Regulation of Hormonal Factors in Aquatic Species Growth

8/3/2019 Regulation of Hormonal Factors in Aquatic Species Growth

http://slidepdf.com/reader/full/regulation-of-hormonal-factors-in-aquatic-species-growth 1/24

1

Regulation of Hormonal Factors in

Aquatic Species Growth

Dr. J.K. Lu

Department of Aquaculture

NTOU

10. 2003

Possible methods applied for fishgrowth manipulation

1) Genetic control- selection breeding2) Envionmental factors (temp, light,photoperiod)

3) Nutritional

4) Endogenous hormonal procedures

5) Genetic engineering

Regulation of Hormonal Factors in

Aquatic Species Aspect

“Smaller fish grow faster than larger one”

“ fish growth does not cease”

Growth aspect

Definition of growth

hyperplasia - cell number increase

hypertrophic - cell size increase

Renewing tissues- continue to proliferate throughout life

- gametes, blood cells, epidermis

- hyperplasia differentiationStatic tissues - permanant loss divide capability

- striated muscle, neurons

- hyperplasia hypertrophic

Expanding tissues - continue dividing until body growthstop

- glands, liver, kidney

- hyperplasia --> differentiation

* Fish: unlimited growth !!!

Evidences:

- kidney (nephrons) continue differentiate

- heart (myoblast maitain same size, cell numberincrease)

- brain (neuron) continue proliferate & differentiate

- retina (new photoreceptors) increase

Lobster, flounder, sturgeon, shark growth is infinity, noaging (immortal)!

Biological clock gene(daf-2 gene): anti-aging gene

- - reduce metabolism rate

- - increase cell repair capabil ity



2

Endogenous Hormonal control of Growth

Hypothalamic hormones related to growth

- GHRH (growth hormone releasing hormone)

- 49 a.a

- GHRIH (growth hormone releasing inhibit

hormone; somatostatin)

Pituitary hormone

- Pituitary growth hormone(GH; somatotrophin)

Mechanisms of Hormone Action

alter target cell activity (i.e., increase or decrease rates of

normal processes)

response is dictated by target cell type

typical changes produced by hormones:

- change plasma membrane permeability and/ormembrane potential by open/close ION CHANNELS

synthesis of proteins (viz., enzymes = regulatory proteins)

activate/deactivate enzymes

induce secretory activity

stimulate mitosis

REGULATION OF GROWTH IN FISH

E N V I R O N M E N T A L F A C T O R S

CSN

HYPOTHALAMUS

- SomatostatinGHRH

PITUITARYT 3

Estrogens

+

G l uco co rt ico i d s+

+

+

Target Tissues

IGF

IGFs

LIVER

C e l l pr ol i l f e r a t ionC e l l M a s s , S i z e I n c r e a s e

GROWTH

Autocrine/Paracrine

GH

Other hormones can promote growth

- Insulin-like growth factors (somatomedins)

- Insulin

- anabolic steroid (testasterone, estradiol)

- Thyroid (T3/T4)



3

Amino Acid Sequence of Growth

Hormone

GH/ReceptorCell Signaling

GH Molecule

1. Growth1. Growth--promoting effects, 2. Protein, lipid, carbohydratepromoting effects, 2. Protein, lipid, carbohydrate

metabolismsmetabolisms

3. Reproduction (3. Reproduction (cogonadotropincogonadotropin) 4. Maintenance immune system) 4. Maintenance immune system

5.5. OsmoregulationOsmoregulation 6. Signal transduction6. Signal transduction

7. Endocrine function (IGF7. Endocrine function (IGF--I)I)

GH actions

Growth Hormone Action

GH & receptors



4

Growth hormone GH; somatotropin)

- adenohypophysis

- in proximal pars distalis (PPD) of anterior lobe

- somatrophes(acidophil cells) produce somatotrophin

- 196 a.a

- single-chain polypeptide

- ~ 22K Dalton

- 2 conserved disulfide bonds

- deamido-N-acety-acidic GH

- phosphorylated GH

- glycosylate GH

Physiological Functions of Growth Hormone Metabolic effects of growth hormone

Promoting protein deposit

Enhance a.a transport through cell membranes

Enhance RNA translation

Increase nuclear transcription of DNA

Decrease catabolism of protein and a.a.

Enhancing fat utilization for energy

Enhancing fat acid covert to acety-CoA

Ketosis

Affect carbohydrate metabolism

Decrease use of glucose for energy enhancing glycogen deposition

Diminish uptake of glucose

Increase insulin secretion and decrease sensitive to insulin

Stimulate cartilage and bone growth

epiphseal cartilage

osteoblast growth

- highly conserved domains (AGH, BGH, CGH, DGH)(13-33,54-94, 113-132, 157-187) are α-helix structure

- AGH, BGH, CGH arranged toward outside, DGH locatesinside

- 3-D structure

- genomic gene about 4.5 - 5 kb, 5 exons, 4 introns

- salmon, trout, tilapia have extra intron in exon V

- Pit-1, thyroid hormone, glucocorticoid regulate GH genetranscription

Mechanisms of growth hormone

enhance fish growth

1. Increase appetite

2. Improve food conversion efficiency

3. Specific dynamic action (metabolic rate)

- protein deamination

- exchange plasma a.a. or structural a.a.



5

GH stimulates muscle hypertophic by regulate proteinsynthesis in muscle

1. Muscle weight

2. Protein content

3. DNA content

4. Activate RNA polymerase

5. Ribosomes

6. Related enzymes

GH stimulates T4 convert into T3

GH can stimulate steroidogenesis in rainbow trout

Synthetic fish growth hormones

- recombinant DNA technology

- expression systems (E coli , yeast, animal cell culture,algae, planktons(rotifer, atemia, dophinia)



6

Effects of Recombinant rtGH on Somatic GrowthPhotoperiod

Pineal Grand ?

GNRHHypothalamus-

P i t u ta r y GTH

Liver

Steroids

+

IGFs

+

+

+

GH

+

Gonads

Gonadotropic Axis Somatotropic Axis

IGFs Growth+

+

GRF

+

Photoperiod

Pineal Grand ?

GNRHHypothalamus

Pi tu ta r y GTH

Liver

Steroids

+

IGFs

+ +

GH

Gonads

Gonadotropic Axis Somatotropic Axis

IGFs Growth

+

+

GRF

+

+

-

+

+

Hormonal administration pathways

immersion

dipping

autoinjection equipment

implant

oral administration*

- deoxycholate (biological detergent

- hydroxypropylmethyl cellulose palate (polymer

matrix, pH dependent polymer)

Genetic engineering - transgenic technology

- GH, IGF, double muscle gene

Insulin

- secreted by pancreatic islet of Langerhans B cells

- 52-a.a.

- 5733 daltons

- A, B chains, C-peptide cleaved during protein process

Precursor hormones of insulin

preproinsulin- signal peptide, B, C, A, D, E domains

proinsulin- B, C, A, D, E domains

mature insulin - B, A chains



7

Insulin processingMolecular Structures of Insulin/IGF FamilyMolecular Structures of Insulin/IGF Family

A 21

A1B30

B1

Insulin

C

B

A

Proinsulin

IGF-I

D

AC

B

IGF-II

CA

BD

A

B

Relaxin

Insulin-like growth factor-I(IGF-I; somatomedinC, sulfation factor)

- secreted by liver

- 70-a.a. (mature form) (immature B, C, A, D, Edomains)

- 4 exons

- 7648 daltons

- mature IGF containing B, C, A, D, domain

IGF systems

IGF1

IGF-I

Humain GPETLCGAELVDALQFVCGDRGFYFNKPT GYGSSSRRAPQTGIVDECCFRSCDLRRLEMYCA PLKPAKSA

Bovin - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -- - - - - - - - - - -

Porc - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -- - - - - - - - - -

Ovin - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -- - - - - - - - - -

Rat - - - - - - - - - - - - - - - - - - -P- - - - - - - - - - - - I - - - - - - - - - - - - - - - - - - - - - - - -T- - - -- -

Souris - - - - - - - - - - - - - - - - - - -P- - - - - - - - - - - - I- - - - - - - - - - - - - - - - - - - - - - - - T-A- - - -

Domaine B C A D



8

IGF-II

Humain AYRPSETLCGGELVDTLQFVCGDRGFYF SRPASRVSRRSR GIVEECCFRSCDLALLRTYCA TPAKSE

Bovin - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - S- -IN - - - - - - -- - - - - - - - - - - - - - - - - - - - - - - - -

Porc - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - N - - - - - - - - -- - - - - - - - - - - - - - - - - - - - - - -

Ovin - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - S- -IN - - - - - - - -- - - - - - - - - - - - - - - - - - - - - A - -

Rat - - - - - - - - - - - - - - - - - - - - - - - - - - - S - - - - - - - - S - -AN - - - - - - - - -- - - - - - - - - - - - - - - - - - - - - - - - -

Souris - - G - G - - - - - - - - - - - - - - - - - - - - -S - - - - - - - - S- -AN - - - - - - -- - - - - - - - - - - - - - - - - - - - - - - - -

Domaine B C A D

InsulinInsulin--like Growth Factorlike Growth Factor--I (IGFI (IGF--I) ActionsI) Actions

Systemic and local effectsSystemic and local effects

Stimulates proliferationStimulates proliferation

Stimulates protein accretionStimulates protein accretion

Stimulates differentiationStimulates differentiation

Metabolic / insulinMetabolic / insulin--like actionslike actions

IGF-I molecule

IGF-I receptorIGF-I Actions

S S

S S

S S

β-chain

α-chain

D

C A

B



9

The IGF system

Insulin like growth factor 1 (IGF-1) and its receptor (IGF-1R) provide a potent proliferative signaling system thatstimulates growth in many different cell types and blocksapoptosis. In vivo IGF-1 acts as an intermediate of manygrowth hormone responses, and may stimulate the growthof some types of cancer. IGF-1 also provides a mitogenicsignal to act as a growth factor for many tissue culturecells. One component of IGF-1 mitogenic signaling isassociation of the receptor tyrosine kinase with Shc, Grb2,and Sos-1 to activate ras and the Map kinase cascade (raf,Mek, Erk).

An endpoint of the Map kinase pathway is modification oftranscription factor activity, such as activation of ELKtranscription factors. Serum response factor (SRF) andAP-1 contribute to mitogenic signaling by many factors.Phosphorylation of IRS-1 and PI3 kinase activation arealso involved in IGF-1 signaling, similar to insulin signaling

Cell Signaling of Growth FactorsCell Signaling of Growth Factors



10

IGF-II

IGF-II (multiplication-stimulating activity; MSA) 67-a.a.

7471 dalton

Dual-effect of GH

Dual-effect: GH first stimulatesprecursor cells to undergo

differentiation, somatomedinsthen

Functions of IGF-1

IGF-1 stimulates - myoblast L6 cell growth (increase cell

mass)

1. Suppression of protein degradation

2. Accelerate a.a uptake

3. Enhance cell proliferation

4. Stimulate differentiation to form postmitotic myotubes

5. Increase creatine kinase (regenerate ATP in muscle)

6. Glucose uptake increase

7. Stimulate myoblast differentiation

8. Mitogenic effect (cell proliferation)

* FGF - mitogenic effects (myoblastproliferation),

- inhibit differentiation

* TGF-β not mitogenic effects- inhibit differentiation

Putative IGF binding domain of IGFBP-6

The survival factor IGF-1 may prolong the life of a cell by ordering thecapture of death agents. Once stimulated by IGF-1 (left), the PI3-Akt pathwaymakes a double arrest. Akt adds phosphate groups to two death proteins,BAD and FKHRL1 (FH), creating binding sites for the bulky protein 14-3-3.Binding by 14-3-3 may prevent BAD and FH from moving around the cell and,consequently, from carrying out their death orders. These death orders may

be executed when the survival factor is withdrawn (right).



11

Thyroid hormones Thyroid hormones (T3/T4 )

3,5,3’ triiodothyronine (T3)3,5,3’ tetraiodothyronine (T4, thyroxine)

T3 is generated from T4 by mondeiodinase systemsin peripheral tissues

T3 is active form thyroid hormone

Physiological effects of thyroid

hormone

T3 enhance general protein synthesis

T3 & glucocorticoids enhance GH transcription

control protein degradation (decrease)

involved metamorphosis processes

limb-bud proliferation

stimulate urea cycle enzyme (ammonia urea)



12

The mutated TR can still bind to the TRE as a homodimer with a normal TR.

This complex can still attract corepressors and because T3 cannot bind it will form a stable inactive complex on the TRE of the gene. When there is some residual T3 binding the homodimer will be released but it is possible that the mutated TR binds the RXR or other cofactor preferentially. In the case where the mutated receptor has been able to heterodimerise and then again binds to the TRE it will block access for the normal receptor heterodimer with coactivator again resulting in silencing of the gene.

Steroid hormones (4 groups)

androgens

estrogens

progestogens

corticosteroids

SIGNALLING THROUGH NUCLEAR RECEPTORS



13

Testosterone & estradial

stimulate anabolic processes in skeletal muscle

increase nitrogen balance via :

food intake (via hypothalumus)

improve food utilization

Mechanisms of steroid improve foodutilization

Increase ACTH androgen releasing(in adrenal)

Increase GH’s production & releasing

Increase insulin’s releasing

Increase producing thyroid hormone

direct tissues utilize non-protein nitrogen

Synthetic steroids

Synthetic steroids (catabolic & androgenic effects)

- 17a-ethynyltestosterone

- 17a-methyltestosterone

Administration of hormonal growth promoters:

- additives

- implant

Condition factor for fish body shape

Condition factor (a) = measure body shape,“ plumpness”

w

A = X 1000

Lb

W= weigh (g)

L = length (cm)

b =contant for specific fish (~ 3)

Skeletal muscle formation

Myogenesis divided by 3 steps:

- myoblast determination

- migration

- differentiation into muscle (morphogenesis)

myoblast (precursor) arise from somites (mesoderm)



14

Myogenesis of skeletal muscle Muscle determined- myoblast commit to muscle (not yet

differentiated) after form neural tube, each somite

differentiate into a Dermomyotome (give rise to muscle &skin)

- Myoblast form at each edge of dermomyotome

- axial myoblast form myotome

- lateral myoblast migrate to limb bud (cell align, formsyncytium), differentiate into muscle cell (multinucleates;

myotube)

Eukaryotic Cell cycle



15

Interaction between Rb（retinoblastomasusceptibility gene）& E2F transcription factor

Myogenic proteins(Muscle Regulatory Factors, MRFs)

4 MRFs:

- myoD (myogenic determination gene D)

- myogenin

- myf5

- mrf4 / herculin/Myf6

Transforming growth factor (TGF-β ) superfamily: secretedgrowth & transformation factors which regulate embryonicdevelopment and tissue homeostasis

Myostatin (GDF-8, growth/differentiation factor-8)

- double muscle gene

- negative regulatory factor

- causes muscle cell hyperplasia & resulting hypertrophy

MyoD family:

(myogenic determination genes)

- basic helix-loop-helix (bHLH) DNA binding transcription

factors (activators)

- form hetero or homo-dimer

- DNA binding domain is consensus domain (CANNTG; E box;

multiple copies in muscle-specific enhancers

- activate muscle-specific genes

Myf5 & MyoD

- expressed in proliferating myoblasts

- for myoblast determination (myoblast formation)

- initiate myoblast differentiation

- convert non-myogenic into myogenic cells

- activate myogenin

myf5 - present in somite before myotome formation

- muscle “determination” step, even somitogenesis

myogenin - promote myoblast differentiation into myotube

- myogenin control myogenic differentiation

- activate Myf4

Mrf4(myf6, herculin)- expressed later in development (formaintenance of muscle cell



16

Myogenin & Myf4 present terminal differentiatingmyocytes & mature myotubes

Myoblast proliferation initiation of differentiationdifferentiation

growth factors regulate (repress) myoblast differentiationby

- activate Id gene

- fos/ras/Jun inhibit MyoD transcription (GF)

- phosphorylation of MyoD (signal transduction)

Double Muscle Cattle

Double-muscled Animals

Double-muscled cattle：

Belgian blue : 11nt deletion in the exon 3

Piedmontese : missense mutation in the exon 3

An increase in muscle mass of about 20%

- Hyperplasia : increase in number of muscle

fibers

- Hypertrophy : increase in muscle fibersindividual diameter

Myostatin

Also called GDF-8 (growth and differentiationfactor-8)

A member of the transforming growth factor β(TGF-β) superfamily

Encoding 376 amino acids

Containing 9 cysteines Forming dimer by disulfide linkage

Double Muscle Gene (Myostatin)

RSRR

N

N

C

C

9 cysteines disulfide linkage

proteolytic sitesignal peptide

Function of Myostatin

Play a role in muscle growth and development

Expressed in developing and adult skeletalmuscle

In early stage -- restricted to the myotomecompartment of developing somites

In later stages and in adult animals -- expressedin many different muscle throughout the body

Function as a negative regulator



17

Myostatin Null Mice

Null mice control

Null mice Control

Immune Stimulant Fusion Protein

Vaccine carrier

DSGLDCDENSSESRCCRYPLTVDFEDFGWD

WIIAPKRYKANYCSGECDYMYLQKYPHTH

LVNKASPRGTAGPCCTPTKMSPINMLYFNG

KEQIIYGKIPSMVVDRCGCS

Myostatin active domain :

Antibody of myostatin

Antibody of myostatin Vaccine carrier (KLH)

MyostatinFusion Protein

Myostatin Vaccination 食慾促進因子-脂肪代謝調控



18

The Neuropeptide Y gene of Chicken and Swine

Neuropeptide Y (NPY) is composed by 36-amino acid. It has been proved that

the injection of NPY at the central nervous system in rat、chicken and pig can

increase food intake.

1. The experiment use three methods to enlarge NPY protein:

(1) Bacterial expression system: (small s cale purification)

(2) Yeast expression systems: (construct yNPY/pGEM-T plasmid)

(3) The peptide synthesizer direct to synthesize NPY peptide.

2. NPY microencapsulation

3. Field trial:

improve the food intake, weight gaining and speed of growth.

NPY

NPY cDNA in marine fishesCobia 151-ta tccaac.aaa gccgacaacc ctggagagga cgcacctgcg gaggagctcg ccaagtatta ttctgcacta agacactaca-250Grouper 151-ta tccaac.aaa gccgacaacc ctggagagga cgcacctgcg gaagagctcg ccaagtatta ttctgcacta agacactaca-250

Cobia 251-tcgacctcat aacacggcag gggtatggga aa-282

Grouper 251-tcaacctcat aacacggcag gggtatggga aa-282

Epinephelus malabaricus

Rachycentron canadum

NYP can stimulate food intake

Molecular Cloning and Expression:The Acrp 30 gene of swine

Molecular cloning and Expression:The ACRP30 gene of Chicken and Swine

ACRP30 (Adipocyte complement-related protein of 30 kDa) is a

secreted serum protein expressed in adipocytes.

To solve the excessive abdominal fat in broiler chicken and swine. The

proposal was conducted to study the lipid metabolism in chicken and

swine. We hope to reduce fat of body:

1. Preparation of the plasmid include the acrp30 gene.

2. Expression of acrp30 recombinant protein.

3. Field trail.

NH2 COOH1 17 45 110 247

Signal sequenceCollagen domain

Globular domain

Non-homologous sequence



19

TheThe acrp acrp 30 gene of Swine30 gene of SwineThe amino acid sequence of the acrp30 gene of different species: (continue)

Human -------------- MLLLGAVLLLLALPGH --- DQETTTQGPGVLLPLPKGACTGWMAG I

Mouse -------------- MLLLQALLFLLILPSHA EDDVTTTEELAPALVPPPKGTCAGWMAG I

pSAE01 ------------------------------------------------------------

pSA E02 MASMTGGQQMGRGS MLLLGAVLLLLALPSL --- GQETT -EKPGALLPMPKGACAGWMAG I

Human PGHPGHNGAPGRDGRDGTPGEKGEKGDPGLIGPKGDIGETGVPGAEGPRGFPGIQGRKGE

Mouse PGHPGHNGTPGRDGRDGTPGEKGEKGDAGLLGPKGETGDVGMTGAEGPRGFPGTPGRKGE

pSAE01 ------ MASMTGGQQMGRGS EKGEKGDTGLTGPKGDTGESGVTGVEG PRGFPGIPGRKGE

pSAE02 PGHPGHNGTPGRDGRDGVPGEKGEKGDTGLTGPKGDTGESGVTGVEGPRGFPGIPGRKGE

Human PGEG AYVYRSAFSVGLETYVTIPNMPIRFTKIFYNQQNHYDGSTGKFHCNIPGLYYFAYH

Mouse PGEA AYVYRSAFSVGLETRVTVPNVPIRFTKIFYNQQNHYDGSTGKFYCNIPGLYYFSYH

pSAE01 PGES AYVYRSAFSVGLETRVTVPNMPIRFT KIFYNQQNHYDVTTGKFHCNIPGLY KLAAA

pSAE02 PGES AYVYRSAFSVGLETRVTVPNMPIRFTKIFYNQQNHYDVTTGKFHCNIPGLYYFSFH

Human ITVYMKDVKVSLFKKDKAMLFTYDQYQENNVDQASGSVLLHLEVGDQVWLQVYGEGERNG

Mouse ITVYMKDVKVSLFKKDKAVLFTYDQYQEKNVDQASGSVLLHLEVGDQVWLQVYGDGDHNG

pSAE01 LEHHHHHH ----- -----------------------------------------------

pSAE02 ITVYLKDVKVSLYKKDKAVLFTYDQYQDKNVDQASGSVLLYLEKGDQVWLQAYGDEENNG

Human LYADNDNDSTFTGFLLYHDTN ----------

Mouse LYADNVNDSTFTGFLLYHDTN ----------

pSAE01 -------------------------------

pSAE02 VYADNVNDSIFTGFL LYQKLAAALEHHHHHH

The acrp30 gene of the swine

1. pSAE01:

size: 122 amino acids, acrp30 fragment (199-484 bp)

MW: 13 kDa, pI=~8.76

vector : pET21a

acrp30 fragment was ligatedwith pET21a in BamHIand Hind III.

N’-MASMTGGQQMGRGSEKGEKGDTGLTGPKGDTGESGVT

GVEGPRGFPGIPGRKGEPGESAYVYRSAFSVGLETRVTVPN

MPIRFTKIFYNQQNHYDVTTGKFHCNIPGLYKLAAALEHH

HHHH-C’

Ps.

white color: amino acids in pET21a

yellow color: collagen repeats

red color: globular domain

The acrp30 gene of the swine

3. pASE02:

Size:267 amino acids, acrp30 fragment (1-719 bp)

MW: 28.9 kDa, pI=~6.4

vector : pET21a

acrp30 fragment was ligatedwith pET21a in BamHIand Hind III.

N’- MASMTGGQQMGRGSMLLLGAVLLLLALPSLGQETTEKPGALLPMPK

GACAGWMAGIPGHPGHNGTPGRDGRDGVPGEKGEKGDTGLTGPKGDTGES

GVTGVEGPRGFPGIPGRKGEPGESAYVYRSAFSVGLETRVTVPNMPIRFT

KIFYNQQNHYDVTTGKFHCNIPGLYYFSFHITVYLKDVKVSLYKKDKAVL

FTYDQYQDKNVDQASGSVLLYLEKGDQVWLQAYGDEENNGVYADNVNDSIFTGFLLYQKLAAALEHHHHHH-c’

Ps.

White color: amino acids in pET21a

Blue color: amino-terminal signal sequence

Green color: unknown

yellow color: collagen repeats

red color: globular domain

TheThe acrp acrp 30 gene of Swine30 gene of Swine

Plasmid pSAE01:

The acrp30 fragment (199-484bp) of swine was ligated with vector

pET21a in BamHIand Hind III.

Insert size is 122 amino acids, pI=8.76, MW=~13kD.

Sty I 58 Xho I 159

Not I 167

Xma I 358

Sma I 360

Nde I 505

Xba I 543

Mlu I 1331

Bcl I 1345

Apa I 1542

EcoR V 1781

HinC II 1837

Ahd I 4325 Bsa I 4386

Pst I 4570

Pvu I 4695

Sca I 4805

Dra III 5468

pSAE01

5709 bp LacI

ori

f1 origin

Amp

s-acrp30

HindIII:HindIII 174

BamHI:BamHI 465


Protein purification and elution of Acrp30 recombinant

protein by Metal affinity chromatography

Talon resins (Sepharose bead bearing the tetradentate

chelator of the Co2+ metal ion)

Elution buffer

Save the samples

Column

Protein purification and elution of Acrp30 recombinant protein (continue)Samples: SACRP30-1 (pSAE01/ E. coli BL21) in 3liter LB/amp broth,

induced by IPTG for 2-3 hr.

Elution buffer:

IMAC-5:IMAC-200,

used gradient buffer by different concentration of the imidizole.(50mM Tris,

pH8.0; 0.5M NaCl, 10% Glycerol, and∆mM imidazole.)

20.7kD

28.8kD

M 1 2 3 4 5 6 7 8 9

13kD

M: prestainedlow-range marker

Lane1: 24.5 mM imidazole.

Lane2: 44mM imidazole.

Lane3: 63.5mM imidazole.






Lane9:180.5mM imidazole.

Result:1. use the elution buffer No.3, including 63.5mM imidazole, then can elute all target protein.

2. mix the fraction 2 & 3, store at -80C for vacuum-dryer.


Figure. 12% SDS-PAGE



20

Drug-delivery techniques

• Microencaplation system

• Implantation systems

• Nano-technology

Liposome preparation

substances acting on the hypothalamic ghrelin receptors can stimulaterelease of growth hormone (GH).



21

Stress

“a state produced by an environmental or other factor

which extends the adaptive responses of an animalbeyond the normal range or which disturbs thenormal functioning to such an extend that in either

case, the chances of survival are significantly

reduced”

- Brett (1958)



22

“Stress is the response of an organism to anydemand placed on it such that it causes anextension of a physiological state beyond thenormal resting state.”

- Selye (1973)

Continuous (chronic) environmental stresses(overcrowding, water quality deterioration,confinement, sub-lethal pollution, social interaction)

may cause stress responses

- increase susceptibility to disease

- suppress the growth rate

- interfere reproduction processes

Acute stress and cortisol

acute stress cause cortisol increase

(stressed fish) cortisol reduce circulating GH level &thyroid hormone

cortisol suppress growth rate, reproductiveprocesses, susceptibility of disease

* Starved fish (chronic stress) incease plasma GH level, butreduce food intake, alos reduce GH receptors in liver

Chronic confinement stress suppress circulationtestosterone & 11-ketotestosterone in maturation

cortisol also inhibit hepatic oestradiol receptors in femalefish

Cortisol act at pituitary suppress GTH secretion, at gonadsuppress steroind hormone

Stress also reduce circulation thyroid hormone

(T3, T4)

Chronic stressful condition

can breakdown fish’s defense mechanisms,and susceptibility to infectious agents.

Hormonal and nervous reactions

ACTH (adrenocorticortropic hormone; from pituitarywhich causes releasing corticosteroid; cortisol).Nervous responses in the alarm reaction also

stimulate release of catecholamines; from chromaffintissue and anterior kidney)



23

ACTH - stress hormone

39 a.a,

single chain peptide

secreted by pitutary anterior lobe

stimulate adrenal cortex secrete glucocorticoid

Glucocorticoid

Cortisol: major adrenal glucocorticoid

- increase gluconeogenesis (in liver), glycogen

synthesis

- suppress host immune response

- decrease anti-inflammatory action

- protein breakdown (in skeletal muscle)

pituitary growth hormone

pituitary - gonadal axis

pituitary - thyroid axis

Other endocrine may involved

- insulin

- prolactin- catecholamines (dopamine, epinephrine,

norepinephrine)

The role of stress in fish disease:

1) The stress concept : Stress is a response in an organismto a change in the environment

GAS (General Adaptation Syndrome): They are of aphysiological and biochemical nature and take place inthree phases:

a) alarm reaction: metabolic and physiological resources aremoblized (return to normal)

b) stage of resistance: After return to normal following the

establishment of a sustainable relationship with theenvironment

c) stage of exhaustion: When the response have notachieved a state of physiological homeostasis and bodilyfunctions become compromised

2) Key stressors in the aquaculture

environment

a) water quality

b) Crowding

c) Handling

d) Disturbance

e) Nutrition

f) Hierarchical - different size fish or densities

3) Consequence of stress

Acute stress - hormonal changes mediate physiologicalchanges primarily in the respiratory, circulatory andosmoregulatory systems.

Respiratory systems- O2 consumption increase

Temperature increase - metabolism increased

Transportation - respiratory stress cause high mortality

High stocking density - toxic metabolic products

increased (NH3 and CO2), can accumulate and contributeto respiratory stress

Respiratory stress - ion and salt imbalance

(osmoregulatory system failure)



4) Reducing stress in the aquacultureenvironment

a) Permit recovery

b) Avoid multiple stressors- reduce handling stress,avoid sudden temperature change, avoid repeated

handling stresses

c) Avoid stressors at high temperature

d) Withdraw food prior to handling

e) Reduce osmotic stress

f) Use of anesthesia

g) Mimic natural environment

h) future development - selective breeding (stress

response is partly heritable characteristic in fish

species

How to reduce fish stress respone

genetic selection genetic engineering

Documents

Regulation of Hormonal Factors in Aquatic Species Growth