cells of ovariectomised rats. This is in contrast to the widely reported occurrence of estradiol receptors in cytosolic fractions214. High-affinity and low-capacily receptors for estradiol have also been reported for hepatocyte plasma membrane fraction^^^^.^^^. E s t r a d i ~ l ~ ~ ~ was demonstrated to alter the morphology and arrest m~tosis in a Chinese hamsler cell line.

There is also evidence that the membrane surface is involved in the progesterone-induced meiosis in Xenopus iaevis oocyteszZ6 and LHRH secretion in uitro by mediobasal hypothalamic slices of ratsZz7. It is known that in endometrial cell suspensions obtained from uteri of ovariectomised rats, estradiol increases CaZf uptake to a large extent within 30 minZo1. In light of the reports that CaZ+ depolymerises r n i c r o t u b ~ l e s ~ ~ ~ ~ ~ ~ ~ , it is possible that Ca2 + may be mediating the effect of estradiol on progesterone s e ~ r e t i o n ' ~ ~ . ' ~ ~ - ' ~ ~ . Based on their experiments with CHO-KI cell lines and the effect of testosterone on the cell morphology, Hsie and PuckZ30 postulated that the effect of testosterone was mediated uiu the microtubules. Using Drosophila melanogaster Kc cells, Sobrier et a1 demonstrated that the insect-moulting hormone, 20-bydroxyecdysone affected actin and tubulin function as well as their b io~ynthes i s~~ ' . It was reported that the synthetic estrogen diethystilhestrol inhibited the in oitro assembly of brain t ~ b u l i n ~ ~ ~ - " ' . Sato r t a1 briefly mentioned that

260 R RAVlNDRA AND C. E. GROSVENOR

estradiol had no effect on tubulin assembly, but did not give details regarding their experimental conditions232.

In an attempt to check whether the effect of estradiol on progesterone secretion by the hamster corpus luteum was due to the direct action of this steroid hormone on the tubulin system, rat brain tubulin was purified by phosphocellulose chromatography and the effects of estradiol on tubulin GTPase activity and assembly were monitored. Brain tubulin was used based on the fact that it is a highly conserved protein and the differences between tubulin from hamster corpus luteum and rat brain could be minimal; also, rat brain is a rich source of this protein, whereas too many hamsters had to be sacrificed to purify a few mg of protein from the corpus luteum. Estradiol inhibited tubulin GTPase activity in a dose-dependent manner, and this inhibition could be overcome by excess GTP, suggesting that the effect of this steroid on tubulin function is reversible. Estradiol also inhibited the assembly in a dose-dependent manner, as monitored by turbidimetric measurements and electron microscopy'99. Taken together, these resuIts'94.'97-199 suggest that estradiol inhibited progesterone secretion by the hamster corpora lutea by interfering with the equilibrium between the soluble and polymerised tubulin pools. It is essential to maintain the equilibrium between soluble and polymerised forms of tubulin in cells, and regulatory mechanisms must exist to prevent all cytoplasmic tubulin from assembling into microtubules. It is tempting to speculate that estradiol may be one such molecule which has a role in regulating tubulin function in vivo.

5. Perspective

Recent work on the interaction of synaptic vesicles and microtubules provided some interesting data regarding the transport of vesicles on microtubules and could very well inspire similar work on hormone secretion via the cytoskeleton (see 235 and 236 for references). In an elegant paper, Gray2" observed that microtubules are in contact with presynaptic dense projections of the central nervous system leading to the suggestion that microtubules translocated the synaptic vesicles. Subsequently, Baines and Bennett236 demonstrated that synapsin I, a synaptic vesicle protein from calf brains, bound saturably to microtubules in vitro. Crosslinking of microtubules by synapsin I was observed by electron microscopy. Thus, synapsin I could play a role in mediating the synaptic vesicle- microtubule interaction. Other models of cell motility in metazoa are actomyosin-based system in muscle237, and the dynein-based system in the axonemes of flagella and cilia2". Another 'motor'system has been recently described in the chick brain239, squid giant axons and bovine brain2". Subsequently this protein was demonstrated to be widely distributed among organisms and cell cultures241. B ~ a d y ' ~ ~ and Vale et aP40, working independently, have reported that this protein is distinct in molecular weight and enzymatic behaviour from myosin or dynein. Vale et proposed the name Kinesin (from the Greek Kinein, to move). In gel filtration columns, both the squid and bovine translocators elute with a n apparent molecular weight of 6M)kDa. The quaternary structure of kinesin is yet to be described. Stoichiometry studies by gel densitometry of the polypeptides in highly purified kinesin preparations indicate that kinesin is a complex of two or three different polypeptides. Addition of kinesin to a mixture of highly purified microtubules (free of microtubule-

CYTOSKELETON AND HORMONE SECRETION 26 1

associated proteins) and membrane organelles from squid axoplasm resulted in the translocation of the organelle on the microtubule as visualised with video-enhanced diffcrcntial interference contrast microscopy. Trypsinisation of the organelles blocked their movement, suggesting that the binding of the organelle may be protein mediated. Latex beads adsorbed to k~nesin could also be translocated along microtubules. Moreover, the movement of microtubules was noted in the presence of kinesin. ATP facilitated the initial interaction between kinesin and microtubules, but the intrinsic ATPase activity of kinesin caused ATP hydrolysis and eventual dissociation of kinesin and microtubules. In the presence of AMP-PNP, a non-hydrolysable analog, kinesin binding to microtubules was enhanced.

Paschal and Vallee242 reported that MAPlC, one of the five high molecular-mass microtubule-associated proteins, has the ability to translocate microtubules. When microtubules were placed on a glars microscope slide coated with MAPlC, microtubule gliding occurred in a continuous, unidirectional manner. MAPlC was demonstrated to be a soluble form of dynein. I t was also shown to be a retrograde translocator i.e., movement from the cell periphery to the cell centre suggesting a role for MAPlC in endocytosis. Kinesin, on the .other hand, was observed to operate in the opposite direction, anterograde, ie., movement from the cell centre to the cell periphery. Thus kinesin appears to be candidate for exocytosis. Future work should reveal if similar 'motor' systems could be characterised in endocrine glands.

Acknowledgements

Part of this work was supported by NIH grant HD-04358 (to CEG). We would like to thank Dr. Aruna Chakravorty for reviewing the manuscript and Ms Becky Lucas, and Sandra Dunn for their help in the preparation of this work. The hamster corpus luteum work was conducted at the Department of Biochemistry, Indian Inslitute of Science, Bangalore, India. Professor ti. Padmanaban's interest in this work and his valuable suggestions have helped immensely.

References

1 . zon, U. Role of cytoskeletal organiralion In the regulation of adenylate cyclase-CAMP by hormones, Endocr. Reu., 1983,4, 1-21.

The role of the cytoskeleton m hormone action, Can. J . Biochem. Cell B i d , 1984, 62, 653-665.

On the mechanism aiactron of ACTH: The role of actin, Endocr. Res., 1984, 10,431-461.

M~crotuhules, Springer-Vcrlag. 1984.

Molecular blology and genettcs of tubuhn, Annu. Rev Emhem., 1985, 54, 331-361.

262 R. RAVINDRA AND C. E. GROSVENOR

6. SNYDER, 3. A. AND

MCINTOSH, J R.

7. KIRSCH~TR, M. W-

8. TIMASHEW, S. N. AND

GRISHAM, L. M.

9. KORN, E. D.

IS. BRYAX, J.

16. TERRY, B. J. AND PURICH, D. L.

20. SHEIANSKI, M. L.,GASKIN, F. AND

Cah-ron, C R.

21. ROTHWELL, S. W., CRASSER, W. A. AND MURPHY, D. B.

22. MCINTOSH, J. R.

23. MARGOLK R. L. AND WILSON, L

28. KARR, T. L. AND PURICH, D. L.

Blochemistry and phys~ology of microtubules, Annu Rw. Bbrrb~ni., 1976.45.699-720.

Mlcrotubulc assembly and nucleation, Inr. Rrr. Csrol., I9?8,54, 1 71.

In tritro assembly of cytoplasm~c m~crotubules, Annu Rm. Biochmi., 1980.49, 565-91.

Actin polymerization and its regulation by profems from nonmuscle cells, Physiol. Rev.. 1982.62, 671-737.

Mechanism of actin polymerizat~on, TIBS, 1982, 7, 55--58.

Actin-bindlng proteins, rbid, 1982, 7, 88-92,

Intermediate filawnrs: A review, 1485, pp 1266. Spnrger-Vcrlg

Concerning the universality of a microtubule antigen in animal cells, J . Cell Biol., 1972,52, 748-754.

Rat brain microtubule protein: purification and derermination of covalently bound phosphate and carbohydrate. Proc. Naln. Aead. Sci. U S A , 197269, 2283-2287.

Preliminary studies on afinity labeling ofthe tubulin.colchicinc binding site, Ann. N.Y. Acad. Scr., 1975, 253, 247-259.

Nucleotide dependent enzymes associated with m~crotubule systems, Ado. Enzymol., 1982, 53, 113-161.

Initiation oi brain tubulin assembly by a high molecular weight flagellar protein factor, J. Cell Bid, 1976,71, 322-331.

Microtubule formation in uitro in solutions containing low calcium concentrations, Science, 1972, 177, 1104-1105.

Visualization of the dynamic instability of individual microtubulo$ by dark-field microscopy, Nature, 1986, 321, b95-607.

Microtubule assembly in the absence of added nucleotldes, Pmc. N a m . Acad. Sci. USA, 1973.70, 765-768.

End-to-end annealing of microtubules in uitro, J. Cell Biol., 1986, 102, 619-627.

M~crotubule catastrophe, Nature, 1984, 312, 196-197

Micratubule treadmills -possible molecular machinery. Nururr, 1981, 293, 705-711.

Head-to-tad polymerization of microtubules in oitro. Electron microscope analysis of seeded assembly. J . Cell Biol.. 1980,84.141-150.

Kinetic analysis oi microtubule self-assembly in uitro, J. Mol. Rial.. 19i7,117, 1-31.

Vlsualizationoi the structural polarity of microtubules. Nature, 1980, 286, 517-519.

Reassembly of flagellar B(rrS) tubulin into singlet microtubules: con- sequences for cytoplasmic microtubule structure and assembly. J. Cell BloL, 1981,89,323-337.

Rings are not microtubule assembly intermediates: An analysis of the lag phase in GTP-dependent self-assembly of bovine brain tubulin, Biochem. Bmphys. Res. Commun., 1980,95, 1885-1889.

CYTOSKELFTON AND HORMONE SECRETION 263

40. W I U ~ N . L., BAMUURG, 1. R., MIZEI., S. B., CRISHAM, L. M.AND CRESWELL K. M.

41. OLMSILU, I. B. AND BORISY, ti. G.

42. WILSON, L.

43. GROSS, P. R. ANU SPINDEL W.

44. MaLAlssE. W. 1.. MALAISE-LAG*+ F., WALKER, M. 0. ANU LACY, P. E.

47. SCHIFF. P. B., FANT, I. AND

Honwlrz, S. B.

48. COLLINS, C. A AND VALLEE. R. B.

49. Koau, E. D

Aswnbly and turnovcr oi detjrosinnted tohulii: in aau, J . Ceil Bid . 1987, 105, 265-276.

Microtubulc dynamics and chromosome motmn vrwalizcd In living anaphase cells, J . Cell Btnl., 1988, 106. 1185-1 192

Dlrect observation ofmicrotubule dynamlcs in hvmgcclis, iNaturp, 1988, 332, 724-726.

Dnect observatron of steady-state microtubule dynam~cs, J. Crll Brol., 1986. 102. IW7-1019.

Microtubule dynrmics in intciphase cells, 1. Crll Blol., 1986, 102, 1020-1031.

Polymeriratlon of tubulin in tie. dmct evuJence ibr aawmhly on to microtubulc ends and from centrosomei, J . Cell Biui., 1985, 100, 1682-1689.

Dynamlc mslabdity ofmicrotubule growth, Nature, 1984,312,237 -242.

Microtubule assembly nucleated by isolated centrosomes, Narure, 1984, 312, 232-237.

Techniques for the study of microtubulc assemhly in uitro, Melh. E~~zymol., 1982, 85, 433 -439.

The colchianc-b~nding protern of mammalian bran and its relatlon to microtubules, Btoehemisrry, 1968, 7, 4466-4479.

The mechanism of actlon of colchicine: colchlcrne bmding properties

01 sca urchin sperm tail outer doublet tubulin, J. Cell B d , 1973, 58, 709-71 9.

Interaction of drugs w t h mmotubule proteins, Fed. Proc., 1974, 33, 158-166.

Action of drugs on microtubules, Lge Sci, 1975, 17, 303-310.

The inhlbitian of mitosis by deuterium, Ann. N.Y. Acod. Sci., 1960,84, 745-754.

Stimulus-secretion coupling of glucose-mduced ~nsulin release V. Pa~ttcipation of a microtubular-m~rofilame~llous system, Dmbetes, 1971.20, 257-265.

Taxol stahilires microtubules in mouse fibroblast wlls, Proc. Norn. Acnd. Sei. USA, 1980, 77, 1561-1565.

Taxol assembles tubulin in the abscnce of exogenous guanosine 5'-triphosphate or microtubular-microfilamentous system, Biochcrnistry, 1981, 20, 3247-3252.

Promotion of microtubule assembly in ultra by taxol, Nature, 1979, m. 665-667.

Temperature-dependent reversible assembly of taxol-treated microtubules, J . Cell Biol., 1987, 105, 2847-2854.

Blochemistry of aclomyosin-dependeilt cell motility (a review], Proc. Nan. Aeud. Sci. USA, 1978.75, 588-599.

264 R. RAVINDRA AND C. E GROSVENOR

50. POLLARD, T. D. AND

WEIHING, R. R.

51. STRAUB, F. B. AND FEuEn, G

52. MARToNon, A,, MOL~NO, C. M. AND GERGELY, ,,

53. Oosawh F. AND KASAI, M.

60. B E N ~ , G . S., FELLINS S. A. AND

H O L ~ H.

61. BENNETT, G. S., FELLINI, S. A,, CRWP,~. M., OTTO, J. I., BRYAN, J. AND HOLTZER, H.

62. FRANKE, W. W., SCHMID, E, OSORN. M. AND WEBE?, K.

65. S T E I ~ R T , P. M., ZACKROFF, R., AYNARDI-WWMAN, M. ~ r n GOLDMAN, R. D.

66. ZIMMERMAN, U-I.P. AND

SCHLWPFU(, W. W.

Actin and myosin and cell movement. CRC Crrt. Rec. Biochum.. 1974, 8 1-65.

Adenosil~etriphosphate, the functional group of sctin. H~ochim. Bh~php. Acta, 19M,4, 455-470.

The binding of dwalent cations to actm, J. BLII. C h m , 19b4, 239, 1057-1064.

Actin. In Subunits in biological syslems, Id.) S. N. Tlmdshell and G. D. Fasman, Dekker, 1971. pi A p. 261.322.

Interaction of actin with annlogs of adenosine triphosphate, Hio- chemistry, 1973, 12, 3927-3932.

The role of the bound nucleotide in the polymerimtion or actin, Biochemistry, 1975, 14, 3250-3256.

The eNects of cytochalasins on actin polymerization and actm ATPasc provide insights into the mechanism of polymerization, i. Biol. Chm., 1980,255,841-844.

Spectrin/actin complex isolated from sheep crythrocytrs accelerntes actin polymerization by simple nucleation. J. Nid. Chmr.. IYXO, 255, 1670-1676.

Methods to measure actin polymerization. Mcrh. Enzymol. 1982, 85, 182-210.

Intermediate filaments: A family of homologous structures. J . Muscle Res. Cell Motri., 1981, 2, 141-166.

Immunofluorescent visualization of 100A Iilaments in different cultured chick embryo cdl types, Differcnriation, 1978. 12. 71.-82.

Dillerences among 100-A filament subunits from dillcrenr cell types, Proc. Natn Acad. Sei. USA, 1978,75,4364-4368.

Direrent mtermediate-sized filaments distinguished by immunofluorescence microscopy, Proc. Notn. Acad. Sri. USA, 1978, 75, 5034-5038.

Intermediate filaments as mechanical integrators of cellular spaa , Nature. 1980, 283,249-256.

Intermediate filaments: a chemically heterogenous, developmentally regulated class of proteins, Annu. Rev. Biochm., 1982.51. 219-250.

Isolation and characterization of intermediate filaments, Meth. Cell. Biol., 1982.24, 399-419.

Characterization of a brain calcium-activated protease that degrades neurofilament proteins, Biochemistry, 1982,21,3977-3983.

Calcium-dependent proteolysis in living cells, Lijp Sci., 1981, 29, 1079-1087.

Comparison of low- and high-calcium requiring forms of the calcium- activated protease with their autowtalytic breakdown products, Biochim. Biophys. Acta, 1982, 709, 166-172


69. GREEN, K. 1. AND GOLDMAN, R. D.

70. SCHLIWA, M. AND

VAN BLERKOM, I.

71. SCHLIWA,M.,PRYZWANSKY,K. B. AND VAN BLERKOM, J

72. WEHLAND, I. AND

WILLINGHAM. M. C.

73. HIROKAWA, N., TILNEY, L. G., Funwhna, K. AND HEUSER, 1. E.

74. HIROKAWA,N., CHENEY, R. E. AND

WaLano, M.

75. FRANKE, W. W., APPELHANS, B., SCHMID, E., FREUDENSTEIN, C., OSBORN, M. AND WEBER, K.

76. BEREITER-HAHN, J., OSBDRN, M., WEBER, K. AND VOTH, M.

77. SCHNITZER. I, FRANKE, W. W. AND SCHACHNER, M.

78. SHARP, G., OSBORN, M. AND

WEBER, K.

79. LAURILA, P., VIRTANEN, I. AND

STENMAN, S.

80. JACOBS, M. AND THOMAS, C.

81. BEYNETT, G. S., TAPSCOTT, S. I., KLEINBART,F.A.,ANTIN, P. B. AND

HOLTZER, H.

82. KNAPP, L. W., O'GUIN, W. M. AND SAWYER, R. H.

83. KNAPP, L W.,O'GUIN, W. M. AND

SAWYER. R H.

84. PHAIKE-WASHINGTON, L., SILVERSTEIN, S. C. AND WANG, E.

85. WANG, E. AND GOLDMAN, R. D.

86. GAWLITTA, W., OSBORN, M. AND

WEBER, K.

The ellects of taxol on cytoskeletal components in cultured fibroblasts and epithelial cells, Cell Motd., 1983, 3, 283-305.

Structural interaction of cytoskcletal components, J . Cell Btoi., 1981, 90,222-235.

Implications of cytoskeletal interactions foi cellular architecture and behavlour, Philos Trans. R. Soc. Lond. B. Bioi Sci., 1982,299,199-205.

A rat monoclonal antibody reacting specifically with the tyrosylated form or u-tubulin, 11. E&cts on cell movement, organization of microtubules and intermediate filaments, and arrangement of Golgi elements, J . Cell Bid, 1983,97, 1476-1490.

Organization of actin, myosin and intermediate filaments in the brush border of intestinal epithelial cells, J. Cell Bzoi., 1982, 94, 425-443.

Location of a protein of the fodrin spectrin-TW 2601240 famrly in the mouse intestinal brush border, Cell, 1983, 32, 953-965.

The organization of cytokeratin filaments in the intestinal epithelium, Eur. J. Cell Biol., 1979, 19, 255-268.

Filament organization and formation 01 microridges at the surface of f ih epidermis, J. Ultrastruct. Res., 1979.69, 316-330.

lmmunocytochemical demonstration or vimentrn in astrocytes and ependymal cells of devebping and adult mouse nervous system, J. Cell Biol., 1981,90,435-447.

Occurrence of two dillerent intermediate lilament proteins in the same filament In s m within a human glioma cell line, Expl. Cell Res., 1982 141, 385-395.

Intermediate filaments in enucleation of human fibroblasts, Expl. Cell Res., 198L 13!,41-46.

The organization of lOnm filaments and microtubules in embryonic neurons from spinal ganglia, J. Neurocytol., 1982, 11, 657-669.

Dllfercnl pro:cinr asroc~ated with lOnm filament, tn cultured u h ~ k nrurons and nonneuronal cells, Snmce. 1981,212, 567-569

Drug-induced alterations of cytokeratin organization in cultured epithelial cells, Science, 1983, 219, 501-503.

Rearrangement of the keratm cytoskeleton after combined treatment with microtubule and microfilament inhibitors, J. Cell Bloi., 1983, 97, 1788-1794.

Phorbol myristate acetate stimulates microtubules and lOnm filament extension and lvsosome redistribution in mouse macroohaaes, J . Cell

Functions of cytoplasmic fibres in intracellular movements in BHK-21 cells, J. Cell Biol., 1978.79, 708-726.

Coilina of intermediate filaments induced by microiniection of a vimentin-specific antibody does not interfere with locomotion and mitosis, Eur. J. Cell Bzol., 1981, 26, 83-90.


88. KLYMKOWSKY, M. W., MILLER. R. H. AND LANE, E. 8.

90. LAZARIDFS, E AND

GRANGER, B. L.

91. TRAUB, P. AND VORGIAS, C. E

93. GEORCAT& S. D. AND BLOBEL, G.

94. GEDRGATOS, S. D. AND B ~ B E L , G.

98. HOWELL, S. L.

99. PELLETIER, G. AND LABRE F.

100. FARQUHAR, M. G.

101. ENoEns, A. C .

102. Smhuss, II1.J. F., SCHULER, L. A,, ROSENBL~M, M. F. AND

TANAKA, T.

103. GEMMELL, R T., STACY, B. D. AYD THORNBURN, G. D.

104. SA~YEF., H. R., A m , Jn. J. H., MCCLELLAY, M. C., SCHMITZ, M. At% NISWENDER, G. D.

105. QUIRK, S. I., WILLCOX, D. L., PARRY, D. M. ~hv THORBuRN, 0. D.

Intermediate filaments in 3T3 cells collapsc after intracellular injection ofamonoclonal anti-intermediate filament antibody, Noture, 1981,291, 249-251.

Morphology, behaviour, and interaction ofcultured epithelial cells after the antibody-induced disruption of keratin filament organization, .l. Cell Biol., 1983, 96,494-509.

Epithelial tonofilaments: investigating their form and function using monoclonal antibodies, Cold Spring Harbor Symp. Quant. Biol., 1982, 46,387-402.

Transcytoplasmic integration in avian erythrocytes and striated muscle: the role of Intermediate filaments, Mod. Cell Btol., 1983, 2, 143-162.

Differential effect of arginmne modification with I, 2-cyclohexanedione on the capacity of vimentin and desmln to assemble into intermediate llaments and to bind to nucleic acids, J . Cell Sci., 1984,65, 1-20,

Intermediate filaments: looking lor a function, Noture, 1987, 329, 392-393.

Two distinct attachment sites for vlmentin along the plasma membrane and the nuclear envelope in avian erythrocytes: A basis for a vectorial assembly of intermediate filaments, J. Cell Biol., 1987, 105, 105-115.

Lamin B constitutes an intermediate filament attachement stte at the nuclear envelope, J . Cell Biol., 1987, 105, 117-125.

Pathways of protein secretion in eukaryotes, Science, 1985,230,25-32.

Role of calcium in the regulation of hormonal secretion, Hormone Res., 1984,ZO. 28-37.

Intracellular aspects of the process of protein synthesis, Science, 1975, 189, 347-358

The mechanism of insulin secretion, Dloberologie, 1984, 26, 319-327.

Anterior pituitary secretary graules. In The secretory granule (ed.) A . M . Poisner and J . M. Trifaro, 1982, pp. 173-212, Elsevier.

Processing ofsecretory products by cells ofthe anterior pituitary gland, Mem. Sot. Endocrinol., 1970, 19, 79-124.

Cytology of the corpus luteum, Biol. Reprod., 1973, 8, 158-182

Cholesterol metabolism by ovarian tissue, Adv. Lipid Rex, 1981, 18, 99-157.

Ultrastructural study of secretory granules in the corpus luteum of the sheep during the estrous cycle, Biol. Reprod., 1974, 1 1 , 447-462.

Secretory granules and progesterone secretion by ovine corpora lutea in aitro, Endocrinology, 1979, 104, 476-486.

Subcellular location of progesterone in the bovine corpus luteum: A biochemical, morphological and cytochemical investigation, Biol. Reprod., 1979,20, 1133-1 145.


106. RUEIN. R. P., SHEILD, B.. MCCAULEY, R. AND

LAYCHOCK, S G

107 GEMMLLL, R. T . LAYPHOCK, S. G. AVD

RIJBIN, R. P.

108 PEAKC€, R B , CRONSHAW, J. A N D

HOLMES, W. W.

112. KLETZEIN, R. F., PERDUE. J. F. AND SPRINGER. A.

11 3. ESTENSEN. R D. AND

PI.AGLMANN, P. G. W.

114. C n c ~ , M. p., LYNN, D. G. AND

I.YNN, W. S.

115 BLOCH, R.

117 LEWIS, C. E., MORRIS, J. F. AND

FINK, G

119 YOUNG, L. S., NAIK, S. I. AND

CLAYTOX, R. N.

ACTH-induced proteln release from the perfused cat adrenal gland: evidence for exocytosis? Endoa.~nolog, 1974.95, 370-378

Ultrastructural and biochemical evidence for a sterod-containing secretory organelle in the perfuscd cat adrenal gland, J Cell Blol., 1977, 72, 209-215.

The fine structure ofthc interrenal cells oftheduck (Anas oiati,rhvnchosl . . . with evldence for the possible exocytotlc release uistemds, Cell Tissue Res., 1977, 183, 203-220.

Etude h~stologique de la transformation du iollicuie preovulatoire en corps janue cycliqur chez la brebls, Ann. Bid. Anim. Bmchim. Btuphys. (Suppl.), 3973, 13, 249-260

Cytochalaain 8: lnhlbitlon of D-2-dcoxy glucose transport mto leukocytes and fibroblasts, Scknee, 1972,176, 1432-1434.

Amodelforthc modcolact~onofcytochalasin B lnhibttion ofD-glucose transport in the human erythrocyte, Con. J Hiochrm. 1975, 53, 1078-1084.

Cytochalasin A and B: inhibition of sugar uptake in cultured cells, J. Bid. Chrm., 1972.247.2964-2966.

Cytochalasin B: Inhibition of glucose and glucosaminc transport, Proc. Natn. Acad. Sci. U S A , 1972, 69, 1430-1434.

Cytochalasin B-sensttive 2-deoxy-D-glucose transport in adipose cell ghosts, J. Biol. Chem., 1973, 248, 3636-3641.

Inhibltian of glucose transport m the human crythrocyte by cytochalasin B, B~ochemistry, 1973, 1 2 4799-4801.

Effects ofmod~fiers of cytoskeletal structures on the dynamics of release of LH from sheep anterior pituitary cells stimulated w~th gonadotrophin-releasing hormone, K' or phorbol ester, J . Endndocr., 1987, 112, 289-298.

The role of microfilaments in the primlng effect of LH-releasing hormone: an ultraslructural study usingcytochalasin B, ibid., 1985,106, 211-218.

Pruning effect of lutemmng hormone relcasmg factor in mtro: role of protcin synthesis, contractile elements CaZ ' and cyclic AMP, hid, 1979,81,223-234 '

Pltultary gonadotrophin-releas~ng hormone reccptor up-rcgulatlon In vitro. dependence on calc~um and m~crotuhule function, ihid., 1985, 101,49-56.

InteractionofGnRH with anterior pituitary 111. Roleofdivalenlcatlons, microtubules and miciotilamenls in the GnRH activated eonadotrooh, B i d Reprod., 1979,21, 1073-1086.

Involvement of microtubule and microfilament system m the GnRH induced relcasc of gonadotropins by rat anterior pituitary cells in culture, FEES Lett., 1979, 104, 410414.

268 R. RAVINDRA AND C. E GROSVENOR

122. KKAICEK, J. ANU

MILLIGAN, J. V.

123. PELLETlEh G. A N D

BORNSTEIN, M. B.

125. SHETERLINE, P. AND

SCHOFIFLD, J. G.

126. SHETERLINE, P., SCHOFIELU, 1. G. AND MIRA, F.

127. MACLEOU, R. M., LEHMEYER, 1. E AND BXUNI, C.

128. LAURIE, F., GAUTHIER, M., PELLETIEK, G., BOKGEAT, P.. LCMAY, A. AND GOUGE, 1. J.

129. GAUNIK, K. M. AND

TASHIIAN. A. I. JR.

130. ANTAKLY, T., PELLETIER, G., ZEYTINCGLU, F. AND LABRIF, F.

132. SUNDBERG D. K., KKULICH. L., FAWCETT, C. P., ILLNEK, P. ANU

M ~ C A N N , S M.

133. SHINO, M. WARCHOL, 1. B. AND

RLNNELS. E. G.

134. HOLCK, S., ALBKECHTSEN, R. AND WEWER, U. M.

135. VALE~TI, C. E., LOMBARUO, R. J., LIBEKTUN, C. AND

CARUINALI, D. P.

136. VALENTI, C. E., VACAS, M. I. AND

CARDINMI. D. P.

137. I'ROUILLAS, J., GIROD, C., SASOUS, F. AND CLAUSTRAT, B.

138. K o v ~ c x K., HDRVATII, E., VANLWN. G. R., REWCASTLE. N. B., EZKIN, C. *NU ROSENBLWM. A. A.

Effcct of colchicrne on in nitro ACTH release indued by high K t and by hypothalamus stalk-median eminence extract, Endocrinoiogy, 1971. 89,408-41 2.

Eflcct of colchicine on rat anterior pttuitary gland in lissue culture, Expl. Cell Res., 1972, 70, 221-223.

The effect oi secretagogues and 2-methyl-pentan-2,4-diol on the minotubule-tubulin equilibrium and the release of growth hormone Rom bovme antenor pituitary slices, Expl Cell Rex, 1977, 104, 1 2 7 134.

Endogenous phosphorylation and dephosphorylation of microtubule- associated proteins isolated from bovine anterior p~tuitary, FEBS LEtt., 1975,56, 297-302.

Colchicine bindlng to bovrne anterior pituitary slices and inh~hition of growth-hormone release, Biochern J., 1975, 148,453-459.

Eflect oiantl-mitotic drugs on the in uitro secretory actlvlty olmammo- trophs and somatotrophs and on their microtubules, Proc. Soc. Expl Bwl. M d . . 1973. 144, 259-267.

Role of mmotubules in basal and stimulated release ofgruwth hormone and prolactin in rat adenohypophysrs in urtro. Endocrrnology, 1973, 93, 903-914.

ElTects of colchicine on the morphology and prolactin secretion ol rat anterior pituitary cells in monolayer culture, Am. J. Anat., 1979, 156, 353-372.

Role of microtubules in the intracellular transport of growth hormone, Cell Trssue Res., 1978, 190, 163-Ill.

Theeffect ofcolchic~ne on the release of rat antenor pituitary hormones rn vitro, Proc. Soc. Expl B i d . Med., 1973, 142, 1097-1100.

Microtubules in prolactin cells oithe rat anterior prtu~tary gland, Proc. Soc. Expl Bioi. Mrd., 1974, 147, 361-365.

Laminin in the antenor pituitary gland of the rat, Lab. Inuert., 1987.56, 481-488.

Changes In total and polymerized tubulin of the medial basal hypothalamus and adenohypophysis of castrated or hormone-injected rats, Experentio, 1980, 36, 1012-1014.

Effects of castration, estradlol and testosterone on tubulin levels of the medial basal hypothaamus and the adenohypophysis of the rat, Experenrio, 1979, 35, 120-122.

The human gonadotrophic adenoma: pathologic diagnosis and hormonal correlations in 26 tumors, Semin Diagnostic Path., 1986, 3, 42-57.

htuitary adenomas associaled with elevated blood follicle-stimulating hormone levels: a h~stologs, immunocytologic, and electron microscopic study of two cases, Fert. Steril, 1978, 29, 622-628.

CYTOSKELETON AND HORMONE SECRETlON 269

139. NIWA, J., MORI, M., MINASE, T. AND HASHI, K.

140. RAVINDRA, R. AND

GnDSvENoR, C. E.


GROSENOR, C. E.


GRosvENoR, C. E.


GROSVENOR, C. E.

145. BURNET, F. R. AND

WAKERLEY, J. B.

146. CHES, H. J., MUELLER, G. P. AND

MEITES, J.

147. GUILLEMAS, R., VARGO, T., ROssrEn, I., MINICK, S., LING, N., Rlvan, C., VALE, W. AND

BLOOM, F.

148. VERLEY, J. M. AND

HOLLENMANN, K. H.

149. RAVINDR~ R. AND

GRDSVENOR, C. E.

150. WHITWORTH, N. S., GRDSVENOR, C. E. AND MESA, F.

151. SHERLINE, P., LEE, Y.-C. AND

JACOBS, L. S.

152. BLOOM, G. S., LUCA, F. C. AND

VALLEE, R. B.

153. BENZONANA, G., DREIPUSS, 1. 1. AND GABBIANG G.

154. OSTLUND, R. E., LEUNG, 1. T. AND KIPNIS, D. M.

155. SHARP, G. W. G., WOLHEIM, C., MULL& W. A., GUTZEIT, A,, TRUEHART, P. A,, BLONDEL, B., ORCI, L. AND RENOLD, A. E.

156. LACY, P. E.

lrnmunofluorescence demonstration of tubulin and actin in estrogen- rnduced rat prolact~noma cells In uitro, Exyl Cell Res., 1985, 161, 517-524.

Microtubules are mobilized in the lactating rat anterior pituitary gland during suckhng, Endocrinology, 1986, 118, 1194-1199.

Soluble and polymerized tubulin levels in the anterior pituitary lobe of the lactating rat dunng suckling, [bid., 1988, 122, 114-119.

Eflect of suckling on pituitary GTPase activlty ln the lactatmg rat, Physiologist, 1985.28, 353 (Abstract).

Suckling inlluences the in uitro assembly of anterior pituitary gland tubulrn into microtubules in the lactating rat, Molec. Cell. Endocr., 1987,54, 231-237.

Stimulation of pituitary prolactln and TSH release in lactating and proestrus rats, Endocnnology, 1974, 94, 503-508.

Plasma concentrations of prolactin and thyrotropin durlng suckling in urethane-anesthetned rats, J. Endocr., 1976,70,429437.

Eflects of L.dopa and somatostatin on suckling-induced release of prolactin and GH, Endocr. Res. Commun., 19?4,1,283-291.

8-endorphin and adrenocorticotropin are secreted concomitantly by the pituitary gland, Sctence, 1977, 197, 1367-1369.

The anterior pituitary of the mouse during pregnancy and lactation: a quantitative electron microscope study. In Lactation, (ed) Falconer, I. R. Pennsylvania State University Press, pp. 93-112.

Dopaminergic regulation of microtubule mobilization in the anterior pituitary during suckling in the rat, 68th Annual Meeting of the Endocrine Society, USA, June 1986, Anaheim, CA, Abstr. # 686.

Autofeedback regulation of prolactin (PRL) secretion: effect of PRL before suckling on the subsequent nursing-induced release of PRL in the lactating rat, Endocrinology, 1981, 108, 1279-1284.

Bindmg of microtubules to pituitary secretory granules and secretory granule membranes, J . Cell Biol., 1977, 72, 380-389.

Identification of high molecular weight microtubule-associated proteins in anterior pituitary tissue and oells using taxol-dependent purification combined with microtubule-associated protein speci~ic antibodies, Biochemistry, 1985,24,4185-4191.

Actin is unevenly distributed in the pituitary gland, Cell Tissue Res., 1979, 200, 123-133.

Muscle actin filaments bind pituitary secretory granules in uitro, J. Cell Biol., 1977, 73, 78-87.

Mechanism of msulin release, Fed. Proc., 1975.34, 1537-1548.

Endocrine secretory mechan~sms, Review, Am. J. Pnthol., 1975, 79, 170-188.


157. PIPELEERS, D. G., PIPELEERS-MARCICHAL, M. A. AND KIPNl$ D. M.

158. SUPKENANT, K. A. AND DENTLER. W. L.

159. MALAISSE, W. J., YALAISSE-LAGAE, F., VAN OBBERGHAN, E., SOMERS, G., DEVIS, G., RAVAZZOLA, M. AND

ORCI, L.

160. MALAISSE, W. 1. AND ORCI, L.

161. MOSKALEWSKI, S., THYBERG, 1. AND Fnamc, U.

162. Eownnos, J C. AND

HOWELL, S. L.

163. Kov~cs , K., HORVATH, E., Szaso, S., DLAU, V. J.,

. CHANG, Y. C., FELDMAN, D. AND

REYNOLDS, E. S.

164. TEMPLE, R. AND WOLFF, J.

167. SACKETI, D. L. AND WOLF?, J.

168. ANNUNZIAM, L., DIRENZO, G. F., SCHETTINI, G , SCAPAGNIN, U. AND PREZIOTI, P.

Microtubule assembly and thz intracellular transport of secretory granules in pancreatic islets, Science, 1976, 191, 88-90.

Association between endocrine pancreattc secretory granules and m uirro assembled microtubules is dependent upon m~crotubule- associated proterns, J. Cell Biol., 1982,93, 164-1 74.

Role of minotubules in the phasic pattern of insulin release, Ann. N.Y. Acad. SCI., 1975,253, 630-652.

The role ofthe cytoskeleton in pancreatic B-cell iunction, Meth. Arhiru. Ewpi Puthol., 1979, 9, 112-136.

In ~ i t ro influence of colchic~ne on the Golgi complex in A- and B-cells of guinea p ~ g pancreatic islets, J. Ultraaruci. Res., 1976, 54, 304-317.

Effects of vinblastine and colchicine on the secretion of glucagon from isolated guinea-pig islets 01 Langerhans, FEBS Lett., 1973, 30, 89-92.

Effect of vlnblastme on the h e structure of the rat adrenal cortex, Horm. Metnb. Rrs., 1975, 7, 365-366.

Stimulation of steroid secretion by antimicrotubular agents, J. B i d Chem., 1973,248, 2691-2698.

The role oftubulin in the steroldogenicresponse of murine adrenal and rat Leydig cells, Endocrinology, 1981, 109, 2261-2263.

Theeffect oicolchicine on thein moo and mvirrosecretion ofaldosterone by rat adrenocortical cells, FEBS Letf, 1982. 140, 77-79.

Cyclic Ah4P-independent stimulation of steroidogenesis in Y-1 adrenal tumor cells by anti-mrtotic agents, Biochirn. Biophys. Acta, 1986, 888, 163-170.

Increased plasma corticosterane and decreased plasma thyroid- stimulat~ng hormone levels in rats treated with vmcn'stine. Cancer Res., 1977,37,2574-2577.

Stimulation of sterold synthesis by normal rat adrenocortical cells in response to antrmlcrotubular agents, Endocnnoloy): 1978, 103, 1281-1288.

Monolayer cultures of normal adult rat adrenocortical cells: steroidogenic responses to nucleotides, bacterial toxins, and antimicrotubular agents, Experientia, 1976, 32,251-253.

Cytochalasin-stimulated steroidogenesls from high density lipoproteins, J . Cell Biol., 1978, 77, 507-516.

Cellular organization for steroidogenesis, Inr. Rev. Cytoi, 1984, 86, 53-95.

Effects of cytochalasin B on unstimulated and adrenocorticotrapin- stimulated adrenocort~cal tumor cells in nitro, Endocrinology, 1982, 111, 1632-1647.

CYTOSKIIPTON AND HORMONE SECRETION 27 I

'Ihe ckct of cytochaias~n D on steroid pioduc:~on and stress fiber argan~zation in cultured bovine adienocort~cal cells, Mo1i.c. Cell. Endocr., 1984, 35. 189-197.

Functional and structural diiferences between cultured uutcr and inner layercells oibov~ne adrenal cortex, Cell Ttssuc Res., 1987,250,507-512.

Morpholog~cal correlates of adrenocorticotroo~n stlmuialed steio~do-

174. RA~NEY, W. E , SHAY, J. W. AND

MASON, .I. L

176. RAINLY. W. E., HORNSUY, P. J AND SHAY, I. W.

177. LOESSER, K. E AND MAIAMED, S. A morphomctrlc analysis 01 adienocortd actin locallzed by immuno- clcctron microscaw the efect o i adrenocorticotromn. Endocnnoioov.

178. CHLIILIY, R. A AND

RAMACHANDRAN, J Regulation of actin in rat adrenocortical cells by cortmtropm, J. Blol. Chem., 1981, 156, 3156-3158.

Purtficat~onot rat adrenocort~cal actm andits use ~n an ~mmunopreapi- tation assay to quantitate cellular actal, Bioclum. Biophys. Acta, 1986, 883, 383-387.

180. Soro, E. A., KLINMAN, If. J. Srnhvss Ill, J. F. AND

PAAVLOVA, L. C .

Gonadotrop~ns and CAMP alter the morphology ol cultured human granulosn cells, B i d R q r o d . , 1986, 34, 559-569.

Microtubulcs and the calcium-dependent rcgulatian of rat granulosa cell steroldogenesls. Biol. Reprod., 1987, 36, 1007-1015.

Microtubules and the gonadotropic regulation of granulosa cell steroidogenesis, Endocrinoiogy, 1987, 120, 819-828.

Thc cytoskcleton and rat granulosa cell steroidogenesis: possible involvement ofm~crotubules and microlilaments. Bioi. Reprod., 1988, 38, 100-108.

184. CAKNECIE, J. A., BYARD, R., DARUICK, I. AND TSANG, B. K.

Culture ofgranulosa cells in collagen gels: the Influence of cell shape on steroidagcnesis, B i d . Reprod., 1988.38, 881-890.

185. ZOR, U., STRULO\IICI, B. AND

LINDER, H. R. Implication ofmicratubules an2 microlilaments in the response of the ovarian adenylate cyclase-cyclic AMP system to gonadotropins and prostaglandm E,, Biochtm. Biophys. Res. Comrnun., 1978.80.983-992

The morphological transformation and inhibition of growth of bovine luteal cells in t1s3ue culture induced by luteinizing hormone and dibutyryl cyclic AMP. Endocrmoiogy, 1975,96,458467.

Receptor-mediated gonadotropin aclm in the ovary, R~ochem. J., 1981, 194, 19- 27.

ElTects of cytochalasin B, colchicine, and vinblastme on progesterone synthesis and secretion by bovine luteal tissue ~n oztro, J. Reprod Fert. 1982,65, 151-156.

189. RODGtRS, R. J., O'SHCA, J. D A N D

FINDLAY,'J. K. Do small and luge luteal cells of the sheep Interact in the production ofprogcslerone? ihld, 1985,75,85-94.

Cytochalasins and colchicine increase the lateral mobillty of hutnan chormnic gonadotropin-occupied luteinizing hormone receptors on ovine luteal cells, Endocrinology, 1988, 122, 261-269.

191. GEMMFLL, R . T . AND STACY, B. D. Effects ofcolch~ine on the ovine corpus lutcurn: role ofmlcrotubules in the secretion of pregesterone, J. Reprod. Fert., 1977, 49, 115-117.


192. HALL, A. K. AND ROBINSON, J.

196 BEATTACHARYYA, B. AND

WOLFF, 1.

206. PIETRAS, R. J., HUTCHENS, T. W. AND SZEOO. C. M.

207. BAULIEU, E. E., GODEAU, F.. SCHORDERET, M. AND

SLATKINE, S. S.

208. MILGROM, E., ATOER, M. AND

BAULIEU, E. E.

209. BAULIEU, E. E., ATOER, M., BEST-BELPOMME, M., CORROL, P., COURVALIN, J. C., M~srrn, J., MILOROM, E., ROBEL, P., ROCHEFORT, H. AND

DE CATOLOGNE. D.

Are microtubules involved in luteolysis? J. Endocrinol., 1978, 79, 42-43P (Abstr).

ENect ofantim~crotubule agents on microtubules and steroidogenesis In luteal cells, Am. 3. Phys., 1982,2433, 380-386.

Involvement of microtubules m the effect of estradiol on progesterone secretion by hamster corpus luteum, J. Indian lnst. Sci., 1983, 64(C), 37-44.

Microtubular proteins in pigeon erythrocyte membranes, Eur. J. Biochem., 1976,71, 177-184.

Membranebound tubulin in brain and thyroid tissue, J. Biol. Chem., 1975,250,7639-7646.

Effect ofestradiol on corpus luteurnfunction in pregnant humster, Ph.D. Thesis, Indian Institute of Science, Bangalore, India, 1982.

ElTect of estradiol on corpus luteum function in pregnant hamster, J. Indian Insr. Scr, 1983, 64(C), 11-25.

ENect ofestradiol on the in uitro assembly of rat brain tubulin, J. Indian Znst. Sci., 1983, 64(C), 27-35.

Cell membrane, a target for steroid hormones, Molec. Cell Endow., 1978,12,247-254.

Endometrial cell calcium and oestrogen action, Nature, 1975, 253, 357-359.

Surface modifications evoked by estradiol and diethystilbestrol in 1sola1.-d end,mclnnl atI1, cvldence f r m lnvn proks dnd errraccll~lar r:lcare 31 l~s010m~1 proleare. tnJccrmo.og.. 19'5, 97. I44-l45J

Specific binding sltes for oestrogen at the outer surfaces of isolated endometnal cells, Nature, 1977, 265, 69-72.

Estrogen receptors in uterine plasma membrane, J 1979, 11, 1471-1483

. Steroid Biochem.,

Metabolic and proliferative responses to estrogen by hepatocytes selected for plasma membrane bind~ng-srte speak for P estradiol-17, J. Cell Physiol., 1979, 98, 145-160.

Hepatocyte plasma membrane subfractions ennched in high-afinity, - . low capacity bmdmg sltes specific for estradiol-17B, Endocrinology, 1978, 102 (Suppl.), 76 (Abstr).

Steroid induced meiotic d~vision in Xenopus laems oocytes: surfdce and calcium, Nature, 1978, 275, 593-595.

Studies on estrogen entry into uterine cells and on estradiol-receptor complexattachment to thenucleus-istheentry ofestrogeninto uterine cells a protein mediated process? Biochim. Biophys. Acta, 1973, 320, 267-283.

Steroid hormone receptors, Vitam. Horm. (N.Y.) 1975.33, 649-736.


210 PECK, E. 1. In., BURGNER, J. AND

CLARK, 1. H.

21 I. MEAN$ A. R. AND

O'MALLEY, B. W.

212 SCHIMKE, R.T., MCKNIGHT,G S., SHAPIRO, D. I., SULLIVAN, D. AND

PALACIOS, R.

213. PALMITER, R. D., MULVIHILL, E. R., MCKNIGHT, GG. S. AND

SENEAR, A. W.

214. GORSKI, J AND GANNON, F.

215. JACKSON, V. AND CHALKLEY, R.

216. WILLIAMS, D. AND GORSKI, J.

217. Glon~l , E. P., MOSES, T. F., GRANT, J. K., SCOTT, R. AND

SINCLAIR, 1.

218. HARRISON, R. W. Ill., FAIRF~ELD, S. AND ORTH. D. N.

219. SZEGD. C. M.

225. WHEELER, W. J. HSU, T. C., TOUSSON, A. AND BRINKLEY, 8. R.

226. BAULIEU, E. E. AND

SLATKINE, S. S.

227 DROUVA, S. V., LAPLANIE, E. AND KORDON. C.

Estrophilic btnding sites of the uterus Relatlon io uptakeandretentlon of estradiol in nitro, Biochemistry, 1973, 12,45964603,

Female steroid hormones and target cell nuclei, Science, 1974, 610-620.

Hormonal regulation of ovalbumin synthesis in the chick oviduct, Rec. Progr. Hormone Res., 1975, 31, 175-211.

Regulat~on of gene expression in the chick oviduct by stenod hormones, Cold Spring Harbor Symp. puont. BloL, 1977, 42, 639-647.

Current models of steroid hormone action: a critique, Annu. Rev. Physiol., 1976, 38, 425-450.

The cytoplasmic estradiol receptors of bovine uterus: their occurrence, interconversion, and binding properties, J. Biol. Chem., 1974, 249, 1627-1636.

Preparation and characterization of free cell suspensions from the rat uterus, Biochemistry, 1973, 12, 297-306.

In uitro studies on the regulation of androgen-tissue relatton in normal canine and hyperplastic human prostate, Molec. Cell. Endocr., 1974, 1, 271-284.

Evidence for glucocorticoid transport into AtT-2Op-I cells, Biochemistry, 1975, 14, 1304-1307.

Lysosotnal function in nucleocytoplasmic communication. In Lysosomes in biology and pathology, (eds) Dingle, 1. T. and Dean, R. T., Vol. 4, pp. 385-477, North Holland, 1975.

Mechanisms of hormone action: parallels in receptor-mediated signal propagation for steroid and peptide erectors, Life Sci., 1984, 35, 2383-2396.

Subcellular distribution of oestrogen receptors, Nature, 1985, 317, 88-89.

Estrogen action at endometrial membranes: alterations in luminal surface detectable within seconds, J . Cell Biol., 1983, 97,679-685.

Specific internalization of estrogen and binding to nuclear matrix in isolated uterine cells, Biochem. Biophys. Res. Commun., 1984,123,84-91.

Receptor identification by density gradient centrifugation, Meth. Enrymol., 1975, %A, 156-166.

Ul101l; ~ n h l b x ~ o n and chroao,ome dlsplvrement mduced by es~radio! m Chmcsr hamster cells, Cell Moril C ~ r o r k ~ l e m n , 198', 7. 235-247.

Steroidal peptidic control mechanisms in membrane of Xenopus lenuis oocytes resuming meiotic division, J. Steroid Biochem. 1983, 19, 139-145.

Progesterone-induced LH-RH release in uitro is an estrogen-as well as Ca" and calmodulin-dependent secretory process, Neuroendocrino- logy, 1985,40, 325-331.

Calcium binding to bovine brain tubulin, FEES Lett., 1975,58,222-225.

274 R. RAVlNDRA AND C E. GROSVENOR

229 ROSENFELD, A. C., ZACKROFF, R. V. AND WElSENl%ERG, R. C.

231. Soenm, M. L., COUDERC, I. L., CHAPEL, S. AND DASTUGUE, B.

232 SATO, Y., M ~ A I , T., TSUMIJRAYA, M., SAITO, H. AND

KODAMA, M.

233. SAm, Y., MURAI, T., ODA, T., SAITO, H., KODAMA, M. AND

HIRATA, A

234. SHARP, D. C AND PARRY, I. M.

236. BAIAT~, A. J. AND BEN~FTT, v. 237. SHEFTZ, M. P. AND

SPUDICH, I. A.

238. GIBBONS, I. R.

239. BRADY S. T.

Magnes~um stimulation of calcium binding to tubulin and calcium induced depolymer~zatlon of microtubules, FEBS Lett., 1976, 65, 144-147.

Morphological transformation of Chinese hamster cells by dibutyryl adenosine cyclic 3':s-monophosphate and testosterone. Proc. Nafn. Acad. Sci. USA, 1971.68, 358-361.

Expression of new Btubulin subunit is induced by 20-hydroxyecdysone in drosophila cultured cells, Biochem. Biophys. Res. Commun., 1986,134, 191-2W.

Disruptive effect of dicthylstdbestrol on microtubules, Gann., 1984,75, 1046-1048.

Inhibition of microtubule polymerization by synthetic estrogens: formation of a ribbon structure, J. Biochem., 1987, 101, 1247-1252.

Diethylstilbestrol: the bind~ng and eilects of diethylstilbestrol upon the polymerization and depolymerization of purified microtubule protein in uitro, Carcinogenesis, 1985, 6, 865-871.

Neurotransmitter release mechanisms and microtubules. Proc. W Soc. Lond., 1983, 218B, 253-258.

Movements ofmyosin-coated fluorescent beads on actin cables in uitro, Nature, 1983, 303, 31-35.

Cilia and flagella of eukaryotes, J. Cell Bid, 1981, 91, 107s-124s.

A novel brain ATPase wtth propertla expected for the fast axonal transport motor, Nature, 1985,317, 73-75.

Identificat~on of a novel force-generating protein, klnesin, involved in mlcrotubule-based matihty. Ce% 1985, 42, 39-50.

Localization of kinesin in cultured cells, J. Cell Bid, 1988, 1193-1204

Retrograde transport by m~notubule-associated proteln MAP IC, Nature, 1987, 330, 181-183.

I. Indian Imt. Sci, July-Aug., 1989,69,275-283 "indian Inst~tute of Science.

ytic algal flora of Phvagmites communis trim

S. G. SARWAR AND D. P. ZUTSHI* Hydrob~ology Research Laboratory, S.P. College, Srinagar 190001, Kashmir (India).

Recaved on Aprd 7, 1988: Revised on September 21, 1988

Abstract

The paper describes the distr~bution and community structure of periphytan on Phrogmztes eommunis Tnn. in three Kashmir Himalayan Valley lakes. One hundred and six tax& representing seven algal classes, were recorded of wh~ch 62 were common to the lakes. Numerically Bac~llariophyceae was dommant both in terms olthe number of taxa and abundance. Its contribut~on to the total population density was almost always more than 50%.

Key words: Community structure, penphyton, bacillar~ophyceae, Kashmir Himalayan Valley lakes.

1. Introduction

The community of microscopic-attached organisms composed of algae, bacteria, fungi, protozoa and small metazoa is called periphyton (syn. aufwuchs). Such communities are responsive to environmental changes and are excellent biological indicators of the degree of eutrophication. In India less attention has been paid to the ecological role of attached algae'-5 relative to the more easily studied phytoplankton. Even this group serves as a sort of storehouse habitat for the accelerated growth in favourable seasons. From June 1982 to May 1983, a detailed qualitative and quantitative investigation was carried out on the algal component of the periphytic flora of an emergent macrophyte (Phragmites communis Trin.) of three Kashmir Himalayan Valley lakes-Dal, Anchar and Waskur. Features of these lakes are described elsewhere5.

2. Material and methods

Sampling was done every month between 1000 and 1200 h IST near the reed belts of the lakes. Surface water samples, collected near the reed belts, were anaiysed following the methods of Mackereth6; APHA7, and Golterman et a18. Since the periphyton attachment to the substrate is effected by mucilage-like polysaccharides, their removal was accomplished by a combination of agitation and acid hydrolysis with FAA (10:7:2:1::95%

'Present address: CORD. The Univers~ty of Kashmir, Srlnagar 190006.

275

276 S. G. SARWAR AND D. P. ZUTSHl

ethano1:water:formalin:glacial acetic acid) following the method of Gough and Woelkerlingg. The macrophyte material was treated with FAA, shaken vigorously for few minutes and then filtered through muslin. The macrophyte fragments, retained by the muslin. were oven-dried at 105°C till constant wei~ht was obtained. Counting of the algal component of the periphytic forms was done every month in a Sedgwick rafter cell and the results are expressed as units per 10 mg dry weight of the macrophytic material. The general principle of huantitative expressions of units has been adopted from Tucker1'. The community coeflicient has been calculated after Taylor".

3. Results and discussion

Average values of various physico-chemical parameters for the three lakes are given in Table I. Major variations in these parameters are not discernible except in the case of conductivity, total alkalinity, calcium, iron and total phosphorus. Calcium and magnesium contents of Anchar and Waskur Lakes were almost double that of Dal Lake.

Sixty-two taxa out of 106 recorded from the investigated lakes were common. They belonged to six classes of algae: Bacillariophyceae, Chlorophyceae, Cyanophyceae, Dinophyceae, Euglenophyceae and Cryptophyceae. In addition, Chrysophyceae was represented by Dinobryon sp. in Dal Lake (Table 11).

The sequence of the dominance of four major algal classes was Bacillariophyceae 1

Table I Average values for various physieo-chemical parameters of investigated lakes

Parameter Lakes

Specific conductivity

pH Total alkal~nity Chloride Calcium Magnesium Sodium Potdssium Dissolved oxygen Silicate Iron Ammonical-nitrogen Nitrate-nitrogen Orthophosphorus Total phosphorus Sulphate

Dal -

133

8.4 71.9 18.9 19.4 4.1 3.3 2.0

11.1 2.0

144 10 48 22 66 0.2

Waskur -

294

PERlPHYTlC ALGAL FLORA O F PHRAGMITES COMMUNIS TRIN. 277

Table I1 Average abundance of periphytic taxa

Name of the taxon Dal lake Anchar lake Waskur lake

A) Bacillariophynae Achnanthes mtnutissima Kutz. Amphora bitumrda Prowse A. normanr Rab. A. oualis Kutz. Asterionellaformosa Hass. Ceratoneis arcus (Ehr.) Kutz. Cocconezs placenrula Ehr. Coscrnodiscus sp. Cyclotella sp. Cymatopbura solea (Breb) W.Sm. Cymbella ofinis Kutz. C. lanceolata (Ehr.) Brun. C. prostrata (Berk.) CI. C. tumida (Breb.) Van Heurck C. turgida (Greg.) CI. C. uentricosa (Kutz.) Meist Diatoma eionyatum (Lyngb.) Ag. D. hiemale (Lyngb.) Herib. Diatomella balfouriana Grev. Diploneis ellrptica (Kutz) CI. Epithemia sorex Kutz. Eunotia diodon Ehr. E. pectinalis (Kutz.) Rab. Frayilaria capucina Desmaz. F. construens (Ehr.) Grun F. crotonensis Kitton F. vaucheriae (Kutr) Peterson Frusrulia rhombordes (Eht) & Toni Gomphoneis herculeanum (Ehr.) CI. Gomphonemn aeuminatum Ehr. var. coronaturn (Ehr.) W Sm. G. angustaturn (Kutz.) Rab. G. augur Ehr. G. consrrietum Ehr. G. geminaturn Ag. G. olivaceum (Lyngb.) Kutz. G. sibrio Ehr. Gyrosigma scolproides (Rab.) CI. Hanrzschia amphioxys (Ehr.) Grun. Melosira sp. Meridion circulare (Grov.) Ag. Nauicula elegantoidrs Hust. N. rhyneocephola Kutz Nauicula spp. Nedium dubium Hust. N~tzschia spp.

278 S. G. SARWAR AND D. P. ZUTSHI

Table I1 (conliauedj

Name of the taxon Dal lake Anchar lake Waskur lake

Pinnularia borealis Ehr. - Rhopaladia gibba (Ehr.) 0. F. Mull. F Stauroneis anceps Ehr. R Synedra spp. F Tabellaria jenestrata (Lyngb.) Kutz. -

B) Cyanophyceae Anabaena sp. R Chroococcus turg~dus (Kutz.) Nag - Coelosphoerium sp. R Gloeotriehia pisum Thuret ex. Born. et Flah R Gomphosphaeria sp. F Lyngbya conrorra Lemm. R Merismopedia eleqans A. 01. - Microcystis aeruginosa Kutr. SD Nostoc sp. R Oscillntoria sp. F Rzuularia sp. R Spirulina sp. R Toiyporhrix tenuis Kutz. -

C) Dinophyceae Glenodinium sp. F Peridinium sp. F

D) Chlorophyceae Ankistrodesmus Jolcatw (Corda) Ralls. - Bulbochaete sp. R Chaetophorn sp. F Charac~um sp. R Clostenum monili/orme (Bory) Ehr. R Coleochaete sp. R Cosmarium botrytis Menegh. R C. granatum Breb. R C. pseudobroomei W o k (Hylan) R C. reneller Wille - C. uexatum W. West R Crucigenia tetrapedia (Kirch.) W. and G. S. West R Euastrum dubium Nag. - Mougeotw sp. R Oedogonium sp. F Pediasrrum duplex Meyen - P. simplex var. duodenorium (Bailey) Rab. R P tetras (Ehr.) Ralfs. R Pieurotoenmm sp. R Scenedesmus acutCrmis Sch. R S. armotus (Chodat) G. M. Sm. - S. bijugatus (Turp.) Kutz. R S. dimorphus (Turp.) Kutz. R S. obl~quus (Turp.) Kutz. R

PEXIPHYTIC ALGAL FLORA O F PIIRAGMUES COM!MUAXT TRIN. 279

TaMe I l (connnwd) p~ .-A

Narnc a( thr lsxon Dal lake Anchar lake Waskur lake

Schwedeno .seiign.a (Sch.) Lrmm. S,pmyyra sp Sioeraslrum sp St~qi.oclunianl luhr~cun, (Ddlw.) f n c s S. renur (Ag) Kutz. Tcmaedrnn tninimum (A. Bi.) Hansg. T. muttrun, (A. Br.) Hansg. T reyulore Kutz. T trilohulatr~m (Rcinsch) Nanag. Ulorhris sp. Zngnniu sp

E) Chrysophyccac Dlnuhrgon sp.

F) Cryptophyccae Crypromonas crow Ehr.

Valucs- Rarc = R - 1- 10 Frcqucnt = F = I I 100 Suh-dominant = SD = 101--500

units/lOmg d.wt

Dominant = D = 501 and ahove

Cyanophyceae > Chlorophyceae > Dinophyceae. Thc ranges and mean values of the percentage composition of major algal classes are prcscnted in Table 111.

The investigation showed the predominance of Bacillariophyceae in the periphyton element. Similar observations were recorded by the authors on artificial and other natural substrate^^-^. The luxuriant growth of diatoms is indicative of the fertility status of these lakes as diatoms have been shown to be sensitive to changes which may occur in aquatic environments and are often considered as reliable indicators of the condition and the quality of the waters in which they liveL2-". One important reason for this is thcir rapid rate of reproduction which allows for significant increases in populations of a given species under favourable conditions while other species concurrently decrease and/or disappear. JorgensenL6 and Vass et a/" also observed high diatom contribution in the periphytic populations.

The species composition of periphyton on P. communis in the investigated lakes is prcscnted in Table IV.

The numerical abundance of periphytic taxa is illustrated in fig. 1. The maximum number of taxa recorded in Dal (Nov.), Anchar (Scpt.) and Waskur (Nov.) lakes was 51,53 and 52, respectively, while their minimum number of 24, 28 and 24 was observed in May 1983.

280 S. G . SARWAK AND D P. ZUTSHl

Table I11 Variations in the percentage composition of major algal classes on Phragmites

Algal class Lake Min.(%) Max.?%)

Bacillariophyceae A B C

Cyanophyccaz A B C

Chlorophyceae A B C

Dinophyceae A B C

A = Dal lake, B = Anchar lake; C = Waskur lake.

Table I V Class~ise distribution of priphytic taxa

Class Lakes

Dal Anchar Waskur

Bacillariophyccae 48 47 48 Chlorophyceae 27 27 23 Cyanophyceae 10 I1 I0 D>nophycme 2 2 2 Crypiophyceae I I I F.uglenophyceae I 1 1 Chryaophyceae I - -

Taial 90 89 85

Cocconeis placentula, Fragilaria spp., Navicula spp. and Oedogonium sp. were the most dominant taxa. Their average per cent contribution to their respective classes is presented in Table V.

Amphora normani. Cymatoplpura solea, Diploneis elliptica, Gomphonemu vihrio, Tolypothrix tenuis, Scenedesmus acutijormis, Stigeoclonium lubricum and Zygnema sp. were some of the rare taxa recorded on Phragmites.

In Dal Lake, the population density showed wide fluctuations in the initial months till it attained a peak of 10,466u/IOmgd.wt. in November 1982. It was mainly constituted by Fragilaria spp. and Oedogonium sp. Thereafter, the density I-emained more or less constant till it registered its lowest value of 627ujIOmg in May 1983. On the other hand, in Anchar lake, the value was very high in June 1982 which was mainly contributed by Cocconeis

PERIPNYTIC ALGAL FLORA OF PHRAGMITES COMAfUNIS TRIN 281

54 C

27

0

FIG. 1 . Time vanations in the number of taxa on Phmgmzes. a = Dal lake: b = Waskur lake; c = Anchar lake. - Total number of taxa. 0 Bacillariophyceae: Dinophyceae;

Chlorophyceae: Chrysophyceae: Cyanophycede; a Cryptophyceae.

282 S G . SARWAR A N D D P. ZUTSHI

Table V Average per cent contribuliaa of dominant genera to their respective classes

Genus Lakes

Dal Anchar Waskur

Cocconers 9.0 12.5 156 Fragllorio 31.5 34.2 18.4 Nariicula 10.0 12 1 7.6 Oedoyonium 36.5 28.3 . 25.6

placenrula. In the ~ubse~uent 'months the values were relatively low recording the lowest value of 670 u/lOmg d.wt. in October 1982. In November 1982, the values showed abrupt rise to register a minor peak of 6.062u/10mg d.wt. It was mainly contributed by Fragilariu spp. and Oedogonium sp. Thereafter, the abundance values gradually decreased till April 1983 when it once again showed an abrupt rise to form a major peak of 9,702 u/10mg d.wt. It was mainly contributed by Fragilaria spp. In the subsequent month, it once again registered a sharp decline to 942u/l0mg d.wt. In Waskur lake, the population density ranged from 650 (October 1982) to 3,850u/10mgd.wt. (June 1982). On mouthly basis wide fluctuations were not observed. The peaks in June 1982 and May 1983 were mainly constituted by Cocconeis placenrula.

Variations in the population density were non-significant. Similar results were also observed on other natural substrates5. The evaluated F value was less than the critical F value (3.26 and 3.27) at 5% level of significance with 2 and 33 degrees of freedom.

The conununity coefficient values of periphyton on Phrugmxes vaned from 22.77% in August 1982 to 31.81% in November 1982 (i: 26.41; S.D. + 2.71). The similarity between the periphyton colonising Phrugn~ites and the phytoplankton of these lakes is a clear indication of their interchangeable nature. Kashmir lakes support a luxuriant macrophytic growth. The periphytic forms are released into the lake waters from the luxuriant macrophytic vegetation due to water currents and anthropogenic activities. It is h~ghly probable that these forms occur in the plankton 'accidentally' after they are washed off or get detachcd from their substrates. Pieczynska" also reported high exchangeability between plankton and periphyton as the latter are loosely associated with the substrate.

Lake to lake variations of periphyton on this substrate did not depict any significant variations. This may be attributed to almost similar physico-chemical characteristics of the lake waters. Variations in some parameters like conductivity, total alkalinity, total phosphorus and calcium content of these lakes result in the variability of species composition and population density.

Acknowledgements

Thanks are due to the Principal, college, Srinagar and the Director, CORD, University of Kashmir, for providing laboratory facilities. This study was made possible by a Teacher Fellowship to SGS by the UGC.

PERlPHYTlC ALGAL FLORA OF PNRAGMITES COMMUNIS TRlN 283

References

2. PHILIPOSL, M. T., NAWY, A. C.. C~nnnAeonru. D. P ~ N D

RAMAKRISHNA. K. V

3. S~ i iwnn , S. G.

4. SARWAR, S. G. AND ZUTSHI, D. P

5. SARWAR, S . G. AND ZUTSHI, D. P

8. GOLTERMAN, H. L., CLYMO, R. S. . AND OHNSTAD. N. A. M.

9. G o u o ~ , S 8. A N D

WOEI.KBRI.ING. W. J.

10. TUCKER. A.

14. CAIRNS, J. Jn.. KAESLAEK, R. L. AND PATRICK, R.

IS. CAIRNS, J. JR., LANZA, G. R. AND PARKER. B C. . .

17. VASS. K. K., BHANOT, K. K. AND

GHOSH, A. N.

Studles on bottom-living diatoms o i a freshwater fish pond, J Bombay Not. Hi.% Soc., 1970, 67, 443-452.

Studles on the distribution in time and space of the periphyton of a perennial pond at Cuttack, Ind~a, Bull. Cenr. InlundFish. Res. Inst., 1976, 21. 43.

Specles composttion and seasonal variations 01 Penphyton on Ceratophyllum demerrusn~ Linn. m Waskurlake, Kashmir, Geobios New Rep., 1987, 6, 114-118.

Primary productivity of periphyton, Geobios, 1987, 14, 127-129.

Studies on periphyton population of H~malayan lakes. 1. Species composition and community structure on natural and artificial substrates, Proe. Indian Natn. Sei. Acad., 1987, 53, 239-243.

Water annlysrs for limnoloqists, 1963,Vol 21, pp. 1-70. Freshwater Biol. Assoc.

Standard methods for the examinntion of water, sewage and industrial wastes, APHA, New York, 1971.

Methods for physical and chemical analysts of Jreshwaters, IBP Handbook No. 8, Blackwell, 1978.

On the removal and quantification of algal aufwuchs lrom macraphyte hosts, Hydrobrolngla, 1976, 48, 203-207.

The relatlon of phytoplankton periodicity to the nature of physico- chemical enwronment with special reference to phosphorus, Am Mid1 Nar., 1957. 57, 300-370.

The planktonic crustaceans of Moncoe Lake; Monroe Country, W.Va., Pmc. W m Virginia Acad. Sci., 1974, 46, 223-229.

The structure of dlatom communities under varying ecological conditions, Ann. N.Y. Acad. Sci., 1963, 108, 359-365.

The structure 01 diatom communlt~es in sirn~lar ecological conditions, Am. Nor., 1968, 102, 173-183.

Occurrence and &str;bution of diatoms and other algae m the Upper Potomac River, Notulae Natur, 1970, 436, 1-12.

Pollution related structural and functional changes m aquatlc communities with emphasis on freshwater algae and protozoa, Proc. Aend. Nat. Sci. Phil., 1972, 124, 79-127.

Diatom perioduty and sllicon assimilation, Densk Bat. Arks., 1957,18, 1-54.

Periphyton production in 2 ponds of brackish water farm at Kakdwlp, West Bengal, Indla, J. Inland Fish. Soc. India, 1978, 10, 32-38.

Investigations on colonization of new substrates by nematodes (Nematoda) and some otherperiphyton organisms, Ekol. Poi, 1964, 12, 185-234.

I. Indianlnrl. Sci., July-Aug., 1989,69,285-290 OIndian institute of Science.

Short Communication

Ecological studies on a desmid bloom

B. JYOTHI, G. SUDHAKAR AND V. VENKATESWARLU

Phycology and River Ecology Laboratory, Department of Botany. Osmania Univenlty, Hydeiabad 500007.

Received on December 19. 1988: Rewed on June 16, 1989

Abstract

The ecology of a uni-algal bloom of Cosniorium t'ariolarum Lund. var. rorundarum (Kreig.) Messik was sludied in relation to certain ahysico-chemical and biochemical variables In evaluatine the collective and individual . . influence of various chemical factors, a new approach has been followed by using multiple regression analysis. Simple correlation analys~s between the physico-chem~cai parameters and algal number has also been made.

Key words: Ecology, bloom, oligotrophy, regression analysis

1. Introduction

It is highly desirable to evaluate the trophic status of water bodies as fresh waters receive enormous quantities of wastes comprising a large number of nutrients in high concentrations which influence rapid multiplication and blooming of certain algae. The factors that influence the development, duration and decline of an algal bloom have been the subject of research investigationst. Prescott' reported that oligotrophic lakes are characterised by Chlorophycean flora with a conspicuous desmid element. Duthie3 studied desmid populations in oligotrophic Welsh lakes to find out the status of desmids in the plankton. According to Brook4 the greatest number of desmid species (i.e., 59%) occur frequently in oligotrophic lakes. Venkateswarlu5 reported Staurastrum tetracerum from oligotrophic habitats. The present paper discusses the ecological charactefistics of Cosmarium variolatum var. rotundaturn and its significance in evaluating the trophic status of the habitat.

2. Material and methods

Surface water samples, altogether 11, were collected from a cement cistern (4m dia and 2m depth) once in three days during April-May 1987. The filtered samples were analysed for various physico-chemical variables by following standard procedures6. Certain biochemical constituents such as proteins and carbohydrates were also estimated7.'. The quantitative estimation of algae was done by using haemocytometer, and the data were statistically analysed by the methods of Snedecor and Cochran9.

3. Results

The results of physico-chemical and biochemical factors are incorporated in Table I. The pH ranged from 8.7 to 9.3 showing highly alkaline nature of the mcdium. The average value of chlorides was 345.9mg/l. Phosphates and nitrates were recorded in low concentrations and ranged from 0.0 to 0.2 and 0.4 to 0.7 mg/l, rcspectively. Dissolved oxygen (D.O.) was high with an average value of7.4mgll. Organic matter was found in considerable quantities, fluctuating between 7.2 and 17.5mg/l. Carbohydrates and proteins varied from 6.0 to 3 1 .Omg/l and 16.0 to 100 mg/l, respectively.

Multiplt regression analysis has been done with phosphates, nitrates and D.O. as independent variables and algal number as dependent variable to find out their collective effect on the growth oPCosmnrium and the overall regression was tested with the help of 'F' value. The overall regression is significant at 1% level of probability and the coefficient of determination (R2) is 0.79. Phosphates, nitrates and D.O. explain the variation in algal number to thc extent of 79.8%. When phosphates are eliminated in the step-down regression analysis there is no drop in R2 value (0.79). This indicates that the effect of phosphates on algal number is negligible and both nitrates and D.O. are required to explain the variation in algal number to the maximum extent. hdividually also, nitrates and D.O. have a significant positive influence on the growth of the alga (fig. 1, Table 11).

4. Discussion

The main conclusion drawn from the various investigations carried out to explore the responses of desmids to the possible chemical conditions was that by far the greatest number oldesmid species occur in acid waters1 (pH 4 7). Recent researches, however, have shown that many species occur often in considerable abundance in alkaline water^^.'^.". In the present study, the pH ranged between 8.7 and 9.3, and it had a positive relation with desmid number and 1s s~gnificant statistically at 5% level of probability. The maximum algal number coincided with the highest value of pH.

Desmids are almost exclusively freshwater algae confined to natural waters with low salinities. However, high concentralions of chlorides were recorded in the present study. Nygaard" also reports the discovery of several Cosmarium species in a lake with high bicarbonate and chloride contents.

In the present investigation the highest algal number coincided with the highest calcium and pH values. Thc alga had a significant positive relation with calcium and negative relation with magnesium. The negative rclation with magnesium might be due to its utilisation in chlorophyll synthesis. Since the average pH of the medium was around 9.0 the alga might have preferred CO, and HCO, as carbon source, thus releasing calciun~ into water13. This explains the positive relation between algal growth and calcium. Gough" also reports that the growth of Cosmarium granatum was better at both higher pH and calcium.

The nitrate concentrations were low, hut were able to influence the growth of the desmid.

Tab

le I

P

hys

ic~

bem

ical

, bio-

cbem

ieal

par

amet

em e

nd a

lgal

num

ber

in t

be h

abita

t (e

xpre

ssed

in m

gA, e

xcep

t pH

and

tem

pera

ture

l

S1

Tem

p

pH

CO

, H

CO

, CI

T

H

Ca

Mg

PO

, N

O,

D.O

. 0 M

. Pr

oter

ns

Car

bo-

Alg

al

no

. ('C

) hy

drat

es

num

ber

(no,

of s

ells

!l)

1.

33

9 96

2.

36

8.7

60

3.

35

9

96

4 31

8.

8 42

5.

34

9.

1 10

2 6.

35

9.

1 84

7.

37

9.

3 84

8.

35

9.

2 78

9.

35

9.

2 56

10

. 35

9.

2 96

11

. 35

5

102

Mea

n 34

.5

9.0

85

SD

. 1.

49 f 0.

1 i 18

0.2

0.2

0.05

0.

05

0.03

0.

09

0.1

0.1

m

trac

er

Nil

N

il

288 0. JYOTHI et a1

FIG. I. Relat~onship between DO, NO,, Ca and algae.

ECOLOGICAL STUDIES ON A DCSMlD BLOOM 289

Table I1 Correlation matrix nf physicoshemical paramears and algal number

xi xi x4 X< X, X, " X, X,, X,, X,, Y - - ~ . ~ - - ~ ~ ~ - ~ . - ~ -- . -

X , 029 041 005 015 -0.03 006 -0.10 --0.01 0.04 -0.35 0.18 -0.01

X, 0 9 0 5 8 0.50 0.31 -0.51 -0.40 -0.39 0.47 0.27 0.12 0.65' X 3 028 048 0.02 -0.22 0.29 --0.29 0.07 0.28 0.40 0.17 Xa - 0 7 5 0.16 -0.10 0.02 078 0.31 0.27 -031 -0.03 x, -0.24 0.19 OW -0.80 -0.21 -0.26 0.32 -0.23 x, -0.62 001 0.50 0.49 0.37 -0.02 0.58' x, 0.73 - 0.34 0.40 0.61 0.40 - 0.72"* X, -002 0.09 -0.50 0.51 -0.45 X, 0.18 0.25 -0.22 0.25 X m 0.01 - 0.03 0.60L X I , - 0 09 0.66* x , 1 - 0.01

X , =Temperature, X, = pH; X, = Carbonates; X, = Bicarbonates; X , =Chlorides; X, =Calelum, X, =

Magnmum; X, = T o l d hardness: X, = Phosphatzs; X,, =Nitrates; X, , = D.O.; X,, =Organic matter; Y = Algal number.

*SignlBcant at 5% level; **S~gnificdnt at 1% level

The positive relation of nitrates and algal growth which is significant at 5% level of probability, might be due to its recycling. The decomposition of nitrogen is favoured as oxygen levels arc increased.

Dissolved oxygen and algal numbers are directly related to each other. The variation in D.O. is due to variation in photosynthetic activity which in turn depends upon the algal number. The relatively high organic content in the water body can be attributed to the metabolic products liberated by the alga which is evident from the presence of high extracellular proteins and carbohydratess.

From the foregoing account it can be concluded that Cosmarium variolatum var. rotundatum prefers oligotrophic habitat as is evident by the presence of low-nutrient (NO, and PO,) content in the medium. Further it can be termed as hard-water inhabiting species due to high water hardness.

Acknowledgements

The financial assistance to BJ and GS by UGC, New Delhi, is greatly acknowledged.' The authors thank the Head, Deptt of Botany, Osmania Uuivers~ty for providing laboratory facilities.

References

The biology of desmids, Botan~cal Monographs, Vol. 16, pp. 196-241, Blackwell. 1981.

7. LOWRY, 0 H.. ROSEIIR~~GH, 8. J., Faun, A. L. AND RANDALL, R. 1.

8. Lo~wus, F. A.

9. SNEUECOR. G. W. AND COCHRAN, W. G.

10. BLAND, R. D. AND BROOK, A J .

B. JYOTHI er 01

Some relationships orphytoplankton oflimnology and aquatic biology. In problems on lake biology. Am. Assoc. Ado. Sci. Puhi.. 1939. 10.65-78.

Some obscivat~ons on the ecology of desmids. J Elol.. 1965. 53, 695- 703.

Planktonic algae as indicators ofiake types with special rererence to the Desmid~aceae, L~mnoi. Oceanoqr, 1965. 10,403-41 1.

Ecology of desmids-I, Stairrostrum retracerum Ralfs, Indian J. Bof., 1965.6.68-73.

Standard merhods for the examination of water and wasre water, 16th edition, American Public Health Association, Washington, 1985.

Protein measurement with the folin phenol reagent, J. Biol. Chem., 1951,193,265-275.

Improvement in anthrone method for determination ofcarbohydrates. And Chem, 1952,24,219-223.

Statistical methods, 6th edn, Oxiord-IBH Publishing Co, New Delhi, 1967.

The spattal distribution of desmtds in lakes in northern Minnesota, USA, Freshwater B d , 1975, 4, 543-556.

The growth of selected desmid (desmidiales, chlorophyta) tana at d~fferent calcium and pH levels, Am. J. Bot, 1977.64, 1297-1299.

Desmids from an Arctic salt lake, Bot. Tidskr., 1976, 71, 84-86.

Influence of chemical speciatmn on the toxicity of heavy mctais to thc microbiota. In Aquatic toxicology, ed. 1. 0. Nriagu, Vol. 13, 1-46, Wiley-lnterscience, 1983.

J liulian lnsz. Sci., July-Aug., 1989,69,291-294. '' Indian Institute of Science

Short Commzsnication

ffect of a commercial growth promoter (trade name Vipol) on fenugreek and its root nodule bacteria

N. KKISHNAN ANI) S. CHANDRASEKARAN Department of Botany, P. S. G College or Arts and Science. Coimbatole 641 014.

Recewed on March 8, 1989; Rcviicd on June 19, 1989

In uirm studies on lrnugrcck Rhizohrum showed that Vipol had neither promotwe nor inhibitory eNect on ~t Vipul spray experlmenfs on lcnugreck plants revealed that a spray of 250ppm of Vlpul two times had s~gnlficantly ~ncreaied the length. dry matter, total N and P contents However, it did not increase the nodule numbers nor prornotcd fcnugruck- Rhxrohiu,n rymb,oss

Key words: Vlpul, fenugreek--Rh~rohiutn, symb~osis

1. Introduction

Fenugreek (Trigonella fnenumyraecum L) is an important spice crop of India'. This work was taken up to assess the effect of Vipul on the growth of fenugreek plants and on its root nodule bacteria Rhhobium meliloli. Vipul (a/i 5000ppm), a plant growth promoter markcted by Godrej Soaps Ltd, Bombay, is an aqueous colloidal emulsion, milky white in colour and is soluble in water; its pH is neutral. It contains different types of alcohols. Triacontanol (24-26%), octacosanol (6-lo%), dotriacontanol (11-15%) and tetrafriacontanol (6-IOU/,) form the major constituents. Triacontanol: rnol. formula is CH,(CH2),,CH,0H; mol.w( is 438. Sterols, namely, lretasitosterol (C,,H5,0). Stigmasitosterol and campesterol are also present but to a smaller extent, around 10%.

Phytohormones arc known to influence nodulation of roots in legumesz. For example, it has bcen found that Indole acetic acid, a growth promoting phytohormone and Colchicine, a plant alkaloid, help in early nodulation in l ~ c e r n e ~ . ~ . Ol late, Triacontanol, a long-chain alcohol, which occurs naturally in plants such as alfa alfa, has gained importance as a plant growth promoter5.

2. Materials a d methods

The root nodule bacteria were isolated from healthy fenugreek plants collected from farmer's lield as per the method of Vincent6.

29 1

292 N. KRISHNAN AND S. CHANDRASEKARAN

One ml of 72-hour old culture of the above isolated Rhizobium sp(log phase cells ca 4 x lo6) was inoculated aseptically into lOOml of sterile yeast extract mannitol broth in 250ml flasks. To these, calculated quantities of Vipul were added aseptically in such a way that the final concentrations obtained were 0,2,10,20 and 40 ppm of the compound. The flasks were incubated at room temperature (30 f 2°C) on rotary shaker. Two replicates were maintained for each concentration of the chemical. The growth of the Rhizobium isolate in culture was assessed at 450nm (Elico calorimeter) at specified intervals.

Studies on the effect of Vipul on fenugreek plants were carried out on plants grown in red soil contained in polythene covers (25 x 18cm). Here pH of the soil was 7.7, E.C. 1.4 x lo3 millimhos; water-holding capacity 36.5%. Fenugreek seeds (Co 1 variety) pelleted with 72- hour old culture of Rhizobium using peat as the carrier and cooked starch as adhesive were sown in the above soil packs. The packets containing the 24-day old plants were grouped under four heads for 0,250,500 and lOOOppm Vipul spray treatment. Vipul was hand- sprayed, on the 24th and 42nd day after sowing, in good weather conditions. Total length, nodule number and plant dry weight were recorded on 11,18 and 28th day after the first spray treatment. The above values for each treatment (control, 250,500,1000 ppm) and for each interval (0,11,18,28th days) are based on six plants spread over three soil packs. The experiment was conducted in triplicate.

Estimation of total nitrogen, total phosphorus, sodium and potassium was done as per standard procedures' on the 28th day after the first spray treatment.

3. Results and discussion

The bacteria showed steady and uniform growth over 72-hour incubation period in control as well as in treated broth without any appreciable difference (Table I).

Fenugreek plants that received the 250ppm Vipul spray recorded the highest growth in terms of total length, dry matter, total nitrogen and phosphorus (Tables I1 and 111). The plants that received 500 and lOOOppm Vipul spray have recorded lower growth rate, dry matter and total N as compared to the untreated control plants in almost aU these

Tsbk I In vim effect of Vipul on R melilori (Mean of two replicates; figures represent OD of culture)

Treatment Hours (in p w )

0 6 I2 24 36 48 b0 72

0 0 0.03 0.15 0.25 0.34 0.38 0.42 0.44 (Control) 2 0 0.03 0.17 0.27 0.35 0.40 0.43 0.44 10 0.02 0.05 0.16 0.25 0.32 0.36 0.42 0.43 20 0.03 0.05 0.15 0.24 0.30 0.32 0.41 0.43 4 0.03 0.04 0.16 0.24 0.30 0.36 0.43 0.40

EFFECT OF VlPUL ON FENUGREEK

- 0

% , f g a g .5 8 3 xg;

2-74 C K A v v v

1 + + + It I I I + I I

294 N. KRISHNAN AND S. CHANDRASEKARAN

treatmcnts and intervals. However, as regards the potassium and sodium contents, the 500ppm Vipd spray was found to be better than the other treatmenls and control plants (Tables I1 and HI).

Any chemical compound sprayed over plant surface can act in more than one way. It can directly influence the plant growth or can be excreted parlially at least, along with other substances as root exudates. It is well known that root exudates have a profound effect on the rhizosphere microflora, rhi~obial population in soil and also on nodulation. It is generally held that phytohormones such as IAA play an inlportant role in nodule initiationg.

Vipul has significant effect on the increase in dry weight, total length, total nitrogen and phosphorus, but it does not correlate with the increase in nodulation. Moreover as seen in Table I, Vipul does not have any promotive effect on the in uitro growth of fenugreek's Rhizobium. These facts would lead us to believe that the above increase in the fenugreek plants may be independent of the symbiotic effect of fenugreek-Rhizobium. Further plant physiological studies are needed to confirm this view.

Acknowledgement

The authors express their thanks to the Principal, YSG College of Arts and Science, Coimbatore for facilities.

References

3. SAKMA, K S B., KUMARI. L. AND

Sueun Rho, N. S.

Research and devclopmcnl in spces. lrdinn Spiccr, 1987, 24. 5- 13

Nodule development and senescence, In Nirmyen fixnrion, ed. W . J . Brouphton. 1983, Vol 3, pp. 153-155, Clarendon Prcss.

Effect of colch~cine and 8-hydroxyquinolme on nodulatlon in some legumes, lrldran J. Agnc. Set., 1973. 43, 203-206.

ElIecl of calchlcin~ and 8-hydroxyqainoline on nodulalion in some legumes, I n d m J . Aync. Sci., 1973, 43, 203-206.

Regulation of plant growth Sci, 1985, 2, 239-289.

with triacontanol. CRC Critical Reu. PI

A mnnuai for :he practical study of root nqduir bacteria, 1970, pp. 164, Blackwell.

Soil chemical onalysis. 1973, pp. 142, 188, 335. 459. Prentice-Hall of India, New Delhi.

Sod microorgonhms and plan1 growrh, 1986, p. 300, Oxford and IBH Publishing. New Delhi.

Development ol legum~nous root nodules, in Ndroyen fixation, ed. W . J. Broughton, 1982, Vol. 2, p. 327, Clarendon Press.

J . Indian lnst %'I.. July-Aug 1989, 69. 295-320. c Indian Insmute or Science

IISc THESES ABSTRACTS

Thesis Abstract (Ph.D.)

Studies on B-DNA: Fibre diffraction and ligand binding by Pradipkumar Parrack Research supervisor: V. Sasisekharan. Department: Molecular Biophysics Unit.

1. Introduction

This thesis consists of two parts. Part I contains X-ray fibre diflraction studies on B-DNA. Part I1 is concerned with the design. synthesis, and physicochemical studies of structural analogues of the oligopeptide antibiotic distamycin, a B-DNA-specific ligand.

2. Fibre diffraction of DNA

A thorough and .)jrcmaiic conformar~onal dnrl!sls zram~ning dl1 poss~blc hel~cal srrucrures i w BD21.A. usmg t he f l c ~ b l c n~dnonucleol~dc as the bcillmg block. wdj lirsr started In Jur laborarory'. As a result of such studies, several right- and left-handed models were generated, which were conastent with the then available X-ray fibre as well as IR dichroism data2. The author started X-ray fibre diffraction of DNA with special emphasis on the B-form. The objective was to supplement the earlier model building work with first-hand fibre data with which different possible models may he compared.

It was observed3 that for the lithium salt of calf-thymus DNA, the diffraction pattern corresponding to the best 'crystalline B-form' cannot be explained in terns of regular helical structures. Additionally, structural transitions in the solid state, as a function of environmental conditions, were also detected highlighting the flexibility inherent in the structure, earlier indicated by conformational calculations from our laboratory.

After an examination of the existing methods for fibre preparation, a method for accurately controlling the salt content in DNA fibres (a necessary factor in determining crystallinity and nature of the X-ray pattern) was developed for the first time ever in our laboratory3. The procedures standardised enabled obtaining crystalline B-DNA patterns with a very high degree of reproducibility. Appearance and disappearance of meridional reflections on the fourth, sixth, and tenth layer lines indicated nonuniformity and variability in the structure3. This was confirmed by sensitive and quantitative measurements of helical parameters from layer-line spacings, using the precision method of X-ray diffraction, for the first time for DNA fibres'. Such variations being present in the best B-patterns, along with the indication of nonhelical nature of the structure, model building studies to explain all the observed data are rendered extremely difficult. It was realised that the X-ray data did not warrant such an accurate solution of the fine structure of B-DNA.

296 lISc THESES ABSTRACTS

3. Ligand-binding studies

In order to study some gross features of the structure (such as handedness) and also to try to 'fix' the structure in one of its microheterogenous states, nonintercalative DNA-binding ligands, netrospin (Nt) and distamycin (Dst), were chosen as model systems. These compounds, which are naturally occurring oligopeptide antibiotics (pyrrolamides), bind to B-DNA with a high specificity toward the DNA secondary structure, AT base pairs, and the minor grooves. Because of their inherent structural complementarity to the B-DNA minor groove, they serve as suitable models hascd on which specific structural probes might be designed.

The author was involved In the initiation ot such an approach to DNA structure, and the work presented in part I1 of the thesis comprises efforts to build up the necessary base for the same. To deslgn structural probes, details of the specific interactions must be known. The program undertaken in our laboratory to this end is aimed at understanding the conformational and chemical basis for the specific interactions of Nt/Dst type of ligands with DNA.

An isohelical analysis on pyrrolamides was carried out with the specific objective of examining their compatibility with the B-DNA structure, and the viability of a pyrrole-8-alanine repeat unit in this regard was recognised. Two such pyrrole-1-ala-containing analogues of distamycin, PPA and PAP, were synthesised. Introduction of the saturated 8-ala moiety results in a decrease iu the extended conjugation (which is present via contiguous pyrrolamides, as in Dst). Detailed spectroscopic studies on the DNA-binding characteristics of PPA and PAP were undertaken to examine the mportant and hitherto unnoticed role of this extended conjugation in Dst-DNA interactions. It is shown that the loss of extended conjugation in the ligands does not influence the specificities of binding6. More detailed investirrations reveal that such conjugation contributes to the stability of the ligand-DNA complex7.

References

1. SAWKW, V. AND

PATTABIRAMAN, N.

2. SA~ISEKHARAN. V., BANSAL, M. AND GUPTA, G.

3. PARRACK, P., DATTA, S 4ND

SASISEKHARAN, V.

4. SUNVARAMOORTHY, M., PARRACK. P. AND

SASISEKHARAN, V.

5. ZIMMER, C. AND WAHNERT, U.

6. PARRACK, P., DASOUPTA., D., AYYER, J. AND SASISEKHARAN, V.

7. DASGUPTA., D.. PARRACK, P. AND

SASI~EKHARAN. V.

Nature, 1978, 275, 159-162.

In NucLic ocids: The uectors of life (eds 8. Pullman and J. Jortner). 1983, pp. 101-111, D. Reidel.

J. B~omol. Strud. Dynam., 1984,2, 149-157.

In B~omoleculor slerrodynamrcs. Val. IV (eds R. H. Sarma and M. H. Sama), 1986, pp. 217-225, Adenine Press, Gudderland, NY.

Prog. Biophys. Mol. Biol., 1986, 47, 31-112.

FEBS Lett., 1987, 212, 297-301.

LiSc THESES ABSTRACTS

Thcsis Abstract (Ph.D.)

Spectroscopic investigations of no antifertility agent, gossypol, and an ionnphore antibiotic Basalocid A by D. S. Sampalh. Research supervisor: P. Balaram. Department: Molecular Biophysics Unit.

1. Introduction

Low molecu13r weight polyfunctional organic natural products constitute a structurally diverse class of molecules w111ch display a remarkable spectrum of biological activities. Such molecules invariably arise as secondary by-products of cellular metabolism and their precise role in the parent system remains obscure. In this study, gossypol and lasalocid A, 1a.o polyorygcnated members of t h~s class have been subjects of detailed biophysical inuestigations.

Cossypol Lasalmd A

2. Experimental procedures

CD spectra were recorded on a JASCO J-ZOA spectropolarimeter. Fluorescence studies were performed either on a Perkin-Elmer MPF-44A spectrofluorimeter or a Hitachi 650-60 spectrofluorimeter. HPLC studies were carried out using an LKB two-pump system with a controller unit. Analyses were carried out on a Lichrosorb reverse-phase C,,(RP-18) colmnn of dimension 4 x 25Omm (particle size, 1Opm) using methanol-water as the eluant. 270MHz 'Hand 67.89MHz 13C NMR spectra were recorded on the Bruker WH-270 FT NMR spectrometer equipped with an Aspect-2000 computer, at the Sophisticated Instruments Facility, Indian Institute of Science, Bangalore.

(+j-Gossypol was extracted from the bark of Thespesla populnea by a modified procedure as described in the literat~re'.~. Amino-acid derivatives employed in thc study wcrc prepared by standard procedures and characterised. Racemic (?)-gossypol acetic acid was obtained as a gift from the National Institute of Child Health (WHO Special Program). Other chemicals were purchased from Sigma Chemical Company, USA.

3. Main results and conclusions

The CD spectral investigations of (+)-gossypol reveal a rich spectrum spanning the range 200- 45Onm (fig. I). There are five principal CD bands situated at - 370 nm (positive; band l), - 340nm (negative; band II), - 300 nm (negative; band III), - 270nm (positive; band IV) and - 230nm (negative; band V). Bands I, IV and V are characterised by high ellipticities whereas bands I1 and 111 exhibit low ellipticitics. Band assignments have been made based on the known electronic transitions

298 llSc THESES ABSTRACTS

Fm. I . CD spectrum of (+)-gossypol (SOpM) in ethanol. Path length, OScm.

of2,t'-binaphthy13, the principal chromophore in gossypol. Band I has been assigned to the 'La+- ' A transition, band 111 to the ' L , c l A transition, band IV to the ' B , t 'A transition and band V to the 'C,+ 'A transition. Band I1 for which there is no clearly distinguishable UV absorption maximum has been assigned to then +n* carbonyl transition. These results have been extended to use gossypol as a CD probe of protein-binding sites.

Optical resolution of racemic (+I-gossypol has been accomplished using a variety of chiral amino compounds. These include L-phenylalaninol, (-)-norepinephrine, (-)-epinephrine, D-$lucosamine, D-galactosamine, D-mannosamine and a dilysine tetrapeptide Boc-Lys-Pro-Aib-Lys-NHMe. Separation of the diastereomeric complexes of (2)-gossypol with the chiral amines is achieved by HPLC on an achiral phase column with methanol-water mixtures of varying composition as the eluant. (-)- or (+)-gossypol can be easily prepared by acid hydrolysis of the respective resolved gossypol adducts. The present procedures differ from the two other procedures published re~ently"~ in the following respects: (i) Both the component enantiomers, (+)- and (-)- of gossypol, could be obtained in good yields from an individual run. (ii)An achiral column suffices to yield a good resolution.

Representative examples depicting the good resolutions obtained are presented in fig. 2.

IISL THESES ABSTRACTS

0 l-TJh+& 0 4 26 30 34 38

- TIME (minutes)-+

FIG. 2. HPLC trace of (a) (2)-gossypol-(-)-norepinephrine adduct and (b)(i)-gossypol-L-phenylalaninol adduct. Column: Lichrosorb RP-18 (4 x ZSOmm, 10pm particle size). Retention time is indicated against the peaks. Dctectlon by UV absorption at 365nm. Elution: (a) Gradient elution with 67-83% MeOH-H,O in 35' followed by 83-95;:, MeOH-H,O in 3'; (b) Gradient elution with 90-95% MeOH-H,O in 15'.

In low concentration regimes (gossypol, $50pM; protein, $50pM) there are no large changes in the CD spectrum of (+)-gossypol on binding to serum albumins. But under high concentration condrtions (gossypol, b 100pM; protein, > 100pM) large changes are observed. Under these conditions 'induced CD' spectra have been observed for racemic (i)-gossypol in the case of HSA and BSA (fig. 3). The spectral characteristics are different for the two proteins. Differential perturbation of the bound-gossypol enantiomers is thought to be responsible for such 'induced CD' spectra.

All the three gossypol forms (+)-, (-)- and (j-) interact with artificial lipid membranes and affect lipid-phase fluidity. At low concentratlons (mole ratios <0.05 with respect to lipid) gossypol has an ordering effect on the membrane lipids, whereas at high concentrations (mole ratios 0.05-0.25, examined in this study) it has a disordering effect. A significant feature of the studies under high concentration conditions is the enantiomeric difference for the gossypol-induced fluidity changes.

The aqueous-phase CD studies of lasalocid A suggest it exists in an open, extended quasilinear

300 IlSc THESES ABSTRACTS

FIG. 3. lnduced CD spectra of(?)-gassypol (335~M) in the presence of (a) HSA (289pM) in phosphate buffer (1 mM, pH 7.0) and, (b) BSA (289 pM) in phosphate buller (1 mM, pH7.41.-Spectra recorded lmmediatcly after miring;---spectra recorded after 72 h of incubation

conformation under such conditions. The resultant exposure of apolar s ~ d e chains and hence unfavourable interactions are suggested to be offset by aggregation of the ionophore into small micellar structures. In micelles and phospholipid dispersions the ionophore appears to largely favour an extended quasilinear conformer. Fluorescene polarisation studics of lasalocid A in water indeed suggest aggregate structures with the aggregation number being - 30. This observation finds further support from gel filtration studies which yield an M , = 18,000 For the molecular species of the ionophore prevalent in water Rased on fluorescence studies an estimate of 0.041M has been arrived at for the critical micellar concentration.

References

1. KING, T.J. AND DE SILVA, L 0. Telmhedron Lett., 1968, 261-263.

2. DAWA, S. C., MIXTI, V. V. S. AND CILII' SCL, 1968, 37, 135 SESHADRI. 1'. R.

3. JAFFE, H. H. A N D ORCHIN, M. Theory and oppllcorions 0fulo.auiolet .speetroscop,py, 1962, Wiley

4. ZHENG, D. K., SI, Y. K.. .I. Chem. Soc.. Chem. Commun., 1985, 168-169. MENG, J. K., ZHOU. J. AND HUANG, L.

5. MATLIN. S. A.. ZHOU, R.. Contmcqtion, 1985, 31, 141-149. BIALY, G . BLYE, R. P., NAQVI, R. H. AND

LINDEBFRO, M. C.

IlSc THESES ABSTRACTS 301


Altered conformations in natural DNA: Studies on supercoiled form V DNA by Yogesh S . Shouche. Research supervisor: S. K. Brahmachari. Department: Molecular Biophysics Unit.

I. Introduction

The demonstration of sequence-dependent conformational flexibility of DNA1 and the discovery of left-handedZ-DNAZpaved the way foradetailedstudy on thepossibleroleforsuchstructuresofDNA. So far thc studies were restricted to synthetic oligo and polynucleotides with very little focus on naturally occurring sequences. In the present work, attempt has been made to identify the sequences that have a potential to adopt unusual structures under the topological stress, which is known to play an important role in gene regulation in uiuo. Supercotling is also known to be an important factor in inducing structural alteration in alternating purine-pyrimidine sequences cloned in circular plasmids.

Form V DNA, prepared by reannealing of two single-stranded complementary c i r c l e ~ ~ , ~ , was used as a model system. In this molecule, due to topological constraint, every right-handed turn is compensated by a left-handed turn or negative supercoiling; as a result up to 40% of the molecule adopts unusual (non-B] conformati~n~.~. In this work, such unusual structures were characterised, their interaction with structure-specific antibodies and transacting factors like Z-DNA binding protein was also investigated. In addition, sequences that adopt unusual structure were mapped using sequence-specilic enzymes like restriction endonucleases and modification methylases. This provided detailed information as to the role of neighbouring sequences on supercoil-induced structural transition in DNA.

2. Materials and methods

Ail the reagents were of Analar grade. Restriction endonucleases and methylases were from New England Biolabs. UV, CD and sedimentation analyses were performed on Beckman DU-8B spcctrophotometer, JASCO-J500 spectropolartmeter and Beckman analytical ultracentrifuge, respectively.

Plasmids were purified by Cscl.Etbr density-gradient centrifugation and by the method developed in our laboratory. Z-DNA antibody raised against Z form of poly (dG-dC) was a gift from Prof. B. D. Stollar. Preparation and purification of pBR322 form V DNA, antibody-binding, 2-D gel electrophoresis restriction endonuclease digestion and methylation experiments were carried out as described earlier4,'.

3. Results and di~ussion

3.1. Preparation ond pur$cation ofform VDNA

Form V DNA was prepared following the method of Stettler et a13. They used DNase I digestion of plasmid in the presence of ethidium bromide (30Opg/m1) to get form I1 DNA having single nick per molecule. Since DNase I clcavage is difficult to control, a new method involving EcoRI digestion in the presence of ethidium bromide (400/1g/ml) was developed taking advantage of the Fdct that EcoRI has a single cleavage site in pBR322. Form V DNA prepared from pBR322 and pBG plasmids gave a


sharp band on electrophoresis Indicating the homogeneous nature of the folded moleculc durmg reannealing It showed a faster electrophoretic mobllity than form T DNA ~ n d ~ c a t ~ v e of higher superco~ling. The yield of form V DNA was found to be 10% of the starting plasmid.

3.2. Characterisation of form V DNA

A deta~led characterisation of form V DNA prepared from plasmids pBR322 and pBG was carried out. Two-dimensional gel electrophoresis and sedimentatron analyses showed that form V DNA is a highly compact supercoiled molecule. Thermal denaturation and circular d~chroism studies suggest that up to 35-40% of the sequences are present in the left-handed helical conformation. Studies using 2-DNA-specific protein and antibody indicate that a major portion ofthe altered structures in form V DNA probably have a left-handed 2 conformation. These structures were found to be stabilised by multivalent cations like hexamme cobalt chloride and deslabilised by DNA unwinding agents like ethidium bromide.

A search of the complete sequence of pBR322 shows that there are very few alternating purine- pyrimidine sequences, which implies that in form V DNA due to zero link, sequences other than these must be adopting Z-hke left-handed helical conformations. Probing the structure adopted by different sequences in form V DNA may reveal the possible conformation that various sequences are capable of adopting under the topological mess.

3.3. Probing of altered structures using single-cut restriction endonucleases

Sequences that adopt unusual structures in form V DNA were mapped using restriction endonucleases, that have single cleavage site in pBR322, as probes. ClaI. EcoRV, EcoRI, SalI. SphI and PvuII sites were completely cleaved, whereas BamHI, AvaI, PstI, Hind111 sites showed resistance to cleavage. These resistant sites became amenable to cleavage after removal of topological strain either by cleavage with other restriction endonucleases or topoisomerase I.

Analysis of flanking sequences at these resistant sites revealed the presence of poiy pyrimidine-poly purine srretches or short stretches of alternating purine-pyrimidine sequences (a few bases out of alternation in some cases) at these locations. These sequences are known to adopt altered conlarmation under the force of super~oi l ing~,~ . Such sequences may be influencing the structure adopted by restriction endonuclease recognition sequence. Using this approach the enzyme recognition sites were divided into two groups: (ilsequences that are cleavable in form V DNA, including those that adopt B or B-DNA-like conformation, and (ii) sequences that are uncleavable by restriction endonucleases. The latter includes Z-DNA, single-stranded DNA and other non-B Structures.

3 4. Probing of nlfered structures using multiple-cut restriction endonucleases

Since the single-site enzymes give information about a very small portion of the molecule, the studies were extended to the enzymes with multiple sites in pBR322. A single hit approach was used to retain the topoioglcal strain at the time of cleavage so that the topological strain would be retained while the enzymes cleave at each of the sites.

All the four sites of enzyme Narl (GGCGCC) and FspI(TGCGCAj showed reduced rate of cleavage in form V DNA as compared to form I DNA. Narl recognition sites 434 and 548 showed signscant resistance to cleavage. These contain an 8-hp alternating purine-pyrimidine sequence and a

IISc THESES AHSrRkCTS 303

ooly purme-poiy pyrimid~ne strctch, respect~vely, in the flanking reglon

Similarly FspI site 1354 that showed resistance to cleavage in form V DNA contained a large stretch of alternating jiunne-pyrimidine sequence. Thus, similar hexanucleotide sequences could adopt different conformations depending on the flanking sequence under identical topological strain.

Geometry of the 2-DNA makes 5' position of the cytosine unavailable for methylation by m e t h y l a ~ e s ' ~ . This h c t was used to may Z-DNA and other unusual structures present In form V DNA. This technique has an advnni~ge over those described earlier because the sequence is modified rather than clcavcd. Hence, the history of the structure of methylatable sites is preserved as a methylation lcvcl even when the superhclical strain In form V is removed by subsequent restriction endonuclease cleavage. Ertcnt of methylst~on of v ~ r i o u s sites was quantitated by incorporation of 'H methyl groups usmg W SAM as substrate for mcthylascs followed by fluorography. After complete or near-complete methylat~on, all the live methylases lested were found to methylate form V DNA to a significantly lower lcvel compared to form 1 DNA. One methylase (M. Hhal) recognising alternating purme- pyrimidine sequence iGCGC) and another (M. Alul) recognislng non-alternating sequence (AGCT) were used to rank their cognate methylation sites based on theextent to which they aremethylatable.

Some sequcnczs In rorm V were shown to be completely in B form while others exlst partly in altered conformation. Modulation eflect of neighbouring sequences was seen as not all the potential Z-forming sequence (alternating purme pyrimidine) of less than 7 bp adopt Z conformation in form V DNA. Reg~ons of ~mperfect alternating purine-pyrimidine structure were sornetlmes capable of adopting altered conformat~on; and poly purine-poly pyrimidine stretches were found to be the second best cand~dntes to adopt unusual structure. In addition, some regions of altered structure had no apparent 2-forming sequence, nor were they in polypurine stretches. Thcse might represent novel Icft-handed helical structures. Large regions of either B or nun-B conformation were not always observed. This was in contrast to the belief that B-Z junctions require a large amount of energy and hence Ions stretches of altered and unaltered conformations would be favoured over short stretches as they mvolve less number of junctions.

4. Conclusions

Using topologically constrained form V DNA as a model system, a detailed study was canled out to look at the probable structure that natural sequence could adopt under high torsional stress. I t could' he demonstrated that the capacity t o flip over to nun-B conformation was highly influenced by the llature of the flanking sequence Sequences other than alternating purine-pyrimidine could adopt non- B conformation, presumably left-handed. Drawing the results from restriction endnnuclease cleavage and methyiatinn studies a hierarchy of altered conformations adopted by DNA sequences in form V was worked out and a map mdicating the regions with structural alterations was developed. We could demonstrate that DNA topology could play a significant role in modulating sequence-dependent conformational transitions and that both right- and left-hauded helical segments can coexist. Most interestingly altered and unaltered conformations appear t o alternate four times in less than 40 nucleotides in the regulatory region ofpBR322. Such an altered conformation in the tetR promoter was shown to inhibit transcription initiation. The combined effect of flanking sequences and negativc supercoiling has a strong effect on structure adopted by a given sequence. Thus formation of unusual structures may play an important role in the regulation of genetic processes in uiuo.

IlSc TIESES ABSTRACTS

References

I SASISEKHAKAV. V . BANSAL, M. BRAHMA?HAR~, S K AND

GUPTA, G

2. RICH, A . NUKIIHEIM, A. AVD

WANG. A 13.-I.

3. STETLER, U. H., WBBEH, I<., KOLLER. T H. AFU WEISSMAN, CH.

4 Suouc~c, Y. S., LATHA, P. K., RAMESH, N , MAJUMDEH, K , MAN~YAN, V. AND

BQAHMACHAI~I, S. K.

5. BRAHMS, S., VLRGYE, I. AND

BRAHhlS. J. G.

6. POHL, F M , THOMAE, R. ANU

DICAPUL, E.

7. BXAH\%ACPAKI. S. K., S ~ o u c w , Y. S.. Cahron, C R ANL, MCCIE~ANI, , M

8. NonnHEIM. A , LnFm, E. M., PECK L. 1. W ~ U G , I C.. STm.mn, B. D AND RICH, A

9 EFSI KATIAUIS, A. AND

CANTOR, C. R.

10. V~nDlMoh, L. AND RICH, A.

Proc. Second SUNYA Conseruntion it, 111v D i a , ~ p l ~ n ~ ~ r!f Bioa~vL~<ular Stcruud>norniis, 1981, Vol. I, p. 123, Adenine PTCS\, New York.

Anttic. Re". Blochem., 1984,53, 791846.

.I. Mol. Bid , 1979, 131, 21-40.

J Moi. Biol. 1982, 162,473-493.

Noturr, 1982, 300, 545 -546.

J. Mol Biol., 1987,193, 201 -211

Nucieic Acids Rri., 1984. 12, 805'3-8072.

Proc. Nan. Acad. Sci., USA, 1984,RI. 3268--3272.

Thesis Abstract (PI1.D.)

Studies on riboflavin-carrier proteins: Physicochemical, biosynthetic and immunological aspecls by Sandhya Visweswariah S. Research supervisor: P. R. Adiga. Department: Biochemistry.

I. Introduction

The mechanism of transport of water-soluble vitamins to the developing mammalian foetus has been little understood, but the discovery of certain vitamin-carrier proteins, and their obligatory requlrernent during pregnancy in rodents', suggests that a mechanism analogous to the avian system is operative in higher animals as well. Thus, a riboflavin-carrier protein (RCP) has been identified in the chicken egg and it has been shown to be involved in providing the vitamin to the developing chick embryo2. Antibodies to the chicken egg white RCP could detect an immunologically cross-reacting protein in pregnant rat serum, and immunoneutralisation of rat RCP was shown t o lead to pregnancy

llSc THESES ABSTRACTS 305

termination in the pregnant rat1. The studies detailed herein are concerned with the detection and subsequent characterisation of RCPs in pregnant primate sera, and a study of their biosynthetlc and molecular characteristics. The fact that these primate RCPs showed immunological cross-reactivity with chlcken RCP prompted the production of monoclonal antibodies (MAbs) to chicken RCP and their subsequent characterlsation and study of their cross-reactivity patterns with the primate RCPs.

2. Experimental procedure

Chicken RCP was purified by the reported procedures and radioimmunoassay was performed as described earlier3. Primale RCPs were isolated from pregnant bonnet monkey serum and pregnant human and umbilical cord sera by high-resolution protein purification techniques such as fast-protein liquid chromatography and affinity chromatography. The isolated proteins were employed in radioimmunoassay and riboflavin-binding studies as described earlier4.

Studies on the hormonal modulation of monkey RCP were performed on bonnet monkeys of the Institute colony. Human sera was obtained from voluntary female donors. Serum samples were assayed for RCP by a heterologous radioimmunoassay, and steroids by standardised methods4.

Monoclonal antibodies to chicken RCP were raised by the hybridoma technique of Kohler and Milstein! Characterisation of these MAbs was performed by established procedures6, and epitope analysis performed by solid-phase bmding assays' and a novel method developed in this thesis involving high-resolution gel filtration.


Initial studies were directed towards the identification of RCPs in pregnant primate sera. Thus, by employing a heterologous radioimmunoassay utilising L2SI-labelled chicken RCP and its antiserum, proteins showing immunological cross-reactivity with chicken RCP were detected in pregnant bonnet monkey sera and human sera. The cross-reacting proteins were barely detected in the sera of male animals suggesting a role for them during pregnancy alone, in a manner analogous to rodent RCP.

Monkey RCP was shown to bind riboflavin with high alfinity, and it was isolated by a series of gel filtration, ion-exchange, and chromatofocusing techniques. The protein was shown to have properties very similar to chicken RCP, such as molecular weight (- 37,000) and pI (-4), and both primate and chicken RCPs had very similar tryptic peptide maps indicating their close sequence homology.

Human RCP was also isolated along similar lines both from human pregnancy and cordsera. These proteins had similar properties to monkey and chicken RCP, and showed a preferential affinity for riboflavin than FMN and FAD. This evolutionary conservation of RCP from birds to primates, both in terms of structure and function, suggests the vital role these proteins may have to play during gestation in higher mammals as well.

Both chicken and rat RCP have been shown to be estrogen-dependent gene products1. In order to study the hormonal modulation of primate RCPs, circulatory concentrations of monkey RCP were monitored during the menstrual cycle and early pregnancy of bonnet monkeys. Consequent to a mid- cycle surge of estrogen, circulatory concentrations of RCP were increased around days 16-18 of the cycle, as well as in early pregnancy. In fact, higher concentrations of RCP could be detected in circulation of immature male and female monkeys following estrogen administration to these animals, in a manner analogous to rat and chicken RCP'. This estrogen-dependent synthesis of monkey RCP again hints at the importance of this protein during pregnancy in monkeys.

Human RCP levels were measured in the sera of female volunteers during the menstrual cycle.

306 llSc THESES ABSTR4CTS

Concomitant with the two elevations of estrogen concentration during the cycle, two pecks of RCP were also observed. again suggesting the estrogcn-dependent synthes~s of human R C P ;is well.

The extensive similarit~es between chicken RCP and prlmatc RCP suggr~ted clow a~n~no-acid sequence homology amongst the pmteins, and their immunolog~cal cross-reactivity was further probed by generating MAbs to chicken RCP. Optimal ~mmunisa t~on protocols 2nd fus~on conditions were standardised and as a result, six MAb-secreting cell lines werc oht:tlned. Thc MAhs werc characterised with respect to sub-class, isotype and light chain type, and the affinities of the antibodies i o chicken RCP estimated by a Scatchard analysis. These six MAbs werc directed lo three differcnc epitopes on chicken RCP, as detected by an epiiope analysis. A novel method of epitope :inalysis was standardised and involved high-resolution gel filtration employing Superose I? in conjunction w ~ t h fast-protein liquid chromatography. The results obtained w ~ t h this mcthod werc conlirmed by the routine solid-phase binding assay. By employ~ng a radioim~nunoassay otilis~np thcse MAbs and 1251- chicken RCP, it was shown that the thrce epitopes defined by these MAbs on chicken I tCP were present on both monkey and human RCP. Evidence was also obtained suggesfing the identity of RCP from human plegnancy sera and umbilical cord sera. However, cerlarn epitopes have divergcd nlorc than others during evolution as suggested by the lower affinities of primate RCPs fck ccrkiin MAhs. These results thus confirm the observat~ons made In the first chilpter of the thests as to the high degree of evolutionary conservation of RCPs, and strongly suggests that the protein-n1cdi;rted vit:~mi~; delivery mechanism 1s operative as in lower mammals.

References

2. WINTER, W. P.. BUSS, E G., CLAOEm, C. 0. AND

BOUCHER, R. V.

3. M ~ T H Y , U S

1 6. GALPKL, G. AND MILSTEIN, C.

7. STAHLI. C., STAEHTLIN, TH. AND

MIGGIANO, V

In M o i c ~ d a r htoloqy d e y g rnarurmon, CIIIA Foundnt~on Symposium. 1983, 98, 1 1 1-136. Pitman. London.

Sludrm oon nhof ln~~in binding pmrern: ph~sirr*./wm.mrlui rlrrd biorvnrb<.lir ospecrs. P h D 'Thesis, Indlan Inst~tulc of Science. Hnngolorc, ladin. 1977.

Srirdies on ummm corrier proferns p h y s i i v c h ~ ~ m n n l , h~o.~~nlheric nnJ /iuncrtonal aapecls. Ph.D. Thesis, lndlnn lnslitute oiSciencc. 8:ing:iloie. India, 1982

Nnrurp (Lond.), 1975, 256, 4%.

Methods Eszymoi.. 1981,17, 3 46

Methods Enzymol, 1983, 92, 26-36.

llSc THESES ABSTRACTS 307

Thesis Abstract (1'h.D.)

Functional and struetud analysis of soybean agglutinin by Must i Joginadha Swamy Research supervisor: A. Surolia. Dcpar tmcnt : Molecular Biophysics Unit.

Lectins are cell agglutinating and carbohydrate-binding proteins of plant and animal origin'. Though first discovered In the late nineteenth century, research on lectins gained momentum only in the 1940s when ~t was shown that certain lectins ngglut~nate human blood groups in a selective manner. Two observations madc in the 1960s that (i) certain lectins induce mitogenicity in lymphocytes. and (li) certam lectins preferentially agglutinate transformed cells as compared to their normal counterparts, led to the widespread use of lectins as tools in biology and medicine, In addition. the use of lectins in the purification and characterisation of glycoconjugates and carbohydrates is being widely exploited. These applications provided the impetus to the pnrification and characterisation of lectins from a variety of sources.

Soybean agglutinin (SBA) is an N-acelylgalactosamine-specific. homotetrameric glycoprotein isolated from the seeds of Glycrnt. mox'. This lectin attracted much attention due to its biological propert~er which include mi(ogenicity, preferential agglutination of transformed cells, a specific recognition of nodulatlng Rhizoblum strains and fractionation of cells. Though a great deal of knowledge has accumulated on biological properties of SBA, which are manifested by its ability, t o hind t o sugars, stud~es reporting the specificity and mechanism of its interaction with various saccharides arc scarcc. We have, therefore, undertaken a detailed thermodynamic and kmetic analysis of saccharide binding t o this lectin.

2. Results and discusion

2.1. Thermodynamic studies on sacchurrde binding to soybean agghtinin

Thermodynamic studies on saccharide binding to SBA have been performed using fluorescence spectroscopy3. Rinding of N-dansylgalactosamine (DnsGaln) to SBA resulted in a large increase in the fluorescence intensity of the sugar, with a coricomttant blue shift of 25 nm in the emission maximum. The changc in fluorescencc intensity and the accompanying blue shift are due to saccharide-specific binding of lhe sugar to the lectin since addition of N-acetylgalactosamine reversed both the effects. By monitoring the flouresccnce changes observed when the sugar was titrated with the lectin, association constanls for the binding of DnsGaln to SRA were determined. From the temperature dependence of the association constants (using van't Hoff plots) the thermodynamic parameters associated with the binding were calculated. Binding of non-fluorescent, inhibitory saccharides was studied by monitoring their ability to displace DnsCaln from ~ t s complex with the lectin. This study shows that in the galactose configuration, C-2 and C-6 hydroxyl groups favour the binding. A hydrophobic substituent at the anomeric position favours binding considerably. The increase in the fluorescence intensity upon binding and thc blue shift in emission maximum indicate that the binding region of SBA is considerably apolar. Thermodynamic parameters obtained for the binding of DnsGaln reveal that the strong binding of this sugar (K,= 1.5 x 106M-') is due to a small entropy value (AS = - 10.9 kJmol- ' K-'). Binding of simple saccharides is mainly governed by enthalpic forces.

308 llSc THESES ABSTRACTS

2.2. Kinetic studies on saccharide binding to SEA

Stopped-now fluorescence and temperature-jump relaxation studies have been performed to study the kinetics of saccharide binding to SBA. In the stopped-flow studies increases in the flourescence intensity of DnsGaln upon its binding to SBA was monitored as a function of time. Decrease in the fluorescence intensity of this sugar due to the dissociation of the protein-sugar complex when it was subjected to temperature perturbation was followed in the T-Jump studies. These studies show that binding of DnsGaln to SBA is a slow process ( k , , = 2.4 x IOS M-'s- ' at 20°C) which is slower than diffusion-controlled processes by 3-4 orders of magnitude. The dissociation of this complex with the rate constants of 0.2s-' at 20°C is one of the slowest observed for lectin-sugar interaction^',^.

2.3. Chemical modification andfluorescence-quenching studies

Chemical modification and fluorescence-quenching studies were carried out to investigate the involvement of tryptophan residues in the activity of SBA. Modification of tryptophan residues with N-bromosuccinimide (NBS) resulted in a loss ofsaccharide binding and hemagglutinating activities of the protein. Modification did not result in any change in the secondary structure of the protein or its state of aggregation as observed from the CD spectra and gel filtration profiles, respectively. Fluorescence quenching by iodide ion showed that some of the tryptophan residues become less accessible to the quencher in the presence of specific sugars like N-acetylgalactosamine. Kinetics of tryptophan modification by NBS, using stopped-flow spectrophotometry showed that there are two types of tryptophan residues in SBA, which are accessible to the reagents with their rate constants differing greatly from each other. In the presence of near-saturating concentrations of N-acetylgalactosamine, the kinetic data yielded three components suggesting that saccharide binding leads to an increased heterogeneity of tryptophan exposure. The kinetic data support the involvement of tryptophan residues in the activity of SBA.

2.4. Studies on the secondary structure of SEA

Prediction of secondary structure4 of SBA and its comparison with that of three other legume lectins, uiz., Concanavalin A, Yiciafaba lectin (Favin) and lenti! lectin (LcL) show that 8-pleated sheet and 8- turn are the predominant secondary structural elements in these lectins. The gross secondary structural content of these lectins is very similar. They contain about 35-45% p-turn, 40-50% j-sheet and 0-10% n-helix and thereby fall into a structurally distinct class of proteins with high 8-sheet and fl-turn content. However, a comparison on a residue-to-residue basis showed that there is a considerably greater similarity in the secondary structure of SBA and Con A and between Favin and LcL than in other pairs suggesting a divergence in the evolution of these lectins5.

2.5. Chemical cross-linking studies

Chemical cross-linking studies have been performed6 to understand the arrangement of subunits in soybean and peanut agglutinin. Cross-linking of these homotetrameric proteins with bifunctional reagents such as dimethyl suberimidate resulted in the formation of covalently coupled oligomeric species. The products formed were analysed by polyacrylamide gel electrophoresis in the presence of sodium dodecylsulphate. The analysis of the relative content of the oligomeric species showed that in both soybean and peanut agglutinin7 the subunits are associated in an isologous fashion (in which there is more than one type of subunit approach). The proteins have a D, symmetry.

11% THESES ABSTRACTS

References

I. SHARON. N AND Lis, H

2. LOTAN. R.. SI~CCLMAN, H. W., Lfi, H , AND SHARON, N.

3 S ~ A M Y , M . J . . SAPTRY, M.V K KHAK, M I, AND Sunol IA. A.

5 SWAMY, M. J.. SASTRY, M. V. K. AND Sunoun. A.

6. HAIDU, J.. RARTHA, f AN0

FRIEVRICK, P.

7. SALUNKE, D. M.. SWAMY, M. J , KHAN, M I., M A N ~ E , S. C , SUKOLIA. A AND VIIAYAN, M

Annu Reu Bmchum. 1986. 55, 35 67.

J Rmi. Chelm.. 1974. 249. 12191224.

tliochrm. J., 1986, 234. 515-522.

Ad" Cnzgmoi., 1978, 47, 45- 148.

J tlio\ii., 1985, 9, 203-212

Eur J. Biochrm.. 1976, 68, 373-383.

J R d Ciwtn., 1985, 260, 13576-13579.


High-pressure and thermal studies on germanium and silicon telluride glasses by S. Asokan. Research supervisor. E. S. R. Gopal . Department: Physics.

I. Introduction

Germanium and silicon chalcogenide glasses have extensive technological applications1. It is highly desirable to understand the physical properties of these materials t o chdracterise them for device appl~cations. In the present study, response to high pressure and thermal crystall~sation behaviour of germanium and silicon telluride glasses was investigated. Such studles throw light on the structure, electronic properties and their dependence on composition of chalcogenide glasses'-4.

2. Experimental programme

Bulk Ge,Te,,,., and Si,Te,,,., glasses have been prepared by the melt-quenching technique. Appropriate amounts of the constituent elements (5 N purity) are sealed in fused silica ampoules (evacuated to and filled with commerc~al argon gas). The ampoules were kept in a rotary furnace at the desired temperature and rotated continuously to homogenisc the melt. After a thorough homogenisation ofthe alloy in the molten state for about 48 hours, the ampoules were quenched in ice water or NaOH + ice-water mixture. The glassy nature of the samples is confirmed by X-ray dilTraction.

The electrical resistivity measurements at high pressures were carried out in a Bridgman a~lvil device up to a pressure of 8 G P a and down to liquid nitrogen temperature. The details of the experimental set-up and the calibration methods have been discussed elsewhere. Structural studies were also performed on samples recovered from the pressure cell.

Thc differential scanning calorimetric studies were performed on a Perkin-Elmer DSC-2 differential scanning calorimeter. These experiments are done at a constant heat~ng rate of 20K min- '. Structural studies were carried out on a Philips X-ray diffractometer.

310 IISc THESES ABSTRACTS

FIG. 1. The variation of electrml reslstinty with pres- FIG. 2. The variation of transirion pressure (P,) with sure for Ge,,Te,,, Ge,,Te,, and Ge,oTe,o glasses. compmtlon for Ge,Te ,,,-, and Si,Tc ,,,., glasses.


The var~ation of electrical resistiwt). with pressure for Ge,Te,,,_, glasses with x 5 20 is shown in fig. 1, indicating that the electrical resist~vity of these glasses decreases nith pressure up to a certain pressure P, At P , there is a sharp, discontinuous transition from the glassy semiconductor to crystaiiine metal states. The behaviour IS similar for all other glasses in the Ge-Te and Si-Te systems with marginal drfferences5? The most interesting aspect is the variation of the transition pressure P , of Ge,Te,,,., and Si,Te,,,_, glasses aith composition x (shown in fig. 2). It is seen from fig. 2 that P , v s x curve of GezTe,,,-, and Si,Te,,,-, glasses shows a maximum at x = 20.

The dc conductivity measurements on these glasses confirm that the matarlal is semiconducting with a single activation energy AE below P, and exhibits a metallic behaviour above P,. The variation of AE as a function ofx in both Ge,Te,,,., and Si,Te,,,_, glasses shows an anomaly al the + = 20 compositmn. Figure 3 shows the variation of AE with x for Ge,Te,,,., glasses. The E u s x curve of Si,Te ,,,_, glasses is similar.

The thermal crystallisation studies on Ge,Te,,,_, and Si,Te,,,-, glasses aiso reveal an interesting feature at the x =20 composition. Ge,Te,,,-, and Si,Te,,,., glasses with x 5 20 show a double- glass transition and double-stage crystallisation. On the other hand, the glasses with x > 20 exhibit a single-glass transition and single-stage crystallisation only7+

The present high pressure and thermal crystallisation studies also reveal the existence of metastable

llSc THESES ABSTRACTS

, 1", , , , , 15 17 19 21 23 25 27

Ge CONCENTRATION (AT%/.)

FIG. 3 The composition dependence of conductivity actlvatlon energ) for Ge,TTe,,,., glasses.

crystalline compounds in the Ge-Te and Si-Te systems.

The important conclusions of the present studies are:

(I) GexTelo,., and SiJe,,,., glasses undergo sharp, discontinuous glassy semiconductor- crystalline metal transition.

(ii) The observed properties like the semiconductor-metal transition pressure; the activat~on energy for electrical conducrion, etc., show an anomaly at a composition x = 20.

(iii) The thermal crystallisation behaviour of these glasses also shows an interesting change at the composition x = 20.

The above results suggest that the x = 20 composition in Ge,Te,oo., and SixTe,o,., systems should have unique structural features. Attempts are being made to explain the x = 20 anomaly in AT'B:6,_, chalcogenide glasses on the basis of a model which proposes a unique non-crystalline

orderirlg state a t x = 20 comprising \ A / ~ - ~ \ A, ' structural units. ' LB-~/ References

3. A s o s ~ s . S., PARTHASARATHY, G. AHU GOPAL. E, S. R.

4. ASOKAY, S., PARTHASARATHY, G., Suseawa. 0. N. AND

GOPAL, E. S: R.

5. ASOKAN, S . , PARTHASARATHY, G A H 0 GOP~L, E. S. R.

Electronic pmce.sse.v In non-rrysrailine materials, 1979. Clarendon.

EBct ($high pressure on chaico,qenide glosses, Bull. Voter. Scl.. 1985,7, 271-302.

Pressure.induced-polymorphous cryslallisalion in bulk Si,,Te,, giass, J . Marer. Sci., 1986, 21. 625-629

Electrical transport and crystallization studies ofglassy semiconducring Si,,Te,, alloy at high pressures, J. Phys. Chem. Solids, 1986, 47, 341-348.

Evidence for a nen metastable crystallme compound in Ge-Te s)slem, .Morer. Res. Bull., 1986, 21, 217-224; 1141-1142.

312 IISc THESES ABSTRACTS

6. ASOKAN, S , PARTHASARATHY, 6. Crystallization of bulk Ge,Te,,,., and Si,Te,,,., glasses at h ~ g h AND GOPAL, E. S. R. pressures and temperatures, Proc. XIV lnt . Congr. on Glass, 1986, 1,

329-336.

7. ASOKAN, S., PARTHASARATHY, G. Crystallizat~on stud~es on bulk SI,T~,~,., glasses, J . Non-cryst. Solids, AND GOPAL, E S. R. 1986,86, 48-64.

8. ASOKAN, S., PARTHASARATHY, G. Cry~talluat~on studies on bulk Ge,Tc,,,-, glasses, Inr. J Rapid AND GOPAL, E. S. R. Solid$, 1987, 26, 257-271.


Studies on laser-induced optical and morphological changes in obliqueliy deposited germanium and lead telluride films by L. Kameswara Rao. Research supervisors: A Selvarajan and M. Ramakrishna Rao (ISU). Department: Electrical Communication Engineering.

1. Introduction

Various properties of the materials, such as optical, electrical, chemical and other properties are known to be highly sensitive to their microstructure. A wide range of microstructures can be created, when the materials are prepared in thih film form, the configuration of which depends upon the preparation method as well as the experimental conditions during deposition. Oblique deposition technique is one such preparation method which is known to give rise to a characteristic tilted . - columnar shape to the microstructure'. Films possessing such microstructures are known to exhibit anomalies in their optical, electrical and physical properties which deviate by a large magnitude from the corresponding normally deposited films. Modification or removal of such a microstructure by external stimulii might result in a large variation in the optical, electrical and other properties and such variations are of scientific and technological importance, in terms of device fabricatioh and applications.

Laser irradiation of semiconductor films has proved to be a fruitful field for both applied and fundamental research2. By virtue of their ability to interact in a highly localised area and with high speed, lasers found their way into semiconductor material processing. Many interesting and 'reproducible material characteristics have been obtained by laser-assisted modification of the microstructure in the semiconductors.

The obliquely deposited films possess an in situ texture, which occurs due to nucleation and growth mechanism during deposition. Laser irradiation in such films might lead to various modifications in their properties and due to the peculiarities of the microstructure, it is expected that laser-induced effects in these films might assume a different nature as well as magnitude. The present investigations are aimed at assessing both qualitatively and semiquantitatively, the nature and magnitude of useful changes that occur in optical, structural, morphological and chemical properties of two such films, uiz., Ge and PbTe.

2. Experimental details

In order to investigate the films as above, two types neodimium lasers were fabricated, using mostly indigenous components. A diagnostic set-up to measure the laser parameters as well as to monitor the onset of laser-induced reactions was also fabricated and used.

IlSc THFSES ABSTRACTS 313

The two lasers, an Nd:YAG and an Nd:glass laser, are excitcd by pulscd-xenon flash laillps a~ ld are operated in 11-ec running mode. Both lasers give a maximum output energy of 250 m~lli Joules (mj), in a pulsewidth of 300 micro seconds (ms). An energy and peak pouer-measuring instrument has been fabricated to monltor the laser beam parameters. A hlgh-speed photodlode assembly with nearly a nanosecond risetime has been fabricated to monitor the pulse shape and duration. The dragnostic set-up used for rnvestigatiotls also facilitates detection of onset of laser-induced opt~cal changes, time-resolved studies on photovoltage as well as time-resolved reflectivity of the sample during laser irradiation. A 20-cm focal length lens is used lo focus the Nd:laser output on to thc film. 3000A-thick films of high-purity Ge and PbTe semiconductors are used for investigations. The films are preparcd at 8 0 angle of deposition in a vacuum of 10-'Torr. The observed reaclioni arc characterised and analysed using clectron microscopy, spectrophotometry, anisotropic transmission of polarised light, laser speckle, Auger and photoelectron spectroscopy and n~icrodensitomctry.


The reactions in both Ge and PbTe films arembserved to proceed in two stages, classified for convenience as low- and high-beam conditions, by virtue of the difference In the nature of the observed reactions.

For Ge films, the irradiation of the films in the power density range of 9-11 Kw/cmZ, gave rise to appreciable optical transmission contrast. In the low-beam conditions, corresponding to power density near 9 Kw/cm2, the observed contrast is nearly 7%, whereas in the high-bmm conditions the observed transm~ssion contrast is nearly 75%. Thc irradiatcd region underwent, simultaneously, structural, morphological and chemical changes. Electron microscopy studies revealed an anlorphous to polycrystalline transformation. Estimation of temperature rise in the irradiated region indicated that the above transformation occurred via solid-phase epitaxy. Spectrophotometric data evaluation revealed that the bandgap of the material increased upon irradiation by nealy @lev. Studies on transmission of polarised light as a function of angle of incidence gave anisotropic peaking at nearly 60", the latter being considered as approximate angle of the micrccolumns present In the film. IJnder low-beam conditions of irradiation, the above anisotropic nature persisted, indicating that columnar collapse 1s not responsible for the observed optical transmission contrast. Resistance measurements indicated an increase in effective resistance, upon irradiation which is considered as due to oxidation contamination of thc film during laser heating. Time-resolved photovoltage studies showed a reversal of sign of the pholovoltage in the film when the power density crossed a threshold value. An examination of Auger and XPS spectra revealed that irradiated region is enriched with GeO phase near low-beam conditions, while GeO, is predominantly present in high-beam conditions. Simple heating of the virgin films in ambient temperature made the film totally transparent with Ge being converted to GeO, phase. This enabled to prepare both positive and negative relief patterns, by suitably tailoring the irradiation conditions. It is concluded that the observed optical transmission contrast and its dependence on laser power density is essent~ally due to the formation of selectwe oxide phases, under different irradiation levels.

The PbTe films showed optical contrast effects in the power density range of 7-9 Kw/cm2. The films exhibited a low-transmission contrast of nearly 4%, with the irradiated region becoming darkened. Relatively high-reflectance contrast of nearly 40% is observed under h~gh-beam conditions with theirradiated rcgion becoming relatively morc transparent.Thespectrophotometricdata revealed that the absorption edge has shifted to longer wavelength. Electron microscopy studies revealed that the films are polycrystalline both before and after irradiat~on, under low-beam conditions, while the films became single crystal under high-beam conditions. Temperature estimations indicated that structural transformations occurred uia liquid-phase epitaxy. From the nature of anisotropic

314 lISc TI-1ESF.S ABSTRACTS

tIansmission ol the polarised light, it is concluded that the columnar features persisted near low-beam conditions while columnar collpase has occurred under high-beam conditions. The resistance change in the film upon irradiation is negligible in the direction of vapour Incidence whilc a slight decrease is observed in the direction perpendicular to the vapour incidence. The observations on the time- resolved photovoltage is slrnllar to that observed in Ge films XPS studies revealed that upon ~rradiation appec~able amount of TeO, and PbO phases are formed in the near-surface region of the film. Heat treatment of films did not reveal any inlerestmg changes. From the above observations it is concluded that the observed transmission contrast is due to photodarkening phenomena in the irradiated regioll and the optical reflectance contrast under high-beam conditions is due to columnar collapse mechanism. The laser speckle stud~es indicated that the morphological changes in the films of both Ge and PbTe upon irradiation arc mostly microscop~c, with little change in the scattering characteristics of the surface.

In summary, obliquely deposited films of both Cie and PbTe exhibit optical contrast, upon laser irradiation, while undergoing, simultaneously, optical. structural, morphological and chemical changes. The mechan~sm responsible for contrast appearance is found to be dependent upon power dens~ty and is different for both the materials. Of the above two, Ge may be preferred for good optical transmission contrast, while PbTe films are better for high-reflectance contrast. Both negative and positive relief patterns can be registered in these films, with no additional treatment in the case of PbTe fiims and with heat treatment in the case of Ge films. A comparison with reported results indicates that these films have high economy of writing energy. The observed results are expected to open new avenues in the fields of optical storage, LCD displays and cold oxidation of semiconductor surfaces for device fabrication.

References

1 DIRKS. A. G. AYD LEAMY, H. I. Columnar microstructure in vapor deposited thm films, Thm Soltd Films, 1977, 47, 219 -233.

Transient annealing of semiconductors by laser, electron beam and radiant heating techniques, Rep. Prog. Phys., 1985,48, 1155- 1233.


Effect of mechanical stress on the properties of defect levels in crystalline silicon by N. Balasubramanyam. Research supervisor: Vikram Knmar. Department: Physics.

I. Introduction

Study of solids under externally applied stress is useful in undcrstanding their electronic structure as well as the fundamentals of the physcal pheuomerua under study. Though the bulk properties of the semiconductors have been studied extensively, relatively little work has been reported on the e&ct of stress on impurity and defect levels. This thesis is devoted to a study of the effects of stress on some technologically important defect levels, namely, the quenched-in levels and thc gold-related levels in silicon and also on meta-semiconductor interface states.

11% THESES ARSTRAC iS

2. Experimental techniques

Current as well as capacitance-voltage measurements are done on ti- and p-type Schottky barriers at varlous hydrostatic pressures. Deep-levd transient spectroscopy1 (DLTS) has been used for the study of the deep levels. A two-channel gated integrator-based DLTS system is used in this work. It is found' that the system parameters such as the gate position ratio, the gate width and the integrator response trme have considerable influence on the sensitivity, resolution and accuracy of the DLTS method. Computer s~mulations have been used to evaluate these effects and suggestions are made Icr the selection of the optimum values of the parameters.

A piston-cylinder type of pressurc cell is used for the applicat~on of hydrostatic pressure. The same cell is modified appropriately Tor the applicalion of uniaxial stress.

3. Effect of pressure on Schottky-barrier heights

The hydrostatic pressure coefficients of the barrier heights of the Schottky contacts on 11- and p-type silicon are determined. It is found that the pressure coefficient of the Schottky-barrier helght 111 the case of n-type silicon is large and comparable to the pressure coefficient of the band gap and m the case of p-type silicon, it is small. These results indicate a pinning of the Fermi level at the surface to the top of the valence band3. This is in accordance with the model involving disorder-induced gap states4 for the interface states at the metal-semiconductor contact.

4. Effect of hydrostatic pressure on deep levels in silicon

The properties of deep levc,ls related to quenched-in defects and gold-rclated defects in silicon are investigated' under atmospheric and hydrostatic pressures. The quenched-in defects are produced by quenching silicon rapidly from high temperatures. Two levels, one at E,-0.32eV and the other at E,.-0.51 eV, are found in quenched n-type silicon. The capture cross-scction of the shallower level decreases with temperature as T-* . This is explajned on the basis of an excited-slate model6 of electron capture at the defect. Both the levels move closer to the conduction band under pressure, the rate or: he movement of the shallower level being slower. Thecapture cross-section of the shallower level increases with pressure. These levels are most likcly related to iron contamination in silicon.

The properties of the gold-related acceptor (Ec-054eV) and donor (EV+0.35eV) levels in silicon are studied under hydrostatic pressure. The pressure coefficlent of the activation energy of the gold acceptor is larger than the pressure coefficient of the silicon band gap. The donor level is weakly pressure dependent. The capture cross-scctions of the levels are essentially pressure independent. From the pressure coefficients of the deep levels the strain coeficients are obtained which arc useful in analysing the uniaxial stress coefficients of deep levels on the basis of specific microscoprc models.

5. Effect of uniaxial stress on deep levels in silicon

The oniaxial stress dependence of the parameters of the gold-related acceptor and donor levels in silicon is studied. The donor level is essentially independent of the muaxial stress in any direction. The capture cross-sections of both the levels are stress independent. Thc activation energy of the acceptor level shows anisotropy in the stress coefficients. The stress coeflicient in the (1 10) direction is positive whereas it is negative in the (100) and ( 1 11) directions of stress. Various mlcroscoPlc models of gold-related defects in silicon are evaluated and a new model ~nvolving a divacancy-gold complex is proposed. The new model can explain the previously published data on these defects as well as the present results.

316 PlSc THESES ABSTRACTS

References

Deep level transienl spectroscopy. A new method to characterize traps In semiconductors, J. Appl. Phys., 1974, 45, 3023-3032.

System effects In double channel gated integrator based deep level transient spectroscopy, J. Appl. Phys., 1988.64, 6311-6314.

Pressure dependence of barr~er heights of Schottky contacts on slhcon, P h p Stat. Sol. (a), 1987, 101, K29-K32.

Correlation beween the location of the ~nterface state minimum at msulator-semiconductor interfaces and Schottky barrler heights, Jop. J. Appi. Phys., 1986,25, L353-L356.

On the quenched-in defects in n-type silicon, Phys Star. (a), 1987, 100, 239-244.


Some studies on holographic interferometry and speckle photography by B. S. Ramprasad. Research supervisors: E. S. Raja Gopal and P. S. Narayanan. Department: Instrumentation and Services Unit.

1. Introduction

Holographic interferometry and its complementary technique of speckle intelrerometry and speckle photography have emerged as powerful techniques for the measurement of small displacements and deformations of opaque objects, besides, the measurement of changes in refractive index in phase objects. In conventional holographic interferometry applied to the measurement of deformation of opaque objects it is not often possible to measure displacements less than half a wavelength of light. Also sometimes there is an ambiguity in the interpretation of fringes. This study presents some solutions to existing problems and proceeds to demonstrate some novel applications of holographic interferometry and speckle photography. The work is divided into two parts. The first part deals with holographic interferometry and the second with speckle photography.

2. Holographic interferometrj

Many of the experiments conducted centre around real-time holographic interferometry. An ellicient kinematically designed liquid gate system for rap~d in-situ processing of real-time holograms using a single solution monobath has been developed'. The zero fringe condition is stable over long periods of time. To measure fractional fringe displacements a holograph~c subtraction technique which overcomes the disadvantages of existing methods has been developed". It uses a half-wave plate to introduce a phase shift of a between exposures. It has been applied to the study of the deflection of a loaded cantilev.qr. Another technique3 of measurement of small displacements is by introducing background fringes in double-exposed holograms (fig. 1). A thin glass wedge used as a wavefront tilter is rotated between exposures. It is shown that the displacement modulates the fringes and displacements as small as 0.07gm can be measured. The wavefront tilter has the added advantage ofeliminatingphase ambiguities. This is confirmed by studying the bending of a strip. The elimination of phase ambiguity has also been achieved by a novel technique of multiplex double-exposure

IlSc IHESES ABSTRACTS

FIG. 1 . Photographs ol rcconstrucliona from double-exposure holograms oi the strip object with (a) no rotation or the wcdgc betwccn cxposurcs and (b) the wedge rotated by 90" between exposures.

FIG. 3. l d i e t i c frmgcs o l a strip clamped a1 both ends and subjected to a couple a l the centre

holographic interferometry. Multiplex hologram o f a thin disc fixed around the periphery and loaded a t the centre ia used to den~onstrate the technique.

While holographic interferometry for opaque objects deals with changes in optical path length due to deformalion o r displacement, holographrc interkrometry of phase objects deals with phase shifts due to the changes of refractive index of the medium or the thickness of the medium (fig. 2). Using real-tirnc holographic interferometry methods have been developed to measure some of the physical paramelers of thin films like the thickness, adhesion4, mtrinsic and thermal stresses.

3. Speckle photography

Speckle pattern photography is a simpler technique compared t o holographic interferometry. .4 double-exposure spccklegram is made by imaging with a lens on a high-resolution photographic plate, light scattered by an object in an undisplaced and a displaced position. Information about the displacement can be extracted by (i) Young's interference frmges obtained by illuminating the specklegram with an unexpanded laser beam or (ii) by a whole-field filtering technique.

Etch depth in metallic specimens has been studiedb using speckle pattern photography and measurements in the range of 50 t o 125jim have been carried out. Most engineering measurements are concerned not with just displacements but strains which are derivatives of displacement. Displacement derivatives can be directly measured usiqg defocussed speckle-pattern photography. The out-of-plane displacement of a strip has been studied theoretically and expertmentally under two conditions of loading, (i) strip fixed a t the longitudinal ends and loaded a t the centre, (ii) strip fixed at the longitudinal ends and subjected to a bending moment. Isothetic fringes showing contours of displacement derivatives have been obtained using the whole-field filtering techn~que (fig. 3).

Some experiments have been conducted to demonstrate the use of speckle photography for phase objects. A novel application Ibr testing of incandescent lamps has been described. lsothetic fr~nges showing changes due to the gradient of refractive index have been obtained. An tnteresting application of speckle photography for the measurement of changes in refractive index of liquid mixtures has been presented. It has been shown that refractive index changes even as small aa 0.0005 can be measured.

318 iiSc THESES ABSTRACTS

References

1 HARIHARAN, P. AND J . P l i p . E. SCL Instrum., 1973, 6, 699-701. RAMPKASAU, B- S.

2 HARIHAKAN, P. A N U J . Phve E. Sc,. Ilutrum., 1972, 5, 976-978. RnMPR&snu, 8. S.

3 Hnnltr~RAN, P. AND . I . Phgs. E - Sci. Instrum., 1973,6, 113-175. RA~IPK~SAU. B. S.

4 RAMPRASAD, 8. S. AND RnnHn. T. S. Appl. Opt., 1978, 17, 2670.

5 Ra~~nasao . B S aho RnaHA, T. S Thui Solid Films, 1978.51. 335


investigations towards the total synthesis of natural and modified steroids by R Saibaba. Research supervisor: T. R. Kasturi. Department: Organic Chemistry.

The general term 'steroids' covers all compounds containing the perhydro-1, 2-cyclopentenophenanthrene nucleus. It includes a w~de variety of naturally occurring substances hke sterols, bile acids, sex hormones, adrenal cortical hormones, cardiac aglycones, sapogenins and alkalotds, which possess important therapeutic properties. The pronoimced physiolog~cal actlvity of the steroid nucleus has made it the target of numerous, often ingenious, synthetic strategies. In the total synthesis of steroids CD-hydrindane precursors can be convenicntly constructed wirh built-in appendages for the formation of A and B rings. These intermediates can be used as potential substrates for asymmetric synthesis of steroid^',^.

The dienealdehyde 1, an important synthon for the total synthesis of steroids was prepared from a known"-ketoester 2 in racernic and chiral forms. Thus, condensation of chlorocthyl methyl ketone with 2-mcthylcyclopentan-1, 3-dione gave the dione 3 which on sodlum borohydrlde reduction, hydroxyl group protection, carhoxylation, followed by esterification afforded the known /I-ketoeskr 2. Sodium borohydride reduction of the compound 2 gave the saturated alcohol 4 resulting from conjugate hydride addition followed by the reduction of the kctone function. The same ester 2 when reduced with LAH yielded the desired allylic alcohol 5 which was oxidised to the diene aldehyde I. Asymmetric synthesis of the aldehyde war realised starting from the chiral 8-ketoester 2, prepared by an aminoacid-catalysed asymmetric aldol cyclisation4.

Investigations towards developing an alternative route to the synthon 1, via the intermediate 6 were also carried out. Although the desired compound 6 could not be prepared by this route, two novel products, 7 and 8, were isolated in the course ofthis study. The structures of these compounds were established unambiguously by spectral (IR, '?I, 'X-NMR and mass) studies. An acceptable mechanism has been formulated for the formation of these two products5.

11% THESES ABSTRACTS 319

Annelation of the dienealdchyde I wlth methyl acetoacetate yielded the tricyclic dienone 9 which was converted to an isomeric dienone ester 10 by hydrogenation of the double bond followed by the introduction of a new double bond using selenium methodology. Condensation with methyl vinyl ketone afforded the desired alkylated product 11.


Tram hydrindanes are of paramount importance in the total synthesis of steroids. Several methods have been developed for the synthesis of rrans hydrindane derivatives6. We have successfully achieved a new synthesis of a novel synthon 12 wlth the trans-CD-ring junction, already built in, as discussed below. The fl-ketoester 2 was hydrogenated and the resulting saturated ketoester was reduced to the alcohol 13. Dehydration of the alcohol 13 t o the unsaturated ester 14 was realised by conversion to corresponding mesylate followed by refluxing in D M F with NaI and pyridine. The ester 14 was transformed to the trans-aldehyde 12 by AIH, reduct~on followed by allylic oxidation. Starting from the optically active P-ketoester 2, and adoptmg the same procedure, the aldehyde 12 was prepared in optically active form.

Many interesting biologically active B-seco-steroidal derivatives have been reported in literature. It has been shown by recent studies7 that 13-ethyl-B-secosteroid derivatives are more potent than the corresponding methyl analogues. In mew of this, various substituted 13-ethyl-B-seco-estrone analogues 15-e were synthesised as follows. Substituted acetophenones 16a-e, synthesised by reported procedures, were converted to the corresponding vinyl carbinols by treatment with vinyl magnesium bromide. Condensation reactions between the vinyl carbinols and 2-ethylcyclopentan-I, 3-dione yielded the secodiones 17a-e, which were cyclised and hydrogenated stereoselectively to the B-seco-steroids 15a-e in excellent yields. The configuration assigned at C-9 and C-14 positions are based on a detailed proton NMR study.

References

1. AKHREM, A. A. AND TITOV, Y. A. Total steroid synthesis, Plenum, 1970

2. BLICKENSTA~F, R. T., GHOSH, A. C. Tofal synthesis oj'steroids, Academic, 1974. AND WOLF, G. C.

3. MCHELL R. A., HAJOS, Z. G., J. Org. Chem, 1975,40, 675-681. COHEN, N., PARRISH, D. R., PORTLAND, L. A., SCIAMANNA, W., Scorr, M. A. AND WEHRLI, P. k

4. HMO$ Z. G. AND PARRISH, D. R. 01g. Syn., 1985,63,26-36.

5. BANERJEE, D. K., KASTURI, T. R., PIOC. Indian Acad. Sci. (Chem. Sei), 1985,95439-446. S*IB.AB~, R. AXD SARKAR, A.

6. COHEN, N. Acc. Chem. Res., !976,9,412-417.

7. JHINGRAN. A. G., GUPTA, R. C., Steroids, !983,42, 627-634. RAY, S., AGARWAL, A. K., SINOH, M. M. AND NINA ANAND

J Indian lnst Scr. July-Aug. 1989, 69. 321-328 "Tnd~an Institute olScience.

BOOK REVIEWS

Quantum mechanics and statistical methods edited by M. M. Suschinskiy (transl. by K. S. Hendzel), [Proceedings of the Lebedev Physics Institute of the Academy of Sciences of the USSR; Series Editor: N. G. Basov; Vol. No. 1731, Nova Science Publishers, Commack, USA, 1988, pp. x 340, $11 1.

After sixty years of intense study, quantum mechanics has the status of a mature fully developed subject with applications extending right from the subnuclear microscop~c world to cosmological macro systems. It is even making inroads Into the explanations of the living processes. However, subtle questions on the foundai~ons or axioms of the subject or on the validity of the computational processes used, remain to be settled. The present book is concerned with the mathematical physical aspects of the latter problem concentrating on the applications to systems with a large number of degrees of freedom, making it necessary to use statistical methods in the computations.

The first article, by L. A. Shelepin, sets the stage by enumetating some select issues in the quantum theory. The measurement problems involving the interaction betweeri the observing processes and the systems under observation, the role of unitary symmetry in enabling the existence of solutions to be proved and the description of dissipative as well as nonlinear systems in quantum mechanics are listed among the issues to be tackled. The relationship with Markov Chains, showing probabilistic links with the past and the future is highlighted, a relationship which repeats many times in the book. The second article, by V. D. Vainshtein and Yu. A. Gol'fand, is concerned with the quantum mechanics of measurement of macro and micro processes. The information theoretic statistical interpretation of the measurement process is analysed as an evolution of the density matrix of the system, especially if the system consists of weakly interacting particles. The incompleteness of the past description and its consequence on the uncertainty of the future trajectories are brought out as the main physical content of the detalled mathematical investigations. The third artlcle by S. A. Reshetnyak, S. M. Kharchev and L. A. Shelepin discusses the asymptotic time evolution of systems undergoing transfomations. The Landau theory of second-order phase transitions is used as the mathematical framework to consider this question of the kinetic temporal behaviour of the order present in the system.

The questions connected with the use of the unitary group theoretic methods are discussed in two articles. The first by V. P. Karasev and N. P. Shchelok starts with the angular momentum theory in the framework of the SU(2) group and considers the general SU(n) group from the point of view of polynominal realisations of the bases of the ~rreducible representations and their applications to many particle quantum systems. The second article by A. L. Shelepin and L. A. Shclepin contmues the probabilistic treatment of the Clehsch-Gordon coefficients of SU(2) and higher dimensional S u b ) groups.

The next art~cle by V. K. Potekhin and L. A. Shelepin applies the group theoretic methods to statistical optics. The polarisation properties of light and the statistical aspects of emission propagation in a medium are considered in the group representation theory. This is pursued further in the art~cle by V. A. Andreev on the use of the inverse scattering techniques in the quantum optics equations. In particular the soliton-type solutions, studied in nonlinear field theories, are found to be

321

3.22 BOOK REVIEWS

present In uilatable or nonlinear situations. The last arttcle by Ye. V. Doktorov and V. L. Man'ko discusses the synchrotron radiation sources and their use in studying the structure, time and space evolution of large molecules or aggrcgate systems. The computations of the overlap integrals needed In thcsc cases are d~scusscd at some length.

The hook thus covers a range ofstud~es in which the authors from FIAN have contributed over the years. The senes of books has the object of drawing attention to thesc contributions and in thls task it has succeeded well The publicat~ons have been in Russian and hance have not recelved the widespread attention of the western scient~sts. The book provides a dipsted version of the studies in an cas~ly accessible form. The only problem is the h~gh cost of the book. Because of the specialised nature and the~mall ctrculation for such booksit appears difficult to reduce the cost.Thus the book IS beyond the reach of most individuals and must depend upon exclustve libraries for the availability.

Department of Physics lndlan Institute of Science Bangalore 560012.

Molecules and radiation-an introduction to modern molecular spectroscopy (2nd edition) by Jeffrey I. Stemfeld. The MIT Press, 5 5 . Hayward Street, Cambridge, Massachusetts 02142, USA, 1985, pp. xv + 485, S4j.13. Indian orders to Affiliated East-West Press Pvt. Ltd., 25, Dr. Mumappa Road, Kilpauk, Madras 600010.

The maln Interest ofthe book lies in molecular dynamics and the interaction of radiation and matter. The concepts and the methods of modern spectroscopy are covered in Chapters 1 to 9 and this forms the basis of the later chapters in which attention is focussed on newer areas of spectroscopy developed during the last 25 years. Chapters 10 to I4 reflect the developments in laser spectroscopy to probe molecular dynamics. The book contains a comprehensive index and is fairly well referenced. There are SIX appendices, which includc a literature survey on spectroscopy.

The book commences with a review of the essential quantum mechanical background and atonllc spcctroscopy (Chapters 1 and 2). A good account of electronic energy levels and electronic transitions and simple molecular orbital theory of diatomic molecules 1s given in Chapter 3. The following chapter covers the rotation and vibration spectra of diatomic molecules. The influence of nuclear spin on rotation and rotation-vibration spectra is discussed. The analysis of the vibrational and rotational energy changes occurnng during an electronic transition of a diatomic molecule is described in Chapter 5. Zeeman, Stark and Faraday effects in m~lecules are also considered.

The elements of molecular symmetry and group theory necessary for the consideration of spectra of polyatomlc molecules are given in Chapter 6. This is followed by a chapter on rotational spectra of polyatomic moleculen. wherein I-douhhng, inversmn doubling, quadrupole and Zeeman effects are briefly discussed The chapter on v~brational spectra of polyatomic molecules deals with normal vibrations in classical mechanics and FG matrix methods. The selection rules for vibrational transitions and rotational fine structure on vibrational bonds are covered. The chapter on electronic spectra of polyatomic molecules describes Walsh rules and the spectra of simple molecules such as iormaldchyde and benzene. Also covered herem are introductory aspects of molecular photoelectron spectroscopy and energy dissipation by non-radiative processes.

The first of the chapters dealing with developments in laser spectroscopy discusses the use of optical pumping to study high-rcsolut~on atomic spectra and relaxation processes. This leads to consideration of molecular beam spectroscopy and then to important types of lasers. The area of

SOOK REVIEWS 323

optical masers is enormous and corntinually developing one. The studies on coherent optical effects are discussed In detail In Chapter 11. Close analogy between magnetic resonance spectroscopy and coherent optical spectroscopy is brought about. The next chapter discusses scveral coherent transient experiments and relatcd processes. Interesting effccts such as optical nutation and photon echoes and their application in FT spectroscopy are discussed. Also considered here are double-resonance and multiphonon spectroscopy. Nonlinear multiple photon processes betwcen atomic and molecular energy lcvels form the subject of Chapter 13. A simple theory ofstimulated Raman effect 1s presented. Methods lor the production of ultrashort light pulses to study molecular processes at p~co-second timc scale are discussed. The book concludes with a chapter projecting the future developments in laser spectroscopy.

This book is a revised edition of the one published in 1974. T h ~ s is one of the very few books that presents, from a consistent theoretical perspective, an introduction hoth to classical atomic and molecular spectroscopy and to the spectroscopic advances made by laser-based methods and thus provides the necessary foundation for modern spectroscopic research. The main area where this book shows ~ t s strength is the unified treatment and breadth of coverage making it very useful to rerve as a textbook. This book is sure to interest post-graduate students and research scholars of chem~stry and physlcs orientation and specialising in molecular spectroscopy and related fields.

Department of Inorganic and Physical Chemistry Indian Institute of Science Bangalore 560 012.

Molecular spectroscopy by P. S. Sindhu. Ta ta McGraw-Hill Publishing Company Ltd., 1214, Asaf Ali Road, New Delhi 110002, 1985, pp. 348, Rs. 39.

Several books are available on molecnlar spectroscopy, and this 1s yet another on the subject It contains ten chapters, commences with a brief introduction to electromagnetic radiation and electromagnetic spectrum followed by elements of atomic spectroscopy. In the next chapter concepts of molecular symmetry which include some information on character tables ofpoint groups are given.

Topics in molecular spectroscopy commence with a chapter on rotational spectra of diatomic and polyatomic molecules. This chapter is followed by chapters which cover vibrational spectroscopy. I n chapter 5, vibrational infrared spectra of diatomic and polyatomic molecules, and vibration-rotation spectra of simple molecules are covered. The chapter on Raman spectroscopy describes classical theory of the Raman effect, vibrational and rotational Raman effect, group theoretical classification of the normal modes according to the molecular point group and classification of vibrations of single crystals. This is folluwed by chapters on electronic spectra of diatomic and polyatomic molecules.

The chapter on spin-resonance spectroscopy covers electron-spin resonance, nuclcar-magnctic resonance and nuclear quadrupole resonance spectroscopic methods. The concluding chapter discusses briefly Mossbauer spectroscopy of chiefly iron compounds.

The book contains n few problems towards the end of each chapter, followed by a few references. There are five appendices (15 pages), so also a subject index.

There is nothing new in this book which 'distinguishes' it from other books currently in use. It appears to lean heavily on C . N. Banwell (Fundamentals of molecular spectroscopy) and I . W . Levin (Molecular spectroscopy). However, it has some serious lapses. Some clearly improper sentences misrepresent the meaning and add to possible source of confusion. Added to this, there are numerous mistakes, typographical as well as scientific. The chapters are not well written and the treatment is

324 BOOK REViEWS

'uneven' For example, without any mention of the slte group or factor group approach the procedure for the analys~s of vlbrat~ons of smgk crystals 1s given. There 1s no mention of the more important resonance Raman among the nonlinear Ramau effects but hyper-laman effect is mentioned. Likewise, the molecular orb~tal treatments given under chaptcrs on electronic spectroscopy lack adequate explanation so also the background of simpler treatments. For these reasons, the book may have very limited interest For the indivrdual reader.

Department of Inorganic and Physical Chemistry lnd~an Institute of Science Bangalore 560012.

Human immunogenetics by Stephen Litwin. Marcel Dekker. Inc., 270 Madison Avenue, New York, NY. 10016, 1989, pp. 828, $150.

Immunogenetics is an interdisciplinary 6eld that draws its strength from both immunology and genetics. This book on human immunogenetics brmgs together diverse new findings in human and mouse ~mmunogenetics. The book is generally well presented but judging from the topics covered, it is a very ambitious one and appears to be written for specialists in the field. This book will not engagc the attention of students for long since it is not meant for them. In fact. the editor's comment, "No attempt will be made to cover basic immunologic or genetic concepts" seems to be significant in this regard. Although relatively up-to-date, experts might feel that the book has lagged behind in some chapters e.g., much more is known today about a, /j and g genes of the T-cell receptor (TCR) than that described. These points apart, the book is a source of very good information and ~n its'enlirety, the topics encompass the major thrust of contemporary immunogenetics. The description of future research trends and developments in many chapters w~ll he very valuable to many readers.

The book contains five sections spearheaded by an excellent first section on immunogenetic tools. T h ~ s chapter deals with a variety of approaches and techniques being used today in the study of cell surface immunogenetics. cloning and immortalisatiol~ of lymphoid cells, acquiring geuetic data and analysis of tumor-associated antigens. Chapter 1 has been well presented and seems to deal briefly with many aspects of inmunology and gives the reader a hint of what follows in other chapters. Table 2 however is unclear whether the comparison was between protems from different organisms or between proteins from the same animal. The importance of restriction fragment length polymorphism (RFLP) and application of recombinant DNA technology in modern immunology has been highlighted in Chapter 2. This chapter on 'Human gene mapping and linkage analysis' does not go into any specific details and can, at best, serve as a preliminary introduction into this complex and fast developing field. Chapter 3 by Henry Winn describes the laws of transplantation and production of congenic mouse strains. Exceptions to these laws that served as a forerunner to natural killer cell (NK cell) discovery have also been described. The use of diagrams would have greatly aided the reader in a better understanding of this chapter. Chapter 4 on maternal-foetal immunogenet~cs is probably the most interesting one in this section and highlights many important observations in a highly controversial but remarkable field. This chapter describes the evidence available to support the varrety of mechanisms that help in the survival of the allogeneic conceptus till term. Due to its attempts to cover a very vast area the chapter by Brian Pollok may appear too simple and shallow in depth to an expert in molecnlar biology. Chapter 6 brings out the salient features of the usefulness of certain specific monoclonal antibodies such as C017-1.4 in analysing tumor antigens. Authors remind the readers about the pitfalls in trying to generate monoclonal antibodies to detect tumor-specific transplantation antigens (TSTA) since these antibodies are most likely to be non-specific in nature.

BOOK REVIEWS 325

Section I1 summarises the nature of cellular interactions and molecular events that occur during immunological activation. The basis for the defects and failure of this complex system that finally leads to autoimmunity and immunodeficiency diseases is described in subsequent chapters. Chapter 7 represents a bird's eye-view of the entire immune system and describes the salient features of the important lymphokines needed for initiation of an immune response. It must, however, be mentioned that many advancements have been made after the hook was written, especially the identification of the lymphocyte homing receptor on endothelial cells and the isolation of the pluripotent stem cell precursor from the bone marrow. Chapter 8 seems to overlap with information given in earlier chapters but the data are presented to benefit a clinical scientist who may be interested in basic immunology. The NK cell, its lineage and cell surface markers are described. The next chapter on 'Antigen-specific immune regulation' by Judith Kapp and Carl Pierce has been written very well and benefits immunologists as well as non-immunologists. The chapter covers a lot of ground and perhaps lays more emphasis on suppressor cells, suppressor factors and their influence on the total immune response. Although taken from different books, the use of appropriate diagrams is a plus point for the chapter. Fill and Tucker have presented most current information on immunoglobulin genes, their regulation and expression on the 'B'-cell surface. Interesting information normally not found in many books such as Kde region participation in K chain deletion has been described. Immunoglobulin gene translocations and the manner in which they cause neoplasms have also been described. Litwin has given an elaborate and exhaustive account on immunoglobulin allotypes in the next chapter. A section on the association of allotypes with specific immune responses and disease states have been included. Chapter 12 on 'Autoimmune states' presents evidences that link immune dysregulation with immunoglobulin genes, MHC and the T-cell receptor. It reviews the association of MHC with selected autoimmune disorders and the use of molecular biological techniques for analysing the association of specific antigenic epitopes with diseases. Although many evidences have been described for the presence of cross-reactive idiotypes in anti-DNA and anti-Sm antibodies the possibility that somatic mutations could convert autoantibodies to normal antibodies has also been covered. The chapter has also a brief description on the regulation and induction of IL-2 synthesis. Richard Hong in his chapter on immunodeficiency diseases does not treat any specific disease in detail but describes the processes involved in the development of a healthy immune response and the production of diseases as a result of genetic and non-genetic disturbances in these processes.

Section I11 deals with the major histocompatibility antigens, their structure, functional aspects, molecular genetics and association with disease and transplantation. This section forms an excellent treatise for immunologists who wish to survey the entire MHC and related antigens. The chapter on t-complex is more developmental biology-oriented than immunogenetics except for its proximal location to MHC genes. The influence that the 't' complex may have on MHC function is as yet unclear.

Information on the structure and function of the MHC antigens of various speciesfrom xenopus to man has been comparatively presented by Btankenhorn. The tables and figures do go a long way in helping the reader in obtaining a clear picture of MHC antigenic structure and terminology. In addition, a description of molecular organisation of individual class I and class I1 genes together with a small section on the evolution of mouse genes does make this chapter very interesting.

The chapters on minor histocompatability locus and QajTl Class I genes will indeed be of great interest for cellular immunologists. The association of xenotropic and ecotropic retroviral sequences with minor H loci serves as an excellent prelude to the next section on viral immunology.

Section IV on the immunogenetics of tumor and viral antigens seems to be the most interesting chapter in the book and represents an area that engages the attention of molecular biologists, oncologists, serologists as well as immunologists and geneticists. The section describes the use of

326 BOOK REVIEWS

serology, cel! biology, and genetic probes in the analysis of infection and tumor-related antigens. The chapters on EB v~rus (Chapter 211, hepatitis virus (Chapter 23) and influenza virus (Chapter 25) describe how both cell- and antibody-medlated immune mechanisms recognise and defend against viral infection. Miiich appears to have deviated from the general trend in this book in that he describes his own work on Hepatitis B antigen most exhaustively. This section has been written very well except for some errors e.g., on p. 500, the statement that IL-2 was discovered by Gallo is obviously a mistake. The complex problems posed by retroviruses are delineated by Wong-Staal and Wolfe and Hardy in Chapters 22 and 24, respectively. The genetic regulation of tumor-associated antigens is described in Chapter 25 on protooncogenes and Chapter 27 on melanoma-associated antigens.

The last section has been devoted to deal with topics of genetic and biomedical importance such as erythrocyte blood groups and serum complement. The blood group antigens which include erythrocytealloantigens (i.e., the ABO system) were the first to be described and is of great significance to clmicians and immunogeneticists. The chapter, in addition to describing the ABO, Lewis and Hh antigens deals with new areas of biological interest-abnormalities and their enzymatic and genetic bases. The polymorphism of MNSs antigens and their antigenic structure i.e., importance of glycophorins is described in the next chapter. Chapter 31 (Tippett) describes the Rh blood group system and its role in causing hemolytic disease of the newborn. The chapter has been approached from the genetic~sts' point of view and could be of great interest to a haemotologist.

The chapters on complement renewed by Schur form an excellent summary of this rapidly developing field and identifies areas of current and future biomedical importance.

Department of Biochemistry Indian Institute of Science Bangalore 560012.

Nucleic acid and monoclonal antibody probes-applications in diagnostic microbiology (Infectious Disease and Therapy Seriesl2) edited by Bala Swaminathan and Gyan Prakash. Marcel Dekker, Inc., 270, Madison Avenue, New York, NY 10016, 1989, pp. 752, $180.

This book covers a wide range of topics under the field of diagnostic probe technology. The individual contributors have made a serious attempt to project the advantages and disadvantages of DNA probes as a rapid diagnostic reagent. The book starts with five articles on the principles and techniques of nucleic-acid detection and characterisation, which include nucle~c-acid hybridisation and plasmid and chromosomal DNA analyses. It then deals with probes for rapid identification of individual pathogens of human and veterinary importance. The rest of the book deals with monoclonal antibody-based immunoassays and follows the same pattern as before, oiz., a section on how to make hybridomas and purify antibodies, followed by the use of monoclonal antibodies for detection of individual human pathogens. lo the concluding chapter, the authors have dealt with recent developments in nucleic acid and monoclona! antibody probe technologies, which have been reported after the main body of the book.

Most chapters in this hook are well written, adequately referred and covers much of the progress made in the area. This allows readers to rapidly gauge the developments made in this area. However, two points stand out. One is the area of diagnostic virology using DNAIantibody probes has been completely ignored. This aspect perhaps has the greatest application in terms of treatment and care of the patient. Furthermore, logical discussions into the utility of various probes in clinical treatment is also not apparent. The second point is the authors' own experience in the experimental protocols for hybridisation should have been highlighted as a major contribution to trouble shooting.

BOOK REVIEWS 327

In summary, while the book should definitely be able to enthuse interest in this field and should be read as a manual before jumping into this area, it falls short as a comprehensive on the bench manual which is useful for trouble shooting. Lastly, it is highly recommended for graduate students to bridge the gap of the application of molecular biology to clinical practice.

Astra Research Centre India Bangalore 560003.

Regulation of gene expression-25 years on edited by I. R. Booth and C. IF. Higgins. Cambridge University Press, Cambridge, CB2 2RU, UK, 1986, pp. 309, $59.50. Indian orders to: Affiliated East-West Press Pvt. Ltd, 25, Dr. Muniappa Road, Kilpauk, Madras 600010.

The revlews on the regulation of gene expression-25 years on cover the recent developments on catabolite repression, RNA polymerase heterogenity, regulation of transcription at the level of initiation, termination, messenger RNA processing and decay, and regulation at the level of translation. Also the involvement of supercoiling of DNA and transposons in the regulation of gene expression are discussed. Topics on the regulation of nitrogen, ' h l 0 and IS10 transposition, plasmid maintenance and gene expression of Streptomyces and Dictyostelium discoideum stand out from the rest of the topics. The reviews presented are mostly on prokaryotes on topics which are unrelated. Though they serve very useful purpose, their scope and contents are very lim~ted. With the advent of recombinant DNA technology, much progress has been made in the understanding of the gene expression and control. However, many areas covering the molecular mechanism of the catabolite repression, basic process in the recognit~on and binding of the RNA polymerase and factors on the promoter, various switches manifested during the life cycle of viruses and microorganisms and the role of many factors involved in gene expression are not clearly understood.

The discussion on the protein-nucleic acid intractions using lambda-phage regulatory protein CIS helps to understand how the various activator/repressor proteins recognise and bind to specific DNA sequences selectively. DNA supercoiling is a potential modulator of gene expression. Much more data are required to establish how the topology of the DNA regulates the gene expression in prokaryotes and eukaryotes. Considerable effort has been put on the regulation by RNA polymerase at the level of initiation and termination of transcription. The RNA polymerase subunits, newer sigma and other factors which modulate gene expression are discussed. The pausing of the RNA polymerases at the stemloop structure, attenuation, ant~termmation and rho-independent termination are described in detail. The secondary structures at the 5'-and ?'-flanking regions of the messenger RNAs and factors which affect the half-life messengers and the specific-binding proteins involved in the modulation of the gene expression are covered in these reviews. The translational regulation and feedback loops are well dealt. There is cocrdination between the ribosomal protein synthesis and assembly of ribosomes. The free ribosomal proteins act as translational repressors on the respective messenger RNAs in E. coli.

The review on the regulation of nitrogen by enteric bacteria contains evidences to show that the product of the gene ntrC (gpntrC) is a DNA-binding protein which can activate and supress transcription. The mechanism and regulation of TnlO and IS10 transposition are described. The molecular mechanisms in the maintenance of plasmid in a cell are not clearly understood. It is of vital importance as many recombinant plasmid DNA molecules, which are engineered to produce very useful products, reject the inserted foreign DNA during the course of multiplication. This aspect is not covered in this review. Streptomyces is an important organism which produces large number of

328 BOOK REVIEWS

antibiotics. The 11fe cycle and the linkage map of this organism are described. In many respects the RNA polymerase and regulation of gene expression of the Streptomyces and E. coli are similar with some differences in codon usage and initiation of transcription and translation. The life cycle, morphogenesis and the control of gene expression during cellular differentiation of Dietyostelium discoideum are covered. It is clear that there is a complete lack of understanding of the molecular mechanisms involved in the transition from one stage to the other, both in the Streptomyees and Dletyoslelum.

Centre for Genetic Engng and Department of Biochemistry Indian Institute of Sc~ence Bangalore 560012.

Published by N. M. Malwad. Executive Editor, Journal ofthe Indian Institute of Science. Bangalore 560012; Typeset and printed at Thomson Press (India) Ltd., Faridabad 121 007, Havana.

Documents

Journal of the Indian Institute of Science Bangalore 560 ...journal.iisc.ernet.in/archives/archives/v9-79/Vol.69(1-6)1989/Vol.69(4...Volume 69 Journal of the Indian Institute of Science