Design, synthesis and molecular docking of pyrazolo [3,4d] thiazole hybrids as potential anti-HIV-1 NNRT inhibitors.

A series of pyrazolo[3.4,d]thiazole hybrids 6 were synthesized from 5-arylidene-2-imino-3-(4-arylthiazol-2-yl)-thiazolidin-4-ones 5. The 5-arylidene-2-imino-3-(4-arylthiazol-2-yl)-thiazolidin-4-ones 5 were synthesized from 2-amino-4-arylthiazoles 1 and 2-chloro-acetamido-4-arylthiazoles 2 via the formation of 2-imino-3-(4-substituted-arylthiazol-2-yl)-thiazolidin-4-ones 3 using substituted aldehydes 4. The 5-acrylidene derivative 5 on cyclisation with phenyl hydrazine give the pyrazolo [3, 4, d] thiazole derivatives 6. The obtained pyrazolo [3.4, d]thiazole derivatives were studied as anti-HIV-1 NNRT inhibitors. It was found that these compounds might have potent RT inhibition activity.

Relative potencies of natural estrogens on vitellogenin and choriogenin levels in the Indian freshwater spotted snakehead, Channa punctata: in vivo and in vitro studies.

The relative efficacies of three natural estrogens viz., estrone (E(1)), estradiol-17beta (E(2)) and estriol (E(3)) to induce synthesis of vitellogenin (Vg) and choriogenin (Chg) were assessed in primary hepatocyte cultures of the Indian freshwater spotted snakehead, Channa punctata. Hepatocytes were isolated from the spotted snakehead liver by a non-enzymatic protocol. Optimum culture conditions were standardized for ensuring their viability and functioning. Isolated hepatocytes were cultured for 48 h for monolayer formation and then exposed to various concentrations (0.001-10 microM) of the three estrogens. Competitive homologous ELISAs, developed and validated for spotted snakehead Vg and Chg were employed to determine the amounts of these two proteins secreted into the culture medium after 48 h of incubation. The results reveal that although all the three estrogens were effective in inducing the production of Vg and Chg in a dose-dependent manner, there were differences in their relative potencies. Of three estrogens, E(1) was the least potent and could induce synthesis of Vg and Chg only at a minimum concentration of 0.5 microM; whereas significant levels of both the proteins were quantified in culture medium by exposing the hepatocytes to E(2) or E(3) even at a concentration of 0.001 microM. All three estrogens were effective in inducing synthesis of Vg and Chg in vivo also. These results suggest the possibility of employing the above in vitro experimental design to monitor the presence of estrogens/estrogen-like chemicals in natural waters, which could interfere with the estrogen receptor system of fish. This study further points to the possibility of using Chg, in addition to Vg, as a parameter for screening various chemicals for their estrogenic activity.

Establishment of ELISA for murrel vitellogenin and choriogenin, as biomarkers of potential endocrine disruption.

Vitellogenin (Vg) and choriogenin (Chg) are sensitive biomarkers for testing endocrine disruption in fish. Therefore, we have developed immunoassays for Vg and Chg in the Indian freshwater murrel, Channa punctatus. Vg is a known precursor of egg-yolk proteins, whereas Chg contributes to the formation of egg-envelope. Vg and Chg were induced in male murrel by administration of estradiol-17beta. Chg had an apparent native molecular mass of 180 kDa. It consisted of a single peptide with a molecular mass of 110 kDa, whereas native Vg protein (530 kDa) contained 175 kDa peptide. Highly specific polyclonal antibodies against purified plasma proteins, Vg and Chg, were employed for developing competitive enzyme linked immunosorbent assays (ELISAs). The sensitivity of Vg assay was 3.9 ng/mL (working range 15-500 ng/mL) and of Chg assay was 1.56 ng/mL (working range 6-200 ng/mL). The inter- and intra-assay variations were well within acceptable limits. The two antisera did not cross-react with male plasma proteins. Antiserum to Vg did not cross-react with Chg. Similarly, antiserum to Chg showed no correlation with Vg. Further, immunofluorescence and Western blotting confirmed the specificity of Vg and Chg antisera.

Vitellogenin exists as charge isomers in the Indian freshwater murrel, Channa punctatus (Bloch).

Three charge isomeric forms of vitellogenin are reported in the blood of estrogen-treated murrel, Channa punctatus on the basis of the results of native and SDS-PAGE, isoelectric focusing, and Ferguson plot analysis. Vitellogenin was induced in adult murrels by exogenous administration of estradiol-17beta and labelled with 32P in vivo. Labelled vitellogenin isolated from other plasma proteins on Ultrogel AcA 34 columns resolve into three bands on native-PAGE. Ferguson plot analysis reveals free electrophoretic mobilities of the three bands as 6.0, 4.5, and 3.7, which are very similar to the isoelectric point values of 5.9, 4.6, and 3.8, respectively. The apparent molecular weight of native murrel vitellogenin is 530 kDa. It consists of a single peptide with a molecular weight of 175 kDa. All the three bands on native PAGE resolves into a single peptide on SDS-PAGE. N-terminal amino acid sequence for murrel vitellogenin peptide is MKAVVLALLL. The protein phosphorus:lipid phosphorus ratio for murrel vitellogenin is 0.9. The total lipid content is 32.8%, consisting of 45% neutral lipids and 30% phospholipids. Phosphatidyl choline is the major phospholipid whereas triglycerides are the major neutral lipids. Results of these analytical analyses indicate that murrel native vitellogenin circulates as three charge isomers; poor in phosphorus but rich in lipids content.

In vitro induction of vitellogenin by estradiol 17 beta in isolated hepatocytes of catfish, Clarias gariepinus.

Vitellogenin is a female-specific calcium-binding glycolipophosphoprotein synthesized in the hepatocytes of fishes. Its synthesis can be induced in fishes of either sex by estradiol or by xenoestrogens. To study the in vitro synthesis of vitellogenin, different culture conditions were set up using the hepatocytes of Clarias gariepinus. The present study reports on a non-enzymatic procedure for isolation and culture of hepatocytes from the liver of the catfish Clarias gariepinus, in order to study the effects of estradiol on vitellogenin synthesis in vitro. The procedure employs chelating properties of ethylenediamine tetracetic acid to achieve cell viability in excess of 95%. Equal numbers of isolated cells were incubated in different culture media viz. RPMI F1640, Medium-199, and Williams' Medium E. At 36 h, cell attachment and monolayer formation is faster in M-199 and Williams' Medium E than in RPMI. In order to study the effects of estradiol on vitellogenin synthesis, the isolated hepatocytes were seeded in Williams' Medium E in 24-well cell culture plates. 17 beta-estradiol (E(2)) was introduced in the culture plates at different concentrations and for different time periods. The media were assayed for vitellogenin using competitive ELISA. Vitellogenin appeared in the medium after 48 h of incubation with 10(-5) M estradiol whereas after 72 h of incubation 5x10(-7) M E(2) could elicit the synthesis.

Immunological identification of two female-specific proteins from the plasma of Indian freshwater murrel, Channa punctatus (Bloch).

Existence of a non-phosphorylated female-specific protein (FS II), in addition to phosphorylated vitellogenin (FS I), in the plasma of murrel by exogenous administration of estradiol-17beta is reported. Polyspecific rabbit antibodies were raised against estrogen-inducible murrel plasma proteins. This antiserum was absorbed with normal male serum in order to obtain female-specific antiserum (FSAS). Radial immunodiffusion studies suggested that both the proteins (FS I and FS II) were present in the plasma of E2-treated and normal vitellogenic females and in the ovarian homogenate from gravid females, but absent in normal male plasma. Autoradiographic experiments demonstrated that phosphorus moiety was attached with FS I only. Further, immunoelectrophoretic analysis and peptide maps supported the observation that FS I and FS II were discrete, unrelated female-specific proteins.

Purification of vitellogenin from the plasma of Indian freshwater murrel, Channa punctatus (Bloch) by different methods: a comparative study.

Plasma from estrogenized, [32P] NaH2PO4-injected murrel, Channa punctatus was collected in the presence of proteolytic inhibitors and subjected to different separation procedures singly or in combination, viz., gel filtration chromatography on Ultrogel AcA 34, ion-exchange chromatography on DEAE sephacel, or selective precipitation with dimethylformamide or with Mg2+: EDTA in order to isolate vitellogenin from other plasma proteins. The results show that chromatography on Ultrogel or DEAE sephacel yields intact vitellogenin whereas prior precipitation with DMF or with Mg2+: EDTA results in either co-precipitation of other plasma proteins or in the cleavage of phosvitin-like material from the native vitellogenin molecule.

Osmoregulation in the stenohaline freshwater catfish, Heteropneustes fossilis (Bloch) in deionized water.

Transfer of the stenohaline catfish, Heteropneustes fossilis from tap water (TW) to deionized water (DW) resulted in an increase in the glomerular filtration rate, urine volume and osmolar and free water clearance. In a closed system, where the DW was renewed only once a day, no change in the plasma osmolality was evident for up to 14 days. When DW was renewed four times a day for 25 days, a significant reduction in the plasma osmolality was observed within 24h. When the fish were transferred back to TW, plasma osmolality increased to normal freshwater level within 24h. These observations suggest the existence of highly efficient branchial mechanisms for active uptake of salts from an exceedingly dilute ambient medium. The fact that prolactin-secreting cells as well as corticotrophs in the pituitary of the fish in DW were highly stimulated suggests the involvement of the hormones in the adaptive responses of the catfish to DW.

Effects of prolactin, adrenocorticotrophin, neurohypophysial peptides, cortisol, and androgens on some osmoregulatory parameters of the hypophysectomized catfish, Heteropneustes fossilis (bloch).

Daily injections of ovine prolactin (PRL), cortisol acetate (FA), arginine vasotocin (AVT), and adrenocorticotrophic hormone (ACTH), at doses ranging from 0.1 to 500 mU/g, 0.2 to 25 micrograms/g, 0.01 to 0.02 micrograms/g, and 5 to 10 mU/g respectively, for 5 days elevated plasma osmolarity and plasma sodium levels in the 3-day hypophysectomized catfish, Heteropneustes fossilis maintained in tap water. PRL enhanced urine production and decreased urine osmolarity and sodium concentration. Administration of FA and AVT increased urine output as well as urine osmolarity and sodium concentration, thereby resulting in severe natriuresis. Simultaneous administration of PRL and FA to hypophysectomized catfish restored plasma osmolarity and sodium concentration to normal levels even in the presence of increased urinary salt loss. However, if the fishes were maintained in deionized water, administration of PRL and FA had no effect on plasma osmolarity or plasma sodium levels suggesting that these hormones increase plasma osmotic pressure by stimulating active uptake of salts from the external medium. Neither PRL nor AVT evoked any increase in plasma cortisol level indicating that their effects on catfish osmoregulation are not mediated through cortisol production. Isotocin, testosterone, and delta 4-androstenedione had no effect on any of the plasmatic or urinary parameters. It is concluded that prolactin, cortisol, and AVT are the principal hormones for osmoionic homeostasis in this catfish and that they act through independent mechanisms.

Gonadotrophin-induced oocyte maturation in the catfish, Heteropneustes fossilis (Bloch), requires steroidogenesis in both interrenal and ovary.

Intact or hypophysectomized catfish, Heteropneustes fossilis, were administered a single injection of ovulating doses of ovine luteinizing hormone (LH: 200 micrograms/100 g body wt) or partially-purified salmon gonadotrophin (SG-G100: 100 micrograms/100 g body wt). Identical groups of catfish were injected with a suboptimal dose of LH (20 micrograms/100 g body wt) or with porcine adrenocorticotrophin (ACTH: 0.25 IU/100 g body wt). At short intervals after hormone administration, plasma and/or ovarian tissue were analyzed for cortisol (F), testosterone (T), and estradiol-17 beta (E2) by radioimmunoassay. Following administration of ovulatory doses of gonadotrophins, plasma levels of the three steroids increased in a sequential manner; high levels were recorded between 15 and 45 min for F and between 45 and 90 min for T and E2. In gonadotrophin-injected catfish, the ovarian content of T and E2 increased during the first 45 min and then declined up to 90 min even as their titers in the plasma were still increasing. When ovarian pieces containing yolky oocytes were incubated in vitro with LH (50 micrograms/ml), levels of T and E2 in the culture medium increased in a sequential manner similar to that observed following in vivo administration of gonadotrophin. No significant change was observed in the levels of any of the three steroids in catfish injected with a suboptimal dose of LH. In catfish treated with ACTH, plasma F levels increased 40-fold, whereas T and E2 levels did not change; ACTH administration had no effect on oocyte maturation. These results suggest that gonadotrophin, at doses sufficient to evoke oocyte maturation, acts at two loci, the interrenal and the ovary. The results also suggest that the failure of ACTH to induce oocyte maturation is due to its inability to act on the ovary.

Effects of hypophysectomy on some osmoregulatory parameters of the catfish, Heteropneustes fossilis (Bloch).

The catfish, H. fossilis, survives for long periods after hypophysectomy, although with impaired osmoregulatory mechanisms. Plasma osmolarity and cortisol levels decline significantly within 2 hr after hypophysectomy and attain the lowest values by about 27 hr. Hypophysectomy also results in a marked decrease in urine flow rate principally due to reduced glomerular filtration. The reduction in the ability of the kidney of hypophysectomized catfish to eliminate water results in hyperhydration of blood and muscle. Urine osmolarity and sodium concentration increase due to reduced tubular reabsorption of sodium. There is, however, no net change in the total urinary sodium loss. The catfish survives in fresh water after hypophysectomy presumably because its tissues can tolerate significant dilution of the body fluids.

Circannual and circadian variations in plasma levels of steroids (cortisol, estradiol-17 beta, estrone, and testosterone) correlated with the annual gonadal cycle in the catfish, Heteropneustes fossilis (Bloch).

Circannual and circadian variations in plasma levels of steroids were estimated by radioimmunoassay in the female and male catfish, Heteropneustes fossilis, over two consecutive annual reproductive cycles. In the female catfish, testosterone (T), estradiol-17 beta (E2), and estrone (E1) were detectable in the plasma only during the reproductively active (preparatory through spawning) period and their levels increased during vitellogenesis. In the fully gravid catfish, when vitellogenesis was nearly complete, levels of E2 declined but those of T continued to increase suggesting a product-precursor relationship between the two steroids. Plasma cortisol (F) was detectable throughout the year and exhibited three peaks coinciding with summer, monsoon, and winter; the first and second peaks coincided with vitellogenesis and spawning, respectively. In the male catfish, changes in plasma T and F levels closely paralleled the seasonal recrudescence and activity of testes and seminal vesicles. After spawning, gonads regressed and levels of sex steroids declined sharply. In the absence of natural spawning due to scanty monsoon rains, as during the second year of this study, gonadal regression was delayed and the sex steroids persisted in the plasma well beyond the normal spawning season. In addition, the first two peaks of F levels merged to form a plateau extending from the preparatory period until the late spawning period. The three sex steroids (T, E2, and E1) exhibited identical circadian rhythms; a major peak occurred at the onset of the dark phase (20:00 hr) and a minor peak was generally observed 4 hr after the onset of the light phase (12:00 hr). The amplitude of rhythms was greatest during the prespawning and the spawning periods. Cortisol peak levels generally alternated with those of sex steroids. Steroid rhythms show rather precise correlations with environmental factors such as photoperiod, temperature, and rainfall as well as with seasonal reproductive activity in both sexes of catfish.