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Biomedical subjects

R S Bhatnagar

Publications and source records attributed to R S Bhatnagar.

At least 37 records · Page 2Linked to original sources
PubMed1993

Regulation of prolyl hydroxylase by ascorbic acid is mediated by polyADP-ribose synthesis.

While it is well known that cellular prolyl hydroxylase activity is increased in the presence of ascorbic acid, the mechanism of this modulation is not fully understood. Ascorbic acid is known to generate reactive oxygen radicals which are involved in the regulation of gene expression through mechanisms involving the synthesis of polyADP-ribose in the nucleus. We examined a possible role for this mechanism in modulating prolyl hydroxylase activity in cultures of human fetal (20 week) and neonatal (foreskin) dermal fibroblasts and IMR-90 fibroblasts. The activity of prolyl hydroxylase in these cells increased in the presence of ascorbate. Ascorbate markedly increased the levels of polyADP-ribose synthetase. The increase in prolyl hydroxylase activity was abolished or decreased by inhibitors of polyADP-ribose synthesis. Our studies suggest that ascorbate may regulate the cellular activity of prolyl hydroxylase by activating epigenetic control mechanisms involving polyADP-ribose.

Ascorbic Acid↗
PubMed1991

Stimulation of collagen synthesis in fibroblast cultures by superoxide.

Exposure of diploid fetal human fibroblasts (IMR-90) to superoxide generated by dihydroxyfumarate resulted in increased collagen synthesis. The synthesis of type III collagen was stimulated to a greater extent than the synthesis of type I collagen. The stimulation of collagen synthesis was abolished by superoxide dismutase. Our observations suggest that superoxide may play a role in the regulation of collagen synthesis and may modulate differential collagen gene expression. These observations may explain the increased synthesis of collagen in tissues following inflammation or exposure to oxidant conditions.

Cells, Cultured↗
PubMed1991

Keratin expression in cultures of adult human epidermal cells.

Keratins are complex fibrous proteins characteristic of epithelial cells. We have developed a procedure that allows us to culture and passage adult human dermal keratinocytes in the absence of mesenchymal substrates. Electron microscopic examination of stratifying cultures showed the presence of numerous filament bundles, desmosomes and electron dense granules. The expression of different classes of keratin was examined by immunofluorescence, SDS-PAGE and immunoblots using monoclonal antibodies. The analysis of water-insoluble proteins revealed the presence of keratins of molecular weights 40 Kda, 50-52 Kda, 56 Kda and 65-67 Kda. Our results indicate that the terminal differentiation of keratinocytes may not require dermal factors.

Blotting, Western↗
PubMed1990

Creatine kinase and lactate dehydrogenase isoenzyme patterns in cultured normal and pathological gingival fibroblasts.

The isoenzyme patterns of creatine kinase (CK) and lactate dehydrogenase (LDH) were examined in cultured fibroblasts derived from the gingival tissue of a healthy person and patients with periodontitis and gingival fibromatosis. Human foreskin fibroblasts were also included for comparison. Cytosol fractions were prepared by sonication and centrifugation. Gingival fibroblasts from normal subjects (A), and from patients with periodontitis (B) or fibromatosis (C), exhibited CK-MM (muscle form) as a predominant isoenzyme and CK-BB (brain form) as a minor fraction. There was no significant difference in specific activities of CK-BB fraction in all 3 types of fibroblasts. However, B fibroblasts contained a higher proportion of MM type isoenzyme than A fibroblasts. C fibroblasts showed the lowest amount of CK-MM isoenzyme as compared to either A or B fibroblasts. In contrast, fibroblasts derived from human foreskin (D) contained only a small amount of CK-BB isoenzyme, but CK-MM was not present. In the studies of LDH isoenzymes, A, B, and C fibroblasts possessed only 3 isoenzymes, namely, LDH-3, LDH-4 and LDH-5. In terms of the relative abundance of M vs H subunits, A, B, and C fibroblasts were shown to have a preponderance of M subunits (85-87% M vs 13-15% H subunits). In comparison, D fibroblasts contained 4 isoenzymes (LDH-2, LDH-3, LDH4, LDH-5) with 73% M and 27% H subunits. The ratio of LDH-5 to LDH-4 was calculated for all fibroblasts tested, and the values were 1.11 in A, 1.39 in B, 1.16 in C and 0.64 in D.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1988

Inter-chain proline:proline contacts contribute to the stability of the triple helical conformation.

The triple helical conformation observed in the collagen group of proteins is related to the presence of large numbers of imino residues and is derived from the stereochemical properties of these residues. The triple helix is stabilized by increasing numbers of these residues. Hydrogen bonds are usually considered to be a major factor in the formation and stability of protein conformation, however, imino residues are not hydrogen bond donors. We have evaluated the role of these residues in stabilizing the triple helix by re-examining two X-ray based structures of the triple helical polypeptide (Pro-Pro-Gly)10 using molecular mechanics calculations. The two minimized structures are comparable in energy and have helical parameters close to the starting values for each starting structure. Our studies suggest that clusters of close van der Waals contacts between proline residues in adjacent chains contribute significantly to the stability of the triple helix. Preliminary NMR studies support this concept. We propose that non-bonded interactions between proline residues may be a significant stabilizing force in the triple helix generated by (Pro-Pro-Gly)10.

Chemical Phenomena↗
PubMed1986

Molecular mechanics studies of dermorphin.

Molecular mechanical simulations have been carried out on dermorphin. Presence of D-Ala2 at the N-terminus and L-Pro6 residue at the C-terminus indicated the probability of beta-turns. From the stereochemical considerations, three types- II', III' and V' - for the beta-turn at the N-terminus of the peptide and two types-I and III- for the C-terminus side of the peptide are possible. In our molecular mechanics calculations, we considered six folded and one extended conformations for dermorphin to asses the relative stabilities. Three of the six folded conformations are lower in energy and have the following general feature-similar in energy, three hydrogen bonds, semirigid beta-sheet segment and favorable Tyr1-Tyr5 interaction. The presence of beta-sheet structure might play a role in mu-receptor selective interaction of dermorphin.

Analgesics, Opioid↗
PubMed1985

Conformational states of Leu5- and Met5-enkephalins in solution.

The molecular conformations of Leu5- and Met5-enkephalins in aqueous and DMSO solutions were investigated by FT-IR and laser Raman spectroscopic methods. The amide I, II, and III regions in the FT-IR spectra of Leu5- and Met5-enkephalins in aqueous solution were analyzed by performing Fourier self-deconvolution of the bands. Leu5-enkephalin in aqueous solution is found to exist in both type II beta-turn and beta-sheet structures, whereas Met5-enkephalin has a lesser tendency to form beta-turn structure in aqueous solution. It is likely that these different conformers of enkephalins might bind to different receptor types.

Chemical Phenomena↗
PubMed1985

Stimulation of poly(ADP-ribose) synthetase activity in the lungs of mice exposed to a low level of ozone.

Toxic effects of O3 are mediated through the formation of free radicals, which can cause DNA strand breaks. Cellular DNA repair is dependent upon the formation of poly(ADP-ribose) (polyADPR) catalyzed by polyADPR synthetase. In order to evaluate whether O3 exposure inflicted DNA damage in lung tissue, we measured the activity of polyADPR synthetase (known to be activated in response to DNA damage) in mouse lungs after exposure to 0.45 ppm (882 micrograms/m3) O3 for up to 7 days. The enzyme activity was stimulated with O3 exposure relative to unexposed controls, showing a 20% (P less than 0.05) increase at Day 5 and 42% (P less than 0.001) at Day 7 of O3 exposure. In addition, the activity of superoxide dismutase (SOD), known to be stimulated in response to production of superoxide anion (.O2-), was measured as an indicator of free radical involvement. Relative to unexposed controls, the SOD activity in exposed animal lungs increased to the peak level at Day 5 (48%, P less than 0.001) and then declined at Day 7 of O3 exposure but was still higher than controls (17%, P less than 0.05). When animals, after 5 days of O3 exposure, were allowed to recover in filtered room air, the activities of both enzymes declined to their respective control values in 6 days. These results suggest a possible temporal relationship between O3 injury and the activities of polyADPR synthetase and a free radical scavenging enzyme, SOD. The stimulation of polyADPR synthetase activity with O3 exposure, reflecting a response to lung cellular DNA repair, may be a sensitive indicator for assessing DNA damage in oxidant injury.

Animals↗
PubMed1985

Poly(ADP-ribose) synthetase activity during bleomycin-induced lung fibrosis in hamsters.

Bleomycin damages cellular DNA and is a potent inducer of pulmonary fibrosis. It has been shown to act through a superoxide-mediated mechanism. We are interested in determining the biochemical mechanisms involved in fibrosis and in this preliminary study we have examined the temporal relationship between early biochemical events associated with DNA damage and fibrosis, in lungs of hamsters after administration of 0.75 unit of bleomycin. The activities of poly(ADP-ribose) synthetase, an enzyme associated with DNA repair, inducible superoxide dismutase (SOD) and prolyl hydroxylase as well as the tissue levels of NAD+ and hydroxyproline in the lung were determined. All three enzyme activities expressed as per milligram DNA or per lung, increased upon bleomycin treatment over the saline-administered controls. Lung poly(ADP-ribose) synthetase activity which is sensitive to DNA breaks, increased first (24% over control in 1 day, P less than 0.0001), attained the maximum value on the 5th day (952% over control, P less than 0.0001), and started to decline thereafter and approached near the control value on 14th day. Bleomycin treatment induced a rapid change in the level of lung NAD+. After 1 day the level of NAD+ was reduced by 42% compared to the control (P less than 0.001), further declined to 65% (P less than 0.001) on the 3rd day, and stayed at that level until the 7th day. On the 14th day, however, the NAD+ level was still lower (29%, P less than 0.05) but approaching the value in the control animals. The activity of prolyl hydroxylase showed significant increase on the 3rd day (50% over control, P less than 0.0001) after bleomycin administration. The enzyme activity continued to increase until the end of the experiment (490% of control, P less than 0.0001, on Day 14). The content of undialyzable hydroxyproline, a marker for collagen, was also increased significantly in the lung tissue on the 3rd day (30% over control, P less than 0.05), continued to increase and reached the highest level on the 14th day (71% over control, P less than 0.001). A significant increase in the activity of SOD (19% over control, P less than 0.001) was seen on the 5th day which continued to increase and attained the highest value on Day 14 (115% over control, P less than 0.0001).(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1984

Rate of viscosity changes in five salivary protein fractions following pH alterations.

The change of viscosity with time following alteration of pH was studied in five protein fractions isolated from human parotid saliva. The results indicate that sudden pH changes may cause rapid charge alterations leading to shock-like expansion or compression of the molecules. This quick reaction seems to be followed by more slowly proceeding conformational alterations probably involving folding, intermingling, and other types of chain interactions. The content of proline residues may influence the rate and extent of such conformational changes.

Humans↗
PubMed1984

Salivary viscosity and lubrication: influence of pH and calcium.

The viscosity of human whole saliva and six salivary protein fractions was studied at different pH levels in the presence and absence of calcium. Changes in pH significantly influenced the viscosity of whole saliva, and centrifugation and filtration removed one of the viscous components. Calcium depletion made the viscosity particularly susceptible to acidification. The protein fractions were obtained from pooled parotid saliva by ion exchange and gel permeation chromatography procedures. Except for a glycoprotein, these fractions generally exhibited the lowest viscosity in the pH region around their isoelectric points. Calcium generally increased the viscosity of these proteins.

Calcium↗
PubMed1980

Genetic control of the murine T lymphocyte proliferative response to collagen: analysis of the molecular and cellular contributions to immunogenicity.

The genetic control of the murine immune response to denatured beef type II collagen was examined in an antigen-presenting cell-dependent T cell proliferation assay. It was found that the response to collagen is under the control of H-2-linked Ir genes, possibly operating at the level of the antigen-presenting macrophage. Utilizing in vitro cross-stimulation with synthetic collagen-like polypeptides, the data suggest that the antigenic determinants stimulating collagen immune T cells are more dependent on primary amino acid sequence and less on conformation. The implications of such findings for Ir gene specificity and other related immunologic functions is discussed.

Animals↗