Effect of sulfate on the osmoregulatory and physio-biochemical responses of GIFT (Oreochromis niloticus) juveniles reared in potassium-deficient medium saline waters.

The inland saline waters were continuously observed to have low potassium concentrations compared to their seawater counterpart of the same salinity. We hypothesize that the toxic effect of sulfate may manifest in low potassium saline (LPSW) waters compared to brackish water of the same salinity. Thus, LC50 trials were performed in GIFT (genetically improved farmed tilapia) fry (0.5 ± 0.02 g) to determine the acute sulfate toxicity in freshwater (FW, 0.5 g L-1), artificial seawater (ASW, 10 g L-1), and LPSW (10 g L-1). The median lethal concentrations (96h LC50) of sulfate ion in FW, LPSW, and ASW for the GIFT were 5.30 g L-1, 2.56 g L-1, and 2.98 g L-1, respectively. A second experiment was conducted for 21 days, exposing fish to a sub-lethal level of sulfate ion (SO42-) concentration (1000 mg L-1, one-fifth of FW LC50) with different types of waters (FW, freshwater, 0.5 g L-1; ASW, artificial seawater, 10 g L-1; LPSW, low potassium saline water, 10 g L-1) with and without sulfate inclusion to constitute the treatments as follows, (FW, FW + SO4, ASW, ASW + SO4, LPSW, LPSW + SO4). The effect of sulfate on GIFT reared in sulfate-rich potassium-deficient medium saline water was evaluated by focusing on the hematological adjustments, stress-induced oxidative damage, and osmoregulatory imbalances. The survival was not altered due to the sulfate concentration and K+ deficiency; however, there were significant changes in branchial NKA (Na+/K+-ATPase) activity and osmolality. The increase in NKA was highest in LPSW treatment, suggesting that internal ionic imbalance was triggered due to an interactive effect of sulfate and K+ deficiency. The cortisol levels showed a pronounced increase due to sulfate inclusion irrespective of K+ deficiency. The antioxidant enzymes, i.e., SOD (superoxide dismutase), catalase, GST (glutathione-S-transferase), and GPX (glutathione peroxidase), reflected a similar pattern of increment in the gills and liver of the LPSW + SO4 groups, suggesting a poor antioxidant status of the exposed group. The hepatic peroxidation status, i.e. TBARS (thiobarbituric acid reactive substances), and the peroxide values were enhanced due to both K+ deficiency and sulfate inclusion, suggesting a possible lipid peroxidation in the liver due to handling the excess sulfate anion concentration. The hematological parameters, including haemoglobin, total erythrocyte count, and hematocrit level, reduced significantly in the LPSW + SO4 group, indicating a reduced blood oxygen capacity due to the sulfate exposure and water potassium deficiency. The hepatic acetylcholine esterase activity was suppressed in all the treatments with sulfate inclusion, while the highest suppression was observed in the LPSW + SO4 group. Thus, it is concluded that sulfate-induced physiological imbalances manifest more in potassium-deficient water, indicating that environmental sulfate is more detrimental to inland saline water than freshwater or brackish water of the same salinity.

Structure-guided approach to modify the substrate specificity of the protein human deglycase-1 (hDJ-1).

Glycation is a non-enzymatic reaction wherein sugars or dicarbonyls such as methylglyoxal (MGO) and glyoxal (GO) react with proteins, leading to protein inactivation. The hydrolysing enzyme human deglycase-1 (hDJ-1) is reported to decrease glycative stress by deglycating the modified proteins, specifically at cysteine, lysine, and arginine sites. This specificity of hDJ-1 is thought to be regulated by its active site cysteine residue (Cys106). Structural analysis of hDJ-1 by molecular docking and simulation studies, however, indicates a possible role of glutamate (Glu18) in determining its substrate specificity. To elucidate this, Glu18 present at the catalytic site of hDJ-1 was modified to aspartate (Asp18) by SDM, and the resultant mutant was termed mutant DJ-1 (mDJ-1). Both hDJ-1 and mDJ-1 were heterologously expressed in Escherichia coli BL21 (DE3) strain and purified to homogeneity. The hDJ-1 showed kcat values of 1.45 × 103 s-1, 3.6 × 102 s-1, and 3.1 × 102 s-1, and Km values 0.181 mM, 18.18 mM, and 12.5 mM for N-acetylcysteine (NacCys), N-acetyllysine (NacLys), and N-acetylarginine (NacArg), respectively. The mDJ-1 showed altered kcat values (8 × 102 s-1, 3.8 × 102 s-1, 4.9 × 102 s-1) and Km values of 0.14 mM, 6.25 mM, 5.88 mM for NacCys, NacLys and NacArg, respectively. A single amino acid change (Glu18 to Asp18) improved the substrate specificity of mDJ-1 toward NacLys and NacArg. Understanding hDJ-1's structure and enhanced functionality will facilitate further exploration of its therapeutic potential for the treatment of glycation-induced diabetic complications.

Structural insight to human Retinoid X receptor alpha-Thyroid hormone receptor beta heterodimer by molecular modelling and MD-simulation studies: role of conserved water molecules.

The Retinoid X receptor alpha-Thyroid hormone receptor beta (RXRα-THRß) heterodimer plays an important role in physiological function of humans specially in the growth and development. Extensive MD-simulation studies on the aquated complexes of modelled RXRα-THRß heterodimer with DNA-duplex have indicated the role of some conserved/semiconserved water molecules in the complexation process in presence or absence of Triiodothyronine (T3) and 9-cis retinoic acid (9CR) in the respective Ligand Binding Domain (LBD) domain. Among the seventeen conserved/semi-conserved water molecules, the W1-W4 water centers have been observed to mediate the interaction between the residues of A-chain (DBD of RXR) to consensus sequence (C-chain) of DNA. The W5-W8 water centers involve in recognition of the residues of B-chain (DBD of THR) to C-chain of DNA. The W9-W13 centers have connected the different residues of B-chain (THR) to D-chain of DNA through H-bonds, whereas W14-W17 water molecules were involved in the interaction of A-chain's (RXR) residues to D-chain of DNA. In our previous study with homodimeric THRß from Rattus norvegicus we have identified fifteen conserved water molecules at the DNA-DBD interface. Moreover, the conformational flexibility of Met313 (in the LBD of THR) from open to close form in presence or absence of T3 molecule in the holo and Apo-protein may provide a plausible rational on the possible role of that residue to acts as gate which could restrict the solvent molecules to enter into the hydrophobic T3-binding pocket of LBD during the absence of ligand molecule and thus could help the stabilization of that domain in THRß structure.Communicated by Ramaswamy H. Sarma.

Taurine and/or inorganic potassium as dietary osmolyte counter the stress and enhance the growth of GIFT reared in ion imbalanced low saline water.

The effects of dietary osmolytes for alleviating osmotic stress and enhancing growth are not well elucidated in fish reared in inland saline water. The present study evaluated the effects of dietary taurine or potassium (K+) individually or in combination on growth, ionic homeostasis, and stress response of GIFT tilapia reared in potassium deficient low saline water (PDLSW, 10 ppt salinity) mimicking inland saline water. Isonitrogenous and isoenergetic diets supplemented with five potassium concentrations (0, 0.3, 0.45, 0.6 and 0.75 %), two taurine (T) concentrations (0.5 and 1.0 %) and two combinations of both (K+ 0.1 % + T 0.5 % and K+ 0.2 % + T 0.5 %) were fed to GIFT juveniles (4.4 ± 0.02 g body weight) and reared in PDLSW for 45 days. The fish fed on the diet fortifying with K+ 0.2 % + T 0.5 % showed the highest growth performance among the controls and other treatment groups. Dietary supplementation had no effects on PDLSW induced increase in osmoregulatory endpoints. The optimum dietary potassium requirement of GIFT reared in PDLSW was 0.57 and 0.599 g/100 g diet. Dietary K+ down-regulated the PDLSW induced expression of NKAa1, AQP1, and ClC2, whereas inhibited taurine-induced up-regulation of AQP1 and CLC2, which is the first report in tilapia. In addition, dietary K+ and taurine modulated antioxidant and metabolic enzyme activities for easing stress and balancing energy requirements. Thus, blending of potassium (0.2 %) and taurine (0.5 %) in the diet appears best to mitigate stress and enhance GIFT growth reared in inland saline water.

Dietary arginine attenuates hypoxia- induced HIF expression, metabolic responses and oxidative stress in Indian Major Carp, Cirrhinus mrigala.

Hypoxia is a common stressor in aquaculture systems, which causes severe physiological disturbances, ultimately leading to mortality or reduced productivity. Arginine, as a precursor of NO, has a role in enhancing oxygen delivery. Thus, an experiment was conducted to evaluate the effect of dietary arginine (Arg) in Cirrhinus mrigala exposed to hypoxia. The fish were fed with different levels of arginine for 60 days and exposed for 72 h to a sublethal level of hypoxia (0.50 ± 0.16 mg/L dissolved oxygen [DO]). The six treatment groups with three replicates were N0 (0% Arg + Normoxia), H0 (0% Arg + Hypoxia), N0.7 (0.70% Arg + Normoxia), H0.7 (0.70% Arg + Hypoxia), N1.4 (1.40% Arg + Normoxia), H1.4 (1.40% Arg + Hypoxia). Eighteen experimental units with twelve animals (5.8 ± 0.18 g) each were used for the trial.The results indicated that supplementation of arginine at 0.7 and 1.4% enhanced the hypoxia tolerance time, although the high dose (1.4%) did not yield any further increments. The exposure to hypoxia up-regulated Hypoxia Inducible Factor (HIF)-1α mRNA expression and supplementation of arginine significantly decreased hypoxia induced up-regulation of HIF at 1.4%. Arginine supplementation partially or completely normalised the hypoxia induced changes in the metabolic enzymes of C. mrigala. The fish exposed to hypoxic conditions exhibited significantly higher (P < 0.05) lipid peroxidation levels than those maintained under normoxic conditions, while arginine feeding significant in reducing lipid peroxidation. Antioxidant enzyme activities were significantly higher (P < 0.05) in hypoxia-exposed carp, indicating increased oxidative stress during the hypoxic exposure, that was improved in Arg-supplemented groups. However, arginine did not modulate erythrocyte countsalthough itreduced the erythrocyte fragility. We conclude arginine supplementation is effective in ameliorating hypoxia induced metabolic alterations and improving antioxidant defences in fish.

Molecular modeling and molecular dynamics simulation studies on thyroid hormone receptor from Rattus norvegicus: role of conserved water molecules.

Thyroid hormone receptor (THR) belongs to the nuclear receptor (NR) superfamily that is activated by binding of appropriate ligand molecules (thyroid hormones). These receptors directly bind to specific DNA sequences for gene expression, which is essential for metabolism, homeostasis, and the development of organisms, making it an important drug target. Extensive MD-simulation studies of triiodothyronine (T3) docked modeled rnTHRß1 structures have indicated the presence of twelve conserved water molecules at the DNA-DBD (DNA binding domain) interface. The W1-W5 water centers have been involved in the recognition between the A-chain of DBD to C-chain of DNA, W6 and W7 mediated the interaction between A-chain of DBD and D-chain of DNA, W8 and W9 recognized the B-chain of DBD and C-chain of DNA, and W9-W12 centers conjugated the residues of B-chain of DBD to D-chain of DNA through hydrogen bonds. The conformation flexibility of Phe272 and Met313 residues in the absence of T3 at the LBD (ligand-binding domain) region have been observed and reported.

Structural insight to hydroxychloroquine-3C-like proteinase complexation from SARS-CoV-2: inhibitor modelling study through molecular docking and MD-simulation study.

The spread of novel coronavirus strain, Severe Acute Respiratory Syndrome 2 (SARS-CoV-2) causes Coronavirus disease (COVID-19) has now spread worldwide and effecting the entire human race. The viral genetic material is transcripted and replicated by 3 C-like protease, as a result, it is an important drug target for COVID-19. Hydroxychloroquine (HCQ) report promising results against this drug target so, we perform molecular docking followed by MD-simulation studies of HCQ and modelled some ligand (Mod-I and Mod-II) molecules with SARS-CoV-2-main protease which reveals the structural organization of the active site residues and presence of a conserve water-mediated catalytic triad that helps in the recognition of Mod-I/II ligand molecules. The study may be helpful to gain a detailed structural insight on the presence of water-mediated catalytic triad which could be useful for inhibitor modelling. Communicated by Ramaswamy H. Sarma.

Hypoxia-mediated inhibition of cholesterol synthesis leads to disruption of nocturnal sex steroidogenesis in the gonad of koi carp, Cyprinus carpio.

Reproductively mature koi carps (Cyprinus carpio) showed a prominent diurnal variation of sex steroids with sustained nocturnal rise. Exposure to chronic hypoxia (DO < 0.8 mg/l) disrupted nocturnal sex steroid production in koi carp gonads. Inhibition of sex steroidogenesis is linked to the down-regulation of HMG-Co A reductase (p < 0.05), which acts as a rate-limiting enzyme in the mevalonate pathway for cholesterol production. HMG-CoA reductase inhibition was obvious in the gonads and liver of both sexes during 18.00 h and 21.00 h resulting in hypocholesterolemia (p < 0.05). The levels of sex steroids, such as estradiol, testosterone, and 11-keto-testosterone in gonads were depleted below the optimum levels owing to disruption of de novo cholesterol synthesis along with attenuation of HDL-cholesterol level in serum. Inhibition of melatonin under hypoxic conditions indicates disruption of melatonin effects on the hypothalamus-pituitary-gonadal (HPG) axis of koi carp. Under severe hypoxic stress, koi carp promoted energy conservation by switching over to the triglyceride (TGA) pathway instead of the mevalonate pathway to suppress cholesterol production. Chronic hypoxia inhibited cholesterol synthesis, a prerequisite for gonadal maturation. It promoted TGA production, as an alternative energy source, suggesting a probable mitigation strategy adopted by hypoxia-tolerant fish to deal with low dissolved oxygen frequently occurring in aquatic bodies.

Dietary GABA enhances hypoxia tolerance of a bottom-dwelling carp, Cirrhinus mrigala by modulating HIF-1α, thyroid hormones and metabolic responses.

The Indian major carp, mrigal (Cirrhinus mrigala), is a bottom-dwelling fish that can survive hypoxic episodes in its natural environment. We hypothesise that it can better survive hypoxic conditions by altering metabolic responses through GABA (Gamma-aminobutyric acid) supplementation. In the first experiment, the hypoxia tolerance time of the fishes was evaluated under extreme anoxic conditions after feeding with GABA, which showed that GABA had improved survival time under hypoxia. To study the response of dietary GABA in hypoxia-exposed fish, the branchial HIF-1α expression levels, serum thyroid hormone levels and hepatic metabolic responses were assessed in the subsequent experiment. The treatment groups were fed for 60 days with experimental diets containing 4 levels of GABA (0.00% G, 0.50% G, 0.75% G and 1.0%G) and were subjected to 72-h hypoxia exposure (0.5 ± 0.02 mg L-1 dissolved oxygen (DO)) whereas a control group was maintained under normoxic conditions (6.0 ± 0.21 mg L-1 DO). The five treatment groups with three replicates were C0 (0% G + normoxia), H0 (0% G + hypoxia), H0.5 (0.50% G + hypoxia), H0.75 (0.75% G + hypoxia) and H1.0 (1.00% G + hypoxia). The results indicated that GABA supplementation triggered downregulation of HIF 1 alpha expression. When compared with the control group, decreased thyroxine (T4) and triiodothyronine (T3) levels were observed in the GABA-fed hypoxic groups. However, TSH (thyroid stimulating hormone) level remained unchanged in all the treatments. The LDH (lactate dehydrogenase) level in hypoxia-exposed groups was decreased by GABA supplementation. Our study demonstrated that GABA supplementation restores acute hypoxia-induced HIF-1α expression, thyroid hormone levels and LDH activities. On the other hand, it enhanced the citrate synthase (CS) activities at 0.5-1.00%, which showed a sharp decline in hypoxia. Hypoxia caused increase in the serum metabolites such as glucose, lactate, cholesterol and triglycerides. However, GABA supplementation was partially effective in reducing glucose and lactate level while triglycerides and cholesterol values remained unchanged. Overall, our results suggested a potential role of GABA in suppressing metabolism during hypoxia exposure, which can increase the chances of survival of the species Cirrhinus mrigala during hypoxia.

Pseudobranch mimics gill in expressing Na+K+-ATPase 1 α-subunit and carbonic anhydrase in concert with H+-ATPase in adult hilsa (Tenualosa ilisha) during river migration.

In hilsa (Tenualosa ilisha), pseudobranch comprises a row of parallel filaments bear numerous leaf-like lamellae arranged on both sides throughout its length. The purpose of this study was to elucidate involvement of pseudobranchial Na+, K+-ATPase (NKA) 1 α-subunit, and carbonic anhydrase (CA) in concert with H+-ATPase (HAT) compared to their branchial counterparts in freshwater acclimation of hilsa during spawning migration from off-shore of the Bay of Bengal to the Bhagirathi-Hooghly zones of the Ganga river system in India. Adult hilsa fish were collected from seawater (SW), freshwater 1 (FW1), and freshwater 2 (FW2) locations, where the salinity level was 26-28‰, 1-5‰, and 0-0.04‰, respectively. Hilsa migrating through freshwater showed a consistent decrease in the plasma osmolality, sodium (Na+) and chloride (Cl-) ion levels indicates unstable ionic homeostasis. The mRNA expression and activity of NKA 1 α-subunit in pseudobranch as well as in true gills declined with the migration to upstream locations. The pseudobranchial CA activity almost mirrors its branchial counterpart most notably while hilsa entered the freshwater zone, in the upstream river suggesting its diverse role in hypo-osmotic regulatory acclimation. Nevertheless, the H+-ATPase activity of both the tissues increased with the freshwater entry and remained similar during up-river movement into the freshwater environment. The results confirm that the pseudobranchial NKA 1 α-subunit mRNA expression and activity mimic its branchial counterpart in the process of ionoregulatory acclimation during migration through salt barriers. Also, the increase in the activities of pseudobranchial and branchial CA in concert with H+-ATPase (HAT) during freshwater acclimation of hilsa suggests their critical involvement in ion uptake.

Alteration in branchial NKA and NKCC ion-transporter expression and ionocyte distribution in adult hilsa during up-river migration.

Hilsa (Tenualosa ilisha) is a clupeid that migrates from the off-shore area through the freshwater river for spawning. The purpose of this study was to investigate the involvement of branchial Na+/K+-ATPase (NKA) and Na+/K+/2Cl- cotransporter (NKCC) in maintaining ionic homeostasis in hilsa while moving across the salt barriers. Hilsa, migrating through marine and brackish waters, did not show any significant decline in NKA activity, plasma osmolality, and plasma ionic concentration. In contrast, all the parameters declined significantly, after the fish reached in freshwater zone of the river. Immunoblotting with NKA α antibody recognized two bands in gill homogenates. The intensity of the higher molecular NKA band decreased, while the other band subsequently increased accompanying the movement of hilsa from marine water (MW) to freshwater. Nevertheless, total NKA expression in marine water did not change prior to freshwater entry. NKCC expression was down-regulated in gill, parallel with NKA activity, as the fish approached to the freshwater stretch of river. The NKA α-1 and NKCC1 protein abundance decreased in freshwater individuals by 40% and 31%, respectively, compared to MW. NKA and NKCC1 were explicitly localized to branchial ionocytes and immunoreactive signal appeared throughout the cytoplasm except for the nucleus and the most apical region indicates a basolateral/tubular distribution. Immunoreactive ionocytes were distributed on the filaments and lamellae; lamellar ionocytes were more in number irrespective of habitat salinity. The decrease in salinity caused a slight reduction in ionocyte number, but not in size and the underlying distribution pattern did not alter. The overall results support previously proposed models that both the ion transporters are involved in maintaining ionic homeostasis and lamellar ionocytes may have the function in hypo-osmoregulation in migrating hilsa, unlike other anadromous teleosts.

Recognition dynamics of dopamine to human Monoamine oxidase B: role of Leu171/Gln206 and conserved water molecules in the active site cavity.

The human Monoamine oxidase (hMAO) metabolizes several biogenic amine neurotransmitters and is involved in different neurological disorders. Extensive MD simulation studies of dopamine-docked hMAO B structures have revealed the stabilization of amino-terminal of the substrate by a direct and water-mediated interaction of catalytic tyrosines, Gln206, and Leu171 residues. The catechol ring of the substrate is stabilized by Leu171(C-H)⋯π(Dop)⋯(H-C) Ile199 interaction. Several conserved water molecules are observed to play a role in the recognition of substrate to the enzyme, where W1 and W2 associate in dopamine- FAD interaction, reversible dynamics of W3 and W4 influenced the coupling of Tyr435 to Trp432 and FAD, and W5 and W8 stabilized the catalytic Tyr188/398 residues. The W6, W7, and W8 water centers are involved in the recognition of catalytic residues and FAD with the N+- site of dopamine through hydrogen bonding interaction. The recognition of substrate to gating residues is made through W9, W10, and W11 water centers. Beside the interplay of water molecules, the catalytic aromatic cage has also been stabilized by π⋯water, π⋯C-H, and π⋯π interactions. The topology of conserved water molecular sites along with the hydration dynamics of catalytic residues, FAD, and dopamine has added a new feature on the substrate binding chemistry in hMAO B which may be useful for substrate analog inhibitor design.

Diel cyclic hypoxia alters plasma lipid dynamics and impairs reproduction in goldfish (Carassius auratus).

Diel cyclic hypoxia occurs with varying frequency and duration in freshwater habitats, yet little is known about its effects on reproduction of freshwater fishes. The present study shows that long-term exposure of goldfish (Carassius auratus) to cyclic hypoxia (0.8 ± 0.2 mg/l dissolved oxygen) for 9 h or more, per day, altered plasma lipid and sex steroid profiles, which in turn directly or indirectly suppressed ovarian growth and viable spermatozoa production. Hypoxia decreased total cholesterol and high density lipoprotein (HDL p < 0.05) and elevated triglycerides (TG; p < 0.05) in both sexes. Plasma steroid concentrations particularly of 17α-hydroxyprogesterone (17-HP), estradiol (E2), testosterone (T) in females, and T and 11-ketotestosterone (11-KT) in males were attenuated under diel hypoxic conditions. Intriguingly, both diel and continuous hypoxia elevated plasma E2 and vitellogenin levels in males. However, neither lipid nor steroid profiles recorded any variation in a dose-dependent manner in response to diel hypoxia. The reduced GSI, decreased number of tertiary oocytes, and motile spermatozoa in hypoxic fish clearly indicate suppression of gametogenesis. Thereby, prolonged diel cyclic hypoxia may affect valuable fishery resources and fish population structure by impairing reproductive performances and inducing estrogenic effects in males.

The putative role of some conserved water molecules in the structure and function of human transthyretin.

Human transthyretin (hTTR) is a multifunctional protein that is involved in several neurodegenerative diseases. Besides the transportation of thyroxin and vitamin A, it is also involved in the proteolysis of apolipoprotein A1 and Aß peptide. Extensive analyses of 32 high-resolution X-ray and neutron diffraction structures of hTTR followed by molecular-dynamics simulation studies using a set of 15 selected structures affirmed the presence of 44 conserved water molecules in its dimeric structure. They are found to play several important roles in the structure and function of the protein. Eight water molecules stabilize the dimeric structure through an extensive hydrogen-bonding network. The absence of some of these water molecules in highly acidic conditions (pH ≤ 4.0) severely affects the interfacial hydrogen-bond network, which may destabilize the native tetrameric structure, leading to its dissociation. Three pairs of conserved water molecules contribute to maintaining the geometry of the ligand-binding cavities. Some other water molecules control the orientation and dynamics of different structural elements of hTTR. This systematic study of the location, absence, networking and interactions of the conserved water molecules may shed some light on various structural and functional aspects of the protein. The present study may also provide some rational clues about the conserved water-mediated architecture and stability of hTTR.

Expression studies on NA+/K(+)-ATPase in gills of Penaeus monodon (Fabricius) acclimated to different salinities.

The decapod crustacean Penaeus monodon survives large fluctuations in salinity through osmoregulation in which Na+/K(+)-ATPase (NKA) activity in the gills plays a central role. Adult P. monodon specimens were gradually acclimatized to 5, 25 and 35 per thousand salinities and maintained for 20 days to observe long-term alterations in NKA expression. Specific NKA activity assayed in gill tissues was found to be 3 folds higher at 5 per thousand compared to 25 per thousand (isosmotic salinity) and 0.48 folds lower at 35 per thousand. The enzyme was immunolocalized in gills using mouse α-5 monoclonal antibody that cross reacts with P. monodon NKA α-subunit. At 5 per thousand the immunopositive cells were distributed on lamellar tips and basal lamellar epithelium of the secondary gill filaments and their number was visibly higher. At both 25 per thousand and 35 per thousand NKA positive cells were observed in the inter-lamellar region but the expression was more pronounced at 25 per thousand. Gill architecture was normal at all salinities. However, the 1.5 fold increase in NKA α-subunit mRNA at 5 per thousand measured by quantitative RT-PCR (qRT-PCR) using EF1α as reference gene was not statistically significant. The study confirms the osmoregulating ability of P. monodon like other crustaceans at lower salinities. It is likely that significant increase in NKA transcript level happens at an earlier time point. At higher salinities all three methods record only marginal or no change from isosmotic controls confirming the hypothesis that the animal largely osmoconforms in hyperosmotic environment.

p53 dependent apoptotic cell death induces embryonic malformation in Carassius auratus under chronic hypoxia.

Hypoxia is a global phenomenon affecting recruitment as well as the embryonic development of aquatic fauna. The present study depicts hypoxia induced disruption of the intrinsic pathway of programmed cell death (PCD), leading to embryonic malformation in the goldfish, Carrasius auratus. Constant hypoxia induced the early expression of pro-apoptotic/tumor suppressor p53 and concomitant expression of the cell death molecule, caspase-3, leading to high level of DNA damage and cell death in hypoxic embryos, as compared to normoxic ones. As a result, the former showed delayed 4 and 64 celled stages and a delay in appearance of epiboly stage. Expression of p53 efficiently switched off expression of the anti-apoptotic Bcl-2 during the initial 12 hours post fertilization (hpf) and caused embryonic cell death. However, after 12 hours, simultaneous downregulation of p53 and Caspase-3 and exponential increase of Bcl-2, caused uncontrolled cell proliferation and prevented essential programmed cell death (PCD), ultimately resulting in significant (p<0.05) embryonic malformation up to 144 hpf. Evidences suggest that uncontrolled cell proliferation after 12 hpf may have been due to downregulation of p53 abundance, which in turn has an influence on upregulation of anti-apoptotic Bcl-2. Therefore, we have been able to show for the first time and propose that hypoxia induced downregulation of p53 beyond 12 hpf, disrupts PCD and leads to failure in normal differentiation, causing malformation in gold fish embryos.

Cdc2-cyclin B-induced G2 to M transition in perch oocyte is dependent on Cdc25.

The G2 to M phase transition in perch oocytes is regulated by maturation promoting factor (MPF), a complex of Cdc2 and cyclin B. In Anabas testudineus, a fresh water perch, 17 alpha,20 beta-dihydroxy-4-pregnen-3-one, the maturation inducing hormone (MIH), induced complete germinal vesicle breakdown (GVBD) of oocytes at 21 h. An unusual cyclin, p30 cyclin B, has been identified in oocyte extract using both monoclonal and polyclonal antibodies. Surprisingly, Cdc2 could not be identified, although a Northern blot with Cdc2 cDNA demonstrated expression of the gene. Purification of MPF through an immunoaffinity column followed by SDS-PAGE showed three proteins, Cdc2, cyclin B, and a 20 kDa fragment, indicating earlier failure in immunodetection may be due to the interference by this fragment. In uninduced oocytes, p30 cyclin B was present, and its expression was increased by MIH. MIH increased p30 cyclin B accumulation at 3 h, a high level which was maintained between 9 and 21 h, but an effective increase in GVBD and H1 kinase activation could only be observed between 15 and 21 h. This delay in active MPF formation was found to be related to the activation of Cdc25, phosphorylation of which was detected at 12 h, and a substantial increase occurred during 15-18 h. Sodium orthovanadate, a tyrosine phosphatase inhibitor, inhibited H1 kinase activity and GVBD, suggesting the requirement of Cdc25 activity in MPF activation. Our results show occurrence of pre-MPF in uninduced oocytes and its conversion to active MPF requires dephosphorylation by Cdc25, the existence of which has not yet been shown in fish.

A new cell secreting insulin.

The pancreatic beta-cell is the only cell in animals that expresses the insulin gene and secretes insulin protein. We have found copious release of immunoreactive and bioactive insulin into the medium from the primary culture of carp adipocytes. Glucose augmented this release to more than 2-fold, and glucose transporter, Glut2, was detected in these cells. These all reflect characteristics of a pancreatic beta-cell. The expression of the adipocyte-specific flotillin gene, the presence of peroxisomal proliferator-activated receptor gamma and Glut4, and the colocalization of insulin and leptin confirmed the identity of these cells as adipocytes. Purified carp adipocyte insulin (AdpInsl) comigrated with porcine and bovine insulin in SDS-PAGE, indicating the similarity of their molecular sizes (5.5 kDa). AdpInsl strongly reduced hyperglycemia in streptozotocin-induced diabetic rats. It also stimulated significantly higher glucose uptake in carp and hamster adipocytes than porcine insulin. Adipocyte RNA hybridized with rat and zebrafish insulin cDNA showing the expression of the insulin gene in this cell. Using oligonucleotide primers designed on the basis of conserved insulin domain, AdpInsl cDNA was reverse transcribed, cloned, and sequenced. The deduced amino acid sequence of AdpInsl A and B chain exhibited 98% homology with zebrafish and more than 70% homology with human, porcine, and murine insulin. To understand the structure-function relationship between AdpInsl and mammalian beta-cell insulin, we have analyzed the amino acid sequences and three-dimensional structure of AdpInsl. In the critical determinant segment for receptor binding, AdpInsl has His at the A8 position instead of Thr in human and porcine insulin, and this attributed greater biological activity to AdpInsl. Our results show that carp adipocyte is a unique cell. As an insulin target cell it can express the insulin gene and secrete highly active insulin protein; thus, it may serve as a natural alternative to pancreatic beta-cell insulin.