Biomedical subjects
Publications and source records attributed to G S Gupta.
Immunocytochemical localization of hexosaminidase activity in human males revealed that the enzyme activity is localized mainly in the Sertoli cells and interstitial tissue of the testis and in the columnar cells of the epididymis. In seminal vesicles, activity was observed around the glandular epithelium in the form of fine granules.
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Glycoproteins were fractionated from placentae of syngeneic mice (CBA female xCBA male - H-2k) and evaluated in vivo and in vitro for the modulation of allo-immune responses against the H-2 incompatible A/J (H-2a) strain. It became apparent that the placenta contained high (309-800 kDA) and low (less than 13 kDA) molecular weight proteins, which favoured Sa1 allograft enhancement, leading to 75-100% lethal tumors, and inhibited mixed lymphocyte cultures (MLC). Though the low molecular weight fraction did not modify the mast cell degranulating (DAAD) antibody response, high molecular weight proteins caused a low production of DAAD allo-antibodies of the IgG1 + IgE isotypes. Moreover, addition of the placental fraction representing a specific band of 119 kDA resulted in the production of allo-immune sera rich in DAAD antibodies, which, however, were not associated with allograft enhancement. Beside these components, placenta is endowed with other proteins which modify humoral immune responses in different ways, as ascertained by the C-mediated cytotoxic antibody assay; fractions, rich in a 105 and 55 kDA bands, were immunosuppressive, whereas another, rich in a specific 100 kDA band, was active in eliciting enhanced production of alloantibodies of the IgG2 isotype. Moreover, the fractions representing specific bands of 50, 68 and 75 kDA, which were most effective in inhibiting MLC, neither caused lethal tumor enhancement nor modified IgG2 antibody responses. Based on in vivo and in vitro modulation of immune responses by placental products, it is concluded that: 1) allograft enhancement and high production of IgG1 antibodies are not linked to the same glycoprotein, 2) the immunomodulators in relation to the protection of viviparity appear to be located at the exclusion limits of Sephacryl S-200 (i.e. greater than 250 kDA and less than 13 kDA proteins) and 3) in vitro assays which depend on the inhibition of MLC by placental components should not be taken as the sole criterion for defining a given immunomodulator.
The effect of gossypol acetic acid, a potent male sterilent was studied on LDH from goat liver (LDH-A4), heart (LDH-B4) and testis (LDH-C4) in vitro. All the preparations of LDH were inhibited by gossypol when the reaction was carried out in pyruvate-lactate (direct) or lactate to pyruvate (reverse) directions. The IC50 of gossypol for the pyruvate oxidation by LDH isozymes varied between 16 and 42 microM in presence of 0.27 mM pyruvate and 0.15 mM NADH at 25 degrees C and pH 7.4 whereas for the lactate oxidation, IC50 was 125 microM in a system containing 3.3 mM lactic acid and 1.8 mM NAD at 25 degrees C and pH 9.0. Reciprocal plots due to Lineweaver-Burk showed that these isozymes are inhibited in a non-competitive manner with respect to pyruvate and lactate, and in a competitive fashion when NAD and NADH were varied as substrates. Ki values of LDH-A4, -B4 and -C4 isozymes in presence of gossypol were 20, 34 and 29 microM against pyruvate; 33, 43 and 45 microM against NADH; 85, 85 and 125 microM against lactate and 94, 108 and 83 microM against NAD respectively.
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Cats were given lindane (1 and 2 mg/kg i.v.) and the levels of glucose in blood and glutathione in blood and liver were measured at 30 min, 1, 2, 4 and 8 h. The results showed a sharp decrease in blood glutathione at an early interval with subsequent recovery, whereas there was no change in liver up to 2 h followed by a significant decrease at later intervals with no sign of recovery. The pesticide also showed a hypoglycemic effect at all intervals.
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Two isoenzymes of N-acetyl-beta-D-glucosaminidase (EC 3.2.1.30) (Hex A and Hex B) from human seminal plasma were purified to homogeneity with specific activities of 26 and 60 units/mg of protein respectively. N-Acetyl-beta-D-glucosaminidase activity was inseparable from N-acetyl-beta-D-galactosaminidase activity in both Hex A and Hex B by various conventional chromatographic procedures. Although Km values of N-acetyl-beta-glucosaminidase activity of Hex A and Hex B were similar (1.33 mM), those of N-acetyl-beta-galactosaminidase activity were 0.14 mM for Hex A and 0.40 mM for Hex B. However, pH optima and temperature optima were identical for N-acetyl-beta-glucosaminidase and N-acetyl-beta-galactosaminidase activities of both isoenzymes; Hex A was far more heat-sensitive than Hex B. Thiol-reactive compounds such as silver salts, mercuric salts, p-chloromercuribenzoate and thimerosal strongly inhibited the N-acetyl-beta-glucosaminidase activities of both isoenzymes. GSH protected the enzyme activities from inactivation caused by these reagents, confirming the presence of thiol groups at the active centres. Inhibitions of N-acetyl-beta-glucosaminidase activities of both isoenzymes by metal salts and organic anions were comparable; acetate and arsenite were effective inhibitors for both isoenzymes. In contrast, inhibitions of N-acetyl-beta-glucosaminidase activities of the two isoenzymes by iodoacetic acid, iodoacetamide and ethylmaleimide were not comparable; Hex B was more susceptible to inhibition by these agents at 20 mM concentration. The N-acetyl-beta-glucosaminidase activities of both isoenzymes are strongly inhibited, in decreasing order, by N-acetyl-galactosamine, mannosamine, disaccharic acid lactone, N-acetylglucosamine and gluconolactone. The Ki values of the N-acetyl-beta-glucosaminidase and N-acetyl-beta-galactosaminidase activities for N-acetylhexosamines and results from mixed-substrate kinetics indicated that the activities for the two substrates are located at different sites in Hex A and at the same site in Hex B. The Mr values of Hex A and Hex B were determined to be 195,000 and 210,000 respectively by gel filtration through Sephadex G-200. SDS/polyacrylamide-gel electrophoresis revealed that Hex A and Hex B are each composed of four subunits corresponding to Mr about 50,000 each. No further polypeptide chain was obtained after reduction and alkylation of Hex A and Hex B with 10 mM-dithiothreitol and 10 mM-iodoacetamide.
The effect of gamma-irradiation on ovine testicular hyaluronidase was studied in aqueous solution. Following irradiation, hyaluronidase is inhibited, and the kinetics of inhibition follow a pattern in which Km and Vmax decline as radiation dose is increased. It was indicated that the binding affinity of the residual activity of hyaluronidase with substrate is enhanced and depends upon radiation damage. Effects of various agents such as pH, salts, PCMB and glutathione on irradiated hyaluronidase have been compared with non-irradiated enzyme. The irradiated hyaluronidase was more sensitive to inhibition by CuSO4 than the non-irradiated enzyme. The residual activity after irradiation is less refractory to FeCl3 inhibition and less sensitive to NaCl stimulation compared to non-irradiated hyaluronidase. pH response curves of ovine testicular hyaluronidase show two maxima which become more evident after irradiation.
Two isozymes of hexosaminidases (Hex) were purified from human seminal plasma and found to be homogeneous as revealed by immunoelectrophoresis and immunodiffusion experiments. Anti-Hex A antibodies were precipitated with isozyme B and vice versa. Though the precipitin arcs were equally stained for protein in these two antigen and antibody systems, enzyme activity was weakly stained in the reverse systems (ie, Anti-Hex A with Hex B and Anti-Hex B with Hex A). Thus, some common sequential antigenic determinants were indicated in two isozymes of Hex. On the other hand, loss of enzyme activity in the precipitate of reverse antigen-antibody complexes as compared to direct system (Anti-Hex A with Hex A and Anti-Hex B with Hex B) revealed that the two isozymes are present in different sequential and conformational states near the active sites. Moreover, antibodies of both forms of Hex cross-reacted with human sperm and reproductive and other vital organs such as kidney, liver, lung, pancreas, and spleen. Though monkey tissues also cross-reacted with human seminal plasma Hex-antisera, mouse, rat, and goat testis and epididymis extracts failed to show any cross-reaction. It is concluded that human Hex(s) are tissue nonspecific but appear to be specific to the human and the subhuman primates.
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