Indian Journal of Biochemistry & Biophysics Vol. 37, August 2000, pp. 235-240

Effect of glyphosate on the activity of DAHP synthase isozymes in callus cultures of groundnut (Arachis hypogaea L.) selected in vitro

Mukesh Jaint and Neera Bhalla-Sarin*

School of Life Sciences, Jawaharl al Nehru University, New Delhi I I 0 067, India

Received 3 I May 1999; revised 10 September /999

Two isozymes of 3-deoxy-o-arabino-heptulosonate-7-phosphate synthase (DAHP synthase or DS, EC 4.1.2.15) from the callus cultures of Arachis hypogaea L. were resolved by DEAE cellulose column chromatography and characterized using selective assays based on divalent cation requirements and regulation by the end-product amino acids. The total extractable activity of DAHP synthase did not show any change in the glyphosate sensitive and tolerant cell lines when compared to 5-enolpyruvylshikimate-3-phosphate synthase (EPSP synthase, EC 2.5. I. I 9), the known target of inhibition by glyphosate. However, the activities of both, DAHP synthase and EPSP synthase were inhibited by glyphosate in vitro. The inhibitory effects of glyphosate on the activities of (a) EPSP synthase and DAHP synthase, and (b) isozymes of DAHP synthase, have been compared. The data indicate that the Co2

+ dependent isozyme of DAHP synthase is a putative target for the action of glyphosate in A. hypogaea.

Shikimic acid pathway, the occurrence of which is restricted to only micro organisms and plants, has been a target for rationally designed herbicides. For instance, the analogues of dehydroshikimic acid, although inhibitory to shikimic acid dehydrogenase, were not herbicidal when applied to the whole plant'. Glyphosate [N-(phosphonomethyl)glycine] (Roundup, Monsanto) is the most widely used herbicide world­wide for the post-emergence control of essentially all annual and perennial grasses, sedges and broad-leaf weeds. Glyphosate inhibited the formation of natural products derived from phenylalanine or chorismic acid, implicating inhibition of aromatic amino acid biosynthesis2

. The in vitro inhibition of conversion of shikimic acid to anthranilic acid in cell free extracts of Aerobacter aerogenes, where EPSP synthase is the target enzyme, was shown by Steinrucken and Amrhein3

. The inhibition of EPSP synthase by glyphosate is compet1t1ve with respect to phosphoenolpyruvate. It i.s known that other enzymes utilising phosphoenolpyruvate are not affected by glyphosate. DAHP synthase catalyses the first reaction of the shikimate pathway involving the condensation of phosphoenolpyruvate and erythrose 4-phosphate and shares the substrate pool of EPSP synthase in its requirement of phosphoenolpyruvate. In the present investigation, the isozymes of DAHP

*Corresponding author. Email: neera@jnuniv.ernet.in trresent address : Department of Plant Genetics, Weizmann Institute of Science, Rehovot 76 I 00, Israel. Email : mukesh.j ain @weizmann.ac.il

synthase from the callus cultures of Arachis hypogaea have been characterized and examined for sensitivity to glyphosate.

Materials and Methods All the chemicals used were obtained from Sigma

Chemical Co., St. Louis, MO, USA and were of analytical grade. The barium salt of shikimate-3-phosphate was prepared according to Coggins et at.

In vitro cultures Proliferative callus cultures of Arachis hypogaea L.

cv. JL-24 were initiated from the leaf explants on semi­solid MS (Murashige and Skoog) medium5 fortified with 2.5% (w/v) sucrose, lOO mg myo-inositol and 1 mg r' each of benzylaminopurine and naphthalene acetic acid. Selection and maintenance of glyphosate tolerant cell lines of Arachis has been described6

. In essence, the glyphosate tolerant cell line (referred to as GR2 in the text), was selected through a step-wise selection protocol by challenging the callus at a sublethal dose of 0 .3 mM glyphosate (i.e. <1Dso =0.5 mM glyphosate). After about six months, giberellic acid was also included in the medium at a concentration of 1 mg r'. This helped in maintaining a healthy green colour of the callus under selection. The selection pressure was increased slowly over a period of two years. Presently GR2 growing at 12 mM glyphosate, shows ca. 20-fold increase in tolerance to the herbicide, and grows at the rate comparable to the control, unselected cell line (data not shown) .

236 INDIAN J 13IOCHEM BIOPHYS, VOL. 37, AUGUST 2000

Isopropylamine salt of glyphosate was used for all experiments . The stock solution was prepared by titrating with I N NaOH until it dissolved completely and making up the fina l volume with double distilled water. The solution was adjusted to neutral pH, passed twice through 0.45 11m Whatman filters and added to the autoclaved medium to the desired concentration .

Fractionation of DAH P synthase isozymes The callus (I 0 g) was frozen in liquid nitrogen and

ground to a very fine powder and extracted with two volumes of buffer A [100 mM EPPS, pH 7.8, I mM

phenylmethylsulfonylfluoride, 10 mM ~-mercapto­ethanol, 5 mM sodium metabisulphite, I mM MnCI 2,

I mM tryptophan and 10% (w/v) polyvinylpyrro­lidone]. The extract was filtered through four layers of cheese cloth and centrifuged at I 0 ,000 g for 30 min at 4°C. The supernatant was desalted on a Sephadex G-25 column (2.5 x 40 em) and fractionated on a DE-52 ion exchange column (2 x 35 em). The isozymes were eluted using a linear gradient of 0-0.3 M NaCI prepared in buffer B [50 mM KH2P04 buffer, pH 7.5, I mM each of phenylmethylsulfonylfluoride and dithiothreitol, 5 mM sodium metabisulfite, 0.1 mM each of MgCb and MnCb, 0.5 mM phosphoenol­

pyruvate, 100 11M tryptophan and 10% (v/v) glycerol]. The fractions containing the highest enzyme activity were pooled and proteins precipitated with 0-75 % (w/v) ammonium sulfate. The protein precipitate was dialysed overnight against three changes of buffer B and stored at -I 0°C. The isozymes of DAHP synthase were assayed and characterized on the basis of divalent cation requirement and end-product feedback regulation according to Sharma et aF. The isozyme peak showing maximum activity in presence of Co2

+ was designated as DS-Co and the one yielding maximum act ivity in presence of Mn2

+ was designated as DS-Mn. DS-Co fraction was further clarified by

centrifugation and applied to a hydroxylapatite column ( 1.5 x 12 em) pre-equilibrated with buffer C [0. 1 M KH2P04 buffer, pH 7 .5, I mM dithiothreitol, 0.1 mM each of MgCI 2 and MnCI 2, 0.5 mM phosphoenolpyruvate, I 00 11M tryptophan and I 0% (v/v) glycerol]. The bound protein was eluted using a linear gradient of 0.1-0.5 M KH2P04 buffer, pH 7.5. I mM dithiothreitol, 0.1 mM each of MgCI 2 and MnCI 2,

100 11M tryptophan and 10% (v/v) glycerol], at 15 ml h( 1 and 2 ml fractions were collected. The fractions with DS-Co activity were pooled in and concentrated

with 35-40% ammonium sulfate precipitation. The protein precipitate was recovered, resuspended in buffer C and dialysed overnight against excess of the same buffer.

Assay of DAHP synthase DAHP synthase activity was assayed spectrophoto­

metrically by a modified procedure of Rubin and Jensen8 and Morris et a/. 9

, as follows: the reaction mixture contained 2 mM phosphoenolpyruvate, 4 mM erythrose 4-phosphate, 50 mM EPPS buffer (pH 8.0), I mM MgCI2, MnCb or CoCI2 (as required) in a final

volume of 250 111. For end-product regulation, I mM of phenylalanine, tryptophan or tyrosine was included. The reaction was initiated by the addition of 150 111 enzyme, incubated at 37°C for 30 min, and terminated with 400 111 of I 0% (w/v) TCA, followed by centrifugation at 3000 g for 3 min. To 250 111 supernatant an equal volume of periodic acid was added and incubated at 37°C for 45 min, followed by addition of 500 111 sodium arsenite. The brown colour thus obtained was allowed to disappear at room temperature, 2 ml thiobarbituric acid was added, and the mixture was incubated at I 00°C for I 0 min. The absorbance of the pink coloured complex (due to formation of DAHP) was read at 549 nm against protein blanks. The 0 min reading was taken as control. For assaying DAHP synthase in column fractions, 0.5 mM Mn2

+ was included. Preliminary standardisation was done by the differential assay procedure of Ganson et a/ 10

• The protein was estimated according to the procedure of Bradford using BSA as the standard 11

.

Assay of EPSP synthase Phosphatase activity was assayed by measuring the

formation of p-nitrophenol from p-nitrophenyl phosphate, as described by Malamy and Horecker12

• The background, non-specific phosphatase activity was determined for correction of the total extractable EPSP synthase activity, since the procedure described measures the rate of appearance of P;, detected spectrophotometrically by the Malachite Green binding assay 13

• EPSP synthase was assayed by the method of Forlani et a/. 14 with slight modification. The protein was extracted by crushing I g callus under liquid nitrogen and suspending in 2 ml of ice cold 50 m-\1 Hepes NaOH (pH 7.0) containing 10% (v/v) glycerol, 5 mM reduced glutathione, 1 mM phenylmethylsulfonylfluoride, 0.1 mM EDTA and 5.0 11M sodium meta-vanadate and 10% (w/v) polyvinylpyrrolidone. The extract was filtered

JAIN & SARIN: EFFECT OF GLYPHOSATE ON THE ACTIVITY OF DAHP SYNTHASE ISOZYMES 237

through four layers of cheese cloth and centrifuged at 10,000 g for 30 min. The reaction was initiated by adding 50 j..LI protein extract in a final volume of 100 j..LI containing 100 mM Hepes NaOH (pH 7.4), I mM each of shikimate-3-phosphate and . phosphoenol­pyruvate, 5.0 j..LM sodium meta-vanadate, and incubated at 37°C for 30 min. The reaction was stopped by adding I ml of a solution containing 9.2 mM Malachite Green dye, 5.0 j..LM sodium meta­vanadate and 0 .2% (w/v) 3-[(3-cholamiodopropyl)­dimethylammonio ]-!-propane sulfonate (CHAPS) to stabilise the colour development, followed, after exactly one min, by 34% (w/v) sodium citrate. After 15 min at room temperature, the samples were read at 660 nm against the unincubated blanks . Under these conditions, the molar absorption coefficient is 75,000 M- 1 cm- 1

• The enzyme activity is expressed as nkat mg-1 protein (where 352 nkat mg·' protein=21.1 j..lmoles P; released min- 1 mg" 1 protein).

Results Partial purification of DAHP synthase isozymes

Two peaks of DAHP synthase were resolved upon DE-52 cellulose chromatography from the callus extract of A. hypogaea (Fig. I). These were characterized on the basis of their divalent cation

O.o7

O.o7

os-eo 0.06 -I O.OS .._,

§ o.os 0\ ...,. V)

~ 0.04

8 fa 0.04

-e ~ O.o3

~ o.oz

0.01

0.00

0 15 30 45 60 7S 90

requirement and regulation by the end-product aromatic amino acids7

. The peaks were pooled separately and concentrated as described earlier in Materials and Methods. The peak eluting first (peak A, specific activity 0.53 ±0.08 nmole mg-1 protein min- 1

) was identified as the DAHP synthase-Co dependent isozyme (DS-Co) since it yielded maximum activity in presence of Co2+ as the cofactor (2.37 ±0.05 nmole mg- 1 protein min-1

). The trailing peak (peak B, specific activity 0.48 ± 0.03 nmole mg-1

protein min·'), eluting at a higher salt concentration, was characterized as DAHP synthase-Mn dependent isozyme (DS-Mn) as it showed maximum specific activity with Mn+2 (1.89 ±0.01 nmole mg·' protein min- 1

). Further, DS-Co proved insensitive to regulation by the end-product aromatic amino acids phenylalanine, tyrosine or tryptophan . In contrast, DS-Mn was activated ca. 2.3-fold (3.76 ±0.03 nmole mg·' protein min-1

) in presence of tyrosine and inhibited by ca. 50% (0.83 ±0.03 nmole mg·' prote in min- 1

) in presence of tryptophan, as compared to the control ( 1.68 ± 0 .00 nmole mg· ' protein min-1

). The feed back insenstttve isozymes of aromatic biosynthesis pathway have been shown to be cytosol localised and are involved in biosynthesis of aromatic amino acids for secondary metabolism by a simple

4.0 "7"'-1 I .._,

3.2 E c

0 ()() N

~ 0.3 2.4 v

0

a 0 -e 0 .2 "" 0 z 1.6 "' ~ ~

0.1

0

105 120 135 150

Fraction number

Fig. I-Resolution of two activ ity peaks of DAHP synthase from callus cultures of Arachis hypogaea after elution with 0-0.3 M NaCl from a DEAE cellulose (DE-52) column. [The protein was extracted under liquid nitrogen and desalted through a Sephadex G-25 column. Crude extract was chromatographed on DE-52 column, and the enzyme activity assayed in 3 ml fractions as described in Materials and Methods. DS-Co and DS-Mn were characterized on the basis of Co2

+ or Mn2+ requirement for maximum activity and regulation by the

end-product aromatic amino acids] .

238 INDIAN J BIOCHEM BIOPHYS, VOL. 37, AUGUST 2000

overflow mechanism. However, the feedback regulated isozymes are present in the chloroplasts and are responsible for the stringently regulated biosynthesis of amino acids required for primary metabolic pathways . Partial purification of DAHP synthase was desired, not only to resolve isozymes, but also to avoid contamination by phenolics, proteases and phosphatases.

Effect of glyphosate selection on the activities of EPSP synthase and DAHP synthase

The specific activities of DAHP synthase and EPSP synthase were compared in glyphosate sensitive and tolerant cell line GR2 (Table I). These two cell lines grow at similar rates and have been described in detail in an earlier report6

. The amount of total extractable protein was comparable between the two cell lines, with or without glyphosate selection. The cell line GR2 showed ca. 1.7-fold increase in the acttvtty of EPSP synthase (specific acttvtty 0.32±0.04 nkat mg-1 protein) when compared to glyphosate sensitive control cell line (specific activity 0.19 ± 0.06 nkat mg-1 prote in). However, no

Table I -Total extractable activities of EPSP synthase and DAHP synthase in glyphosate sensitive control and tolerant, GR2 cell lines. [The values in parentheses denote the % enzyme activity with respect to the glyphosate sensitive control cell line].

Enzyme EPSP synthase* DAHP synthase** (Peak A) DS-Co (Peak B) DS-Mn

Contro l cell line 0. 19 ±0.06 ( 100) 1.25 ± 0.10 ( 100) 0.65 ± 0.07 ( I 00) 0.52 ± 0.05 ( I 00)

GR2 0.32 ± 0.04 ( 168)

1. 20 ± 0.09 (96) 0.72 ± 0.01 ( II 0) 0.49 ± 0.06 (94)

*nkat mg-1 protein ; **nmole mg· ' protein min- 1

100

] 90 c 80 0 0 ..... 70 0

~ 60 "' ~ 50 :>.. ... ·;;: 40 .B ~ 30 II)

~ 20 c 10 J.J.l

0 0 0.1

Glyphosate (p.M)

significant difference in the total extractable activity of DAHP synthase was discernible between the two cell lines (specific activity 1.20 ± 0 .09 and 1.25 ± 0.10 nmole mg- 1 protein min-1

, respectively). Similarly the activities of DAHP synthase isozymes, DS-Co and DS-Mn, were also comparable in the two cell lines . The specific activities of DS--Co in the glyphosate sensitive cell line and tolerant, GR2 cell line were 0.65±0.07 and 0.72±0.01 nmole mg-1 protein min-1

,

respectively . The specific activity of DS-Mn was 0.52±0.05 and 0.49±0.06 nmole mg" 1 protein min- 1

,

in the control and GR2 cell line, respectively . The data indicate that EPSP synthase is the target for glyphosate action in A. hypogaea, that is over­expressed in the GR2 cell line, and that the expression of other enzymes of shikimic acid pathway are not affected by glyphosate. These findings are consistent with our earlier repore.

Earlier Pinto et al. 15 reported increase in the DAHP synthase activity, when potato (Solanum tuberosum) cells grown in suspension cultures were treated with sub lethal doses of glyphosate. The rel ative increase in the activity was a reflection of glyphosate concentration, and glyphosate had no effect on DAHP synthase activity in vitro. However, our results indicate that glyphosate inhibits the activity of EPSP synthase as well as DAHP synthase in vitro by 50% of the control in presence of 2 ~-tM and I mM glyphosate under standard assay conditions of substrate saturation (Fig. 2) . In contrast to its effect on EPSP synthase, the glyphosate concentration required for 50% inhibition of DAHP synthase activity is 1000 fold higher.

(b)

0.1 1.0 10 100

Glyphosate (m.M)

Fig. 2 -In vitro inhibition of (a) EPSP synthase and (b) DAHP synthase activities by glyphosate. [The control values fo r EPSP synth ase and DAHP synthase were 0.22 ± 0.0 I nkat mg· ' protein and 1. 35 ± 0.03 nmole mg·' protein min· ' , respecti vely).

JAIN & SARIN: EFFECT OF GL YPHOSA TE ON THE ACTIVITY OF DAHP SYNTHASE ISOZYMES 239

Glyphosate induced inhibition of Co-dependent isozyme of DAH P synthase

The inhibitory action of glyphosate (l mM) on the two DAHP synthase isozymes recovered from the DE-52 column was tested. The Co- dependent isozyme (peak A, DS-Co) was much more strongly inhibited (56%) by glyphosate than the Mn-dependent isozyme (peak B, DS-Mn) (12%) (Table 2). Inclusion of 1 mM Co2

+ (for peak A, DS-Co) or Mn2+ (for peak

B, DS-Mn) resulted in elevation of DS-Co and DS­Mn activities by ca. 330% and 278%, respectively. However, for peak A, the residual enzyme activity upon addition of l mM glyphosate dropped from ca. 44% (control A) to 26% (control B). Thus, in the presence of glyphosate, the extent of activation of DS­Co by Co2

+ was significantly reduced from ca. 330%

Table 2- Inhibitory effect of glyphosate on the activities of DAHP synthase isozymes in vitro, in glyphosate tolerant cell line GR2, in presence and absence of divalent cations. [Reaction mixtures contained 2 mM phosphoenolpyruvate, 4 mM erythrose 4-phosphate and 50 mM EPPS buffer (pH 8.0), and I mM glyphosate and/or Co2

+ or Mn2+ as indicated. Control A was

without glyphosate or ani: divalent cation; Control B and C included either Co2

+ or Mn + respectively. The enzyme activity is expressed as nmole mg- 1 protein min-1

• The values in parentheses denote the % enzyme activity] .

Control A (no ion) Glyphosate EDTA Control B (+Co2+) Glyphosate + Co2

+

EDTA Control C ( + Mn2+) Glyphosate + Mn2

+

EDTA

Peak A (DS-Co)

0.62 ± 0.07 ( I 00) 0.27 ± 0.03 (43 .5 ) 0.60 ± 0.02 (96.8) 2.05 ± 0.08 (I 00) 0.54 ± 0.03 (26.3) 1.74 ± 0.04 (84 .9) 1.09 ± 0.03 (I 00)

0.30 ± 0.01 (27.5) 0.94 ± 0.03 (86.2)

(a)

0.7

"j 0.5 ~

0.3

-I 0

Peak B (DS-Mn)

0.51 ± 0.02 (I 00) 0.45 ± 0.02 (88.2) 0.49 ± 0.0 I (96. 1) 0.85 ± 0.00 (I 00) 0.43 ± 0.01 (50.6) 0.73 ± 0.04 (85.9) 1.42 ± 0.0 I (I 00) 1.25 ± 0.05 (88.0) 1.22 ± 0.02 (85 .9)

0

2

[rnME4Pr1

3 4

(control A) to ca. 200% (control B). The inhibitory effects of glyphosate on the activity of DS-Mn (peak B) were less dramatic both in presence and in absence of Mn2

+. In view of the metal chelating properties of glyphosate16

, the effect of EDTA was also investigated to preclude the possibility of sequestration or chelation of the activating cation by glyphosate. The activity of peak A (DS-Co) was inhibited ca. 15% by l mM EDTA as compared to ca. 74% loss in presence of glyphosate (control B) . Further, the in vitro inhibition of DS-Co activity by EDTA was also seen in the case of DS-Mn, and evident irrespective of the presence of either Co2

+ or Mn2+ as the activating cation.

The kinetics of the Co-dependent isozyme recovered from the hydroxylapatite column, free of contaminating phosphatase activity, was studied (Fig. 3). From the Lineweaver-Burk plot, glyphosate was found to be a competitive inhibitor of DS-Co with respect to erythrose 4-phosphate. The inhibition of phosphoenolpyruvate by glyphosate was non­competitive. Earlier, Co2

+ activated isozymes of DAHP synthase from mung bean (Vigna radiata) 11

and tobacco (Nicotiana sylvestris) 18 had been reported to be inhibited by glyphosate. The inhibition of DAHP synthase in Nicotiana sylvestris was competitive with respect to erythrose 4-phosphate.

Discussion Glyphosate is an effective broad spectrum

herbicide as well as a potent inhibitor of microbial growth. Our results indicate that EPSP synthase is the prime target for the phytotoxic action of glyphosate in groundnut consistent with earlier reports6

. However, considering the vast diversity in the isozymes of

(b) 0.7

j 0.5 ~ 0

0

0.3 0

-2 0 2 4 6 8

[rnMPEPr1

Fig. 3 - Double reciprocal plots Co-dependent isozyme of DAHP synthase (DS-Co) with (a) erythrose 4-phosphate and (b) phosphoenolpyruate as the vari able substrates. [Partially purified enzyme fraction recovered from the hydroxylapatite column was assayed as described in Materials and Methods in absence (e) or presence (0 ) of 1 mM glyphosate].

240 INDIAN J BIOCHEM BJOPHYS, VOL. 37, AUGUST 2000

aromatic pathway enzymes and their regulation, the extraordinary effectiveness of glyphosate against both plants and microorganisms warrants a detailed examination. We are reporting the resolution of two isozymes of DAHP synthase, viz. DS-Co and DS-Mn, from the callus cultures of A. hypogaea, for the first time. The detailed purification protocol and kinetic analysis of these isozymes will be presented elsewhere (manuscript under preparation) . The isozymes of DAHP synthase have earlier been characterized in leaves of Vigna radiata8

, Daucus 19 S . . l B . l carota , pmacw o eracea, rasstca o eracea,

Glycine max, Medicago sativa, Cucurbita pepo, Triticum aestivum and Secale cereale, in suspension

I f N . . l . 10 20 . b f cu tures o tcotwna sy vestns · , 111 tu ers o Solanum tuberosum9 and in leaves and callus cultures of Brassica juncea7

'21

. Enzymes needed for the production of aromatic amino acids have been demonstrated within the plastids of these plant species. The cytosolic isozymes, being unregulated, are assumed to provide the precursors for secondary metabolic pathways by a simple overflow mechanism. The resolution of DAHP synthase isozymes in the cytosolic and plastidic compartments in a number of plant species has been instrumental in formulating the "dual pathway hypothesis of aromatic biosynthesis'm. A co-ordinate regulation between members of . isozyme pairs for the key enzymes in the synthesis of primary and secondary metabolites is indicated.

Our data show that the Co2+ specific isozyme of

DAHP synthase is a possible target of glyphosate inhibition in groundnut. Although glyphosate is a metal chelator, the inhibition of DS-Co can not be a result of cobalt chelation. Since cobalt stimulates activity of DS-Co17, the sequestration of activating cation should result in non-competiti ve inhibition by glyphosate with both erythrose 4-phosphate and phosphoenolpyruvate as substrates. However, in the present study, the inhibition of DS-Co is competitive with respect to erythrose 4-phosphate. General inhibition of DAHP synthase activity by glyphosate in vivo seems improbable since accumulation of high levels of shikimate-3-phosphate and shikimic acid has been reported in a variety of species23

. Since the Mn2+

stimulated isozyme, DS-Mn, is not susceptible to glyphosate, in vivo inhibition of only DS-Co, may not be deleterious , and the glyphosate action at the DS-Co step may be bypassed through the activity of DS-Mn isozyme. The activity of DS-Mn, however, may allow futile loss of energy rich substrates erythrose 4-phosphate and phosphoenolpyruvate into the aromatic

pathway, resulting in a possible energy drain24. In

plants like V. radiata, DS-Mn is feed back inhibited by the pathway intermediate L-arogenate rather than the end-products of the pathwa/7. Glyphosate induced inhibition of EPSP synthase depletes cellular levels of L-arogenate, which normally inhibits the activity of DS-Mn. The di stinctly incomplete ability of aromatic amino acids to reverse glyphosate induced growth inhibition in groundnut6 and many other

25 h' h h . systems supports t IS ypot es1s.

Acknowledgement The UGC grant No . 3-36/94(SRII) to NBS and

CSIR Senior Research Fellowship to MJ are gratefully acknowledged.

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