Biomedical subjects
Publications and source records attributed to C Gupta.
Home made weaning foods developed from locally available foods like bajra, barley, green gram (Vigna radiata L.), amaranth grain (Amaranthus sp.) and jaggery using household technologies like roasting and malting had a PER ranging from 2.04 to 2.13, BV 79.56 to 80.68, NPU 66.75 to 67.86, NPR 2.13 to 2.76 and PRE 34.18 to 44.18. The values were comparable to that of cerelac--a commercial weaning food.
Recent observations from this laboratory indicated a role of prostaglandin E2 (PGE2) in masculine differentiation of the external genitalia of the fetal mouse, induced by fetal testosterone. In this communication, we further investigated the role played by PGE2 in the testosterone-induced differentiation of the internal genital tract (Wolffian duct) of the fetal mouse. Using in vitro organ culture bioassay of Wolffian duct differentiation, we determined the effect of a PG-depleting agent, namely, anti-PGE2 antibody, and of inhibitors of PG synthesis for their ability to prevent Wolffian duct differentiation in the presence of testosterone. We demonstrated that anti-PGE2 antibody inhibited Wolffian duct differentiation in a dose-dependent manner in embryonic male explants containing fetal testes. At 1:10 dilution, the antibody inhibited the appearance of the entire Wolffian duct as well as growth of the specimen. At 1:100 dilution, however, only development of the Wolffian duct was prevented, as indicated by the absence of regions of the Wolffian duct or by the presence of epithelial disintegration throughout the ductal lumen. The antibody at 1:1000 dilution produced no significant effect on the appearance of the Wolffian duct. PGE2 (10 micrograms/ml) replacement in the medium prevented Wolffian duct disintegration induced by anti-PGE2. We next determined whether the testosterone-dependent Wolffian duct differentiation requires ongoing PG synthesis within the reproductive tract and analyzed the effects of the compounds inhibiting PG synthesis at the level of phospholipase A2 (PLA2) namely, cortisone and dexamethasone-and of those inhibiting at the level of cyclooxygenase-namely, aspirin and indomethacin. We have demonstrated that both PLA2 and cyclooxygenase inhibitors inhibited Wolffian duct differentiation in the male explant, i.e., in the presence of testis. These compounds also prevented the appearance of the Wolffian duct in female explants induced by exogenous testosterone. PGE2 added in the medium blocked the anti-masculinizing effects of PG synthesis inhibitors both in the male and female specimens. Thus, it appears that PG synthesis plays a role in the testosterone-induced masculine differentiation of the Wolffian duct.
Low cost weaning mixtures were prepared by mixing (i) malted pearl millet (Penicitum typhidium L), roasted amaranth (Amaranthus sp.); roasted green gram (Vigna radiata); jaggery and (ii) malted barley (Dehusked barley); roasted amaranth grain; roasted green gram; jaggery in proportion 60:20:40:45 wt/wt and were nutritionally evaluated. Both the blends had a nutrient composition within the range prescribed by the Indian Standard Institute (ISI) for processed weaning foods. The processing of grains resulted in lower levels of phytic acid, polyphenols and saponins and higher in vitro protein digestibility than those of the raw grains used for preparing mixtures. Both the mixtures were acceptable to trained panelists and children.
EGF is known to modulate a variety of cellular functions including differentiation. The aim of this investigation was to determine the role of EGF in androgen-induced masculine differentiation. Accordingly, a series of experiments were designed and the results are summarized as described below. (1) We found that the specific deprivation of EGF using anti-EGF serum during the period of masculine differentiation in an organ culture bioassay system resulted in the disintegration of the Wolffian system in a dose-dependent manner. (2) Exogenous EGF supplemented in the above experiment corrected the anti-EGF effect, suggesting a specific role of EGF. (3) Anti-EGF serum was also found to disrupt the differentiation even in the presence of exogenous testosterone, suggesting an effect independent of testosterone synthesis. (4) EGF was found to have a direct masculinizing effect both in vivo and in vitro; however, it was not able to mimic all masculinizing effects of testosterone. The mesonephric segment of the Wolffian duct was retained by EGF in the female fetal tract under in vitro conditions, and under in vivo conditions EGF was able to increase anogenital distance and to induce epididymis in some female fetal mice. (5) We were able to detect an EGF-like material in the fetal genital tract during differentiation and found that the level of this material increased with advancement of differentiation. Thus, it appears from the above results that EGF plays a role in testosterone-induced reproductive tract differentiation.
Recently we have identified a protein fraction (55-63 K) from male and testosterone-exposed female mouse genital tract, which stimulates phospholipase A2 (PLA2) and induces masculine differentiation in an undifferentiated mouse genital explant, suggesting a role of this protein in the action of testosterone. In the current study we have further investigated the role of this protein by determining whether anti-masculinizing agents, namely, estradiol and cyproterone acetate, have any effect on the production of this protein. The results described here indicate that a protein fraction containing PLA2 stimulatory activity was present in both control male and estradiol- or cyproterone acetate-exposed male fetal genital tract. However the specific activity of the PLA2-stimulatory protein was significantly higher in the control males than in the experimental males. We did not find any major difference in the behavior of this protein fraction in various chromatographic steps except that in CM-sepharose column; the PLA2-stimulatory activity from the male preparation was eluted in two overlapping peaks with 0.3 and 0.25 M NaCl and that from the treated males was eluted only with 0.25 M NaCl. The SDS-gel analysis of this protein fraction revealed a doublet band (55 and 63 K) in control samples and primarily a 63 K band in experimental samples. The protein fraction from all these sources showed a significant difference in their biological activity. The control male preparation induced Wolffian duct whereas the estradiol sample was completely ineffective and the cyproterone acetate sample was partially effective in inducing Wolffian duct. Thus, it appears that the protein fraction has a role in the masculinizing action of testosterone.
The present study was designed to test whether phospholipase-A2 stimulatory protein (PLSP) has any role during androgen-induced masculine differentiation. Thus, an investigation was made to identify such a protein in the fetal genital tract and to test whether this protein can produce masculinization in vitro. Fetal tracts (15/batch) containing genital ducts and urogenital sinus from male, female, and testosterone-exposed (40 mg/kg.day, from days 13-17 of gestation) female embryonic mice on day 18 of gestation were fractionated using Bio-Rad P-100 gel filtration, DEAE-cellulose, and carboxymethyl-Sephadex chromatography. Phospholipase-A2 (PLA2) stimulatory activity was identified at every step of purification. The final preparation stimulated both bee venom and mouse genital PLA2; however, it had no effect on PLC. The preparation lost its PLA2 stimulatory activity after pronase treatment. The partially purified PLSP fraction produced two bands (63K and 55K), as determined by sodium dodecyl sulfate-gel electrophoresis, and its PLA2 stimulatory activity appeared at the region of 55K on a P-100 gel filtration column. PLSP was also identified in female and testosterone-exposed female genital tracts. However, the specific activity of the female PLSP was much lower than that of the male or testosterone-exposed females. Sodium dodecyl sulfate-gel analysis of 2-3 micrograms partially purified PLSP revealed the presence of a faint 55K band in the females compared to the presence of a darker 55K band in the male and testosterone-exposed female. The intensity of the 63K band was similar in both sexes. PLSP from the male and testosterone-exposed females maintained and stimulated the Wolffian duct, whereas PLSP from the female tract had no masculinizing effect. Thus, the masculinizing activity of the PLSP preparation appears to correlate with its PLA2 stimulatory activity and 55K band intensity, suggesting the role of PLA2 stimulatory protein in masculine differentiation.
The effect of feeding a choline deficient (CD) diet, an efficient liver tumor promoting regimen, on the prostaglandin metabolism in the liver of male Sprague-Dawley rats was investigated. The possible biological significance of the alteration was examined using hypolipidemic peroxisome proliferators and modifiers of prostaglandin metabolism such as indomethacin and menhaden oil in the short term assay of the induction of enzyme altered foci in the liver. A CD diet, when fed for 10-30 days, induced 2-2.5 times increases in the levels of prostaglandin E2 (PGE2) in the liver, while the hypolipidemic peroxisome proliferators, 4-chloro-6(2,3-xylidino) pyrimidinylthio(N-hydroxyethyl)-acetamide (BR931) and di(2-ethylhexyl)-phthalate (DEHP), markedly reduced the levels of this metabolite. The addition of BR931 or indomethacin to a CD diet suppressed the diet-induced elevations of PGE2 and a substitution of fats in a CD diet with menhaden oil had the same effect. Furthermore, both indomethacin and menhaden oil added to a CD diet suppressed the induction of gamma-glutamyltranspeptidase positive hepatocyte foci in the liver of rats initiated with a single dose of diethylnitrosamine after 8 weeks of the dietary promotion. The results suggest that altered prostaglandin metabolism may be involved in the liver tumor promoting effect of a CD diet.
The present study was designed to test the hypothesis that the stimulation of arachidonic acid prostaglandins (PG) mediates the masculinizing effects of testosterone in the fetal mouse. We examined a number of situations in which masculine organization was either induced or inhibited by external agents and determined whether PG levels in the fetal genital tracts were altered in a manner coincident with those situations. We show here that both PGE2 and 6-keto-PGF1 alpha levels in the male fetal genital tracts increased with the advancement of masculine differentiation. PG levels in males (1.2 +/- 0.3 pg PGE2 and 0.1 +/- 0.03 pg 6-keto-PGF1 alpha on day 14 of gestation) increased to 2.1 +/- 0.3 pg PGE2 and 1.2 +/- 0.3 PG 6-keto-PGF1 alpha on day 18 of gestation. The PG levels in the male genital tract were significantly higher than the PG levels in the female genital tract throughout the period of sexual differentiation. Before this period, however, such as on day 14 of gestation (when male and female fetuses have identical genital tracts) we found no difference in their PG levels. Moreover, administration of testosterone (40 mg/kg) during the critical period of masculine differentiation (days 13-17 of gestation) significantly increased PG levels (approximately 2-fold) of female fetuses. Furthermore, we found that cyproterone acetate (20 mg/kg), an inhibitor of masculine differentiation, on days 13-17 of gestation deceased PG levels in male fetuses. In addition, administration of PG synthesis inhibitors, namely indomethacin (1 mg/kg) and aspirin (100 mg/kg), inhibited both resting and androgen-inducible PG levels in male and female fetuses. Considering these results together, it may be concluded that PGs have a critical role in the masculinizing action of fetal testosterone.
The masculinizing effects of prostaglandins (PGS) PGE2 and PGF2 alpha on mouse fetal genital tract differentiation were studied both in vivo and in vitro. Prenatal exposure to PGE2 and PGF2 alpha on days 11-17 of gestation (the critical period of the differentiation) increased the anogenital distance of the female fetuses in a dose-dependent manner. PGE2 also increased the anogenital distance of male fetuses in the presence of an inhibitor of testosterone synthesis, namely estradiol (2 mg/kg.day), and in the androgen-insensitive Tfmy males. Internally, PGE2 induced the epididymal duct in the females, estrogen-exposed males, and Tfmy males. However, no other changes were noticed in the internal genital tract of these fetuses. To avoid the problems associated with the placental transfer of any external agent, we also studied the effect of PGE2 in an in vitro system. Female genital ducts on day 13 of gestation were cultured in the presence and absence of different concentrations of PGE2 for a total of 6 days. PGE2 at doses 0.2 and 1 microgram/ml induced and stimulated the Wolffian and epididymal ducts. Thus, PGs appear to have a masculinizing role in androgen-induced sexual differentiation.
The effect of two liver tumor-promoting regimens, a choline-deficient (CD) and a phenobarbital (.06% PB) diet, on the level of epidermal growth factor (EGF) receptor in rat hepatocytes was examined at 3, 10, and 28 days of feeding. Both diets produced a significant decrease in the number of cell surface receptors at 10 and 28 days of treatment. When PB was included in a CD diet, the decrease in the receptor number was evident even after 3 days feeding of the combined diet. Neither diet alone had any effect on the binding at that time. Along with the changes in the receptor number, the binding affinity of EGF to its receptor was also altered by these diets. Furthermore, PB and PB plus CD diets also decreased the EGF binding at the intracellular sites whereas CD diet showed no effects indicating that the decrease in surface binding of EGF by the promoter-treated hepatocytes was not due to rapid internalization of the receptors. The reduced level of hepatocyte surface EGF receptors represents the common property shared by two diverse types of the liver tumor promoters, and may thus be related to the tumor-promoting ability of these agents.
Dietary administration of 4-chloro-6-(2,3-xylidino)-2-pyrimidinylthio(N-beta-hydroxyethyl) acetamide(BR931) and di(2-ethylhexyl)phthalate (DEHP), hypolipidemic agents, to rats for 3-35 days induced a marked reduction in the hepatocyte surface and intracellular binding of [125I]EGF without affecting its binding affinity. The reduction was apparent after 3 days' feeding of BR931 and the magnitude of the reduction was consistently higher in hepatocytes of BR931-treated rats than those of DEHP-treated rats. The liver extracts and the sera from rats fed BR931 or DEHP for 4 weeks showed no inhibitory effects on the EGF binding of hepatocytes from rats fed a basal diet. Possible significance of the changes in relation to their hepatocarcinogenic action was discussed.
Exposure of rats to phenobarbital (PB), a tumor promoter in the two-stage hepatocarcinogenesis model, in their diet (0.06%) induces alterations in insulin receptors in the hepatocytes. There is a decrease both in the number of receptors and the dissociation constant (Kd) when compared with animals fed control laboratory diet. The number of insulin receptors/cell and the Kd were respectively: 183,000 +/- 19,000 and 15.3 +/- 2.5 nM for controls; 47,000 +/- 5000 and 2.8 +/- 0.3 nM for PB. The glycogen synthesis in response to insulin was found to be unresponsive in the hepatocytes from rats exposed to PB. Glucagon receptors on hepatocytes, however, were unaltered in animals treated with PB or fed a choline-deficient (CD) diet and the glucagon-stimulated glycogenolytic responses were also comparable to the controls. There is, therefore, a selective alteration in the hepatocyte surface membrane receptors. Both PB and CD have been shown to reduce the hepatic cell membrane receptors for epidermal growth factor, indicating that the two different tumor promoters alter peptide receptors with endogenous protein kinase activities. This similar though selective effect of the tumor promoters on cell surface receptors may be of significance in their action in carcinogenesis by having an effect on the alteration of regulation of cell growth and metabolism.
We have investigated whether the androgen-induced masculine differentiation of the sex organs involves an induction of phospholipases. We have measured phosphatidylinositol-specific phospholipase C and phosphatidylcholine-specific phospholipase A2 in the reproductive tract of male and female mouse (CD-I) fetuses at the 18th day of gestation. We report here that (1) the activity of these two enzymes is higher in the male genitalia than in the female genitalia; (2) exogenous testosterone at the 13th to 17th day of pregnancy induces both phospholipase A2 and phospholipase C in the female fetal genitalia; and (3) prenatal administration of cyproterone acetate, an antiandrogen, known to produce feminized males, completely prevents the stimulation of phospholipase A2 and C by testosterone in the female fetuses. In the male fetuses, however, cyproterone acetate inhibits the PLC activity but is unable to alter phospholipase A2 activity. These findings provide evidence that the mechanism by which testosterone organizes the genitalia may involve a modification of phospholipases A2 and C.
Our recent studies of the teratogenic mechanisms of phenytoin (DPH) and glucocorticoids in mice have indicated that DPH utilizes the anti-inflammatory pathway of glucocorticoids in producing congenital defects, such as cleft palate. This pathway is influenced by H-2 and H-3 histocompatibility-linked genes in the mouse, such that congenic strains have H-2 or H-3 alleles that confer susceptibility to DPH-induced congenital defects, and susceptible H-2 congenic strains have high glucocorticoid receptor levels. However, other H-2 or H-3 alleles confer resistance to these defects in their otherwise genetically identical congenic partner strains, and "resistant" H-2 alleles are associated with low levels of these receptors. To determine whether this animal work is applicable to the human, we have sought to investigate whether the level of glucocorticoid receptors in circulating lymphocytes of children with the fetal hydantoin syndrome (FHS) is as it is in the animals. We found that children with FHS had glucocorticoid receptor levels significantly elevated above those of unaffected children with similar DPH exposure in control families. The receptor level of affected children was also significantly elevated above that of fathers of children with the FHS and of fathers and mothers of control children. These findings are consistent with those documented in the animal models and suggest that an elevated level of glucocorticoid receptors in lymphocytes may be a marker for susceptibility to the FHS syndrome.
Bioavailability of co-trimoxazole suspension was determined with and without concurrent administration of pectin and kaolin in 8 volunteers. Twenty ml suspension of co-trimoxazole containing 160 mg trimethoprim (TMP) and 800 mg sulphamethoxazole (SMX) and co-trimoxazole suspension along with 20 ml of pectin-kaolin suspension were administered in a random order with 7 days interval. Plasma estimation of trimethoprim and sulphonamide was carried out at serial intervals. Area under curve (AUC) and Cmax of TMP were significantly higher when co-trimoxazole suspension alone was used. No statistically significant changes were observed in case of sulphamethoxazole. Clinical study is necessary to verify whether concurrent administration of co-trimoxazole and pectin-kaolin leads to loss of antibacterial efficacy.
A rapid method for the assay of phospholipase A2 has been developed using a radioactive substrate, L-alpha-dipalmitoyl-(2-[9,10(N)-3H]palmitoyl)-phosphatidylcholine. The substrate diluted with cold carrier (1 mM) is dissolved in 80% ethanol containing 25 mM sodium deoxycholate. The enzymatic reaction is performed in 1.0 ml 0.1 M glycine-NaOH buffer, pH 9.0, containing 2 mumol CaCl2, 10 micrograms bovine serum albumin, 2.5 mumol sodium deoxycholate, 0.01 unit (or less) phospholipase A2, and 40-100 nmol substrate. The enzymatic reaction is terminated by adding 0.2 ml 5% Triton X-100 solution containing 40 mumol EDTA. The product of the enzymatic reaction, radioactive palmitic acid, is extracted by 10 ml hexane containing 0.1% acetic acid in the presence of anhydrous sodium sulfate (0.5 g/ml). Activity of phospholipase A2 is directly determined from the radioactivity in the hexane extract. The present method achieves a quick separation of the radioactive product, [3H]palmitic acid, from the radioactive substrate, L-alpha-dipalmitoyl-(2-[3H]palmitoyl)-phosphatidylcholine, without the need of separation by TLC.
We have evaluated whether the arachidonic acid cascade may be involved in the folding and fusion of the penis and scrotum in masculine differentiation, a possibility raised by recent observations of the involvement of the arachidonic acid cascade in the analogous embryonic processes of elevation and fusion of the palatal shelves and of folding and fusion of the neural tube. To test this hypothesis, during embryonic masculine differentiation in mice of the B10.A strain, we administered certain agents that produce blockade of masculinization. We report that arachidonic acid can reverse the inhibition of masculine development in male embryos produced by estradiol-17 beta or by cyproterone acetate, an androgen receptor-site blocker, and that such reversal can be prevented by an inhibitor of cyclooxygenase, such as indomethacin. We have also found that agents that block the arachidonic acid cascade at the level of phospholipase A2 (cortisone, phenytoin) or at the level of cyclooxygenase (indomethacin, aspirin) also block masculine differentiation and that such antimasculinization is reversed by arachidonic acid. The masculinization of male embryos is inhibited by indomethacin and aspirin, and the masculinization of female embryos produced by exogenous testosterone is prevented by indomethacin. These findings provide evidence that the mechanism by which testosterone organizes the genitalia involves a role of the arachidonic acid cascade leading to prostaglandins at a critical period of development and that interference with testosterone synthesis or action leads to a teratogenic deficiency of arachidonic acid during this time in the genital anlagen.
We studied the glucocorticoid receptor complexes of pulmonary and thymic cytosols of female A/J and CD-1 mice and of hepatoma G2 cells by two column-chromatographic systems, using both [3H]dexamethasone (DEX) and [3H]phenytoin (DPH) as ligands. Three DNA-cellulose adsorbable [3H]DEX-receptor complexes were separated in each system. Molecular sieving gave a 7-, a 5.4-, and a 3.5-nm complex (Stokes radii), and DEAE-Sephadex A-50 chromatography gave a complex eluting in the wash, one at 0.14 M KCl, and one at 0.20 M KCl by a KCl gradient. DPH blocked the binding of the 7- and 3.5-nm, wash, and 0.14 M KCl [3H]DEX complexes. Only two DNA-cellulose adsorbable [3H]DPH complexes, each blocked by DEX, were obtained in each system: a 7- and a 3.5-nm, a wash, and a 0.14 M KCl complex. Thus, there is a common receptor for both DPH and DEX. This receptor has two properties which distinguish it from the 5.4-nm DEX-specific receptor: (i) it binds with a variety of steroids other than glucocorticoids and DPH, and (ii) it rebinds new [3H]DEX or [3H]DPH after loss of ligand during chromatographic separation. These results indicate that DPH binds to receptor IB and not to receptor II of Litwack. [G. Litwack, 1976, in Glutathion: Metabolism and Function (Arias, I.M., and Jakoby, W.B., eds.), pp. 285-299, Raven Press, New York]. We have also found that hepatoma G2 cells have only receptor II. DPH affects neither the induction of tyrosine aminotransferase by DEX nor the basal level of this enzyme in these cells. Moreover, neither DEX nor DPH inhibits the release of [3H]arachidonic acid prelabeled in these cells, as they do in thymocytes which have the common receptor. Thus, it appears that glucocorticoid receptor IB binds DEX and DPH as glucocorticoid agonists mediating the anti-inflammatory and teratogenic action of these drugs, while receptor II apparently is responsible for the induction of tyrosine aminotransferase by DEX.