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Biomedical subjects

K S Bhat

Publications and source records attributed to K S Bhat.

At least 19 recordsLinked to original sources
PubMed1998

UVB irradiation alters the activities and kinetic properties of the enzymes of energy metabolism in rat lens during aging.

Ultraviolet (UV) radiation is one of the major risk factors of cataract (loss of eye-lens transparency). The influence of UVB radiation (300 nm; 100 microW cm-2) on the activity and apparent kinetic constants (Km and Vmax) of rat lens hexokinase (HK;EC 2.7.1.1), phosphofructokinase (PFK; EC 2.7.1.11), isocitrate dehydrogenase (ICDH; EC 1.1.1.41) and malate dehydrogenase (MDH; EC 1.1.1.37) of energy metabolism has been investigated by irradiating the lens homogenate of three- and 12-month-old rats. In the three-month-old group specific activities of HK and PFK are reduced by 56 and 43%, respectively, and there is no change in ICDH and MDH activities after a 24 h exposure. On the other hand, in the 12-month-old group the decreases are 72, 71, 24 and 16% for HK, PFK, ICDH and MDH, respectively. UVB irradiation increases the apparent Km of HK and PFK (in both age groups), whereas the Km of ICDH and MDH is not altered. While the decrease in Vmax of these enzymes due to UVB exposure is only marginal in three-month-old rats, it is more pronounced (significant) in 12-month-old rats. A similar decrease in enzyme activities of HK and PFK is also observed upon UVB exposure of the intact rat lens. The photoinduced changes in energy metabolism may in turn have a bearing on lens transparency, particularly at an older age.

Aging↗
PubMed1998

Synergistic effect of UVB radiation and age on HMPS enzymes in rat lens homogenate.

The behaviour of rat lenticular enzymes, glucose-6-phosphate dehydrogenase (G6PD, EC: 1.1.1.49) and 6-phosphogluconate dehydrogenase (6PGD, EC: 1.1.1.44) as a function of age and UVB irradiation (in vitro) was investigated by irradiating the lens homogenate from 3- and 12-month-old rats at 300 nm (100 microW cm-2). In the 3-month-old group the specific activities of G6PD and 6PGD were reduced by 26% and 42%, respectively, after 24 h of irradiation, whereas in the 12-month-old group the decrease was 38% and 49% respectively, which suggests that the susceptibility of HMPS enzymes to UVB damage is higher in older lenses. The decrease in specific activity was associated with a change in apparent K(m) and Vmax (marginal in 3 months and significant in 12 months) of these enzymes due to UVB irradiation. UVB irradiation also decreased the levels of NADPH and NADPH/NADP ratio. These changes, altered activities of G6PD and 6PGD and altered levels of NADPH, may in turn have a bearing on lens transparency.

Aging↗
PubMed1998

Flavin nucleotides in human lens: regional distribution in brunescent cataracts.

The biochemical mechanism(s) underlying brunescent cataracts remain unclear. Oxidative stress due to reactive oxygen species may have a role in the pigmentation process in eye lens. We have analysed human cataractous lenses for flavins by high-performance liquid chromatography (HPLC), since flavins are light sensitive and act as endogenous sensitizers generating reactive oxygen species in the eye. The most significant observation in this study is that higher levels of flavin nucleotides occur in brown lens compared to yellow lens. The concentration of flavin nucleotides (flavin monouncleotide, FMN + flavin adenine dinucleotide, FAD) was highest in the nuclear region of the lens followed by the cortical and capsule-epithelial regions. However, the ratio of FAD/FMN was lowest in the nuclear region of the lens followed by other regions. On the other hand, riboflavin was not detected in any of the lens (cataractous) regions. These results suggest that the observed increase in flavin nucleotides in the ocular tissue could contribute towards deepening of lens pigmentation.

Biomarkers↗
PubMed1995

Effect of four vehicles on the pH of calcium hydroxide and the release of calcium ion.

Nonsetting calcium hydroxide pastes are commonly used in endodontic practice. The desired therapeutic effect of such a paste is in part dependent on the dissociation of calcium hydroxide into hydroxy ion and calcium ion. This in turn is influenced by the vehicle used to make the paste. This investigation attempts to quantitatively estimate the release of hydroxyl ion and calcium ion from pastes made by using distilled water, normal saline solution, camphorated monochlorophenol and also an experimental vehicle, propylene glycol. The results of this study indicate that propylene glycol induces the most favorable release characteristics of the two ionic species.

Calcium↗
PubMed1993

Generation of a plasmid vector for deletion cloning by rapid multiple site-directed mutagenesis.

The construction of a new plasmid vector, devoid of all MboI (GATC) and TspEI (AATT) restriction sites, is described. The lack of these two frequent-cutting restriction sites is a unique feature among plasmids. This new plasmid, pBRkanf1-, allows selective fragmentation of a cloned insert. As a result, the vector offers an alternative strategy to create overlapping and sequentially deleted subclones. In addition, the construction of the new plasmid required the development of a rapid and accurate multiple site-directed mutagenesis procedure. The mutagenesis method uses a combination of DNA amplification and chain extension by DNA polymerase. By this method, mutations are created progressively from one end of a DNA molecule to the other.

Base Sequence↗
PubMed1992

Secondary structure of the pentraxin female protein in water determined by infrared spectroscopy: effects of calcium and phosphorylcholine.

The secondary structure of hamster female protein in aqueous solutions in the presence or absence of calcium and phosphorylcholine has been investigated using Fourier transform infrared spectroscopy. Our present studies provide the first evaluation of the secondary structure of FP and its calcium- and phosphorylcholine-dependent conformational changes. Quantitative analysis indicated that FP is composed of 50% beta-sheet, 11% alpha-helix, 29% beta-turn, and 10% random structures. Calcium- and phosphorylcholine-dependent infrared spectral changes were observed in regions assigned to beta-sheet, alpha-helix, turn, and random structures. The infrared-based secondary structure compositions were used as constraints to compute theoretical locations for the different secondary structures along the amino acid sequence of the FP protein. Two putative calcium-binding sites were proposed for FP (residues 93-109 and 150-168) as well as other members of the pentraxin family on the basis of the theoretical secondary structure predictions and the similarity in sequence between the pentraxins and EF-hand calcium-binding proteins. The changes in protein conformation detected upon binding of calcium and phosphorylcholine provide a mechanism for the effects of these ligands on physiologically important properties of the protein, e.g., activation of complement and association with amyloids.

Alpha-Globulins↗
PubMed1991

Secondary structure analysis of the scrapie-associated protein PrP 27-30 in water by infrared spectroscopy.

A protease-resistant form of the protein PrP (PrP-res) accumulates in tissues of mammals infected with scrapie, Creutzfeldt-Jakob disease, and related transmissible neurodegenerative diseases. This abnormal form of PrP can aggregate into insoluble amyloid-like fibrils and plaques and has been identified as the major component of brain fractions enriched for scrapie infectivity. Using a recently developed technique in Fourier transform infrared spectroscopy which allows protein conformational analysis in aqueous media, we have studied the secondary structure of the proteinase K resistant core of PrP-res (PrP-res 27-30) as it exists in highly infectious fibril preparations. Second-derivative analysis of the infrared spectra has enabled us to quantitate the relative amounts of different secondary structures in the PrP-res aggregates. The analysis indicated that PrP-res 27-30 is predominantly composed of beta-sheet (47%), which is consistent with its amyloid-like properties. In addition, significant amounts of turn (31%) and alpha-helix (17%) were identified, indicating that amyloid-like fibrils need not be exclusively beta-sheet. The infrared-based secondary structure compositions were then used as constraints to improve the theoretical localization of the secondary structures within PrP-res 27-30.

Acetylglucosamine↗
PubMed1991

Effect of pigmentation on glutathione redox cycle antioxidant defense in whole as well as different regions of human cataractous lens.

'Reactive oxygen species', generated within the lens, are implicated in the deepening of nuclear pigmentation leading to browning of the human cataractous lens. The present study, which was carried out in senile brunescent cataracts, deals with changes in the antioxidant glutathione redox cycle defense system during the progression of browning (yellow to brown) in different regions of the human lens. A significant reduction in the levels of reduced glutathione was noted in the cortical and nuclear regions of the brown lens as compared to yellow lens, despite normal glutathione reductase activity. The rate limiting enzyme of the hexosemonophosphate shunt pathway, glucose 6-phosphate dehydrogenase, and the important oxidant scavenger enzyme, glutathione peroxidase, showed significant changes in opposite directions (glucose 6-phosphate dehydrogenase increased and glutathione peroxidase decreased), only in the cortical region of the brown lens as compared to yellow lens. The significance of the present findings in relation to the overall biochemical mechanisms underlying browning of the lens tissue in human cataracts is discussed.

Aged↗
PubMed1991

The opacity proteins of Neisseria gonorrhoeae strain MS11 are encoded by a family of 11 complete genes.

Variants of Neisseria gonorrhoeae MS11 show distinct colony morphologies because of the expression of a class of surface components called opacity (Opa, PII) proteins. Southern analyses combined with molecular cloning of genomic DNA from a single variant of MS11 has identified 11 opa genes contained in separate loci. These opa genes code for distinct opacity proteins which are distinguishable at their variable domains. The opa gene analyses were also extended to divergent variants of MS11. These studies have shown that, during in vitro and in vivo culture, 10 of the 11 opa genes did not undergo significant change in their primary sequence. However, in these variants, one gene (opaE) underwent non-reciprocal inter-opa recombinations to generate newer Opa variants. Phylogenic analysis of the opa gene sequences suggests that the opa gene family have evolved by a combination of gene duplication, gene replacement and partial inter-opa recombination events.

Amino Acid Sequence↗
PubMed1991

Prevalence of gene sequences coding for hypervariable regions of Opa (protein II) in Neisseria gonorrhoeae.

Opas (protein IIs) are a family of surface-exposed proteins of Neisseria gonorrhoeae. Each strain of N. gonorrhoeae has multiple (10-11) genes encoding for Opas. Identifiable elements in opa genes include the coding repeat within the signal sequence, conserve 5' and 3' regions, and hypervariable regions (HV1 and HV2) located within the structural gene. N. gonorrhoeae strains appear to have many biological properties in common that are either HV-region-mediated or associated with the presence of specific HV regions, suggesting that HV regions could be found in many clinical isolates. Oligonucleotides from three source strains representing three conserved regions of opa, 12 HV1 regions, and 14 HV2 regions were used by dot blot analysis to probe 120 clinical isolates of N. gonorrhoeae. The probe for the coding repeat hybridized to all 120 strains, the 3' conserved-region probe reacted with 98% of the strains, and the 5' conserved-region probe with 90% of the strains. Nine HV1 probes hybridized to 3.3-39.2% of the strains, and 13 of the HV2 probes hybridized to 1.7-25% of the isolates. Analysis of the number of probes that hybridized to each of the isolates showed that 19% did not hybridize with any of the HV1 probes and 25% did not hybridize with any of the HV2 probes. Approximately three-quarters of the isolates hybridized with one, two or three of the HV1 probes or one, two or three of the HV2 probes; 89% of the isolates hybridized to least one HV1 or one HV2 probe. The data indicate that some genes encoding HV regions of N. gonorrhoeae Opa proteins are widely distributed in nature.

Bacterial Outer Membrane Proteins↗
PubMed1989

Characterization of primary transcripts and identification of transcription initiation sites on the heavy and light strands of mouse mitochondrial DNA.

Total RNA from Ehrlich ascites mitochondria pretreated with RNase-free DNase was capped in vitro with [alpha-32P]GTP and guanylyl transferase. The cappable RNAs representing the primary transcripts show a heterogeneous size distribution with four major species of 46, 63, 94, and 152 nucleotides and four minor species of 19, 24, 104, and 790 nucleotides in size. Hybridization with the D-loop DNA probes shows that the 19-nucleotide-long capped RNA is coded by the H-strand of mitochondrial DNA while the rest are coded by the L-strand. S1 nuclease mapping and primer extension analyses suggest the occurrence of a transcription initiation of H-strand at about 19 nucleotides upstream from the start of the tRNA(Phe) gene. All of the L-strand cappable RNAs have a common 5' end mapping to nucleotide 16,183 +/- 5 of the genome. The 3' ends of four major cappable RNA species line up to the conserved sequence boxes, putative start sites of DH-DNA; and in fact about 2% of these cappable species are found to exist as DNA-linked RNA under steady-state conditions. The 3' end of the 790-nucleotide cappable RNA lies close to the start of the tRNA(Pro) gene, suggesting that it may be the true precursor of L-strand transcript endonucleolytically processed at the 3' end. The level of L-strand-coded cappable RNAs varies markedly under different growth conditions. Treatment with cycloheximide results in a reduction while chloramphenicol caused over 3-fold induction, suggesting that these "primer" RNAs may have an additional regulatory function.

Animals↗