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Biomedical subjects

R K Singhal

Publications and source records attributed to R K Singhal.

At least 19 recordsLinked to original sources
PubMed2005

The use of ultra filtration in trace metal speciation studies in sea water.

During this work, size fractionation technique "ultra filtration" is used in speciation studies of trace elements in the coastal sea water. Filtration is the most commonly used method to fractionate trace metal species, but often only "dissolved" and "particulate" fraction. The purpose of the present study is to determine colloidal and suspended particulate concentrations of Fe, Zn, Cu, Ni, and Mn in sea water. Suspended particulate matter were separated in three different size groups namely (>2.7 microm, <2.7->0.45 microm and <0.45->0.22 microm) by suction filtration using cellulose acetate and nitrate filter membranes. Thereafter to concentrate the solution with colloidal particle <0.22 microm-1.1 nm (0.5 k Nominal Molecular Weight cut-off Limit {NMWL}), the solution obtained from filtration through <0.22 microm, is sequentially passed through the ultra-filtration membranes having pore diameters of 14 nm (300 k NMWL), 3.1 nm (50 k NMWL), 2.2 nm (30 k NMWL), 1.6 nm (10 k NMWL) and 1.1 nm (0.5 k NMWL) by using Stirred Ultra-filtration Cells, operating in concentration mode. The concentration of Fe, Zn, Cu, Ni, and Mn were measured in suspended and dissolved fraction by ion chromatography, ICP-AES and Atomic Absorption Spectrometer. The salinity of the solution in various dissolved fractions of sequential filtration varies between 30.89-34.22 parts per thousand. The maximum concentrations of colloidal Zn, Cu, Ni and Mn in dissolved fraction were in <2.2->1.6 nm fraction. In case of Fe, colloidal fractions <2.2->1.6 nm and <1.6-<1.1 nm shows higher concentration. The concentration of Zn, Cu, Ni and Mn increase with decrease in size in suspended particulate matter, while the reverse is observed in case of Fe. This size separation data that specifies the partitioning of metals between dissolved and suspended solid phases is necessary for developing physically based models of metal transport in aquatic system.

Benzopyrans↗
PubMed2001

Simultaneous measurement of XANES in halide-intercalated BSCCO(2212) using electron and fluorescence yield to compare their performance.

Total Yield with an escape depth of approximately 100-200 A is known to be rather surface sensitive. Fluorescence Yield, on the other hand, with an escape depth of approximately 1000-2000 A is relatively less prone to surface effects but necessitates some corrections to obtain the true signal. Both have their plus and minus points and, if used with care, yield reliable data. In the present experiment both the techniques have been simultaneously employed for measuring orientation dependent O K and the Cu L3 edges from an uncleaved surface of I(2)BSCCO(2212) single crystal to compare the performance of the two modes of detection. Despite glaring differences in intensities the results from the two appear to show reasonable agreement in respect of relative intensities of the spectral features.

Journal Article↗
PubMed2001

Polarization-dependent XANES study of Bi2Sr2Ca(1-x)Pr(x)Cu2O8-delta insulating single crystal.

Pr doping has been extensively studied in cuprate superconductors to understand the mechanism of quenching of superconductivity in cuprate perovskites. Experience has revealed that it acts differently in different cuprate perovskites. We have made high resolution polarization XANES measurements on a Pr-doped single crystal within the ab-plane on the Cu K and Pr L III absorption edges to ascertain the valence state of Pr cation and thereby try learning how it is quenching superconductivity in the present case. Our results show that besides the Pr quenching holes the possibility of its localizing some holes through hybridization with O 2p and Cu 3d cannot be ruled out.

Journal Article↗
PubMed2001

O K and Cu LIII edge study of itinerant holes in I2-, Hgl2- and HgBr2- intercalated BSCCO(2212) single crystals.

Intercalation effect on BSCCO (2212) system, although it increases the interlayer distance and the c-axis remarkably, produces only a small change in the transition temperature. Thus, amongst other things, intercalation provides an effective method to investigate the influence of the interblock coupling. Electrons are transferred from the host Cu-O2 layers to the guest molecules I2, HgBr2, HgI2 leading to evolution of the Tc. For this we have made high resolution XANES study on the O K and Cu L3 edges to estimate the density of the doping holes. We attempt on basis of our and earlier results the evolution of Tc in these as also the much larger decrease produced in Tc for I2-intercalation for which the increase in basal spacing is the smallest of the three halides.

Journal Article↗
PubMed2000

Modulation of base excision repair by low density lipoprotein, oxidized low density lipoprotein and antioxidants in mouse monocytes.

In the present study, we found that oxidized low density lipoprotein, but not low density lipoprotein, down-regulated base excision repair activity in extracts of mouse monocyte cell line PU5-1.8. An enzyme required in this pathway, DNA polymerase beta, was also down-regulated. In contrast, treatment of monocytes with a combination of ascorbate and alpha-tocopherol up-regulated base excision repair activity and expression of DNA polymerase beta. Co-treatment of monocytes with antioxidants plus oxidized low density lipoprotein prevented down-regulation by oxidized low density lipoprotein. Oxidative DNA damage, as measured by 8-hydroxyguanine accumulation in genomic DNA, was found in cells treated with oxidized low density lipoprotein; 8-hydroxyguanine was not found in the cells treated with low density lipoprotein, antioxidants or oxidized low density lipoprotein plus antioxidants. These results establish a linkage between the DNA base excision repair pathway, oxidative DNA damage and oxidized low density lipoprotein treatment in mouse monocytes. Since oxidized low density lipoprotein is implicated in chronic disease conditions such as atherogenesis, these findings facilitate understanding of genetic toxicology mechanisms related to human health and disease.

Animals↗
PubMed2000

Glutathione ethyl ester supplementation prevents mortality in newborn rats exposed to hyperoxia.

Human premature neonates suffer from respiratory distress syndrome due to immature lungs and require assisted ventilation with high concentrations of oxygen. Hyperoxic exposure and/or antioxidant deficiency causes an increase in the lung levels of reactive oxygen species (ROS) leading to oxidative stress-induced cellular damage. In this study, we explored the protective role of the nonenzymatic antioxidant glutathione, by administering glutathione ethyl ester (GSHEE), in newborn rats exposed to hyperoxia (>95% FiO(2)). Our results show that GSHEE supplementation (5 mmol/kg/day) prevents mortality in newborn rats exposed to hyperoxia. We further show that delayed GSHEE supplementation in newborn rats, pre-exposed to hyperoxia for 4 days, also prevents death. Electron microscopic studies on the lung of GSHEE-treated hyperoxic rats showed normal histology and an absence of the marked swelling and degeneration of mitochondria and lamellar bodies, which are typically observed in the hyperoxic lungs of newborn rats. Furthermore, there were no apparent differences in weight gain or general appearance/activity among room air and hyperoxic GSHEE-supplemented animals when monitored, post-treatment, in room air for 30 days. Our results show a preventive/therapeutic role of GSHEE supplementation against mortality caused in newborn rats due to hyperoxic exposure, and may further be applicable to a variety of degenerative diseases that are caused as a result of ROS accumulation.

Animals↗
PubMed1998

Up-regulation of base excision repair correlates with enhanced protection against a DNA damaging agent in mouse cell lines.

DNA polymerase beta is required in mammalian cells for the predominant pathway of base excision repair involving single nucleotide gap filling DNA synthesis. Here we examine the relationship between oxidative stress, cellular levels of DNA polymerase beta and base excision repair capacity in vitro , using mouse monocytes and either wild-type mouse fibroblasts or those deleted of the DNA polymerase beta gene. Treatment with an oxidative stress-inducing agent such as hydrogen peroxide, 3-morpholinosydnonimine, xanthine/xanthine oxidase or lipopolysaccharide was found to increase the level of DNA polymerase beta in both monocytes and fibroblasts. Base excision repair capacity in vitro , as measured in crude cell extracts, was also increased by lipopolysaccharide treatment in both cell types. In monocytes lipopolysaccharide-mediated up-regulation of the base excision repair system correlated with increased resistance to the monofunctional DNA alkylating agent methyl methanesulfonate. By making use of a quantitative PCR assay to detect lesions in genomic DNA we show that lipopolysaccharide treatment of fibroblast cells reduces the incidence of spontaneous DNA lesions. This effect may be due to the enhanced DNA polymerase beta-dependent base excision repair capacity of the cells, because a similar decrease in DNA lesions was not observed in cells deficient in base excision repair by virtue of DNA polymerase beta gene deletion. Similarly, fibroblasts treated with lipopolysaccharide were more resistant to methyl methanesulfonate than untreated cells. This effect was not observed in cells deleted of the DNA polymerase beta gene. These results suggest that the DNA polymerase beta-dependent base excision repair pathway can be up-regulated by oxidative stress-inducing agents in mouse cell lines.

Animals↗
PubMed1996

Specific interaction of DNA polymerase beta and DNA ligase I in a multiprotein base excision repair complex from bovine testis.

Base excision repair (BER) is a cellular defense mechanism repairing modified bases in DNA. Recently, a G:U repair reaction has been reconstituted with several purified enzymes from Escherichia coli (Dianov, G., and Lindahl, T.(1994) Curr. Biol. 4, 1069-1076). Using bovine testis crude nuclear extract, we have shown that G:U is repaired efficiently in vitro, and DNA polymerase beta (beta-pol) is responsible for the single nucleotide gap-filling synthesis (Singhal, R. K., Prasad, R., and Wilson, S. H.(1995) J. Biol. Chem. 270, 949-957). To investigate potential interaction of beta-pol with other BER protein(s), we developed affinity chromatography matrices by cross-linking purified rat beta-pol or antibody against beta-pol to solid supports. Crude nuclear extract from bovine testis was applied to these affinity columns, which were then extensively washed. Proteins that bound specifically to the affinity columns were co-eluted in a complex with beta-pol. This complex had a molecular mass of approximately 180 kDa and was able to conduct the complete uracil-initiated BER reaction. The BER complex contained both beta-pol and DNA ligase I. An antibody to beta-pol was able to shift the complex in sucrose gradients to a much larger molecular mass (>300 kDa) that again contained both beta-pol and DNA ligase I. Furthermore, DNA ligase I and beta-pol were co-immunoprecipitated from the testis nuclear extract with anti beta-pol IgG. Thus, we conclude that beta-pol and DNA ligase I are components of a multiprotein complex that performs BER.

Animals↗
PubMed1996

Requirement of mammalian DNA polymerase-beta in base-excision repair.

Synthesis of DNA by DNA polymerase-beta is distributive on single-stranded DNA templates, but short DNA gaps with a 5' PO4 in the gap are filled processively to completion. In vitro studies have suggested a role of beta-polymerase in different types of DNA repair. However, the significance of these studies to the in vivo role of beta-polymerase has remained unclear. Because genetic studies are essential for determining the physiological role of a gene, we established embryonic fibroblast cell lines homozygous for a deletion mutation in the gene encoding DNA polymerase-beta. Extracts from these cell lines were found to be defective in uracil-initiated base-excision repair. The beta-polymerase-deleted cells are normal in viability and growth characteristics, although they exhibit increased sensitivity to monofunctional DNA-alkylating agents, but not to other DNA-damaging agents. Both the deficiency in base-excision repair and hypersensitivity to DNA-alkylating agents are rescued following stable transfection with a wild-type beta-polymerase minitransgene. These studies demonstrate that beta-polymerase functions specifically in base-excision repair in vivo.

Animals↗
PubMed1995

Specific inhibition of DNA polymerase beta by its 14 kDa domain: role of single- and double-stranded DNA binding and 5'-phosphate recognition.

DNA polymerase beta (beta-polymerase) has been implicated in short-patch DNA synthesis in the DNA repair pathway known as base excision repair. The native 39 kDa enzyme is organized into four structurally and functionally distinct domains. In an effort to examine this enzyme as a potential therapeutic target, we analyzed the effect of various beta-polymerase domains on the activity of the enzyme in vitro. We show that the 14 kDa N-terminal segment of beta-polymerase, which binds to both single- and double-stranded DNA, but lacks DNA polymerase activity, inhibits beta-polymerase activity in vitro. Most importantly, the 8, 27 and 31 kDa domains of beta-polymerase do not inhibit beta-polymerase activity, demonstrating that the inhibition by the 14 kDa domain is specific. The inhibition of beta-polymerase activity in vitro is abolished by increasing the concentrations of both of the substrates (template-primer and deoxynucleoside triphosphate). In contrast, an in vitro base excision repair assay is inhibited in a domain specific manner by the 14 kDa domain even in the presence of saturating substrates. The inhibition of beta-polymerase activity by the 14 kDa domain appears specific to beta-polymerase as this domain does not inhibit either mammalian DNA polymerase alpha or Escherichia coli polymerase I (Klenow fragment). These data suggest that the 14 kDa domain could be used as a potential inhibitor of intracellular beta-polymerase and that it may provide a means for sensitizing cells to therapeutically relevant DNA damaging agents.

Base Sequence↗
PubMed1995

DNA polymerase beta conducts the gap-filling step in uracil-initiated base excision repair in a bovine testis nuclear extract.

The G:U mismatch in genomic DNA mainly arises from deamination of cytosine residues and is repaired by the base excision repair pathway. We found that a bovine testis crude nuclear extract conducts uracil-initiated base excision repair in vitro. A 51-base pair synthetic DNA substrate containing a single G:U mismatch was used, and incorporation of dCMP during repair was exclusively to replace uracil. A neutralizing polyclonal antibody against DNA polymerase beta (beta-pol) inhibited the repair reaction. ddCTP also inhibited the repair reaction, whereas aphidicolin had no significant effect, suggesting that activity of beta-pol was required. Next, the base excision repair system was reconstituted using partially purified components. Several of the enzymatic activities required were resolved, such that DNA ligase and the uracil-DNA glycosylase/apurinic/apyrimidinic endonuclease activities were separated from the DNA polymerase requirement. We found that purified beta-pol could restore full DNA repair activity to the DNA polymerase-depleted fraction, whereas purified DNA polymerases alpha, delta, and epsilon could not. These results with purified proteins corroborated results obtained with the crude extract and indicate that beta-pol is responsible for the single-nucleotide gap filling reaction involved in this in vitro base excision repair system.

Animals↗
PubMed1994

Laboratory evaluation of interception and translocation of 131I in fenugreek and okra plants.

The work reported here deals with the study of interception and translocation of airborne 131I in fenugreek (Trigonella foenum-graecum) and okra (Hibiscus esculentus), two very common vegetables in India. Activity was injected into the experimental chamber in the form of iodide aerosols having a size distribution of 0.3 to 5.0 microns (AMAD). Samples of plants were collected over a period of a few days at different time intervals after injection of the aerosols. Evaluation of interception with deposition and translocation of 131I was done from the activity measured in air and in plant parts. For the deposition factor, the values are 1.22 m3 kg-1 for fenugreek leaves, and 1.49 m3 kg-1 for the plant as a whole. For okra plants these values are 0.02 and 0.16 m3 kg-1 in edible okra and leaves, respectively. For the okra plant as a whole, the value is 0.19 m3 kg-1. The translocation factors vary from 0.62 to 0.86 and 0.47 to 0.87 for leaves and stem, respectively, in the case of fenugreek plants. For okra plants, the translocation factor varies from 0.21 to 0.82, 0.53 to 0.93, and 0.42 to 0.81 in edible okra, leaves, and stems, respectively.

Aerosols↗
PubMed1993

Yeast open reading frame YCR14C encodes a DNA beta-polymerase-like enzyme.

We have shown by activity gel that overexpression in E. coli of a yeast chromosome 3 open reading frame (ORF) designated YCR14C and bearing homology to mammalian DNA polymerases beta results in a new DNA polymerase in the host cells. The molecular mass of this enzyme corresponded to the YCR14C-predicted 67 kDa protein, and NH2-terminal amino acid sequencing confirmed that the expressed protein was encoded by the yeast ORF. This new yeast DNA polymerase was purified to homogeneity from E.coli. In a fashion similar to that of mammalian beta-polymerases, the purified yeast enzyme exhibited distributive DNA synthesis on DNA substrate with a single-stranded template and processive gap-filling synthesis on a short-gapped DNA substrate. Activity of this yeast beta-polymerase-like enzyme was sensitive to the beta-polymerase inhibitor ddNTP and resistant to both 1 mM NEM and neutralizing antibody to E. coli DNA polymerase I. These results, therefore, indicate that YCR14C encodes a DNA beta-polymerase-like enzyme in yeast, and we name it DNA polymerase IV. Yeast strains harboring a deletion mutation of the pol IV gene are viable, they exhibit no increase in sensitivity to ultraviolet light, ionizing radiation or alkylating agents, and sporulation and spore viability are not affected in the mutant.

Amino Acid Sequence↗
PubMed1993

Short gap-filling synthesis by DNA polymerase beta is processive.

Mammalian DNA polymerase beta (beta-pol) is considered to be part of the DNA repair machinery. However, there have been conceptual problems with this idea because beta-pol does not completely fill some gapped substrates, and unlike other DNA polymerases, beta-pol adds just one dNMP for each cycle of binding to the DNA substrate, incorporation, and product release (distributive synthesis). To examine the questions of complete gap filling and gap recognition by beta-pol, we designed template-primer substrates with a range of gap sizes from 1 to 53 template residues between the 3'-OH primer and a downstream polynucleotide. M13mp18(+) single-stranded DNA was used as template, and synthetic deoxyoligonucleotides were used as primers and downstream polynucleotides. We find that beta-pol can completely fill gaps and that the gap-filling activity on substrates with gaps of up to 6 nucleotides is highly processive rather than distributive; processive synthesis to fill a gap strictly required 5'-phosphate on the 5'-moiety of the gap. Similar specificity for filling short gaps was not observed with other DNA polymerases. These results on substrate specificity and the mechanism of beta-pol suggest distinct requirements for the role of beta-pol in vivo.

Animals↗
PubMed1992

The REV3 gene of Saccharomyces cerevisiae is transcriptionally regulated more like a repair gene than one encoding a DNA polymerase.

We measured the relative steady-state levels of the mRNA transcribed from the Saccharomyces cerevisiae REV3 gene in cells at different stages of the mitotic and meiotic cycles, and after UV irradiation. This gene is thought to encode a DNA polymerase concerned only with a specific recovery function, the replication on mutagen-damaged templates that produces damaged-induced mutations. In keeping with this proposed function, the REV3 gene showed no evidence of the periodic transcription at the G1/S boundary of the mitotic and meiotic cycle that occurs with genes encoding replication enzymes. However, levels of REV3 mRNA were much increased in late meiotic cells, like those of transcripts of some other DNA repair-related genes. Steady-state levels of REV3 transcript were increased only slightly in response to UV irradiation.

Amino Acid Sequence↗
PubMed1989

Stabilization, purification, and characterization of glutamate synthase from Clostridium pasteurianum.

Clostridium pasteurianum possesses a high level of glutamate synthase (EC 1.4.1.14) activity and cell yield when grown on 4 mM ammonium chloride and molasses as the sole nitrogen and carbon sources, respectively. The enzyme activity is stabilized by addition of alpha-ketoglutarate, EDTA, and 2-mercaptoethanol. Ammonium sulfate precipitation and single-step combined gel and ion-exchange chromatography followed by fractional dialysis yield a homogeneous protein with 40% recovery of the glutamate synthase activity. The native enzyme (Mr congruent to 590,000) gives five different subunits (as dimers) upon SDS gel electrophoresis. The enzyme has been characterized for pH and temperature optimum, substrate specificity, Kmapp values, energy of activation, half-life, and thermal stabilization. Metal ions and citric acid cycle metabolites do not affect the enzyme activity. Glutamate synthase shows fluorescence maximum at 370 nm when excited at 280 nm. The fluorescence is quenched upon the addition of NADH. Spectroscopic examination of the enzyme gave absorption maximum at 280 and none at 380 and 440 nm, indicating the absence of iron and flavin. The absence of iron and flavin was also confirmed by atomic absorption, chemical analysis, and fluoroscopy, respectively. The C. pasteurianum enzyme differs from that of other aerobic bacterial sources.

Chemical Precipitation↗