P. R. Gupta

Calreticulin transacetylase catalyzed modification of the TNF-α mediated pathway in the human peripheral blood mononuclear cells by polyphenolic acetates

Polyphenols, coumarin (1,2-benzopyrone) and chromone (1,4-benzopyrone), are naturally occurring constituent of variety of plant species. They have attracted immense interest because of their diverse pharmacological activities. Not much was known about biological activities of acetyl derivative (polyphenolic acetates) of parent polyphenols. In previous investigations, we have conclusively established calreticulin transacetylase catalyzed activation of endothelial nitric oxide synthase (eNOS) by polyphenolic acetates. In the present work, calreticulin transacetylase of human peripheral blood mononuclear cells was characterized with respect to specificity for various polyphenolic acetates and its role in the activation of TNF-alpha induced nitric oxide synthase (iNOS). Peripheral blood mononuclear cells incubated with a model polyphenolic acetate, 7,8-diacetoxy-4-methylcoumarin (DAMC), along with L-arginine caused activation of NOS. The incubation of peripheral blood mononuclear cells with TNF-alpha and DAMC resulted in increased production of NO as compared to TNF-alpha alone. This increased NO production was attenuated by l-Nomega-nitro-L-arginine methyl ester (L-NAME), a well known non-specific inhibitor of NOS, and 1400W (N-[3-(aminomethyl) benzyl] acetamidine), a specific inhibitor of human iNOS. These results substantiate the CRTAase catalyzed activation of iNOS. Further, expression of NOS isoforms by semi-quantitative PCR and real-time RT-PCR confirms the preponderance of iNOS in TNF-alpha treated peripheral blood mononuclear cells over the untreated one. It was also observed that polyphenolic acetates inhibit TNF-alpha mediated release of IL-6 from peripheral blood mononuclear cells.

Changes in Protein Profile of Erythrocyte Membrane in Bronchial Asthma

Objectives. Erythrocyte membrane proteins reflect the prototype of multifunctional proteins of various erythroid and non-erythroid cells, which demonstrate various cellular functions. The protein profile of cells changes in various diseases. Therefore, the objective of this study was to understand the changes in protein profile of erythrocyte membranes in bronchial asthma. Methods. The study included 20 patients of bronchial asthma and 20 healthy subjects. Erythrocytes were isolated from peripheral blood, membranes were prepared followed by the determination of protein contents, and protein profile was assessed using SDS-PAGE. Results. In bronchial asthma, the protein contents of erythrocyte membranes in asthmatic patients were significantly higher (p < .005) than in healthy controls. Analysis of protein profile showed absence of the proteins, namely, band 4.2 and adducin subunit-II, and appearance of protein bands of molecular weights corresponding to galectin-3, glyceraldehyde 3-phosphate dehydrogenase, β-actin, dematin, band 4.1, and adducin (subunit-I) in asthmatic patients when compared with healthy controls. Conclusions. In asthma, there are quantitative and qualitative changes in proteins of erythrocyte membranes. The absence of band 4.2 protein may cause impairment of the erythrocyte membrane integrity, and presence of galectin-3 may lead to the activation of various inflammatory cells. The altered protein profile may possibly lead to altered response of the inflammatory cells to the asthmogenic stimuli, which may be responsible for pathophysiology and manifestation of the symptoms of bronchial asthma.

Sphingomyelin metabolism in erythrocyte membrane in asthma

Background. Sphingomyelin (SM), a major lipid constituent of outer leaflet of plasma membranes, with cholesterol, constitutes microdomains, which are termed as lipid rafts. These rafts provide support to proteins, receptors, enzymes, and so on and organize and orient them to conduct cellular functions including transmembrane signaling to substances in external milieu. The SM contents are regulated by its metabolism, changes in which may affect the composition of lipid rafts and cell response to the triggers of asthma which may lead to the pathophysiology. For studying changes in membranes, erythrocytes, which contain lipid rafts, are considered to be the best cell type. Hence, this study was conducted on plasma membrane of erythrocytes of asthmatic patients. Objective. The objective is to understand the changes in SM metabolism in asthma. Methods. The study included 50 subjects (25 asthmatics and 25 healthy subjects). Erythrocytes were isolated from the peripheral blood and membrane prepared. This was followed by determination of total cholesterol, phospholipids, SM, and sphingomyelinase activity. P < .05 was considered significant. Results and conclusions In asthmatics, there was a significant decrease in cholesterol contents (p < .05), decrease in total phospholipid contents (p < .005), increase in SM (p < .01), decrease in cholesterol: SM ratio (p < .001) and increase in sphingomyelinase activity (p < .001) in erythrocyte membranes. We conclude that in asthma, the increase in SM contents is associated with increased sphingomyelinase activity which shows an imbalance in SM metabolism, directed toward its accumulation. The ratio of cholesterol to SM, critical for maintenance of lipid rafts, was significantly lower in asthmatics. This indicates changes in structure of lipid rafts which may lead to the pathophysiology and development of asthma. Regulation of SM metabolism may help in disease regulation and its control.

Comparison of protein acetyltransferase action of CRTAase with the prototypes of HAT.

Our laboratory is credited for the discovery of enzymatic acetylation of protein, a phenomenon unknown till we identified an enzyme termed acetoxy drug: protein transacetylase (TAase), catalyzing the transfer of acetyl group from polyphenolic acetates to receptor proteins (RP). Later, TAase was identified as calreticulin (CR), an endoplasmic reticulum luminal protein. CR was termed calreticulin transacetylase (CRTAase). Our persistent study revealed that CR like other families of histone acetyltransferases (HATs) such as p300, Rtt109, PCAF, and ESA1, undergoes autoacetylation. The autoacetylated CR was characterized as a stable intermediate in CRTAase catalyzed protein acetylation, and similar was the case with ESA1. The autoacetylation of CR like that of HATs was found to enhance protein-protein interaction. CR like HAT-1, CBP, and p300 mediated the acylation of RP utilizing acetyl CoA and propionyl CoA as the substrates. The similarities between CRTAase and HATs in mediating protein acylation are highlighted in this review.

Application of Rough Sets in diagnosis of the depressive state of mind

Rough Set Theory is an emerging rule based soft computing methodology that employs approximations of crisp concepts. It has been used widely for knowledge discovery in real life data-centric applications that typically include uncertain or incomplete data. This paper describes an application of rough sets in identifying depressive episodes in the field of psychiatry. The core concepts of rough sets such as Reduct and Core are used to reduce the number of descriptive attributes based on their relative significance. The reduced information system yields a compact set of high-strength rules that identify the state of mind of a person to categorize new patients with high accuracy. We illustrate how Rough Sets can find symbolic and easily readable rules that could be used fruitfully by psychiatrists for clinical diagnosis.