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Biomedical subjects

M Gupta

Publications and source records attributed to M Gupta.

At least 253 records · Page 14Linked to original sources
PubMed1995

Eukaryotic DNA topoisomerases mediated DNA cleavage induced by a new inhibitor: NSC 665517.

A compound with a novel structure, NSC 665517, was tested in the National Cancer Institute Preclinical Drug Discovery Screen. With the COMPARE algorithm, the pattern of differential cytotoxicity for NSC 665517 most closely resembled those of known topoisomerase II (top2) inhibitors. In vitro data showed that NSC 665517 induced DNA cleavage in the presence of top2 and topoisomerase I (top1) (at a higher concentration). The minimum concentration required to induce top2 cleavage was 0.5 microM. A substantial decrease in top2-induced cleavage by NSC 665517 was seen when the reaction mixtures were shifted to elevated temperature (55 degrees), suggesting that top2-induced cleavage occurs through the mechanism of stabilizing the reversible enzyme/DNA complex and inhibiting religation. The DNA cleavage pattern induced by NSC 665517 with top2 was different than that of other known top2 inhibitors, including etoposide, mitoxantrone, anthracyclines, amsacrine, and ellipticine. top2 cleavage sites induced by NSC 665517 showed strong preference for G located 3' to the top2-mediated DNA cleavage (position +1). NSC 665517 produced limited DNA unwinding at high drug concentration. DNA damage analyzed in KB cells by alkaline elution showed that NSC 665517 induced strand break. Data from the cytotoxicity in KB-V1 overexpressing P-glycoprotein and COMPARE analysis with rhodamine efflux assay indicated that NSC 665517 is a substrate of P-glycoprotein. These results strongly suggest that NSC 665517 is a novel topoisomerase-targeted drug. Preclinical evaluation of NSC 665517 as an antitumor agent is under way.

Antineoplastic Agents↗
PubMed1995

Influence of Ca2+ on microsomal lipid peroxidation.

Lipid peroxidation in microsomes prepared from liver of mice was initiated by NADPH, ascorbic acid and ferrous ions. The presence of Ca2+ modulated the lipid peroxidation in all these three systems. The mode and magnitude depend on the system and concentration of cofactors used for initiation of lipid peroxidation. In ascorbate system, Ca2+ enhanced the lipid peroxidation up to 30 microM concentration of ascorbic acid and beyond 30 microM concentration it inhibited. Ca2+ increased NADPH-dependent lipid peroxidation at all concentrations. Depending on concentration of Fe2+, lipid peroxidation was either decreased or increased in presence of Ca2+. It suggested that the in vitro findings may be cautiously extrapolated to the animal systems. In absence of cofactors, Ca2+ enhanced lipid peroxidation. EGTA inhibited Ca2+-enhanced lipid peroxidation. However in presence of ionophore A23187, Ca2+ potentiated lipid peroxidation. Since Ca2+ has a closed-shell electronic state and lacks electronic transitions, it may not participate directly in lipid peroxidation process. The effect of Ca2+ on lipid peroxidation may be through some biochemical processes or its interactions with membranes leading to various changes in their characteristics.

Animals↗
PubMed1995

Procoagulant activity after exposure of monocyte-derived macrophages to minimally oxidized low density lipoprotein. Co-localization of tissue factor antigen and nascent fibrin fibers at the cell surface.

The role of tissue factor (TF) as an initiator of the thrombotic complications secondary to atherosclerosis has been acknowledged, and in situ expression of TF activity by monocyte-derived macrophages and lesion-associated macrophage foam cells has been documented. Macrophages express TF activity upon exposure in vitro to either oxidized low density lipoprotein LDL (Ox-LDL) or endotoxin (lipopolysaccharide). This activity has been associated with membrane vesicles that apparently are shed after procoagulant expression. The present study based upon the correlative use of an enzyme-linked coagulant assay and three-dimensional multi-antigen, immunogold electron microscopy, reports the ultrastructural localization of TF antigen and spatially correlates TF with OX-LDL binding and the presence of nascent fibrin polymers on the plasma membrane of cultured macrophages. Pigeon monocyte/macrophages, after a 4-hour induction with lipopolysaccharide (2 micrograms/ml) or minimally oxidized LDL (50 micrograms/ml; thiobarbituric acid reducing substance, 5 to 8 nmol/mg protein) were incubated for 40 minutes in a Tris-buffered medium containing factors VII, V, X, II, and I before either assaying for coagulant activity or processing for gold-colloid cytochemistry. TF activity, as measured by enzyme-linked coagulant assay peaked 6 hours after agonist exposure with lipopolysaccharide and Ox-LDL giving, respectively, 115- and 60-fold stimulation as compared with control. This activity corresponded to the elaboration of membrane ruffles and microvilli on the cell surfaces. Through correlative immunogold cytochemistry (15-nm-diameter colloid) and gold-ligand cytochemistry (30-nm-diameter colloid), TF antigen (83%) and Ox-LDL (78%) were primarily associated with the membrane ruffles and microvilli. Multi-antigen immunogold cytochemistry when used in conjunction with ligand-gold cytochemistry documented co-localization of Ox-LDL (22-nm gold), TF antigen (15-nm gold) and a delicate three-dimensional network of short fibrin fibers that were decorated in a linear fashion with the immunogold probes (30-nm gold). These results provide evidence that TF antigen is located at selected regions on the cell surfaces. Furthermore, these same regions provide binding sites for agonist uptake and organization sites for fibrin polymerization. Hypothetically, the localized membrane regions could be shed from the cell surface as a means for regulating coagulation potential.

Animals↗
PubMed1995

The effect of newer generation lithotripsy upon renal function assessed by nuclear scintigraphy.

PURPOSE: We studied the effect of second generation lithotripsy on renal function. MATERIALS AND METHODS: We evaluated 42 patients with unilateral renal calculi by nuclear renography, serum creatinine levels, renal ultrasonography and plain radiographs. RESULTS: There was no significant change in glomerular filtration rate at 1 or 3 months. Split function of the treated kidneys was lower at 1 month (mean 47.2%, p = 0.01) and 3 months (47.3%, p = 0.01) than before treatment (49.1%). A greater than 5% decrease in split function of the treated kidney occurred at 1 month in 6 patients (16.2%) and at 3 months in 3. Of the patients 23 (62.2%) were stone-free and 11 had residual fragments less than 4 mm., with a 19% retreatment rate for an overall success rate of 91.9%. CONCLUSIONS: Newer generation lithotriptors may limit renal damage while permitting satisfactory treatment of renal calculi.

Adult↗
PubMed1995

Normal parameters of ventricular system in healthy infants.

Six hundred healthy inborn newborns and infants upto the age of 18 months were studied. Cranial sonography was performed by real time 2D scanner with 5 MHz transducer and images were obtained through anterior fontanelle and temporo-squamal suture. Various parameters related to ventricular system were measured for different ages till the fontanelle remained open. These values will prove useful for diagnosing hydrocephalus at an early stage of the disease and also to find out the blocks at various levels in ventricular system.

Age Factors↗
PubMed1995

Ultrasonic diagnosis of intracranial hemorrhage in high risk neonates.

One hundred and eleven high risk neonates were subjected to (cranial ultrasound (CR-USG) Caranial sonography was performed by 2D realtime scanner with 5 MHz transducer through anterior and posterior fontanelle and temporo-squamal suture. One quarter of these neonates developed intracranial hemorrhage (ICH) within 120 hours of birth. Of them 42.8% neonates recovered completely, 21.4% developed ventriculomegaly, 21.4% neonates expired, 10.4% developed pseudo-porencephalic cysts and 3.5% developed aqueductal block. It is concluded that CR-USG is a useful technique for detection and monitoring of complications of ICH and at least one screening sonogram is essential in the first week of life of all high risk neonates.

Cerebral Hemorrhage↗
PubMed1994

DNA topoisomerases I & II cleavage sites in the type 1 human immunodeficiency virus (HIV-1) DNA promoter region.

Topoisomerase sites were mapped in the 5'-long terminal repeat of HIV-1 DNA by agarose and sequencing gel electrophoresis. Topoisomerase II sites were observed in the absence and presence of teniposide and amsacrine in the transcription initiation region and the TATA box, consistent with a possible role of topoisomerase II in transcription. The NF-kB and Sp1 regions were poorly cleaved. Topoisomerase I sites were relatively unfrequent even in the presence of camptothecin. They were absent in the core promoter and were concentrated in the TAR and the upstream region near the junction with the host DNA.

Amsacrine↗
PubMed1994

An E-box/M-CAT hybrid motif and cognate binding protein(s) regulate the basal muscle-specific and cAMP-inducible expression of the rat cardiac alpha-myosin heavy chain gene.

Expression of the cardiac myosin heavy chain (MHC) genes is regulated developmentally and by numerous epigenetic factors. Here we report the identification of a cis-regulatory element and cognate nuclear binding protein(s) responsible for cAMP-induced expression of the rat cardiac alpha-MHC gene. By Northern blot analysis, we found that, in primary cultures of fetal rat heart myocytes, the elevation of intracellular levels of cAMP results in up-regulation of alpha-MHC and down-regulation of beta-MHC mRNA expression. This effect of cAMP was dependent upon the basal level of expression of both MHC transcripts and was sensitive to cycloheximide. In transient expression analysis employing a series of alpha-MHC/CAT constructs, we identified a 31-base pair fragment located in the immediate upstream region (-71 to -40), which confers both muscle-specific and cAMP-inducible expression of the gene. Within this 31-base pair fragment there are two regions, an AT-rich portion and a hybrid motif which contains overlapping sequences of E-box and M-CAT binding sites (GGCACGTGGAATG). By substitution mutation analysis, both elements were found important for the basal muscle-specific expression; however, the cAMP-inducible expression of the gene is conferred only by the E-box/M-CAT hybrid motif (EM element). Using mobility gel shift competition assay, immunoblotting, and UV-cross-linking analyses, we found that a protein binding to the EM element is indistinguishable from the transcription enhancer factor-1 (TEF-1) in terms of sequence recognition, molecular mass, and immunoreactivity. Methylation interference and point mutation analyses indicate that, besides M-CAT sequences, center CG dinucleotides of the E-box motif CACGTG are essential for protein binding to the EM element and for its functional activity. Furthermore, our data also show that, in addition to TEF-1, another HF-1a-related factor may be recognized by the alpha-MHC gene EM element. These results are first to demonstrate transcriptional activation of a sarcomeric gene by cAMP and support the role of TEF-1 and HF-1a-like factors in the regulation of alpha-MHC gene expression in cardiac myocytes.

Animals↗
PubMed1994

Methylphosphonodiester substitution near the conserved CA dinucleotide in the HIV LTR alters both extent of 3'-processing and choice of nucleophile by HIV-1 integrase.

We present evidence suggesting that the 3'-processing activity of HIV-1 integrase is dramatically affected by electrostatic and/or steric perturbations 3' to the conserved CA dinucleotide. When the phosphodiester bond 3' to the scissile phosphodiester is replaced by a methylphosphonodiester linkage, 3'-processing decreases by two orders of magnitude. This block of cleavage can be somewhat overcome by increasing the pH of the reaction. Labeling of the substrates at the 3'-end revealed blockage of water and glycerol, but stimulation of the viral DNA 3'-hydroxyl, acting as the nucleophile with the methylphosphonodiester substrate. Interestingly, a circular trinucleotide was formed using the phosphodiester and methylphosphonodiester substrates when the terminal nucleotide was 3'-deoxyadenosine but not 2'-deoxyadenosine. Mutagenesis of the enzyme active site has previously been shown to alter the choice of nucleophile in the 3'-processing reaction. Taken together, the results in this study suggest that 'mutagenesis' of the DNA backbone can also alter the choice of nucleophile.

Base Sequence↗
PubMed1994

Inhibition of human immunodeficiency virus type 1 integrase by 3'-azido-3'-deoxythymidylate.

The effects of 3'-azido-3'-deoxythymidine (AZT) and three of its intracellular metabolites, azido- thymidine mono-, di-, and triphosphates, on the human immunodeficiency virus type 1 integrase have been determined. AZT mono-, di-, and triphosphate have an IC50 for integration between 110 and 150 microM, whereas AZT does not inhibit the integrase. The inhibition by AZT monophosphate can be partially reversed by coincubation with either thymidine monophosphate or 2',3'-dideoxythymidine monophosphate, suggesting that either of these monophosphates can bind to the integrase but that the azido group at the 3' position could be responsible for the inhibition. Integrase inhibition is associated with reduced enzyme-DNA binding but does not appear to be competitive with respect to the DNA substrate. Inhibition of an integrase deletion mutant containing only amino acids 50-212 suggests that these nucleotides bind in the catalytic core. Concentrations up to 1 mM AZT monophosphate can accumulate in vivo, indicating that integrase inhibition may contribute to the antiviral effects of AZT. The increasing incidence of AZT-resistant virus strains may, therefore, be associated with mutations not only in the reverse transcriptase but also in the human immunodeficiency virus integrase. Finally, these observations suggest that additional strategies for antiviral drug development could be based upon nucleotide analogs as inhibitors of human immunodeficiency virus integrase.

Base Sequence↗
PubMed1994

Mutations of vaccinia virus DNA topoisomerase I that stabilize the cleavage complex.

Two mutations in vaccinia virus topoisomerase I, K167D and G226N, have been characterized. SOS induction was observed in Escherichia coli expressing vaccinia topoisomerase I with either one of these mutations. The mutant enzymes were purified to homogeneity and compared with the wild type enzyme for relaxation activity and the partial activities of substrate binding, site-specific DNA cleavage and DNA religation to determine the mechanism of SOS induction. The K167D mutant enzyme had reduced binding affinity for the DNA substrate with a Kapp that was 10-fold higher than wild type. Nevertheless, in reactions with high enzyme concentration, its substrate cleavage activity was 90% that of wild type. The G226N mutant enzyme had virtually wild type binding and cleavage activities. However, intermolecular religation by these two mutants were observed to be significantly reduced. The cleavage complexes formed with the K167D and G226N mutants were more stable to high salt than the wild type cleavable complex. We propose that these mutants in vivo induce the SOS response in E. coli due to the shift of topoisomerase cleavage-religation equilibrium towards cleavage and increased stability of the cleavage complex. The mutation thus has a similar effect as the topoisomerase-targeting inhibitors that turn topoisomerases into DNA damaging agents.

Base Sequence↗
PubMed1994

Ultrastructural localization of tissue factor on monocyte-derived macrophages and macrophage foam cells associated with atherosclerotic lesions.

The expression of tissue factor (TF) antigen by circulating monocytes, cultured macrophages, and macrophages associated with atherosclerotic lesions was ultrastructurally analysed using immunogold labeling. A subpopulation of macrophages associated with the intimal surface overlying lesions had a significant TF expression. Macrophages and macrophage foam cells that projected from the intima into the arterial lumen also expressed a high level of TF (14-fold increase over control). In contrast, circulating monocytes and macrophages in culture did not express TF above background control levels. This TF expression by macrophages in vivo but not by macrophages cultured from either normal or hypercholesterolemic animals suggests that monocyte activation and macrophage transition, as measured by TF expression, is lesion-dependent and not stimulated solely by intimal attachment, surface migration, or hypercholesterolemia. These results further suggest that macrophages and foam cells associated with early lesions of atherosclerosis can initiate fibrin formation, which could contribute to lesion complications and transition to a fibromuscular stage.

Animals↗