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Biomedical subjects

A Dey

Publications and source records attributed to A Dey.

At least 37 records · Page 2Linked to original sources
PubMed2000

A bromodomain protein, MCAP, associates with mitotic chromosomes and affects G(2)-to-M transition.

We describe a novel nuclear factor called mitotic chromosome-associated protein (MCAP), which belongs to the poorly understood BET subgroup of the bromodomain superfamily. Expression of the 200-kDa MCAP was linked to cell division, as it was induced by growth stimulation and repressed by growth inhibition. The most notable feature of MCAP was its association with chromosomes during mitosis, observed at a time when the majority of nuclear regulatory factors were released into the cytoplasm, coinciding with global cessation of transcription. Indicative of its predominant interaction with euchromatin, MCAP localized on mitotic chromosomes with exquisite specificity: (i) MCAP-chromosome association became evident subsequent to the initiation of histone H3 phosphorylation and early chromosomal condensation; and (ii) MCAP was absent from centromeres, the sites of heterochromatin. Supporting a role for MCAP in G(2)/M transition, microinjection of anti-MCAP antibody into HeLa cell nuclei completely inhibited the entry into mitosis, without abrogating the ongoing DNA replication. These results suggest that MCAP plays a role in a process governing chromosomal dynamics during mitosis.

Amino Acid Sequence↗
PubMed1999

Ligand-induced recruitment of a histone deacetylase in the negative-feedback regulation of the thyrotropin beta gene.

We have investigated ligand-dependent negative regulation of the thyroid-stimulating hormone beta (TSHbeta) gene. Thyroid hormone (T3) markedly repressed activity of the TSHbeta promoter that had been stably integrated into GH(3 )pituitary cells, through the conserved negative regulatory element (NRE) in the promoter. By DNA affinity binding assay, we show that the NRE constitutively binds to the histone deacetylase 1 (HDAC1) present in GH(3 )cells. Significantly, upon addition of T3, the NRE further recruited the thyroid hormone receptor (TRbeta) and another deacetylase, HDAC2. This recruitment coincided with an alteration of in vivo chromatin structure, as revealed by changes in restriction site accessibility. Supporting the direct interaction between TR and HDAC, in vitro assays showed that TR, through its DNA binding domain, strongly bound to HDAC2. Consistent with the role for HDACs in negative regulation, an inhibitor of the enzymes, trichostatin A, attenuated T3-dependent promoter repression. We suggest that ligand-dependent histone deacetylase recruitment is a mechanism of the negative-feedback regulation, a critical function of the pituitary-thyroid axis.

Base Sequence↗
PubMed1999

Tissue- and cell type-specific expression of cytochrome P450 1A1 and cytochrome P450 1A2 mRNA in the mouse localized in situ hybridization.

We used in situ hybridization to examine organ- and cell type-specific constitutive and 3-methylcholanthrene (3MC)-inducible cytochrome P450 (CYP)1A1 and CYP1A2 mRNA expression in various tissues of the C57BL/6N mouse. In situ hybridization was carried out 10 hr after the mice had received intraperitoneal 3MC, or vehicle alone. We detected levels of 3MC-induced CYP1A1 mRNA in: liver (centrilobular, more so than periportal, regions); lung (Clara Type II cells much more than Type I epithelial cells); brain, especially endothelial cells lining the vascular surface of the choroid plexus; the digestive tract (duodenum > jejunum > ileum > colon > esophagus > stomach--in particular, the villous epithelium, plus cells surrounding glands in the lamina propria); renal corpuscles of the kidney; the ovary (medulla more so than cortex); and the endothelial cells of blood vessels throughout the animal. Constitutive CYP1A1 mRNA was not detectable by in situ hybridization in any of these tissues. In contrast, constitutive CYP1A2 mRNA was measurable in liver, and 3MC-inducible CYP1A2 mRNA was observed only in liver, lung, and duodenum (having cell-type locations similar to those of CYP1A1); the other above-mentioned tissues were negative for CYP1A2 mRNA. These data demonstrate the striking differences in tissue- and cell type-specific expression between the two members of the mouse Cypla subfamily. Because of the ubiquitous nature of 3MC-inducible CYP1A1 throughout the animal rather than just "portals of entry," these results support our hypothesis that CYP1A1, induced by particular endogenous signals in various tissues and cell types, might participate in one or more critical life processes--in addition to its well-established role of metabolism of polycyclic hydrocarbons, certain drugs, and other environmental pollutants.

Animals↗
PubMed1999

Osmotic response element is required for the induction of aldose reductase by tumor necrosis factor-alpha.

Induction of aldose reductase (AR) was observed in human cells treated with tumor necrosis factor-alpha (TNF-alpha). AR protein expression increased severalfold in human liver cells after 1 day of exposure to 100 units/ml TNF-alpha. An increase in AR transcripts was also observed in human liver cells after 3 h of TNF-alpha treatment, reaching a maximum level of 11-fold at 48 h. Among the three inflammatory cytokines: TNF-alpha, interleukin-1, and interferon-gamma, TNF-alpha (100 units/ml) gave the most induction of AR. Differences in the pattern of AR induction were observed in human liver, lens, and retinal pigment epithelial cells with increasing concentrations of TNF-alpha. A similar pattern of AR promoter response was observed between TNF-alpha and osmotically stressed human liver cells. The deletion of the osmotic response element (ORE) abolished the induction by TNF-alpha and osmotic stress. A point mutation that converts ORE to a nuclear factor-kappaB (NF-kappaB) sequence abolished the osmotic response but maintained the TNF-alpha response. Electrophoretic gel mobility shift assays showed two NF-kappaB proteins, p50 and p52, capable of binding ORE sequence, and gel shift Western assay detected NF-kappaB proteins p50 and p65 in the ORE complex. Inhibitors of NF-kappaB signaling, lactacystin, and MG132 abolished the AR promoter response to TNF-alpha.

Aldehyde Reductase↗
PubMed1999

Gamma interferon induces expression of Mad1 gene in macrophage, which inhibits colony-stimulating factor-1-dependent mitogenesis.

Gamma interferon (IFN-gamma) has long been known as an antiproliferative cytokine. The mechanism of its action, however, remains elusive. Monocytes and macrophages are primary targets of IFN-gamma. To understand the antiproliferative signaling of IFNgamma, we studied the effect of IFNgamma on expression of c-Myc, Mad1, Max, cyclin D1, and cyclin D2 genes in both a macrophage cell line and in primary bone marrow-derived macrophages (BMM) in response to colony-stimulating factor-1 (CSF-1). We found that whereas IFNgamma inhibits CSF-1-stimulated c-Myc gene expression, it induces Mad1 expression. Induction of Mad1 mRNA could be detected as early as 90 min following IFNgamma treatment and was maintained for at least 15 h. These results suggest that IFNgamma treatment could shift the Myc-Max complex to the Mad1-Max complex in cells. The levels of Max, cyclin D1, and cyclin D2, however, remained unchanged. Enforced ectopic expression of Mad1 in the cells results in inhibition of [3H]thymidine incorporation and proliferation in response to CSF-1. This study suggests a mechanism by which IFNgamma inhibits CSF-1-stimulated proliferation of macrophages, i.e., by elevating the Mad1 level in the cells.

Animals↗
PubMed1999

The translational hop junction and the 5' transcriptional start site for the Prevotella loescheii adhesin encoded by plaA.

The Prevotella loescheii adhesin gene, plaA, contains a coding gap between a small open reading frame (ORF-1) and a large open reading frame (ORF-2). Translation of the plaA mRNA requires bypassing this 29-nt coding gap on the plaA transcript. We have determined the N-terminal peptide sequence of the SO34 adhesin beyond the gap sequence. This sequence shows that the peptide junction between ORF-1 and ORF-2 is continuous in the adhesin and supports the conclusion that synthesis of the SO34 adhesin occurs by a ribosomal hop mechanism. To elucidate upstream signals, we used the 5' RACE (rapid amplification of cDNA ends) technique to map the start point of the plaA mRNA. DNA sequencing of plasmids with the 5' RACE products placed the 5' end of plaA mRNA 270 nt upstream from the plaA start codon. A region corresponding to a Bacteroides fragilis promoter consensus sequence precedes this start site.

Adhesins, Bacterial↗
PubMed1999

Pharmacokinetics of clindamycin HCl administered intravenously, intramuscularly and subcutaneously to dogs.

A buffered aqueous solution of clindamycin Hcl (200 mg/mL) was injected intravenously (i.v.) intramuscularly (i.m.) and subcutaneously (s.c.) in a non-randomized, partial cross-over trial involving six male and six female dogs. Blood samples were collected at conventional, predetermined time periods and serum drug concentrations were determined by microbiological assay. Dogs were observed clinically for signs of pain, and activity of serum creatine phosphokinase (CPK) was monitored after i.m. dosing. The i.v. data from five of the dogs best fitted a two-compartment open-system pharmacokinetic model whereas a non-compartment model was most suitable for analysis of the data from the remaining seven dogs. The mean i.v. elimination half-life (t1/2 beta) and the mean residence time (MRT) were 124 and 143 min, respectively. The mean volume of distribution at steady state (Vss) was 0.86 L/kg. Little pain was recorded upon i.m. injection; mean peak serum drug concentration (Cmax) was 4.4 micrograms/mL, the elimination half-life (t1/2el) was 247 min and the calculated bioavailability (F) was 115% of the i.v. dose. Serum CPK activity was elevated to 25-fold the pretreatment level in samples collected 4, 8 and 12 h after i.m. injection. Pain was not recorded after s.c. drug administration; the mean Cmax of 20.8 micrograms/mL was significantly greater than the corresponding value for the i.m. route, and F was 310%. The s.c. route appears to be superior to the i.m. route in terms of local tolerance and serum drug level; a 10 mg/kg SID treatment regimen is suggested for treatment of canine infections due to clindamycin sensitive bacteria.

Absorption↗
PubMed1999

Mutant analysis of Prevotella sp. plaA-lacZ fusion protein expression in Escherichia coli: support for an essential role of the stem-loop.

This study investigated the involvement of RNA folding in the synthesis of a fusion protein with beta-galactosidase activity. The coding gap region of the Prevotella loescheii adhesin gene plaA was fused in-frame with the Escherichia coli lacZ gene on plasmid pSK105. N-Terminal sequencing of the expressed plaA-lacZ protein indicated that it resulted from translational initiation at a fortuitous ribosomal-binding site within the plaA sequence at nt 570. Specific mutations were introduced in the stem-loop region that precedes the gap sequence. Analysis of stem-loop mutants, together with the introduction of compensatory mutations that restored activity, supports a requirement for stem-loop formation within the plaA sequence preceding the translational initiation site. A mutation reducing the predicted size of the loop, but preserving the stem structure, inactivated fusion protein synthesis. A suppressor mutation predicted to restore the size of the loop restored efficient fusion protein synthesis. In addition, the sequence preceding the translational start site of the plaA-lacZ fusion has several similarities to sequences that function as translational enhancers in prokaryotes. These include a stem-loop structure, an A-U rich region preceding the initiation codon, and a region of homology to 16S rRNA.

Adhesins, Bacterial↗
PubMed1998

Markedly increased constitutive CYP1A1 mRNA levels in the fertilized ovum of the mouse.

Using a highly sensitive RT-PCR technique that measures mRNA (cDNA)-to-DNA ratios, we are able to detect constitutive CYP1A1 mRNA in adult mouse liver as well as in the oocyte. Twelve hours after fertilization of the ovum, there is a more than 100-fold increase in constitutive CYP1A1 mRNA levels; this dramatic increase completely disappears by the 2-cell stage at gestational day 1.5 (GD1.5), as well as in the blastocyst at GD3.5. The CYP1A1 enzyme has been shown to remove an endogenous ligand for the Ah receptor (AHR), and the AHR is known to play a role in cell cycle regulation and apoptosis. We therefore postulate that this striking abundance of constitutive CYP1A1 mRNA in the fertilized ovum at GD0.5 might be important for maintaining sufficient amounts of the CYP1A1 enzyme during the transition from maternal to zygotic control (GD0.5 to GD1.5 embryo); availability of catalytically active CYP1A1 would ensure that any (exogenous or endogenous) AHR ligand be rapidly degraded, so that all undesirable AHR-mediated gene transcription would be prevented during these very critical moments of early mammalian embryogenesis.

Animals↗
PubMed1998

Sonic and mechanical toothbrushes. An in vitro study showing altered microbial surface structures but lack of effect on viability.

The purpose of the present study was to compare the in vitro effects of a mechanical and a sonic toothbrush on the viability of Actinomyces viscosus, the rationale being that induction of irreparable microbial damage resulting from aggressive mechanical action or sonic energy, may inhibit or disrupt the process of successional colonization. Cultures of A. viscosus were grown to a standardized optical density and subdivided into 3 treatment groups of 20 specimens each. Treatment groups consisted of an untreated control and exposure to a mechanical or sonic toothbrush for 15, 30, 45, and 60 s. Subsequent to the prescribed treatment, samples were taken from each specimen dish, subcultured, and the number of CFUs determined. Additional samples were obtained for negative staining and examination by electron microscopy. The mean number of CFUs for each treatment group at each treatment interval were statistically analyzed by ANOVA and multiple pairwise comparisons. Results showed a significant main effect for toothbrushes (p<0.0001) and exposure time (p<0.01), but only marginal significance for the interaction of toothbrush with exposure time (p<0.055). Post-hoc tests showed a significantly greater number of CFUs for the sonic toothbrush compared to both the untreated control and mechanical toothbrush groups. Electron microscopic examination revealed a decrease in aggregation tendency and loss of fimbriae in the sonic toothbrush group. Based on the lack of morphologic evidence that would indicate cell damage and the increase in CFUs over that of the control group, it appeared that neither the mechanical or sonic toothbrushes affected cell viability.

Actinomyces viscosus↗
PubMed1998

Detection of Helicobacter pylori associated antigen and heat shock protein 60 on follicular dendritic cells in the germinal centres of low grade B cell lymphoma of gastric mucosa associated lymphoid tissue (MALT).

AIMS: To investigate the localisation of Helicobacter pylori antigens and the expression of human heat shock proteins (HSP) in stomachs affected by MALT lymphoma. METHODS: Surgically resected stomachs from 24 patients with MALT lymphoma were immunostained with anti-H pylori rabbit antibodies (ORP-1 and ORP-2) and anti-human HSP60 mouse monoclonal antibodies (mAb) (LK-1 and LK-2). RESULTS: Follicular dendritic cells of germinal centres in the stomachs affected by MALT lymphoma were immunostained with anti-H pylori polyclonal antibodies and with anti-human HSP60 mAb, as were the epithelial cells. None of the lymph node samples reacted. CONCLUSIONS: Human HSP60, which cross reacts with anti-H pylori polyclonal antibodies, is often expressed on follicular dendritic cells in gastric MALT lymphoma tissues and may be aetiologically relevant to lymphomagenesis of MALT lymphoma.

Antigens, Bacterial↗
PubMed1998

Antibody and cytokine responses in Helicobacter pylori-infected various mouse strains.

Helicobacter pylori (H. pylori) infection in the stomach is etiologically closely associated with chronic active gastritis, peptic ulcer, gastric cancer and gastric mucosa-associated lymphoid tissue lymphoma. In this study, we examined the antibody responses and cytokine profiles of three strains of mice (BALB/c, C3H/He, and C57BL/6) infected with H. pylori. Following this, correlations between host-immune reactions and intensity of inflammation were analyzed. H. pylori (ATCC43504) was intragastrically administered once a week to the mice from 4 weeks of age, and they were sacrificed at the ages of 4 and 7 months. In these mice, we examined the histology of the stomach, antibody titers against H. pylori, and serum levels of cytokines (IL-4, IL-10, TNF-alpha, IL-2 and Interferon-gamma). In BALB/c mice, inflammation of the stomach was minimal. Inflammation was observed in 63.6% of C57BL/6 mice and 33.3% of C3h/He mice. In C57BL/6 and C3H/He mice, all the cytokines tended to increase. In contrast, BALB/c mice were inactive in cytokine production except for IL-2. Two C3H/He mice developed severe inflammation with lymph follicles; one showed a response largely typical of Th-1, and the other showed a response largely typical of Th-2. Although a definite correlation was not shown between Th-1/Th-2 response evaluated by cytokine production and intensity of inflammation, it appears that in H. pylori-induced inflammation both cell-mediated (Th-1) and humoral (Th-2) immunity play a role in pathogenesis.

Animals↗
PubMed1998

Modeling all-cause mortality: projections of the impact of smoking cessation based on the NHEFS. NHANES I Epidemiologic Follow-up Study.

OBJECTIVES: A model that relates clinical risk factors to subsequent mortality was used to simulate the impact of smoking cessation. METHODS: Survivor functions derived from multivariate hazard regressions fitted to data from the first National Health and Nutrition Examination Survey (NHANES I) Epidemiologic Followup Study, a longitudinal survey of a representative sample of US adults, were used to project deaths from all causes. RESULTS: Validation tests showed that the hazard regressions agreed with the risk relationships reported by others, that projected deaths for baseline risk factors closely matched observed mortality, and that the projections attributed deaths to the appropriate levels of important risk factors. Projections of the impact of smoking cessation showed that the number of cumulative deaths would be 15% lower after 5 years and 11% lower after 20 years. CONCLUSIONS: The model produced realistic projections of the effects of risk factor modification on subsequent mortality in adults, Comparison of the projections for smoking cessation with estimates of the risk attributable to smoking published by the Centers for Disease Control and Prevention suggests that cessation could capture most of the benefit possible from eliminating smoking.

Aged↗
PubMed1997

A 150-base pair 5' region of the MHC class I HLA-B7 gene is sufficient to direct tissue-specific expression and locus control region activity: the alpha site determines efficient expression and in vivo occupancy at multiple cis-active sites throughout this region.

To characterize cis- and trans-acting mechanisms that regulate MHC class I transcription during development and in adult tissues, we have used transgenic mice to study a series of human MHC (HLA)-B7 class I gene constructs. Previous studies identified the 5' -0.66-kb to -0.075-kb region as sufficient to direct appropriate and efficient tissue-specific levels of HLA-B7 RNA relative to H-2 class I. Results here show that DNA 5' of -0.26 kb is not required for any aspect of expression. As the expression level correlated with the transgene copy number, was comparable to H-2 or a per-gene copy basis and was independent of integration site, the -0.075 to -0.26-kb segment also functions as a locus control region. With this region, sequences 3' of -0.075 kb, possibly at the promoter, appear to direct the appropriate tissue distribution. Of conserved sequences in the -0.075 to -0.26-kb region, enhancer B box is nonessential. In contrast, in vivo "footprinting" implicated region I/ enhancer A/NF-kappaB, IFN consensus/response sequence, and alpha in class I regulation as they are "occupied" in a tissue-specific pattern that correlates with expression. Mutation of alpha leads to decreased expression and loss of occupancy not only at alpha but also at region I/enhancer A/NF-kappaB and IFN consensus/response sequence. Thus, site alpha is an essential class I regulatory element, the dominant function of which is to mediate tissue-specific occupancy at multiple adjacent cis-active sites, possibly by facilitating stable synergistic interactions between factors at these distinct elements.

Animals↗
PubMed1997

HPV16 E6 oncoprotein stimulates the transforming growth factor-beta 1 promoter in fibroblasts through a specific GC-rich sequence.

Human papillomaviruses (HPV) have been etiologically linked to human cervical cancer. Transforming growth factor-beta 1 (TGF-beta 1) is a cytokine which is a potent growth inhibitor of most epithelial, endothelial, lymphoid, and myeloid cells, but is mitogenic for mesenchymal cells and bone cells. In this study, we analyzed the effects of HPV 16 oncoproteins E6 and E7 on the TGF-beta 1 promoter. The results showed that the HPV 16 E6 significantly induced (sixfold) the TGF-beta 1 promoter activity while HPV 16 E7 showed no significant effect. The E6 effect was cell type-specific and was observed only in the fibroblast cell lines, not in epithelial cells. Promoter analysis revealed that a 9-bp sequence, GGGGCGGGG, representing the consensus Sp1-binding site between -109 and -100 of the TGF-beta 1 promoter, was the major target for E6-mediated transactivation. Mutation analysis of the E6 polypeptide showed that the retention of amino acids between 123 and 136 of the HPV 16 E6 protein was critical for the transactivation of the TGF-beta 1 promoter. Previous studies have shown that the adenovirus 12S E1A oncoprotein represses the TGF-beta 1 promoter by targeting an adjacent (-90 to -81) but different GC-rich sequence (TGGGTGGGG). These studies provide evidence that variant GC-rich promoter elements are not functionally identical and are differentially regulated by the DNA virus oncoproteins.

3T3 Cells↗
PubMed1997

Genomic footprinting of retinoic acid regulated promoters in embryonal carcinoma cells.

Retinoic acid (RA) treatment of embryonal carcinoma (EC) cells initiates a cascade of alterations in gene regulation, leading to their differentiation into various cell types. In P19 EC cells RA treatment stimulates induction of the RAR beta gene, while it represses Oct3/4 gene expression. Here we present dimethylsulfate-based genomic footprinting analyses of these two genes. We found that the RAR beta promoter is not occupied prior to RA treatment, but following RA treatment all regulatory elements in this promoter become occupied. On the other hand, the Oct3/4 promoter is occupied at all three known elements before RA treatment, but this occupancy is coordinately lost following the treatment. Thus, factor occupancy coincides with expression of the genes. It is likely that the presence of factor binding or its absence revealed here represents a mechanism of the regulated expression of these genes in vivo. Our results demonstrate the power of genomic footprinting for studying regulatory events for transcription in vivo. In contrast, with in vitro protein-DNA binding assay, factors for both promoters are present in these cells regardless of RA treatment. It has been shown that RA receptor (RAR) and retinoid X receptor (RXR), by heterodimerization, mediate the RA action in EC cells. To elucidate the role of RAR/RXR heterodimers in the RAR beta promoter occupancy in vivo, genomic footprinting has been performed in P19 cells stably expressing dominant negative mutants of RXR. Two such mutants, lacking either the DNA binding domain or the C-terminal activation domain, inhibit RA induction of the RAR beta gene in these cells. RA-induced factor occupancy is also markedly inhibited at all elements in the RAR beta promoter in these cells. Our results show that binding of liganded RAR/RXR heterodimers to RARE is required for other factors to gain access to their respective elements in the promoter.

Animals↗
PubMed1997

Induction of MHC class I expression by the MHC class II transactivator CIITA.

Major histocompatibility complex (MHC) class I-deficient cell lines were used to demonstrate that the MHC class II transactivator (CIITA) can induce surface expression of MHC class I molecules. CIITA induces the promoter of MHC class I heavy chain genes. The site alpha DNA element is the target for CIITA-induced transactivation of class I. In addition, interferon-gamma (IFNgamma)-induced MHC class I expression also requires an intact site alpha. The G3A cell line, which is defective in CIITA induction, does not induce MHC class I antigen and promoter in response to IFNgamma. Trans-dominant-negative forms of CIITA reduce class I MHC promoter function and surface antigen expression. Collectively, these data argue that CIITA has a role in class I MHC gene induction.

Breast Neoplasms↗