Biomedical subjects

G Gupta

Publications and source records attributed to G Gupta.

At least 91 records · Page 5Linked to original sources

PubMed1998

Contribution of the CXC chemokines IP-10 and Mig to the antitumor effects of IL-12.

The mechanisms by which interleukin-12 (IL-12) exerts antitumor effects have been difficult to dissect. In this study, we examined the potential contribution of the chemokines interferon-gamma-inducible protein-10 (IP-10) and Mig to the antitumor effects of IL-12. Using an athymic mouse model, local inoculations with IL-12 consistently produced tumor size reductions associated with characteristic tumor necrosis and vascular damage. These effects were indistinguishable from those produced by IP-10 or Mig injected locally in the same tumor model. Local and systemic treatment with IL-12 was associated with expression of the interferon-gamma (IFN-gamma), IP-10, and Mig genes and proteins in the tumor. Levels of IP-10 and Mig expression in the tumor, the liver, and the kidney were inversely correlated with tumor size. Administration in vivo of neutralizing antibodies to IP-10 and Mig reduced substantially the antitumor effects of IL-12 inoculated locally into the tumors. These results support the notion that IP-10 and Mig contribute to the antitumor effects of IL-12 through their inhibitory effects on tumor vasculature.

Animals↗

PubMed1998

Fragile X DNA triplet repeats, (GCC)n, form hairpins with single hydrogen-bonded cytosine.cytosine mispairs at the CpG sites: isotope-edited nuclear magnetic resonance spectroscopy on (GCC)n with selective 15N4-labeled cytosine bases.

Here, we provide a direct proof that the formation of hairpins by (GCC)n at the 5'-UTR of the FMR-1 gene offers a mechanism for CpG hypermethylation associated with the fragile X syndrome. For this, we have performed hetero-nuclear (15N-1H) magnetic resonance spectroscopy to probe the structure of the CpG sites in the (GCC)n hairpins that are 15N-labeled at the amino (N4) groups of specific cytosine bases. Analyses of chemical shift, pH-induced chemical exchange, and NOE pattern of the (15N-labeled) amino protons of cytosine bases reveal that the cytosine bases at the CpG sites are intrahelical and well-stacked with the neighboring G.C base-pairs in the stem of these hairpins and probably form single hydrogen-bonded C.C mispairs. Measurements of pH-dependent 1H line-width also demonstrate that the C.C mispairs are more susceptible to open-closure than the G.C base-pairs. Thus, the Cs at the CpG sites of the (GCC)n hairpin are "flipped out" more easily to the activated state than those in the corresponding Watson-Crick duplex, (GCC)n. (GGC)n and this makes the hairpin a better target for methylation by the human methyltransferase, the enzyme that methylates the Cs at the CpG sites.

5' Untranslated Regions↗

PubMed1998

GAA instability in Friedreich's Ataxia shares a common, DNA-directed and intraallelic mechanism with other trinucleotide diseases.

We show that GAA instability in Friedreich's Ataxia is a DNA-directed mutation caused by improper DNA structure at the repeat region. Unlike CAG or CGG repeats, which form hairpins, GAA repeats form a YRY triple helix containing non-Watson-Crick pairs. As with hairpins, triplex mediates intergenerational instability in 96% of transmissions. In families with Friedreich's Ataxia, the only recessive trinucleotide disease, GAA instability is not a function of the number of long alleles, ruling out homologous recombination or gene conversion as a major mechanism. The similarity of mutation pattern among triple repeat-related diseases indicates that all trinucleotide instability occurs by a common, intraallelic mechanism that depends on DNA structure. Secondary structure mediates instability by creating strong polymerase pause sites at or within the repeats, facilitating slippage or sister chromatid exchange.

Alleles↗

PubMed1998

Solution structures of the Huntington's disease DNA triplets, (CAG)n.

Highly polymorphic DNA triplet repeats, (CAG)n, are located inside the first exon of the Huntington's disease gene. Inordinate expansion of this repeat is correlated with the onset and progression of the disease. NMR spectroscopy, gel electrophoresis, digestion by single-strand specific P1 enzyme, and in vitro replication assay have been used to investigate the structural basis of (CAG)n expansion. Nondenaturing gel electrophoresis and 1D 1H NMR studies of (CAG)5 and (CAG)6 reveal the presence of hairpins and mismatched duplexes as the major and minor populations respectively. However, at high DNA concentrations (i.e., 1.0-2.0 mM that is typically required for 2D NMR experiments) both (CAG)5 and (CAG)6 exist predominantly in mismatched duplex forms. Mismatched duplex structures of (CAG)5 and (CAG)6 are useful, because they adequately model the stem of the biologically relevant hairpins formed by (CAG)n. We, therefore, performed detailed NMR spectroscopic studies on the duplexes of (CAG)5 and (CAG)6. We also studied a model duplex, (CGCAGCG)2 that contains the underlined building block of the duplex. This duplex shows the following structural characteristics: (i) all the nucleotides are in (C2'-endo, anti) conformations, (ii) mismatched A x A base pairs are flanked by two Watson-Crick G x C base pairs and (iii) A x A base pairs are stably stacked (and intra-helical) and are formed by a single N6-H--N1 hydrogen bond. The nature of A x A pairing is confirmed by temperature-dependent HMQC and HMQC-NOESY experiments on the [(CA*G)5]2 duplex where the adenines are 15N-labeled at N6. Temperature- and pH-dependent imino proton spectra, nondenaturing electrophoresis, and P1 digestion data demonstrate that under a wide range of solution conditions longer (CAG)n repeats (n> or =10) exist exclusively in hairpin conformation with two single-stranded loops. Finally, an in vitro replication assay with (CAG)8,21 inserts in the M13 single-stranded DNA templates shows a replication bypass for the (CAG)21 insert but not for the (CAG)8 insert in the template. This demonstrates that for a sufficiently long insert (n=21 in this case), a hairpin is formed by the (CAG)n even in presence of its complementary strand. This observation implies that the formation of hairpin by the (CAG)n may cause slippage during replication and thus may explain the observed length polymorphism.

DNA↗

PubMed1998

Hairpin induced slippage and hyper-methylation of the fragile X DNA triplets.

The fragile X triplet repeats, (GCC)n x (GGC)n are located at the 5' untranslated region of the FMR-1 gene. Inordinate repeat expansion and hyper-methylation of the CpG islands inside the repeat lead to the suppression of the FMR-1 gene and the subsequent onset and progression of the disease. Previously, we have shown that the (GCC)n strand of the fragile X repeat readily forms hairpin structures under physiological conditions (Chen et al., Proc. Natl. Acad. Sci. USA, 92:5199-5203, 1995: Mariappan et al., Nucl. Acid Res. 24:784-792, 1996). Here, we show by an in vitro assay that formation of the (GCC)n hairpins leads to slippage during replication. The slippage structure is a three-way junction with two Watson-Crick, (GCC)n x (GGC)n, arms and a third (GCC)n hairpin arm. Formation of such slippage structures during replication may explain the observed length polymorphism of the fragile X repeat. We have also studied the substrate efficiency of these three-way junctions toward the human methyltransferase. the enzyme that methylates the CpG sites in DNA. These methylation studies show that the slippage structures induced by the (GCC)n hairpins are 10-15 times more efficient substrates than either the corresponding Watson-Crick duplexes or the (GCC)n hairpins. We demonstrate by appropriate designs that the exceptional substrate efficiency of the three-way junction slippage structures is due to two factors: (i) the presence of the (GCC)n hairpin in which CpG sites are more accessible for methylation than the CpG sites in the Watson-Crick duplex and (ii) the ability of the (GCC)n hairpin in these three-way junctions to move along the Watson-Crick arms that facilitates conversion of low-affinity Watson-Crick CpG sites into high-affinity hairpin CpG sites. Therefore, we suggest that the formation of the (GCC)n hairpins during replication can explain both length polymorphism and hyper-methylation of the fragile X repeats.

Base Sequence↗

PubMed1998

Bioaccumulation and biosorption of lead by poultry litter microorganisms.

Microorganisms are known to interact with metals through a number of mechanisms, including binding the metals to their cells' walls and intracellular accumulation. Poultry litter has a high density of various microorganisms along with many nutrients. The objective of this research was to study the removal of Pb from an aqueous solution by the microorganisms found in poultry litter under an aerobic environment. A Pb(NO3)2 solution was treated with the aqueous extract of either the nonradiated litter or gamma-radiated litter in order to differentiate between the removal of Pb through bioaccumulation (by the living organisms) and biosorption (by the dead organisms). Lead removal was measured using anodic stripping voltammetry. Both the nonradiated and the gamma-radiated litter removed significant amounts of Pb. After 14 d, the amount of Pb removed through bioaccumulation by the nonradiated litter and through biosorption by the radiated litter was about 44 and 30%, respectively. There was no increase in Pb removal between 14 and 60 d. The biomass density of the aqueous extract of poultry litter increased significantly in the presence of 400 ppb Pb(NO3)2.

Absorption↗

PubMed1998

Lipid metabolising enzymes in isolated rat testicular germ cells and changes associated with meiosis.

To assess the lipid metabolising potential of testicular germ cells undergoing meiosis, spermatocytes and spermatids were isolated from adult rat testis and purified by centrifugal elutriation followed by density gradient centrifugation. Seven key enzymes of lipid metabolism (namely beta-hydroxybutyrate dehydrogenase, carnitine acetyl transferase, ATP citrate lyase, hydroxyacyl-CoA dehydrogenase, glycerol 3-phosphate dehydrogenase, acetyl-CoA carboxylase and long chain acyl-CoA synthetase) were assayed in cell homogenates. The results indicated that germ cells possess the key enzymes for de novo synthesis and oxidation of fatty acids. The significant increase in activities of anabolic enzymes and decrease in activities of catabolic enzymes in post-meiotic germ cells indicated a shift in lipid metabolism towards fatty acid synthesis during meiosis. Long chain acyl-CoA synthetase activity was not detected in the two cell types. The study indicates a major reorganization of fatty acid turnover during meiosis with equilibrium shifting in favour of synthesis.

3-Hydroxyacyl CoA Dehydrogenases↗

PubMed1998

Genetic mapping of soluble guanylyl cyclase genes: implications for linkage to blood pressure in the Dahl rat.

The nitric oxide (NO) signaling system, consisting of NO synthases, soluble guanylyl cyclase, and cGMP, plays a prominent role in salt handling and regulation of blood pressure. Soluble guanylyl cyclases are heme-containing heterodimers (alpha/beta). The alpha1/beta1 isoform has greater NO sensitivity than the alpha1/beta2. It has recently been shown that expression of the beta subunits is altered in the kidney of the Dahl salt-sensitive rat, ie, the beta1 subunit is decreased and the beta2 subunit increased. However, whether soluble guanylyl cyclase is linked to salt sensitivity is not known. In the present study, we investigated linkage of guanylyl cyclase genes to blood pressure. Alpha1 and beta1 gene loci for soluble guanylyl cyclase were mapped to rat chromosome 2, and the beta2 gene locus was mapped to rat chromosome 5 using fluorescent in situ metaphase hybridization. By use of a rat radiation hybrid panel, the gene loci were then further mapped with respect to known quantitative trait locus markers of salt-sensitive hypertension in the Dahl rat on chromosomes 2 and 5. Genes for alpha1 and beta1 were closely linked by two-point analysis to Na+,K+-ATPase alpha1 isoform (LOD of 15.1 and 14.0, respectively) and calmodulin-dependent protein kinase II-delta loci (LOD of 14.3 and 12.9, respectively), which have been previously shown to flank a quantitative trait locus for blood pressure in the Dahl rat. The alpha1 and beta1 genes were closely linked (LOD of 11.3; theta, 0.4). The beta2 gene locus was closely linked to the endothelin-2 (ET-2) locus (LOD of 13.0), which has been shown to cosegregate with blood pressure. We conclude that soluble guanylyl cyclase subunit loci, ie, alpha1, beta1, and beta2, are good candidates for genes controlling salt-sensitive hypertension in the Dahl rat.

Animals↗

PubMed1998

Changes in carbohydrate metabolism of testicular germ cells during meiosis in the rat.

A study was undertaken to estimate the activities of the key enzymes of glycolysis, the pentose phosphate pathway and the tricarboxylic acid (TCA) cycle in purified rat spermatocytes and spermatids, which have been shown to die in glucose-containing medium and require lactate/pyruvate for maintaining normal ATP concentrations. The aim was to elucidate the changes in the glycolytic and oxidative potential of germ cells undergoing meiosis. Pachytene spermatocytes and round spermatids from adult rat testis were purified to approximately 90% purity by trypsin digestion followed by a combination of centrifugal elutriation and Percoll density gradient centrifugation. After the purity and viability of these cells had been established, their contents of hexokinase, phosphofructokinase, lactate dehydrogenase (LDH) and LDH-X of glycolysis, glucose 6-phosphate dehydrogenase of the pentose phosphate pathway and citrate synthase, aconitase, malate dehydrogenase and 2-oxoglutarate dehydrogenase of the TCA cycle were estimated. These enzymes were also estimated in epididymal spermatozoa for comparison with the testicular germ cells. The results indicate greater activity of glycolytic and pentose phosphate pathway enzymes in spermatocytes than in spermatids, which exhibited greater activity of TCA cycle enzymes than the former. The difference in activity was statistically significant for most of the enzymes studied. In contrast, spermatozoa exhibited markedly greater activity of glycolytic enzymes and significantly lower activity of pentose phosphate pathway and TCA cycle enzymes than did the testicular germ cells. We conclude that the unusual dependence of spermatids exclusively on lactate may be due to their lower glycolytic potential, whereas spermatocytes with comparatively greater glycolytic activity have an intermediate dependence on lactate and are therefore able to utilise lactate, pyruvate, or both, while retaining a better ability to utilise glucose. Spermatozoa with the greatest glycolytic potential and the lowest TCA cycle activity appear to be 'programmed' to utilise exclusively glucose/fructose for energy.

Animals↗

PubMed1997

Cystosine-rich strands of the insulin minisatellite adopt hairpins with intercalated cytosine+.cytosine pairs.

Previously, we reported the high resolution NMR structure of the hairpin G-quartet structure formed by the G-rich strand of the insulin minisatellite of repeat sequence, (ACAG4TGTG4/TGTC4ACAC4) located upstream of the human insulin gene. Here, we report structural studies on the C-rich strand of this insulin minisatellite. First, we show by high resolution NMR that (C4TGTC4) forms a hairpin dimer with intercalated C+.C pairs (referred to as the hairpin i-motif); 340 NOE distance constraints uniquely define the nature of hairpin folding and the pattern of C+.C intercalation. Second, we show by one-dimensional NMR spectroscopy and molecular modeling studies that (C4TGTC4ACA4TGTC4) forms an intramolecularly folded hairpin with intercalated C+.C pairs. Third, we demonstrate by in vitro replication studies that several such hairpin i-motifs are present in long (C4TGTC4ACA)n (n>/=6) sequences, even in the presence of their complementary strands. Finally, we discuss structural and biological significance of the hairpin i-motifs formed by the C-rich strands of the insulin minisatellite.

Base Sequence↗

PubMed1997

The beta2 subunit inhibits stimulation of the alpha1/beta1 form of soluble guanylyl cyclase by nitric oxide. Potential relevance to regulation of blood pressure.

Cytosolic guanylyl cyclases (GTP pyrophosphate-lyase [cyclizing; EC 4.6.1.2]), primary receptors for nitric oxide (NO) generated by NO synthases, are obligate heterodimers consisting of an alpha and a beta subunit. The alpha1/beta1 form of guanylyl cyclase has the greatest activity and is considered the universal form. An isomer of the beta1 subunit, i.e., beta2, has been detected in the liver and kidney, however, its role is not known. In this study, we investigated the function of beta2. Immunoprecipitation experiments showed that the beta2 subunit forms a heterodimer with the alpha1 subunit. NO-stimulated cGMP formation in COS 7 cells cotransfected with the alpha1 and beta2 subunits was approximately 1/3 of that when alpha1 and beta1 subunits were cotransfected. The beta2 subunit inhibited NO-stimulated activity of the alpha1/beta1 form of guanylyl cyclase and NO-stimulated cGMP formation in cultured smooth muscle cells. Our results provide the first evidence that the beta2 subunit can regulate NO sensitivity of the alpha1/beta1 form of guanylyl cyclase. Northern analysis for guanylyl cyclase subunits was performed on RNA from kidneys of Dahl salt-sensitive rats, which have been shown to have decreased renal sensitivity to NO. Compared to the Dahl salt-resistant rat, message for beta2 was increased, beta1 was decreased, and alpha1 was unchanged. These results suggest a molecular basis for decreased renal guanylyl cyclase activity, i.e. , an increase in the alpha1/beta2 heterodimer, and decrease in the alpha1/beta1 heterodimer.

Animals↗

PubMed1997

Role of the p53 tumor suppressor gene in the tumorigenicity of Burkitt's lymphoma cells.

Burkitt's lymphoma (BL) cell lines carry a translocated c-myc gene and, in 60-80% of cases, exhibit mutations in the p53 tumor suppressor gene. We examined the potential role of the p53 gene in BL tumorigenicity using an in vitro assay that measures p53-dependent cell cycle arrest in the G1 phase of the cell cycle and an in vivo athymic murine model that detects differences in the tumorigenicity of BL cell lines. A highly significant inverse correlation was found between the ability of BL cells to arrest in G1 after irradiation and their tumorigenicity in athymic mice, consistent with the notion that loss of p53 function is associated with increased tumorigenicity. Inactivation of wild-type (wt) p53 function by expression of the human papillomavirus E6 protein in the AG876V BL cell line, which carries both wt and mutant p53 proteins, rendered the cell line significantly more tumorigenic in athymic mice. Transfection of the wt p53 gene into the p53 mutant and highly tumorigenic BL-41 cell line caused it to acquire wt p53 function and rendered it less tumorigenic in mice. In addition to confirming a role for the loss of p53 function in tumor progression, the data demonstrate that wt p53 protein can reduce BL tumorigenicity in vivo.

Animals↗

PubMed1997

Mig, the monokine induced by interferon-gamma, promotes tumor necrosis in vivo.

Mig, the monokine induced by interferon-gamma, is a CXC chemokine active as a chemoattractant for activated T cells. Mig is related functionally to interferon-inducible protein 10 (IP-10), with which it shares a receptor, CXCR3. Previously, IP-10 was found to have antitumor activity in vivo. In the present study, murine Mig RNA was found to be expressed at higher levels in regressing Burkitt's lymphoma tumors established in nude mice compared with progressively growing tumors. Daily inoculations of purified recombinant human Mig into Burkitt's tumors growing subcutaneously in nude mice consistently caused tumor necrosis associated with extensive vascular damage. These effects were indistinguishable from those produced by intratumor inoculations of Burkitt's tumors with IP-10. These results support the notion that Mig, like IP-10, has antitumor activity in vivo.

Animals↗

PubMed1997

Effect of river and wetland sediments on toxicity of metolachlor.

Metolachlor is a preplant, preemergent herbicide applied to corn and soybean fields. Agricultural runoff after application can cause the herbicide to enter into natural waters. The objective of this study was to determine the effect of river and wetland sediments on the toxicity of metolachlor. Sediments from a river and a wetland (separately) were mixed with Ottawa sand (0, 25, 50, 75, and 100%). Metolachlor (in water) and sediment were mixed in an orbital shaker for 8 hr; the mixture was centrifuged, and the supernatant liquid was tested for toxicity (EC50%) using the marine luminescent bacteria Vibrio fischeri (Microtox). The toxicity (EC50%) of metolachlor with the river sediment was 64.61. Metolachlor after interaction with the wetland sediment demonstrated no toxicity possibly due to increased adsorption on the higher amount of organic matter (10 times) and clay (3.5 times) present in the wetland sediment than the river sediment.

Acetamides↗

PubMed1997

Expression and purification of soluble, active heterodimeric guanylyl cyclase from baculovirus.

A method for expression and purification of active cytosolic heterodimeric histidine (His)-tagged guanylyl cyclase of the alpha 1/beta 1 isoform has been developed using recombinant baculovirus-transfected insect cells. Confirmation of expression of active cyclase was obtained by both Western analysis and enzymatic activity. A His tag on the COOH-terminus of the alpha 1 and beta 1 subunits allowed rapid purification of the heterodimeric form of guanylyl cyclase in a single affinity step using a nickel column. A second gel-filtration step was applied to reconstitute into the complex heme, a required cofactor. This was confirmed spectroscopically by absorbance in the Soret region. Like enzyme purified from tissue, the activity of recombinant guanylyl cyclase was increased by protoporphyrin IX and inhibited by both Zn- and Sn-protoporphyrin. The method described here should provide a general approach for the expression and purification of alternate forms of cytosolic guanylyl cyclase and facilitate mechanistic and structural studies of this important family of enzymes. Furthermore, the procedure demonstrates the utility of the His-tag system to purify multimeric proteins.

Animals↗

PubMed1997

Effect of antispermatogenic compound CDRI-84/35 on marker enzymes of rat testis cells. A study on site of action.

Marker enzymes of Sertoli and germ cells were estimated to study the mechanism of action of antispermatogenic compound CDRI 84/35 in adult male rat testis. Animals were killed after 22, 41, and 64 days of treatment with antispermatogenic dose of CDRI 84/35 in order to evaluate the effect of the compound on spermatid, spermatocyte, and spermatogonial stages, respectively. Studies were also extended to a recovery period of 90 days. Results indicate a direction action of the compound on germ cells, with no apparent effect on Sertoli cells. Studies also show a massive depletion of postmeiotic germ cells after the treatment, with some damage to premeiotic germ cells as well. Reversibility of the compound was partial, with the marker enzymes of pre- and postmeiotic germ cells not being restored to control levels after withdrawal of treatment.

Animals↗

PubMed1997

Surveillance of colonization and infection with Staphylococcus aureus susceptible or resistant to methicillin in a community skilled-nursing facility.

BACKGROUND: Methicillin-resistant Staphylococcus aureus (MRSA) is an important nosocomial pathogen in acute care hospitals and long-term care facilities. Few studies have been reported in private skilled nursing facilities (SNFs) not experiencing outbreaks of infections caused by MRSA. METHODS: From a 149-bed SNF with no outbreaks, we report a 1-year prospective surveillance study of S. aureus colonization and infection, with focus on S. aureus phenotypes, both methicillin susceptible (MS) and methicillin resistant (MR). Nasal and stool or rectal screening cultures were done on admission, and all patients underwent screening on at least a quarterly basis for 1 year. RESULTS: Overall, 35% of patients were colonized at least once with S. aureus, (72% MS, 25% MR, and 3% mixed phenotypes), 94% of the MRSA were ciprofloxacin resistant. Nasal colonization with any S. aureus was more frequent, but 13% of patients had positive results only in rectal specimens. Twenty-one percent of the newly admitted and 15% of continuing patients acquired colonization during their stay in the SNE Colonization was transient or persistent, persisted longer in the nares compared with colonization in rectal specimens, and was more stable for methicillin-susceptible S. aureus. Nine percent of patients had development of infection with S. aureus. There was no indication that MRSA colonization led to more infections than methicillin-susceptible S. aureus. Of the 13 infected patients in whom cultures had previously been obtained, seven (54%) had been colonized by the same phenotype strains. CONCLUSIONS: In this private SNF, endemic S. aureus infections occur at a low frequency, reflecting a moderate level of colonization with S. aureus. However, a trend showing gradual increases in frequencies of colonization and infection is of concern and suggests that in this SNF, future intervention could become warranted.

Aged↗

PubMed1997

Novel 29 kDa heparin-binding lectin from human foetal brain.

Heparin inhibitable lectins are physiologically important because of their interactions with extracellular matrix and with other cell surface glycoconjugates. However, due to the unstable nature of these animal lectins, it becomes necessary to purify them in the shortest possible time. In the present study, a chromatographic procedure was developed to separate heparin inhibitable lectin activity. Lectin activities from human foetal brain were separated on a Q-Sepharose column employing different equilibration conditions. When proteins were loaded on to a phosphate-buffered saline (PBS) equilibrated column and eluted with salt gradient, only one lectin peak was obtained. However, when proteins were loaded on to a hypotonic equilibrated column and eluted with a salt gradient, four lectin peaks were obtained. The lectin peak obtained from the PBS equilibrated column was characterized as heparin inhibitable lectin. On SDS-PAGE analysis, it gave a single band of 29 kDa. For optimum lectin activity, a pH of around 7.0 was required. Lectin activity was stimulated by Mn++; amino acid composition was different from other known lectins. The lectin was particularly rich in acidic amino acids. Regional distribution of 29 kDa lectin in different foetal brain regions gave the highest content in the cerebral cortex, showing a caudoroastral distribution. Determination of the subcellular distribution of the lectin in the foetal cerebral cortex gave the highest value with a mitochondrial fraction.

Animals↗