Biomedical subjects

R Dey

Publications and source records attributed to R Dey.

At least 19 recordsLinked to original sources

PubMed2001

Isolation and partial sequencing of potentially odontoblast-specific/enriched rat cDNA clones obtained by suppression subtractive hybridization.

Odontoblasts, which are responsible for dentine formation, are known to synthesize unique gene products such as dentine sialophosphoprotein. To further identify and clone novel odontoblast-specific genes, a suppression subtractive hybridization technique was used here. Differentially or predominantly expressed cDNAs in odontoblasts of rat incisors were obtained by subtracting the common cDNAs expressed in odontoblasts, osteoblasts and pulp cells. Clones were then partially sequenced and analysed for nucleotide sequence homology by the basic local alignment search tool program. From a total of 1290 clones analysed, 538 odontoblast-enriched clones were identified in the subtracted cDNA library. Out of 538 clones, 498 clones (92.6%) demonstrated high identity with genes in the GenBank database. In contrast, 31 clones (5.7%) showed low sequence identity with known genes, among which 18 clones (3.3%) were observed more than once, thereby possibly representing odontoblast-specific/enriched genes. The majority (390 clones; 72.5%) of the clones with high homology to known genes were found to be the rat/mouse dentine sialophosphate by dot-blot analysis (326 clones) and sequencing (64 clones). The second highest enrichment (39 clones) was for phosphate-regulating gene with homology to endopeptidase on the X-chromosome, which codes for a neutral endopeptidase. After suppression subtractive hybridization, several cDNAs that are commonly present in osteoblasts and odontoblasts appeared unsuppressed. Therefore, a rat odontoblast-specific/enriched subtraction cDNA library has been created from which a number of potentially novel genes for odontoblasts could be identified.

Animals↗

PubMed2000

Functional constraints of nuclear-mitochondrial DNA interactions in xenomitochondrial rodent cell lines.

The co-evolution of nuclear and mitochondrial genomes in vertebrates led to more than 100 specific interactions that are crucial for an optimized ATP generation. These interactions have been examined by introducing rat mtDNA into mouse cells devoid of mitochondrial DNA (mtDNA). When mtDNA-less cells derived from the common mouse (Mus musculus domesticus) were fused to cytoplasts prepared from Mus musculus, Mus spretus, or rat (Rattus norvegicus), a comparable number of respiring clones could be obtained. Mouse xenomitochondrial cybrids harboring rat mtDNA had a slower growth rate in medium containing galactose as the carbon source, suggesting a defect in oxidative phosphorylation. These clones respired approximately 50% less than the parental mouse cells or xenomitochondrial cybrids harboring Mus spretus mtDNA. The activities of respiratory complexes I and IV were approximately 50% lower, but mitochondrial protein synthesis was unaffected. The defects in complexes I and IV were associated with decreased steady-state levels of respective subunits suggesting problems in assembly. We also showed that the presence of 10% mouse mtDNA co-existing with rat mtDNA was sufficient to restore respiration to normal levels. Our results suggest that evolutionary distance alone is not a precise predictor of nuclear-mitochondrial interactions as previously suggested for primates.

3T3 Cells↗

PubMed2000

Human satellite 3 (HS3) binding protein from the nuclear matrix: isolation and binding properties.

Satellite DNA (satDNA) is the main component of residual DNA in nuclear matrix (NM) preparations. Gel mobility shift assay (GMSA) revealed specific human satellite 3 (HS3) binding activity in NM extracts. An HS3 binding protein was purified using diethylaminoethyl (DEAE)-cellulose and preparative GMSA. The binding was specific, although other satDNA fragments compete to some extent for the binding. DNase I footprinting and methylation interference revealed multiple points of protection distributed throughout the HS3 fragment with periodicity of about 10 bp, mostly inside an AT island. Polyclonal antibodies (AB) were raised against HS3-protein complexes cut from the preparative GMSA gel. On immunoblots, AB recognise a protein, which is not lamin, with apparent molecular mass 70 kDa, the same as revealed by purification (p70). In in situ nuclear matrix preparations combined immunofluorescence (AB) and fluorescent in situ hybridisation (HS3) shows that HS3 and p70 areas correspond to each other. The localisation of this protein detected with AB in interphase nuclei coincides with the heterochromatic regions which surround nucleoli in correspondence with the known HS3 position in the nuclei.

Antibodies↗

PubMed2000

Lack of oxidative phosphorylation and low mitochondrial membrane potential decrease susceptibility to apoptosis and do not modulate the protective effect of Bcl-x(L) in osteosarcoma cells.

We explored the role of low mitochondrial membrane potential (DeltaPsim) and the lack of oxidative phosphorylation in apoptosis by assessing the susceptibility of osteosarcoma cell lines with and without mitochondrial DNA to staurosporine-induced death. Our cells without mitochondrial DNA had low DeltaPsim and no functional oxidative phosphorylation. Contrary to our expectation, these cells were more resistant to staurosporine-induced death than were the parental cells. This reduced susceptibility was associated with decreased activation of caspase 3 but not with the mitochondrial permeability transition pore or cytochrome c release from the mitochondria. Apoptosis in both cell lines was associated with an increase in DeltaPsim. Bcl-x(L) could protect both cell types against caspase 3 activation and apoptosis by a mechanism that does not appear to be mediated by mitochondrial function or modulation of DeltaPsim. Nevertheless, we found that Bcl-x(L) expression can stimulate cell respiration in cells with mitochondrial DNA. Our results showed that the lack of functional oxidative phosphorylation and/or low mitochondrial membrane potential are associated with an antiapoptotic effect, possibly contributing to the development of some types of cancer. It also reinforces a model in which Bcl-x(L) can exert an antiapoptotic effect by stimulating oxidative phosphorylation and/or inhibiting caspase activation.

Adenosine Triphosphate↗

PubMed2000

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Administration, Cutaneous↗

PubMed2000

Antifungal characteristics of a fluorescent Pseudomonas strain involved in the biological control of Rhizoctonia solani.

A plant growth-promoting isolate of a fluorescent Pseudomonas spp. EM85 was found strongly antagonistic to Rhizoctonia solani, a causal agent of damping-off of cotton. The isolate produced HCN (HCN+), siderophore (Sid+), fluorescent pigments (Flu+) and antifungal antibiotics (Afa+). Tn5::lacZ mutagenesis of isolate EM85 resulted in the production of a series of mutants with altered production of HCN, siderophore, fluorescent pigments and antifungal antibiotics. Characterisation of these mutants revealed that the fluorescent pigment produced in PDA and the siderophore produced in CAS agar were not the same. Afa- and Flu- mutants had a smaller inhibition zone when grown with Rhizoctonia solani than the EM85 wild type. Sid- and HCN mutants failed to inhibit the pathogen in vitro. In a pot experiment, mutants deficient in HCN and siderophore production could suppress the damping-off disease by 52%. However, mutants deficient in fluorescent pigments and antifungal antibiotics failed to reduce the disease severity. Treatments with mutants that produced enhanced amounts of fluorescent pigments and antibiotics compared with EM85 wild type, exhibited an increase in biocontrol efficiency. Monitoring of the mutants in the rhizosphere using the lacZ marker showed identical proliferation of mutants and wild type. Purified antifungal compounds (fluorescent pigment and antibiotic) also inhibited the fungus appreciably in a TLC bioassay. Thus, the results indicate that fluorescent pigment and antifungal antibiotic of the fluorescent Pseudomonas spp. EM85 might be involved in the biological suppression of Rhizoctonia-induced damping-off of cotton.

Antibiosis↗

PubMed2000

A novel myopathy-associated mitochondrial DNA mutation altering the conserved size of the tRNA(Gln) anticodon loop.

We report a novel mitochondrial DNA alteration in a 12-year-old boy with myopathy. We identified a single nucleotide insertion (an adenine) in the mitochondrial tRNA-glutamine gene. This addition of an additional adenine in a polyadenine stretch (at mitochondrial DNA positions 4366-4369), alters the length of the evolutionary conserved anticodon loop from seven to eight bases. The nt-4370 addition was heteroplasmic and was abundant in the patient's muscle. Lower proportions of mutated mitochondrial DNA were observed in skin fibroblasts, but were below detectable levels in white blood cells. A muscle biopsy of the patient showed ragged red fibers and an unusually high percentage of cytochrome c oxidase-deficient fibers (89%). The pathogenicity of the mutation was also evident by the fact that fibers harboring lower levels of the mutation showed normal cytochrome c oxidase activity. The insertion in the anticodon loop of tRNA(Gln) gene identified in our patient may provide a unique tool to study protein synthesis in human mitochondria.

Adenine↗

PubMed2000

Cytochrome c oxidase assembly in primates is sensitive to small evolutionary variations in amino acid sequence.

Respiring mitochondria require many interactions between nuclear and mitochondrial genomes. Although mitochondrial DNA (mtDNA) from the gorilla and the chimpanzee are able to restore oxidative phosphorylation in a human cell, mtDNAs from more distant primate species are functionally incompatible with human nuclear genes. Using microcell-mediated chromosome and mitochondria transfer, we introduced and maintained a functional orangutan mtDNA in a human nuclear background. However, partial oxidative phosphorylation function was restored only in the presence of most orangutan chromosomes, suggesting that human oxidative phosphorylation-related nuclear-coded genes are not able to replace many orangutan ones. The respiratory capacity of these hybrids was decreased by 65%-80%, and cytochrome c oxidase (COX) activity was decreased by 85%-95%. The function of other respiratory complexes was not significantly altered. The translation of mtDNA-coded COX subunits was normal, but their steady-state levels were approximately 10% of normal ones. Nuclear-coded COX subunits were loosely associated with mitochondrial membranes, a characteristic of COX assembly-defective mutants. Our results suggest that many human nuclear-coded genes not only cannot replace the orangutan counterparts, but also exert a specific interference at the level of COX assembly. This cellular model underscores the precision of COX assembly in mammals and sheds light on the nature of nuclear-mtDNA coevolutionary constraints.

Animals↗

PubMed1996

Tissue-specific expression of the human pyruvate dehydrogenase alpha (Pdha-1)/chloramphenicol acetyltransferase fusion gene in transgenic mice.

A chimeric gene (-763/+33Pdha-1/CAT) containing -763/+33 nucleotides o f the human pyruvate dehydrogenase gene (Pdha-1) and the structural gene of chloramphenicol acetyltransferase (CAT) was used to generate transgenic mice. CAT activity was detected predominantly in the brain followed in decreasing order by adipose tissue, spleen, heart, kidney and liver. Developmental expression of CAT activity in the testes was similar to that of the endogenous Pdha-1 subunit expression in the testes. Dietary regulation of the transgene was comparable to the expression of endogenous pyruvate dehydrogenase complex in kidney and adipose tissue. Thus, the -763/+33 bp segment of the human Pdha-1 gene is transcriptionally active in vivo and can direct the expression of CAT in several tissues.

Adipose Tissue↗

PubMed1995

Cyclic AMP mediates change in superoxide dismutase activity to monitor host-parasite interaction in Leishmania donovani.

The present study is designed to understand the role of cyclic AMP (cAMP) in host-parasite interaction involving Leishmania donovani, the causative agent for Kala-azar. When Leishmania promastigotes or macrophages were pretreated with dibutyryl cAMP or theophylline and epinephrine, which are well defined initiators for cAMP release, a key enzyme of the oxygen defense system, superoxide dismutase (SOD), was inhibited. At the same time, parasite interaction was considerably reduced to the level of 54.5% and 46.2%, respectively, for pretreated promastigotes. Internalization of the organisms in phagolysosomes was similarly affected. Dibutyryl cAMP-treated promastigotes in the presence of SOD, on the other hand, restored in vitro infection to the normal level. At least 50% less cAMP entered into Leishmania promastigotes when SOD was added to the incubation system containing dibutyryl cAMP. Data reveal that cAMP perturbs the Leishmania-macrophage interaction through inhibition of SOD, pointing to the importance of a promastigote enzyme for the survival of this pathogen within phagolysosomes.

Animals↗

PubMed1994

Leishmanial glycosomes contain superoxide dismutase.

In this work we report that superoxide dismutase is entrapped in a microbody-like organelle, the glycosome, present in Leishmania spp. Studies on the sensitivity of the enzyme to various inhibitors indicated that glycosomal superoxide dismutase is predominantly of the Cu/Zu type. Localization of superoxide dismutase in glycosomes points to the major importance of this organelle in the survival of this pathogen, suggesting a new approach towards chemotherapy for leishmaniasis.

Animals↗

PubMed1994

Effect of vitamin A supplementation on diarrhoea and acute respiratory tract infection in children. A double blind placebo controlled trial in a Calcutta slum community.

To determine the effect of a massive single oral dose of vitamin A (200,000 IU) supplementation on diarrhoea and acute respiratory infection (ARI), a double-blind placebo controlled trial involving 174 children under six years of age (excluding infants) was carried out in a Calcutta slum community. Ninety-one children received vitamin A supplementation (experimental group) and 83 children received a placebo (control group). All the children were followed up for six months by active fortnightly surveillance for occurrence of diarrhoea or ARI and their duration. There was no statistically significant difference in the incidence of diarrhoeal episodes or ARI. However, there was a significant difference (p < 0.05) in the average duration of diarrhoea per episode (2.1 vs. 3 days) between the experimental and control groups. Possible beneficial effects of a single oral dose of vitamin A supplementation on the incidence of diarrhoea and ARI could not be demonstrated in the present study.

Acute Disease↗

PubMed1993

Calcium dependent thiol protease caldonopain and its specific endogenous inhibitor in Leishmania donovani.

A calcium dependent proteolytic enzyme was detected in the lysed promastigotes of Leishmania donovani, the causative agent of kala-azar. The protease was able to hydrolyse an added substrate, azocasein and showed high affinity for calcium. Rate of azocasein digestion was primarily slow but boosted up after eight hours. It was not inactivated when heated at 55 degrees C for 15 min at pH 7.4. Sulfhydryl reagents significantly reduced the enzymic activity but trypsin-like protease inhibitors hardly had any effect. The enzyme was not sensitive to calmodulin from a heterologous source but registered low activity when treated with chlorpromazine. The caseinolytic activity was stimulated when leishmanial cells were preincubated with ionophore A23187 in presence of 1 mM Ca2+. The enzyme is named caldonopain due to its similarity with a general class of calcium dependent protease calpain present in different tissues and cells. Caldonopain was found to be localized in cytosol along with its specific endogenous inhibitor caldonostatin. The ratio of caldonopain-caldonostatin unit was higher in the infected macrophage compared to the parasitic protozoa and Balb/c macrophage alone. It may be postulated that the amount of both calcium and its protein inhibitor may have a direct impact on the caldonopain-induced biological process to regulate cellular action of this pathogen.

Animals↗

PubMed1992

Molecular cloning and analysis of an eIF-4A-related rat liver nuclear protein.

In this paper, we report the cloning and analysis of a cDNA encoding a protein of M(r) congruent to 47,000 (p47), which is localized to the nucleus of rat hepatocytes. The cDNA showed 37% overall sequence identity with a mouse translation initiation factor, eIF-4A, which belongs to a family of putative ATP-dependent RNA helicases. We raised polyclonal antibodies against the fusion protein and by indirect immunofluorescence on primary cultures of hepatocytes have demonstrated that p47 is located in the nucleus. Although only approximately 27% of hepatocytes showed this nuclear staining, most of the nuclei in proliferating transformed cell lines such as 3T3, PtK-1, and Hela were fluorescently labeled. Studies on serum-starved cells in culture indicated that p47 was expressed in a cell cycle-dependent manner. Northern analyses demonstrated that the levels of p47 mRNA were high in fetal liver and dropped significantly after birth to low levels in adult liver. Our data suggest that p47 is developmentally regulated in rat liver at the mRNA level.

Amino Acid Sequence↗

PubMed1992

The effect of aging on glomerular hemodynamics in the rat.

Glomerular hemodynamics were measured in male Sprague-Dawley rats, aged 4 to 5 months (young) or 20 to 22 months (old). Body weight (BW) and left kidney weights (KW) were higher in old rats than young (BW: 507 +/- 12 g v 342 +/- 11 g, P less than 0.001; KW: 2.0 +/- 0.1 g v 1.3 +/- 0.1 g, P less than 0.001). Arterial blood pressure (AP) was slightly higher in old rats, but within the normotensive range (106 +/- 4 mm Hg v 94 +/- 4 mm Hg, P less than 0.05). Glomerular filtration rate (GFR; factored for KW) was lower in old versus young rats (0.67 +/- 0.05 mL/min/gKW v 1.00 +/- 0.08 mL/min/gKW, P less than 0.02). The cortical surface of the kidney in old (but not young) rats showed marked heterogeneity and single-nephron (SN)GFR was measured only in filtering nephrons and was higher and more variable in old versus young rats. Glomerular blood pressure (PGC) was unchanged in old compared with young rats (53 +/- 4 mm Hg v 55 +/- 2 mm Hg). There was a significantly greater level of glomerular sclerosis (in outer cortical glomeruli) in old versus young rats, and glomerular volume was substantially greater in old rats. This study suggests that age-related glomerulopathy is not primarily mediated by glomerular capillary hypertension.

Aging↗

PubMed1991

Molecular cloning of matrin 3. A 125-kilodalton protein of the nuclear matrix contains an extensive acidic domain.

We report here the cloning and sequencing of matrin 3, an acidic internal matrix protein, from a rat insuloma cDNA library. The nucleotide sequence has a single open reading frame encoding a polypeptide of 845 amino acids. The Genbank and National Biomedical Research Foundation databases did not contain any sequences similar to that of matrin 3. The primary structure consists of 33% charged residues and is generally hydrophilic. The amino-terminal region (residues 1-120) is positively charged and contains a large number of amino acids with free hydroxyl groups (26 of the first 100 residues) as in the lamins and several non-lamin intermediate filament proteins. A highly acidic domain (approximately 170 amino acids) near the carboxyl terminus, in which 32% of the amino acid residues are acidic (Glu or Asp), is a characteristic found in other nuclear proteins (Earnshaw, W. C. (1987) J. Cell Biol. 105, 1479-1482). A putative nuclear targeting signal sequence (Ser-Lys-Lys-Lys-Leu-Lys-Lys-Val-Glu) is located in the middle of the highly acidic domain. The corresponding human deduced partial amino acid sequence is 96% identical to the rat sequence, indicating that matrin 3 is a highly conserved protein.

Amino Acid Sequence↗

PubMed1989

The behavior of centrosomes in multinucleate cells formed after colcemid treatment.

Multinucleate PtK1 cells were generated by treating the cells with colcemid for up to 60 h. Cells with scattered chromosomes reconstructed nuclear envelopes around these chromosomes. After recovery of up to 36 h these multinucleate cells went into mitosis. In such cells mainly two types of spindles are found: a bipolar spindle with some "accessory" half-spindles and multipolar mitotic apparatus with several equally-sized half-spindles ordered in an irregular way. Ultrastructural studies revealed centrosomes within such spindles which had not developed a microtubular connection to chromosomes and obviously could not act as mitotic pole. This result is interpreted in the way that centrosomes undergo a maturation cycle. Immature centrosomes cannot form mitotic poles. The asynchrony of the cycles of the multiple centrosomes seems to be generated by an uneven distribution of special factor(s).

Animals↗

PubMed1985

Evidence for the existence of substance P in the prepubertal rat ovary. II. Immunocytochemical localization.

Nerve fibers containing substance P (SP) were localized in ovaries from juvenile and peripubertal rats by immunofluorescence. These fibers were closely associated with the theca externa of antral follicles, as well as being in the interstitial tissue and within the tunica adventitia of small blood vessels, mostly arterioles. Consistently, the greatest amount of SP immunoreactivity was observed surrounding the ovarian vasculature. Substance P was not detected in cells or within the corpora lutea (CL). Additionally, the peripubertal animals seemed to have a greater concentration of ovarian SP than the juvenile animals. Possible functional roles for this peptide in the ovary are discussed.

Animals↗