Biomedical subjects
S Kumar
Publications and source records attributed to S Kumar.
Comparison of flow cytometric DNA content analysis of fresh and formalin-fixed paraffin-embedded solid neoplasms.
In an attempt to compare results of fresh and fixed tissue flow cytometric DNA analysis we analyzed paraffin blocks of 87 solid tumors; 67 of these were DNA aneuploid and 20 were DNA euploid by fresh flow cytometric analysis. Forty-two aneuploid tumors and all diploid tumors were concordant (71.3% concordance), whereas 25 of 67 (37%) aneuploid tumors appeared diploid on fixed tissue analysis. Reasons for discordance included loss of small aneuploid peaks, merging of G1/G0 peaks of diploid and aneuploid stemlines giving a broad G1/G0 peak, and masking of near-tetraploid aneuploid peaks by the G2/M peak in fixed tissue. S-phase fraction could be determined in 51 of 67 (76%) aneuploid cases analyzed fresh and 29 of 42 (69%) concordant aneuploid cases analyzed from fixed tissues. All parameters were consistently higher in the fixed tissue: S-phase fraction (mean = 18.29% versus 12.82%), coefficient of variation of diploid G1/G0 peak (5.62% versus 2.11%), and DNA index (1.79 versus 1.69). We conclude that significant differences exist between flow cytometric data obtained from analysis of fresh versus fixed tissues and therefore prognostic correlations made on the basis of results of fixed tissue flow cytometric analysis may not necessarily parallel those of fresh tissue studies.
A comparative evaluation of three transfection procedures as assessed by resistance to G418 conferred to HEPG2 cells.
Three commonly used transfection techniques (electroporation, calcium phosphate precipitation and scrape loading) and a novel procedure combining the latter two methods were evaluated and conditions optimised for successful transfection of human HepG2 cells with plasmid DNA incorporating a mouse MHC Class I gene and a selectable marker (neomycin transferase gene) conferring resistance to G418. While transfection with linear DNA by scrape-loading gave satisfactory results, transfer of cloned circular DNA by electroporation, calcium phosphate precipitation or a combined use of scrape-loading and calcium phosphate gave best results for HepG2 cells.
Detection of tuberculous IgG antibodies using Mycobacterium tuberculosis H37Ra, excretory secretory antigen and tuberculin-purified protein derivative.
Different antigen preparations, viz. excretory-secretory antigen (ES Ag), phosphate buffer saline soluble antigen (PBS-S Ag) and sodium dodecyl sulphate soluble antigen (SDS-S Ag) of M. tuberculosis (M.tb) H37Ra strain along with tuberculin purified protein derivative (PPD) were employed in stick indirect ELISA to detect IgG antibodies in sera of sputum positive pulmonary tuberculosis cases. Sera from healthy individuals and individuals with diseases other than tuberculosis (cross-reacting diseases) were used as controls. ES antigen and PPD showed higher antibody titres in tuberculosis cases (GMT-1378 each) compared to PBS-S Ag (GMT-454) and SDS-S Ag (GMT-974). Thereafter, an extensive study was done analysing higher number of sera in each group for the detection of tuberculous IgG antibodies using ES Ag and PPD. The ES Ag showed better sensitivity (87%) and specificity (85%) compared to the sensitivity (73%) and specificity (78%) achieved with PPD. The ES Ag also showed higher IgG antibody titre (GMT-1068) than PPD (GMT-721). From the present study it can be envisaged that ES Ag has high diagnostic potential in tuberculosis.
The impact of five-month basic military training on the body weight and body fat of 197 moderately to severely obese Singaporean males aged 17 to 19 years.
The impact of five-month basic military training on body weight and fat mass was studied in 197 male obese recruits, aged 17 to 19 years old. The training programme of five days per week lasted for 20 weeks. Of these sessions, 57% could be considered to be physically intense for the obese subjects. No dietary restriction was imposed. The cohort was divided into three categories based on their percentage body fat, i.e. %BF 24 to < 30, %BF 30 to < 35 and %BF > or = 35. The overall mean weight loss was 12.5 kg or 0.63 kg per week. Mean weight losses for each category were 10.7 kg, 13.1 kg and 16.1 kg, respectively. Most of this weight was lost in the 2nd month of training. The weight loss is attributed to a decrease of body fat as determined by skinfold measurements. Attrition rate, due to training injuries resulting in temporary disabilities, was negligible at 6.1%. In conclusion, this study shows that intense physical activity over a duration of 20 weeks is effective in reducing the body fat of our obese subjects and in training obese subjects for combat duties.
Effects of hormones and cysteine protease modulators on infection of HepG2 cells by Plasmodium berghei sporozoites in vitro determined by ELISA immunoassay.
An enzyme-linked immunosorbent assay (ELISA) detecting a Plasmodium berghei liver-stage-specific protein Pbl-1 is described. The quantitative detection limits ranged from 0.01 to 0.05 microgram of parasite protein. Qualitatively the assay detected as little as 0.001 microgram Pbl-1 per well. Using the ELISA dexamethasone and insulin together was shown to promote higher parasite infections in HepG2 cells compared to unsupplemented medium. Anti-cowpea-protease cysteine inhibitor significantly increased hepatocyte invasion as compared to controls, whereas a significant decrease was recorded in the presence of the protease inhibitor E64. Partial involvement of cysteine proteases in HepG2 invasion by P. berghei sporozoites is therefore suggested.
The viral and cellular Rel oncoproteins induce the differentiation of P19 embryonal carcinoma cells.
Rel proteins have been implicated in the differentiation and transformation of lymphoid cells. Despite their significant sequence homology to the Drosophila Dorsal morphogen, a functional role for Rel proteins in early development has yet to be established. Here we show that expression of the v- or c-rel genes in P19 embryonal carcinoma (EC) cells leads to drastic morphological changes accompanied by the expression of several differentiation-specific antigens. In contrast, v-Rel mutants defective for DNA-binding as well as those lacking the F9 cell-specific transcription activation region of v-Rel were equally unable to promote P19 cell differentiation. Our results demonstrate that transcriptional activation by Rel proteins correlates with Rel-induced differentiation of EC cells, and raise the intriguing possibility that Rel proteins may play an important role in early developmental processes.
Analysis of the RNA-recognition motif and RS and RGG domains: conservation in metazoan pre-mRNA splicing factors.
We present a systematic analysis of sequence motifs found in metazoan protein factors involved in constitutive pre-mRNA splicing and in alternative splicing regulation. Using profile analysis we constructed a database enriched in protein sequences containing one or more presumptive copies of the RNA-recognition motif (RRM). We provide an accurate alignment of RRMs and structure-based criteria for identifying new RRMs, including many that lack the prototype RNP-1 submotif. We present a comprehensive table of 125 sequences containing 252 RRMs, including 22 previously unreported RRMs in 17 proteins. The presence of a putative RRM in these proteins, which are implicated in a variety of cellular processes, strongly suggests that their function involves binding to RNA. Unreported homologies in the RRM-enriched database to the metazoan SR family of splicing factors are described for an Arg-rich human nuclear protein and two yeast proteins (S. pombe mei2 and S. cerevisiae Npl3). We have rigorously tested the phylogenetic relationships of a large sample of RRMs. This analysis indicates that the RRM is an ancient conserved region (ACR) that has diversified by duplication of genes and intragenic domains. Statistical analyses and classification of repeated Arg-Ser (RS) and RGG domains in various protein splicing factors are presented.
Searching for replication origins in mammalian DNA.
The attempts at identifying precise replication origins (ori) in mammalian DNA have been pursued mainly through physico-chemical and biochemical approaches, in view of the essential failure of the search for autonomously replicating sequences in cultured cells. These approaches involve the mapping of short stretches of nascent DNA, the identification of the regions where either leading or lagging strands switch polarity, or the localization of replication intermediates by two-dimensional gel electrophoresis. Due to the complexity of animal cell genomes, most of these studies have been performed on amplified domains and with the use of synchronization procedures. The results obtained have been controversial. In order to avoid the use of experimental procedures potentially affecting the physiological mechanism of DNA replication, we have developed a method for the localization of ori in single-copy loci in exponentially growing cells. This method entails the absolute quantification of the abundance of selected DNA fragments along a genomic region within samples of newly synthesized DNA by competitive polymerase chain reaction (PCR); the latter is immune to all the uncontrollable variables which severely affect the reproducibility of conventional PCR. The application of this method to SV40 ori-driven plasmid replication precisely identifies the known ori localization. Using the same approach, we have mapped an ori for bi-directional DNA replication in a 13.7-kb locus of human chromosome 19 encoding lamin B2.
Morphology and ordering in thin liquid-crystal films on flat glass substrates.
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I kappa B alpha-mediated inhibition of v-Rel DNA binding requires direct interaction with the RXXRXRXXC Rel/kappa B DNA-binding motif.
Rel family proteins bind to kappa B DNA sites, form heterodimers with one another, and modulate expression of genes linked to kappa B motifs. I kappa B factors associate with Rel proteins, inhibit Rel DNA binding in vitro, and displace DNA from DNA-bound Rel complexes. We have investigated the mechanism by which the p40/I kappa B alpha inhibitor interfers with Rel DNA-binding activity. Here, we report that p40 contacts the RXXRXRXXC DNA-binding motif conserved in all Rel family proteins, in addition to associating with the nuclear localizing sequence. Competition assays with a Rel-derived peptide comprising the DNA-binding region specifically alleviated p40-mediated inhibition of v-Rel DNA-binding activity, whereas a covalently modified Rel peptide was inactive. Combined, these results indicate that I kappa B alpha interaction with the RXXRXRXXC motif is required for inhibition of v-Rel DNA binding and suggest that nuclear I kappa B factors may be critical for regulating transcription by Rel family proteins.
Prognostic value of blood vessel density in ischaemic stroke.
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Theoretical evidence for the correlation between hole density and Tc in high-Tc Tl-based superconductors.
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Cloning of a cDNA which encodes a novel ubiquitin-like protein.
The cloning of a mouse cDNA, Nedd-8, which encodes a small novel protein of 81 amino acid residues with a relative molecular mass of 9 kDa, is described. The putative Nedd-8 product is approximately 60% identical to ubiquitin protein. The 0.6 kb mRNA for Nedd-8 gene can be detected in various mouse tissues and cell lines derived from various sources. Nedd-8 probe hybridizes to genomic DNA from various vertebrate species as well as yeast, indicating high degree of interspecies conservation. These data suggest that like ubiquitin, the product of this gene may play some essential role in eukaryotic cellular metabolism.
Crystal structure of the HhaI DNA methyltransferase complexed with S-adenosyl-L-methionine.
The first three-dimensional structure of a DNA methyltransferase is presented. The crystal structure of the DNA (cytosine-5)-methyltransferase, M.HhaI (recognition sequence: GCGC), complexed with S-adenosyl-L-methionine has been determined and refined at 2.5 A resolution. The core of the structure is dominated by sequence motifs conserved among all DNA (cytosine-5)-methyltransferases, and these are responsible for cofactor binding and methyltransferase function.
X-ray-diffraction characterization and sound-velocity measurements of W/Ni multilayers.
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Photoinduced antiferromagnetic interlayer coupling in Fe/(Fe-Si) superlattices.
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A monoclonal antibody detects heterogeneity in vascular endothelium of tumours and normal tissues.
A new murine monoclonal antibody (MAb), E-9, has been raised using tissue-cultured human umbilical vein endothelial cells. The antigen recognized by this MAb is a peptide of 170 kDa under non-reducing conditions and 96 kDa under reducing conditions. MAb E-9 showed marked heterogeneity in its distribution in various tissues. The antigen recognized by it was present in vascular endothelial cells of all tumours, foetal organs and in regenerating and inflamed tissues. It stained a few normal tissues. However, with the exception of tonsils, staining tended to be weak and limited to a few blood vessels, as revealed by double staining using pan-endothelial antibody (CD31) and antibody to von Willebrand factor, another marker of vascular endothelium. Surprisingly, blood vessels within the placental villi were completely negative. The function of the antigen recognized by MAb E-9 is not known, but its evaluation and use should increase our understanding of angiogenesis.