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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
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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)
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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
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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.
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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)
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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
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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
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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
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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
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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).
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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)
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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
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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)
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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
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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
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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
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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
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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
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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
TheThe acrp acrp 30 gene of Swine30 gene of Swine
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.
Lane4: 83mM imidazole.
Lane5: 102.5mM imidazole.
Lane6: 122mM imidazole.
Lane7: 141.5mM imidazole.
Lane8: 161mM 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.
TheThe acrp acrp 30 gene of Swine30 gene of Swine
Figure. 12% SDS-PAGE
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Drug-delivery techniques
• Microencaplation system
• Implantation systems
• Nano-technology
Liposome preparation
substances acting on the hypothalamic ghrelin receptors can stimulaterelease of growth hormone (GH).
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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)
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“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)
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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)
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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