Biomedical subjects

I Neverova

Publications and source records attributed to I Neverova.

4 recordsLinked to original sources

PubMed2001

Proteomic analysis of pharmacologically preconditioned cardiomyocytes reveals novel phosphorylation of myosin light chain 1.

Proteomic analysis of rabbit ventricular myocytes revealed a novel posttranslational modification to myosin light chain 1 (MLC1), consisting of phosphorylation at two sites. Subproteomic extraction to isolate myofilament-enriched fractions enabled determination of the extent of phosphorylation, which increased from 25.7+/-1.6% to 34.0+/-2.7% (mean+/-SE, n=4; P<0.05) after adenosine treatment at levels sufficient to pharmacologically precondition the myocytes (100 micromol/L). Mass spectrometry of MLC1 tryptic digests identified two peptide fragments modified by phosphorylation. These two phosphopeptides were characterized by peptide mass fingerprinting to determine the phosphorylation sites within rabbit ventricular MLC1, which correspond to Thr69 and Ser200 of rat MLC1, and to Thr64 and Ser194 or 195 of human MLC1. This proteomic analysis of preconditioned myocardium has revealed a previously unsuspected in vivo posttranslational modification to MLC1.

Adenosine↗

PubMed2001

Cardiovascular proteomics: evolution and potential.

The development of proteomics is a timely one for cardiovascular research. Analyses at the organ, subcellular, and molecular levels have revealed dynamic, complex, and subtle intracellular processes associated with heart and vascular disease. The power and flexibility of proteomic analyses, which facilitate protein separation, identification, and characterization, should hasten our understanding of these processes at the protein level. Properly applied, proteomics provides researchers with cellular protein "inventories" at specific moments in time, making it ideal for documenting protein modification due to a particular disease, condition, or treatment. This is accomplished through the establishment of species- and tissue-specific protein databases, providing a foundation for subsequent proteomic studies. Evolution of proteomic techniques has permitted more thorough investigation into molecular mechanisms underlying cardiovascular disease, facilitating identification not only of modified proteins but also of the nature of their modification. Continued development should lead to functional proteomic studies, in which identification of protein modification, in conjunction with functional data from established biochemical and physiological methods, has the ability to further our understanding of the interplay between proteome change and cardiovascular disease.

Animals↗

PubMed1997

Optimization of the isolation and effective use of mRNA from rat mast cells.

To define the molecular regulation of mast cell phenotype and function optimized procedures must be available to study mRNA from mast cells freshly isolated from tissues. However, rat peritoneal mast cells (PMC) contain large amounts of the proteoglycan heparin, and unfortunately, this molecule which is a potent inhibitor of reverse transcriptase (RT) and Taq polymerase and thus RT-PCR, copurifies with RNA. Here we describe an optimized protocol for extracting and amplifying RNA from rat PMC. Mast cells were isolated from rat peritoneum and a method modified from that of Chomczynski and Sacchi (1987) was used to extract the RNA. Following the removal of heparin by heparinase digestion, first strand cDNA synthesis was primed with oligo-dT and the resulting cDNA was quantified by rapid paper chromatography. The use of a detection system for the reverse transcription reaction ensured that the production of cDNA had occurred and allowed subsequent PCR testing to be optimal. cDNA thus produced can be used to detect relatively specific (histidine decarboxylase) and non-specific (beta-actin) mast cell products. Our PCR studies have shown a 300-fold increase in sensitivity over RNA processed by other methods.

Animals↗

PubMed1994

A spectrophotometric assay for glucosidase I.

A spectrophotometric assay for glucosidase I using the synthetic trisaccharide alpha-D-Glc 1-->2 alpha-D-Glc 1-->3 alpha-D-Glc-O(CH2)8COOCH3 is reported. The terminal glucose is released from the substrate by the enzyme and quantitated using glucose oxidase, peroxidase, and o-dianisidine. The trisaccharide is specific for glucosidase I and provides all the necessary structural features for correct interaction in the enzyme active site. The utility of the assay for monitoring enzyme activity during isolation and for use in kinetic and inhibition studies (i.e., with 1-deoxynorjirimycin) is demonstrated.

1-Deoxynojirimycin↗