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Y Sharma

Publications and source records attributed to Y Sharma.

34 records · Page 2Linked to original sources
PubMed1996

A conformational study of corneal dermatan sulfate proteoglycan using fluorescence spectroscopy.

DSPG is a major proteoglycan of the corneal stroma and is thought to be important for the transparency of the tissue. We have studied its conformation by exploring the microenvironment and dynamics of its lone Tryptophan (Trp) residue using steady state and time resolved fluorescence spectroscopy. DSPG exhibits a doublet Trp fluorescence emission. Such a doublet emission has been observed earlier in the copper protein azurin and in avian lens delta-crystallin. Unlike the above cases where the doublet emission is thought to arise due to vibronic structure or the location of Trp at the interface of interacting subunits, fluorescence quenching, denaturation studies and ANS binding with DSPG indicate the location of Trp at two different environments. Such a situation could arise from the differential glycosylation of the core protein or due to duplexation and aggregation of the glycosaminoglycan chains.

Animals↗
PubMed1996

Fluorescence properties of isolated intact normal human corneas.

We have examined the fluorescence properties of excised intact normal human corneas from over a hundred donors, using synchronous excitation fluorescence spectroscopy. In some of the corneas from the donors, a fluorophore with an excitation band centered at 330 nm was observed. This fluorophore does not seem to correspond to the dityrosine moiety or to any photoproducts of tryptophan. Isolated corneas irradiated with light of 295 nm wavelength do not produce any fluorescent photoproducts, suggesting that the intact tissue has endogenous quenchers, radical scavengers and antioxidants that inhibit its photodamage. The non-tryptophan fluorophores that accumulate in some corneas thus appear to arise largely from the nonenzymatic glycosylation (glycation) of the constituent proteins as similar fluorophores are detected in the corneas of rats in which diabetes is induced.

Adult↗
PubMed1996

Calcium binding properties of beta-crystallins.

beta-Crystallins, the oligomeric proteins of the eye lens, were found to bind calcium ions with low affinity, in the millimolar range. Calcium was found to induce conformational changes in the secondary and tertiary structure of beta-crystallins. While these changes in conformation are seen in low ionic strength media, they are masked when the protein is dissolved in an intermediate ionic strength medium, where oligomers of lower molecular weight are formed. The calcium binding takes place in these conditions. The behaviour of this protein with respect to calcium binding is reviewed.

Animals↗
PubMed1996

Effect of UVB radiation on corneal aldehyde dehydrogenase.

PURPOSE: A Class 3 aldehyde dehydrogenase happens to be a major soluble protein constituent of the cornea. Its role is conjectured to be manifold: to protect the tissue from oxidative damage by eliminating the toxic aldehydes produced upon lipid peroxidation under oxidative stress, to act as an UV-absorber, and to maintain the level of the coenzyme NADH in the cornea. We have studied the effect of UVB on the structure and enzyme activity of corneal aldehyde dehydrogenase. METHODS: Aldehyde dehydrogenase was irradiated at 295 nm for varying periods of time and change in its enzyme activity assayed. The structural changes in the molecule accompanying irradiation were monitored using fluorescence and circular dichroism spectroscopy, and its hydrodynamic behavior and surface hydrophobicity studied using gel filtration chromatography and binding of the hydrophobic fluorophore ANS. The protective ability of aldehyde dehydrogenase in preventing aggregation of photolabile proteins, such as Gamma-crystallin of the eye lens, was studied by monitoring the scattering value of the test protein with irradiation by UVB. RESULTS: Aldehyde dehydrogenase is seen to undergo photodamage with alterations in its quaternary structure, though no significant change is noticed in the peptide chain conformation. Under such conditions the molecule continues to act as a protectant by preventing aggregation of photolabile proteins such as the eye lens Gamma-crystallin. CONCLUSIONS: Our earlier studies have shown that the free sulfhydryl groups are important for the antioxidant abilities of aldehyde dehydrogenase. Its protective ability towards photoaggregation of Gamma-crystallin seen here might arise both due to: (i) oxyradical quenching and (ii) the increased surface hydrophobicity of the molecule upon irradiation, which allows it to bind to, and thus inhibit the aggregation of interacting proteins.

Aldehyde Dehydrogenase↗
PubMed1994

In situ fluorescence spectroscopic studies on bovine cornea.

The cornea is a transparent ocular tissue and its transparency is thought to be a result of intramolecular interactions and the supramolecular organization of its protein constituents. We have studied the intrinsic fluorescence properties of intact bovine corneas and compared these with that of the opaque sclera. It was observed that with increasing excitation wavelengths the emission maxima shifted toward the red edge exhibiting the phenomenon of red edge excitation shift, which is indicative of immobilization of the constituent fluorophores. The magnitude of the shift increased after photodamage by irradiation at 295 nm. Many of the spectral characteristics of the cornea are shown to be due to its proteoglycans, which show surprisingly significant red edge excitation shift in solution.

Animals↗
PubMed1993

Studies on the interaction of the dye, stains-all, with individual calcium-binding domains of calmodulin.

We show that the calcium-mimic dye, Stains-all, is a convenient probe to study the structural features of the individual calcium-binding sites of calmodulin (CaM) and related calcium-binding proteins (CaBP). These peptides bind the dye in their calcium-binding sites, and induce a circular dichroism (CD) band in the bound dye in the 620 nm (J band) region, which is abolished upon the addition of calcium. Replacement of Asp by Asn in the + x position of the weaker calcium-binding site (site I of CaM) abolishes the dye binding, while the same change in the higher affinity site IV attenuates the binding of the dye and does not abolish it. Replacement of Tyr in site IV with Trp does not distort the geometry, although it increases the dye binding affinity.

Amino Acid Sequence↗
PubMed1993

Alteration of dynamic quaternary structure and calcium-binding ability of beta-crystallin by light.

beta-crystallin, one of the three main constituent proteins of the eye lens, exists as an equilibrium population of oligomeric (beta H), trimeric (beta L1) and dimeric (beta L2) species. This equilibrium is dependent on various factors such as the protein concentration, ionic strength and pH of the medium. We have studied the effect of ultraviolet B radiation on the aggregational patterns of beta-crystallin, using size-exclusion chromatography. Irradiation of a solution of beta H-crystallin at 295 nm for about 30 min causes the deaggregation of the hexameric population into dimers. Irradiation for a longer time, however, produces cross-linked high molecular weight products. Irradiation of a beta L2 solution for 30 min does not perturb the elution profile, while irradiation for a longer time increases the content of beta L1 (trimeric) crystallin. Irradiation also causes a decrease in the calcium-binding affinity of the beta-crystallins.

Animals↗
PubMed1992

A critical review of prescriptions in internal medicine.

A study was undertaken to assess the prescribing trends in inpatients of the Internal Medicine Department of Government General Hospital which is attached to Guntur Medical College, Guntur. Prescriptions for 250 patients were audited under various heads like details of drugs used, generic versus trade name prescribing, dosage, dosage form, dose interval, drug of choice and duration of treatment. Among 250 prescriptions audited, 7 percent used generic names, 22 percent trade names, and 71 percent combinations of both generic and trade names. Dosages and dosage intervals were appropriate. In 75 percent of the audited cases, the duration of treatment was appropriate; 25 percent did not specify the duration. The choice of drugs was proper in 84 percent and inappropriate in 16 percent of cases. Certain deficiencies in the prescribing trends are analysed.

Cross-Sectional Studies↗
PubMed1991

Conformation of alginate and pectate chains monitored by the binding of the dye stains-all.

The carbocyanine dye Stains-all displays spectral colour shifts or metachromasia upon binding to proteins, polysaccharides and other anionic substrates. The conformational status of the binding region of the substrate appears to govern the metachromatic features of the bound Stains-all. We have used this property to derive information about the conformational differences between the two anionic polysaccharides alginate and pectate. The stronger induction of circular dichroism in the 500 nm region of the dye by pectate is indicative of a greater extent of helical order in this polymer in solution than in the alginate or perhaps even hyaluronate chains.

Alginates↗
PubMed1989

Binding site conformation dictates the color of the dye stains-all. A study of the binding of this dye to the eye lens proteins crystallins.

The interaction of the cationic carbocyanine dye Stains-all (1-ethyl-2-[3-(1-ethyl-naphthol[1,2-d]thiazolin-2-ylidene)-2- methylpropenyl]naphthol[1,2-d]thiazolium bromide) with the eye lens proteins crystallins has been studied. alpha- and gamma-crystallins do not bind the dye, while beta- and delta-crystallins do, consistent with the fact that the latter two proteins bind the calcium ion. beta-Crystallin resembles parvalbumin in that it induces only the J-band of the bound dye. delta-crystallin, on the other hand, induces only the gamma-band. Analysis of the metachromasia induced in the dye by these and other proteins suggests that Stains-all is responsive to the conformational status of the region to which it binds in a protein. The J-band of the dye is activated when it binds to a globular domain, and the gamma-band is activated when it binds to a helical stretch of the protein.

Animals↗
PubMed1989

Calcium ion binding to delta- and to beta-crystallins. The presence of the "EF-hand" motif in delta-crystallin that aids in calcium ion binding.

Abnormal levels of endogenous calcium ions are known to induce eye lens opacity, and a variety of causative factors has been proposed, including calcium-mediated aggregation and precipitation of the lens proteins crystallins. We have specifically looked in some detail at the interaction of Ca2+ with various crystallins and its consequences. Lenses incubated in solutions containing 10 mM Ca2+ or 5 mM Tb3+ opacified. Fluorescence titration of crystallins with TbCl3 revealed that this ion binds to delta- and beta-crystallins in solution. Equilibrium dialysis showed that four Ca2+ ions bind to one delta-crystallin tetramer with an affinity of 4.3 x 10(3) M-1. Analysis of the amino acid sequence of delta-crystallin reveals the presence of a calmodulin-type "helix-loop-helix" or "EF-hand" calcium ion binding conformational motif in the region comprising residues 300-350. This is a novel feature of the molecule not reported so far. No other crystallins appear to have this motif. beta-Crystallin also binds four Ca2+ ions/aggregate unit of mass 160 kDa, with an affinity of 2.6 x 10(3) M-1, presumably in the midregion of the molecule that is rich in anionic and polar residues. Circular dichroism spectroscopy shows that the binding of calcium ion leads to subtle conformational changes in the molecules, notably in the tertiary structure.

Amino Acid Sequence↗
PubMedDate not supplied

Prevalence of Helicobacter pylori in Dhulikhel Hospital, Kathmandu University Teaching Hospital: a retrospective histopathologic study.

OBJECTIVE: The main objective of this study was to determine prevalence of Helicobacter pylori infection in Dhulikhel Hospital, Kathmandu University Teaching Hospital. METHODOLOGY: Endoscopic antral biopsies from January 1, 2004 to August 31, 2005 were studied retrospectively. Hematoxylin and eosin and Giemsa stained histological sections were examined. RESULTS: Out of 224 patients (Male 125 and female 99) who underwent endoscopic biopsy and included in the study, a total of 76 (33.9%) patients (Male 50 and female 26) were infected by H. pylori. The mean age of the H. pylori infected patients was 40.2 years (SD 16.0). The infection by H. pylori was significantly higher in males than females (p<0.05) with male to female ratio of 1.9:1. The most common (31.3%) histopathologic finding was chronic superficial gastritis followed by normal histology (30.8%). A total of 10 cases (4.5%) of adenocarcinoma and 16 cases (7.1%) of intestinal metaplasia were detected and neither of them showed presence of H. pylori. Two cases (0.9%) were diagnosed as suspected malignancy and both were negative for H. pylori infection. Among gastritis, H. pylori was most frequently observed in chronic active gastritis (86.1%). Two of 69 cases (2.9%) of normal gastric mucosa showed H. pylori.

Adolescent↗
PubMedDate not supplied

Assessment of observer variability in the classification of human cataracts.

An in vitro cataract classification system was developed in our laboratories and used to demonstrate a relationship between sustained aspirin intake and the apparent deceleration or retardation of human cataract formation. The purpose of this investigation was to assess the reliability of this cataract classification schema. Sets of extracted human cataractous lenses, which had been photographed in vitro, were randomly assigned to five observers. The task was to classify the lenses on the basis of nuclear and cortical involvement, as reflected in color and area changes along five groupings. Assessments were made on the basis of both intraobserver and interobserver agreement levels, corrected for chance (weighted kappa values). All five examiners evidenced levels of intraobserver agreement which ranged between "Good" and "Fair" and "Excellent" (.46-.83). Each of the five observers was ranked on the basis of his agreement levels with the remaining four observers. The results followed a predictable pattern such that the more experienced the observer in classifying cataracts, the more consistent his rankings vis-à-vis the remaining four evaluators. These results are discussed in the general context of observer variability studies in the field of medicine.

Aged↗