Biomedical subjects

M R Sharma

Publications and source records attributed to M R Sharma.

10 recordsLinked to original sources

PubMed2001

Three-dimensional reconstruction of the recombinant type 3 ryanodine receptor and localization of its amino terminus.

Recombinant type 3 ryanodine receptor (RyR3) has been purified in quantities sufficient for structural characterization by cryoelectron microscopy and three-dimensional (3D) reconstruction. Two cDNAs were prepared and expressed in HEK293 cells, one encoding the wild-type RyR3 and the other encoding RyR3 containing glutathione S-transferase (GST) fused to its amino terminus (GST-RyR3). RyR3 was purified from detergent-solubilized transfected cells by affinity chromatography using 12.6-kDa FK506-binding protein in the form of a GST fusion as the affinity ligand. Purification of GST-RyR3 was achieved by affinity chromatography by using glutathione-Sepharose. Purified recombinant RyR3 and GST-RyR3 proteins exhibited high-affinity [(3)H]ryanodine binding that was sensitive to activation by Ca(2+) and caffeine and to inhibition by Mg(2+). 3D reconstructions of both recombinant RyR3 and GST-RyR3 appeared very similar to that of the native RyR3 purified from bovine diaphragm. Comparison of the 3D reconstructions of RyR3 and GST-RyR3 revealed that the GST domains and, hence, the amino termini of the RyR3 subunits are located in the "clamp" structures that form the corners of the square-shaped cytoplasmic region of homotetrameric RyR3. This study describes the 3D reconstruction of a recombinant ryanodine receptor and it demonstrates the potential of this technology for characterizing functional and structural perturbations introduced by site-directed mutagenesis.

Cell Line↗

PubMed2000

Three-dimensional structure of ryanodine receptor isoform three in two conformational states as visualized by cryo-electron microscopy.

Using cryo-electron microscopy and single particle image processing techniques, we present the first three-dimensional reconstructions of isoform 3 of the ryanodine receptor/calcium release channel (RyR3). Reconstructions were carried out on images obtained from a purified, detergent-solubilized receptor for two different buffer conditions, which were expected to favor open and closed functional states of the channel. As for the heart (RyR2) and skeletal muscle (RyR1) receptor isoforms, RyR3 is a homotetrameric complex comprising two main components, a multidomain cytoplasmic assembly and a smaller ( approximately 20% of the total mass) transmembrane region. Although the isoforms show structural similarities, consistent with the approximately 70% overall sequence identity of the isoforms, detailed comparisons of RyR3 with RyR1 showed one region of highly significant difference between them. This difference indicated additional mass present in RyR1, and it likely corresponds to a region of the RyR1 sequence (residues 1303-1406, known as diversity region 2) that is absent from RyR3. The reconstructions of RyR3 determined under "open" and "closed" conditions were similar to each other in overall architecture. A difference map computed between the two reconstructions reveals subtle changes in conformation at several widely dispersed locations in the receptor, the most prominent of which is a approximately 4 degrees rotation of the transmembrane region with respect to the cytoplasmic assembly.

Animals↗

PubMed2000

Amino acid residues 4425-4621 localized on the three-dimensional structure of the skeletal muscle ryanodine receptor.

We have localized a region contained within the sequence of amino acid residues 4425-4621 on the three-dimensional structure of the skeletal muscle ryanodine receptor (RyR). Mouse monoclonal antibodies raised against a peptide comprising these residues have been complexed with ryanodine receptors and imaged in the frozen-hydrated state by cryoelectron microscopy. These images, along with images of antibody-free ryanodine receptor, were used to compute two-dimensional averaged images and three-dimensional reconstructions. Two-dimensional averages of immunocomplexes in which the ryanodine receptor was in the fourfold symmetrical orientation disclosed four symmetrical regions of density located on the edges of the receptor's cytoplasmic assembly that were absent from control averages of receptor without added antibody. Three-dimensional reconstructions revealed the antibody-binding sites to be on the so-called handle domains of the ryanodine receptor's cytoplasmic assembly, near their junction with the transmembrane assembly. This study is the first to demonstrate epitope mapping on the three-dimensional structure of the ryanodine receptor.

Animals↗

PubMed1998

Interactions of gender, growth hormone, and phenobarbital induction on murine Cyp2b expression.

The interactions of gender, growth hormone, and phenobarbital induction on Cyp2b expression were examined in phenotypically normal (lit/+) and growth-hormone deficient "little" (lit/lit) mice. Using an immunocrossreactive monoclonal antibody designed to identify rat CYP2B1 and 2B2 proteins, we observed three hepatic Cyp2b proteins in control (lit/+) females, but only two proteins, one at trace levels, in control males. Phenobarbital administration to lit/+ mice increased the expression of the two Cyp2b isoforms in the males by 3- to 4-fold, but produced an approximately 75% reduction in the female-expressed proteins. Whereas growth hormone depletion (lit/lit) had no effect on the expression profile of Cyp2b proteins in females, it had a de-repressive effect in males, resulting in the expression of three proteins at concentrations now comparable to those observed in female liver. Generally, phenobarbital had no inductive effects in the lit/lit mice of both sexes. In all groups, transcript levels measured by a CYP2B1 probe were in agreement with the protein findings. In contrast, Cyp2b mRNA identified by an oligonucleotide probe for CYP2B2 were repressed completely by growth hormone in both sexes, and was expressed as a female-predominant transcript in the lit/lit mice. In spite of an apparent high degree of sequence homology between the rat CYP2B and murine Cyp2b gene families, the present findings highlight fundamental differences in their constitutive and gender-dependent expression, growth hormone regulation, and phenobarbital inducibility.

Animals↗

PubMed1998

Cryoelectron microscopy and image analysis of the cardiac ryanodine receptor.

The three-dimensional structure of the cardiac muscle ryanodine receptor (RyR2) is described and compared with its skeletal muscle isoform (RyR1). Previously, structural studies of RyR2 have not been as informative as those for RyR1 because optimal conditions for electron microscopy, which require low levels of phospholipid, are destabilizing for RyR2. A simple procedure was devised for diluting RyR2 (in phospholipid-containing buffer) into a lipid-free buffer directly on the electron microscope grid, followed by freezing within a few seconds. Cryoelectron microscopy of RyR2 so prepared yielded images of sufficient quality for analysis by single particle image processing. Averaged projection images for RyR2, as well as for RyR1, prepared under the same conditions, were found to be nearly identical in overall dimensions and appearance at the resolution attained, approximately 30 A. An initial three-dimensional reconstruction of RyR2 was determined (resolution approximately 41 A) and compared with previously reported reconstructions of RyR1. Although they looked similar, which is consistent with the similarity found for the projection images, and with expectations based on the 66% amino acid sequence identity of the two isoforms, structural differences near the corners of the cytoplasmic assembly were observed in both two- and three-dimensional studies.

Animals↗

PubMed1998

A comparison of cytochrome P450-dependent testosterone 2alpha-hydroxylase in rat (P4502C11) and mouse (P4502alpha).

Hepatic P450 2C11 in the rat and P450(2alpha) in the mouse are unique in being the only isoforms in their respective species with testosterone 2alpha-hydroxylase activity. Comparing gender differences, tissue distribution and physicochemical properties, we investigated whether this uncommon catalytic activity shared by the two isoforms is dependent upon a high degree of homology. Using additional substrates (e.g. androstenedione, hexobarbital), we observed that P450(2alpha) and P450 2C11 produced no metabolites in common. Moreover, concentrations of antisera prepared against purified P450(2alpha) that inhibited 95% of P450(2alpha)-dependent testosterone 2alpha-hydroxylase activity had only a minimal inhibitory effect (< 20%) on P450 2C11-dependent testosterone 2alpha-hydroxylase and were similarly unreactive to the rat isoform isolated on Western blots. Comparison of the isoforms' N-terminal amino acid residues and two internal peptide fragments indicated almost no sequence homology (< 4%). Gender-dependent tissue expression levels of P450(2alpha) and P450 2C11 revealed additional dichotomies. Whereas hepatic P450(2alpha) was moderately female-predominant (M/F; 0.62), hepatic P450 2C11 was clearly male-specific (M/F; 32.9). Murine P450(2alpha) mRNA was equally and substantially expressed in liver, kidney and brain; by contrast earlier studies reported that rat P450 2C11 was exclusively expressed in liver. The present results indicate that the unique testosterone 2alpha-hydroxylase activities of P450(2alpha) and P450 2C11 are expressed by two very different proteins exhibiting minimal homology.

Amino Acid Sequence↗

PubMed1996

Purification and characterization of constituent androstenedione 15 alpha-hydroxylase (cytochrome P450(15 alpha AD)) from mouse liver. Sex- and tissue-dependent expression.

Hepatic microsomal androstenedione 15 alpha-hydroxylase (i.e.cytochrome P450(15)alpha AD was purified from female CD-1 mice. Protein purification was monitored in eluates from Fractogel, DEAE-Sephacel, and hydroxylapatite columns at heme absorbing 417 nm, by cytochrome P450 content, reactivity to monoclonal antibody against female-specific rat cytochrome P450 2C12, and androstenedione 15 alpha-hydroxylase activity. The catalytic activity for androgens of the purified cytochrome P450(15)alpha AD, exhibiting a high degree of regioselectivity and stereospecificity, was restricted to the 7 alpha- and 15 alpha-hydroxylation of androstenedione, representing, respectively, > 5% and > 93% of the total metabolites. Polyclonal antibodies against cytochrome P450(15)alpha AD exhibited a concentration-dependent and very selective inhibition of hepatic microsomal androstenedione 7 alpha- and 15 alpha-hydroxylation and a 60% inhibition of benzphetamine demethylation, the latter drug appearing to be a much more effective substrate than androgens. Cytochrome P450(15)alpha AD accounted for about 3% of the total P450 in female mouse liver microsomes. The apparent subunit molecular weight of P450(15)alpha AD was 53,000, and the protein appeared as a single band or sodium dodecyl sulfate-polyacrylamide gels. The isoform was intensely expressed in both liver and lung of CD-1 female mice and was female-predominant in the livers of five or eight strains examined; it was sex-independent in the remaining three strains. Amino-terminal sequence analysis indicates that cytochrome P450(15)alpha AD is a member of the murine cytochrome P450 2c subfamily.

Amino Acid Sequence↗

PubMed1995

Simulataneous isolation of NADPH-cytochrome P-450 reductase and cytochrome P-450 using tentacle ion-exchange chromatography and interspecies comparison of the reductase activity.

Using the same initial Fractogel (tentacle) ion-exchange chromatography to isolate murine cytochrome P-450, mouse hepatic NADPH-cytochrome P-450 reductase (EC 1.6.2.4) was simultaneously isolated from solubilized liver microsomes and purified on a DE-52 column to a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme had a molecular mass of 77 kD, and its specific activity was 25.4 mumol.min-1.mg protein-1. Purified constitutive mouse liver NADPH-cytochrome P-450 reductase was successfully reconstituted in vitro with dilauroylphosphatidyl-choline and constitutive purified mouse testosterone 2 alpha-hydroxylase (cytochrome P-450(2)alpha) with an observed activity of 13.8 nmol.min-1.nmol P-450-1. Although the partially purified reductase obtained from the Fractogel column was contaminated by significant levels of two unidentified proteins, it was as equally effective in the reconstituted system as the DE-52-derived purified reductase. Lastly, we found that rat and mouse NADPH-cytochrome P-450 reductases were similarly effective in supporting the catalytic activity of rat cytochrome P-450 2B1, but the murine reductase was 50% more effective than the rat reductase in a reconstituted system containing mouse cytochrome P-450(2)alpha.

Animals↗

PubMed1994

Neuropathological studies on Plasmodium yoelii nigeriensis-induced malaria in mice.

Malaria infection in mice was produced by intraperitoneal inoculation of 10(6) erythrocytes parasitized with Plasmodium yoelii nigeriensis, a virulent strain of murine malaria. About one week after infection parasitaemia ranged between 60 and 80%, and 100% mortality was observed. Infected animals were killed 6 days after infection to allow the examination of brain tissue. Electron microscopical observations revealed marked damage to cerebral vascular vessel walls with separation of muscular layers, media and adventitia. The endothelial cell layer was discontinuous in places. Activated fibroblast cells producing collagen fibres were seen around the necrotic region of cerebral vasculature. Some parasitized erythrocytes were also seen attached to the endothelial cell lining. Cerebral oedema was prominent around the blood vessels.

Animals↗

PubMed1992

Ascorbic acid status in uremics.

Plasma levels of ascorbic acid (AA) and dehydroascorbic acid (DHA) were estimated in 27 patients of end stage renal failure (ESRF) on standard conservative therapy (group A) and 9 patients of ESRF on maintenance haemodialysis (MHD; group B). Fourteen healthy subjects matched for age and sex served as control (group C). The dietary intake of vitamin C was significantly decreased in group A than in group B compared to control. Similarly, plasma AA was significantly lowered to 0.801 +/- 0.283 mg per cent in group A compared to 1.421 +/- 0.47 mg per cent in control. While it was just lowered to 1.058 +/- 0.272 mg per cent in group B. Although plasma level of DHA was raised to 0.243 +/- 0.486 mg per cent and 0.166 +/- 0.54 mg per cent in groups A and B respectively, the increase was not statistically significant. In our present study, the DHA/AA ratio was found to be inversely proportional to the plasma AA. Further, this ratio has been claimed to be a better indicator of overall reducing atmosphere (i.e., profile of vitamin C) of the body.

Ascorbic Acid↗