Bioinformatic Analysis, Structure Modeling and Active Site Prediction of Aquaporin Protein from Catfish Heteropneustes fossilis

Aquaporins (AQPs) are membrane channel proteins which facilitate the rapid transport of water across cellular membrane and are of fundamental importance to the control of cell volume and transcellular water traffic. In the present study using bioinformatic tools an in silico modeling and analysis of aquaporin protein sequences of catfish Heteropneustes fossilis was conducted. Primary structure prediction and physiochemical characterization were performed by computing theoretical isoelectric point (pI), molecular weight, extinction coefficient, instability index and aliphatic index. Secondary structure assessment of aquaporin protein of Indian catfish using GOR IV reveals greater percentage of residues as alpha helix and random coils against the beta sheets. After performing homology modeling using MODELLER, a 3D structure of aquaporin of Indian catfish have been predicted from its amino acid sequence. After the prediction structure has been validated through various validation tools. This homology modeling based structure will provide an insite to its functional aspect and further studies which are based on tertiary structure of protein. Active site of aquaporin protein has been predicted using DoGSite scorer. Analysis of aquaporin active site will provide an insight to more précised functional characterization of this protein. In future, present study will allow the designing of mutant and inhibitors for the study of physiological role of aquaporin protein in Heteropneustes fossilis

Selection of Appropriate Inhibitor for Ovarian Type Cytochrome P450 Aromatase Gene of Heteropneustesfossilis with the Application of Homology Modeling, Molecular Docking and ADMET Analysis

Background: Cytochrome P450 aromatase (Cyp P450 arom) is an important steroidogenic enzyme responsible for conversion of androgens to estrogens and therefore it plays a critical role in vertebrate reproduction. In contrast to vertebrates, teleost fish have two distinct forms of aromatase. One form predominates in ovaries (ovarian type;cyp19a1a), while the other form prevails in brain (brain aromatase;cyp19a1b). Aromatase is highly present during the differentiation of ovaries. It is also susceptible to environmental influences, particularly temperature and xenoestrogens, environmental natural and synthetic pollutants. Methods: In the present study, by applying bioinformatics approach we investigated relationship between catfish ovarian aromatase to its inhibitor related compounds or drugs. The three dimensional (3D) structure of cyp19a1ais not predicted experimentally, so its structure is modelled by using comparative modelling approach and further validated by various tools like RAMPAGE, ProSa, Errat. Various bioinformatics approaches such asUniprot, Homology modeling, Molecular docking, ADMET analysis were followed for the selection of appropriate aromatase inhibitor. We mainly emphasised on Docking that was carried out by Autodock 4.2. The ligand molecules docked into the structures of macromolecular targets (aromatase) and as a result of docking we came to know about different Binding Energies of different inhibitor molecules and lastly Docked structures were analysed by Protein-Ligand Interaction Profiler. Results: Predicted protein model described in this work may be further used for finding interactions with other proteins involved in different types of diseases. Among the drugs Exemestane is highly structurally similar to known active compounds. The selected compounds were further analysed and refined using drug-likeness and ADMET (Absorption, Distribution, Metabolism, Excretion and Toxicity) analysis and with the help of tools we predicted about pharmacokinetic, metabolic, toxicity endpoints. Conclusion: The study showed that how in silico approaches will further increase our ability in the discovery of appropriate drug in the form of Exemestane (which is most stable and perfect aromatase inhibitor among various selected inhibitors). Overall, findings of this study may be helpful in designing the novel therapeutic targets to cure aromatase related cancer disorders

Inflammatory cytokine profile of VEGF and IL-6 from the endometrium of women with and without endometriosis

Background: Endometriosis affects an estimated 176 million women worldwide, which constitutes 10% of the total female population causing debilitating symptoms of pelvic pain and infertility, which limits the quality of reproductive life of affected women. The present study aimed to assess the serum level of VEGF and pro-inflammatory marker IL-6 in cases and normal healthy control groups.Methods: VEGF and IL-6 serum levels were measured by competitive sandwich enzyme-linked immunosorbent assay (ELISA) from 34 subjects with surgically confirmed endometriosis, 16 subjects with adenomyosis and 18 similar  age matched healthy controls with no clinical evidence of the disease or detectable endometriotic lesions at the time of surgical examination. Patients were characterized in terms of their socio-demographic and clinically diagnosed characteristics, including the validated infertility and chronic disease registries.Results: The mean serum level of VEGF was significantly (p<0.0001) higher in case of endometriosis (3887 ng/l) followed by adenomyosis (2588 ng/l) group and lower in case of normal healthy control (665 ng/l) group. The mean serum IL-6 was found to be highly significant (p<0.0001) in case of endometriosis (90.49 pg/ml) followed by adenomyosis (70.37 pg/ml) group and lower in case of normal healthy control (22.97 pg/ml) group.Conclusions: VEGF and IL-6 seems to play a crucial role in the implantation and development of endometriotic lesions, and the disease is associated with significant modulations in the serum levels of VEGF. IL-6 can be a reliable non-invasive diagnostic marker for endometriosis

Exposure of 4-Nonylphenol: Effect on Physiology and Reproductivity of Male Mice

Introduction: The number and accumulation of hazardous chemicals are increasing gradually in the environment. These chemicals directly or indirectly affect the human health, and now-a-day’s these mostly include Endocrine-Disrupting Chemicals (EDCs). These EDC’s interfere with the normal hormonal signaling, leading to reproductive or physiological imbalance causing different diseases. Studies suggest that in these EDCs, 4-Nonylphenol (4-NP) is most commonly used after industrialisation and it is hazardous for all of us. But the complete or detailed studies regarding its effects on reproduction and physiology, mode of action, pathway followed and other related causative factors are yet to be studied. Aim: To examine the multigenerational effects of environmental EDC 4-NP on reproductive, histological and hormonal fitness of male mice exposed for 21 days. Materials and Methods: Male mice were given oral doses 21.25 and 85.0 mg/kg Body Weight (BW) of 4-NP for 21 days. After 21 days BW, organ weight, sperm viability tests were done to analyse the difference with respect to control group. Further histopathological [via Haematoxylin-Eosin (H&E), Periodic Acid Schiff (PAS) stain and Immunohistochemistry (IHC) techniques] and stress parameters (Superoxide Dismutase (SOD) and Lipid Peroxidation (LPO)) examinations were done in different organ tissue samples for detailed study. Results: The results showed that in male mice, 4-NP had a significant negative effect on the weight of its body organs. It further has an adverse effect on spermatogenesis and sperm quality with a resultant impact on male mice fertility with hormonal imbalance. Histopathological examination of the liver and testes biopsy samples illustrates that exposure to 4-NP at high doses induces occurrence of anomalies. 4-NP also causes decrease in SOD and increase in LPO, a parameter for increasing toxicity in the organs

Molecular and functional characterization of catfish (Heteropneustes fossilis) aquaporin-1b: Changes in expression during ovarian development and hormone-induced follicular maturation

10 pages, 6 figures.The oocytes of the freshwater catfish Heteropneustes fossilis hydrate during hormone-induced meiotic maturation. To investigate if this process may be mediated by aquaporins (AQPs), as it occurs in marine fish producing highly hydrated eggs, the cloning of ovarian AQPs in catfish was carried out. Using degenerate primers for conserved domains of the major intrinsic protein (MIP) family, and 5′ and 3′end amplification procedures, a full-length cDNA encoding for an AQP1-like protein was isolated. The predicted protein showed the typical six transmembrane domains and two Asn-Pro-Ala (NPA) motifs conserved among the members of the AQP superfamily. Phylogenetic analysis indicated that the catfish AQP clustered with the teleost-specific aquaporin-1b subfamily, and accordingly it was termed HfAqp1b. Heterologous expression in Xenopus laevis oocytes indicated that HfAqp1b encoded for a functional AQP, water permeability being enhanced by cAMP. Site-directed mutagenesis revealed that cAMP induced the translocation of HfAqp1b into the oocyte plasma membrane most likely through the phosphorylation of HfAqp1b Ser227. In adult catfish, hfaqp1b transcripts were detected exclusively in ovary and brain and showed significant seasonal variations; in the ovary, hfaqp1b was maximally expressed during the pre-spawning period, whereas in the brain the highest expression was detected during spawning. In vitro stimulation of isolated catfish ovarian follicles with vasotocin (VT) or human chorionic gonadotropin (hCG), which induce oocyte maturation and hydration, elevated the hfaqp1b transcript levels after 6 or 16 h of incubation, respectively. These results suggest that HfAqp1b may play a role during VT- and hCG-induced oocyte hydration in catfish, and that VT may regulate HfAqp1b at the transcriptional and post-translational level in a manner similar to the vasopressin-dependent mammalian AQP2.This work was partially supported by grants from the DST and CSIR, New Delhi, to KPJ, and by a grant from the Spanish Ministry of Education and Science (AGL2007-60262-QUACYTE) to JC. FC was supported by a postdoctoral fellowship from Juan de la Cierva Programme (Spanish Ministry of Education and Science).Peer reviewe

Molecular and functional characterization of catfish (Heteropneustes fossilis) aquaporin-1b: Changes in expression during ovarian development and hormone-induced follicular maturation

10 pages, 6 figuresThe oocytes of the freshwater catfish Heteropneustes fossilis hydrate during hormone-induced meiotic maturation. To investigate if this process may be mediated by aquaporins (AQPs), as it occurs in marine fish producing highly hydrated eggs, the cloning of ovarian AQPs in catfish was carried out. Using degenerate primers for conserved domains of the major intrinsic protein (MIP) family, and 5′ and 3′end amplification procedures, a full-length cDNA encoding for an AQP1-like protein was isolated. The predicted protein showed the typical six transmembrane domains and two Asn-Pro-Ala (NPA) motifs conserved among the members of the AQP superfamily. Phylogenetic analysis indicated that the catfish AQP clustered with the teleost-specific aquaporin-1b subfamily, and accordingly it was termed HfAqp1b. Heterologous expression in Xenopus laevis oocytes indicated that HfAqp1b encoded for a functional AQP, water permeability being enhanced by cAMP. Site-directed mutagenesis revealed that cAMP induced the translocation of HfAqp1b into the oocyte plasma membrane most likely through the phosphorylation of HfAqp1b Ser227. In adult catfish, hfaqp1b transcripts were detected exclusively in ovary and brain and showed significant seasonal variations; in the ovary, hfaqp1b was maximally expressed during the pre-spawning period, whereas in the brain the highest expression was detected during spawning. In vitro stimulation of isolated catfish ovarian follicles with vasotocin (VT) or human chorionic gonadotropin (hCG), which induce oocyte maturation and hydration, elevated the hfaqp1b transcript levels after 6 or 16 h of incubation, respectively. These results suggest that HfAqp1b may play a role during VT- and hCG-induced oocyte hydration in catfish, and that VT may regulate HfAqp1b at the transcriptional and post-translational level in a manner similar to the vasopressin-dependent mammalian AQP2This work was partially supported by grants from the DST and CSIR, New Delhi, to KPJ, and by a grant from the Spanish Ministry of Education and Science (AGL2007-60262-QUACYTE) to JC. FC was supported by a postdoctoral fellowship from Juan de la Cierva Programme (Spanish Ministry of Education and Science)Peer reviewe