Biomedical subjects
Publications and source records attributed to K D Sharma.
Bioassay studies were carried out to assess the toxicity of distillery effluent on seed germination, seedling growth and pigment contents. Higher concentrations (> 5%) of effluent were found to be toxic but however, can be used for irrigational purpose after proper dilution.
In order to establish base-line data on angiogenic factors in development of mesenchymal tumors, expressions of vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) in implanted MT-8 and MT-9 tumors, both derived from a transplantable malignant fibrous histiocytoma (MFH) in the F344 rat, were investigated by immunohistochemistry and Western blotting method. MT-8 and MT-9 tumors were developed in syngeneic rats by implant of a tumor tissue fragment. MT-8 tumors were examined on post-implantation (PI) days 3, 6, 9 and 17, and MT-9 tumors were on PI days 9, 14, 17 and 23. The growth of MT-8 tumors was faster than that of MT-9 tumors. Histologically, MT-8 tumors were features of undifferentiated sarcomas, whereas MT-9 tumors exhibited a typical storiform growth pattern of MFH. Immunohistochemically, all cells constituting MT-8 and MT-9 tumors reacted with antibodies to VEGF and bFGF, indicating production of these factors by mesenchymal neoplastic cells. However, there were no marked differences in these immunoreactions between tumors examined. Thus, the bands obtained in the Western blotting methods were densitometrically scanned. The expression levels of VEGF and bFGF gradually increased PI day 3 to 9 in MT-8 tumors and PI day 9 to 17 in MT-9 tumors. On last examination day, the levels of bFGF in both tumors and of VEGF in MT-9 tumors decreased, but the VEGF expression level in MT-8 tumors was still increased. These findings indicated that VEGF and bFGF may contribute cooperatively to angiogenesis in an early growth of mesenchymal tumor development.
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Diphtheria toxin receptor (DTR), which is identical to the membrane-anchored form of heparin-binding EGF-like growth factor (proHB-EGF), has a high affinity for heparin. We studied the effect of heparin-like molecules on the binding of diphtheria toxin (DT) to DTR/proHB-EGF. Mutant Chinese hamster ovary (CHO) cells deficient in heparan sulfate (HS) proteoglycans were about 15 times less sensitive to DT than wild type CHO-K1 cells. When free heparan sulfate or heparin was added to the culture medium, DT sensitivity of the mutant cells was fully restored. Studies of binding of 125I-labeled DT to HS-deficient CHO cells transfected with human DTR/proHB-EGF cDNA indicated that the increased sensitivity to DT after addition of heparin is due to increased binding of DT to cells. Vero cells display a relatively large amount of heparan sulfate residues compared to CHO-K1 cells or L cells. Enhancement of DT binding by the addition of heparin was also observed with CHO-K1 cells and L cells that had been transfected with human DTR/proHB-EGF cDNA, but the degree of enhancement was less than that observed with the HS-deficient CHO cells. Addition of heparin did not affect DT binding or DT sensitivity of Vero cells. Heparin-dependent binding was observed when intact Vero cells were treated with heparitinase or when the cell membrane was solubilized with a neutral detergent. Scatchard plot analysis for the binding of DT to a recombinant HB-EGF in vitro and to L cells expressing human DTR/proHB-EGF revealed that heparin increases the affinity of DTR/proHB-EGF for DT but does not change the number of binding sites. Although DRAP27/CD9 is known to enhance DT binding to DTR/proHB-EGF, the results indicate that heparin and DRAP27/CD9 increase DT binding by independent mechanisms. Thus, heparin-like molecules, probably in the form of heparan sulfate proteoglycan on the cell surface, are a third factor required for maximal DT binding activity of cells.
Resonance Raman spectra are reported for catalases from bovine liver, the ascomycete fungus Aspergillus niger, and the bacterium Micrococcus luteus. The vibrational frequencies of the oxidation-, spin-, and coordination number-sensitive spectral bands are indicative of high spin pentacoordinate hemes in the resting ferric enzymes of each of these organisms. This result is in accord with the crystal structure of bovine catalase (Fita, I., and Rossmann, M.G. (1985) J. Mol. Biol. 185, 21-37). In contrast, the crystallographic study of catalase from the ascomycete Penicillium vitale (Vainshtein, B. K., Melik-Adamyan, W. R., Barynin, V. V., Vagin, A.A., Grebenko, A. I., Borisov, V. V., Bartels, K. S., Fita, I., and Rossmann, M. G. (1986) J. Mol. Biol. 188, 49-61) showed electron density on the distal side of the heme which could imply the presence of a sixth ligand, possibly a water molecule. However, both of these crystallographic studies showed the proximal ligand in catalase to be a tyrosine. The present study confirms tyrosinate coordination in each of the three catalases from the appearance of selected resonance-enhanced tyrosine vibrational modes. The most characteristic band is the tyrosinate ring mode at approximately 1612 cm-1 which is maximally enhanced with 488.0 nm excitation. The appearance of tyrosinate modes at 1607 and 1245 cm-1 in the resonance Raman spectra of M. luteus cyano catalase serves to identify tyrosine as an axial ligand in bacterial as well as eukaryotic catalases. Unlike non-heme iron tyrosinate proteins, whose resonance Raman spectra are dominated by several intense bands diagnostic of tyrosine ligation, the heme-linked tyrosine modes are not easily distinguished from the large number of porphyrin vibrations.
The copper binding site of amicyanin from Paracoccus denitrificans has been examined by resonance Raman spectroscopy. The pattern of vibrational modes is clearly similar to those of the blue copper proteins azurin and plastocyanin. Intense resonance-enhanced peaks are observed at 377, 392, and 430 cm-1 as well as weaker overtones and combination bands in the high frequency region. Most of the peaks below 500 cm-1 shift 0.5-1.5 cm-1 to lower energy when the protein is exposed to D2O. Based on the pattern of conserved amino acids, the axial type EPR spectrum, and the resonance Raman spectrum, it is proposed that the copper binding site in amicyanin contains a Cu(II) ion in a distorted trigonal planar geometry with one cysteine and two histidine ligands and an axial methionine ligand at a considerably longer distance. Furthermore, the presence of multiple intense Raman peaks in the 400 cm-1 region which are sensitive to deuterium substitution leads to the conclusion that the Cu-S stretch is coupled with internal ligand vibrational modes and that the sulfur of the cysteine ligand is likely to be hydrogen-bonded to the polypeptide backbone.
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The effects of malarial infection, induced by infecting the mouse with Plasmodium berghei (30-40% parasitemia), on hepatic superoxide dismutase (SODs) were studied. Total SOD and Cu-Zn SOD activities were found to be significantly reduced. Mn SOD activity, however, was not found to be altered.
Serum antibodies in suspected angiostrongyliasis patient were detected by ELISA. The antibody titre was 1:51,200 in the serum and 1:6,400 in CSF with preadult A. cantonensis antigen. Other tests like AGD and CIEP failed to show any positive reaction with both preadult and adult worm antigens. Experimental infection with 100 A. cantonensis larvae in albino rats indicated positive CIEP reaction in serum from the day 5 to 375 after infection. No precipitin line was seen on the other hand, in AGD during observation period. Different rat groups infected with larval doses of 100, 500, 2,000, and 5,000 showed positive CIEP reaction, on the 21st day of infection when preadult worms were seen in CNS. There was no CIEP reaction when a low dose of 15 larvae was used. Cerebral fluid of rats infected with heavy dose of 5,000 larvae showed positive CIEP reaction on the 21st day.
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The metabolites of some test fungi adversely affected the seed germination. The maximum reduction was caused by a culture filtrate of Penicillium citrinum followed by Fusarium moniliforme and F. equiseti, while the minimum was caused by Mucor sp. A gradual increase in effective metabolite production was observed by all the fungi up to 15 days.
Albino mice were infected per os with 1000 infective larvae of Ancylostoma caninum and pulmonary superoxide dismutase activity was studied after completion of lung migration. The total superoxide dismutase activity per mg of lung protein was elevated by 15% in infected mice. Of the two forms of superoxide dismutases studied, that containing Cu-Zn was elevated by 44%. This suggests that A. caninum in mice may result in histopathological changes leading to transient pulmonary oxygen toxicity.
In studies of the role of surface antigens of Shigella flexneri in HeLa cell invasion, three antisera were employed to block the invasion. Antisera against live (ALS) and boiled (ABS) S. flexneri blocked invasion very effectively. Reduction in the numbers of intracellular shigellae was always accompanied by reduction in the number adherent to the cells, indicating the importance of adhesion in the invasive process. Anti-live absorbed antiserum (ALAS) prepared by exhaustive absorption of ALS with boiled S. flexneri blocked adhesion and invasion at dilutions of 20 or 50; the efficiency of the absorption was indicated by absence of agglutinating and anti-lipopolysaccharide (LPS) antibodies. S. flexneri LPS did not block adhesion and invasion even at a concentration of 1.0 mg/ml. Hence it was concluded that heat-labile surface antigens are important in adhesion and invasion of HeLa cells by S. flexneri. Antiserum against heat stable antigen (ABS) probably blocks adhesion by steric hindrance.
The distribution of preadult Angiostrongylus cantonensis helminths in various regions of the central nervous system of rats, infected with varying doses of larvae, was studied. A dose of 10 000 larvae proved to be lethal; the rats showed paralytic symptoms and loss of vision prior to death. A very low dose of 15 larvae also proved to be infective to the central nervous system. Incidence of eye involvement was observed in two rats receiving a dose of 500 larvae. Maximum number of helminths were recovered from the cerebral hemisphere region but their involvement per unit weight of the tissue was found to be highest in the region of medulla oblongata.
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