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A Dey

Publications and source records attributed to A Dey.

At least 55 records · Page 3Linked to original sources
PubMed1997

Retinoid-induced chromatin structure alterations in the retinoic acid receptor beta2 promoter.

Transcription of the retinoic acid receptor beta2 (RARbeta2) gene is induced by retinoic acid (RA) in mouse P19 embryonal carcinoma (EC) cells. Here we studied RA-induced chromatin structure alterations in the endogenous RARbeta2 promoter and in an integrated, multicopy RARbeta2 promoter in EC cells. RA markedly increased restriction site accessibility within the promoter, including a site near the RA responsive element (RARE) to which the nuclear receptor retinoid X receptor (RXR)-RAR heterodimer binds. These changes coincided with RA-induced alterations in the DNase I hypersensitivity pattern in and around the promoter. These changes became undetectable upon removal of RA, which coincided with the extinction of transcription. Analyses with receptor-selective ligands and an antagonist showed that increase in restriction site accessibility correlates with transcriptional activation, which parallels the RA-induced in vivo footprint of the promoter. Despite these changes, the micrococcal nuclease digestion profile of this promoter was not altered by RA. These results indicate that concurrent with the binding of the RXR-RAR heterodimer to the RARE, the local chromatin structure undergoes dynamic, reversible changes in and around the promoter without globally affecting the nucleosomal organization.

Animals↗
PubMed1997

Retinoid X receptor (RXR) within the RXR-retinoic acid receptor heterodimer binds its ligand and enhances retinoid-dependent gene expression.

Retinoic acid receptor (RAR) and retinoid X receptor (RXR) form heterodimers and regulate retinoid-mediated gene expression. We studied binding of RXR- and RAR-selective ligands to the RXR-RAR heterodimer and subsequent transcription. In limited proteolysis analyses, both RXR and RAR in the heterodimer bound their respective ligands and underwent a conformational change in the presence of a retinoic acid-responsive element. In reporter analyses, the RAR ligand (but not the RXR ligand), when added singly, activated transcription, but coaddition of the two ligands led to synergistic activation of transcription. This activation required the AF-2 domain of both RXR and RAR. Genomic footprinting analysis was performed with P19 embryonal carcinoma cells, in which transcription of the RARbeta gene is induced upon retinoid addition. Paralleling the reporter activation data, only the RAR ligand induced in vivo occupancy of the RARbeta2 promoter when added singly. However, at suboptimal concentrations of RAR ligand, coaddition of the RXR ligand increased the stability of promoter occupancy. Thus, liganded RXR and RAR both participate in transcription. Finally, when these ligands were tested for teratogenic effects on zebra fish and Xenopus embryos, we found that coadministration of the RXR and RAR ligands caused more severe abnormalities in these embryos than either ligand alone, providing biological support for the synergistic action of the two ligands.

Animals↗
PubMed1996

Retinoic acid-mediated down-regulation of Oct3/4 coincides with the loss of promoter occupancy in vivo.

Oct3/4, a hallmark of the earliest stages of embryogenesis, is expressed in undifferentiated embryonal carcinoma (EC) and embryonic stem (ES) cells. Oct3/4 gene expression is dependent on the promoter region, the proximal enhancer and the newly identified distal enhancer. We have analysed in vivo occupancy of these elements. In undifferentiated EC and ES cells, strong footprints were detected at specific sites of all three regulatory elements. These were promptly lost upon RA treatment in ES cells and in P19 EC cells, in parallel with sharply reduced Oct3/4 mRNA levels. Thus, the occupancy of regulatory elements is coupled with Oct3/4 expression, and RA treatment causes coordinated factor displacement, leading to extinction of gene activity. In F9 EC cells, footprint was first abolished at the proximal enhancer. However, this loss of binding site occupancy did not result in a decrease in Oct3/4 mRNA levels. The partial factor displacement seen in F9 EC cells, combined with the observation that EC and ES cells utilize the proximal and distal enhancers in differential manner, indicate the complex pattern of Oct3/4 gene regulation, which could reflect a cell type- and lineage-specific expression of the gene in vivo.

Animals↗
PubMed1996

Inhibition of ligand induced promoter occupancy in vivo by a dominant negative RXR.

BACKGROUND: Retinoid X receptors (RXRs) heterodimerize with other nuclear hormone receptors and control ligand mediated transcription. To address how RXRs function as heterodimers, we investigated activities of truncated RXR alpha and RXR beta that lack approximately 20 conserved C-terminal amino acids. RESULTS: The truncated RXRs formed heterodimers and bound to respective DNA elements in vitro. By transient reporter assays we found that these RXRs act as dominant negative receptors and inhibit ligand dependent transcription by the retinoic acid receptor (RAR) and vitamin D receptor. P19 embryonal carcinoma cells stably expressing the truncated RXR beta (termed delta C2) were deficient in activating the endogenous RAR beta gene and an RA responsive reporter. To study the dominant negative activity of delta C2 further, genomic footprinting analysis was performed for the RAR beta2 promoter. In control P19 clones, the RA responsive element (RARE) and other elements in the promoter were protected after RA treatment. However, in delta C2 clones RA-induced protection was markedly inhibited at all elements. CONCLUSIONS: These results indicate that the C-terminal region of RXR is required for full RARE occupancy in vivo, a RA dependent process that leads to the recruitment of other factors to the promoter and the subsequent transcriptional activation. Thus, RXRs play an integral role in ligand dependent transcription.

Animals↗
PubMed1996

Plantar ulceration in patients with leprosy.

A controlled trial was implemented to investigate the hypothesis that plantar ulcer sites associated with leprosy, in subjects who were supplied with foot orthoses, would heal more quickly than those in a control group. The population was comprised of individuals with impairments associated with leprosy, living near a leprosy hospital in India. Ulcer area was compared in the two groups and assessed at three-monthly intervals from July 1993 to March 1994. At the March 1994 assessment 58% of ulcers in the experimental group had healed but only 14% in the control group. At this point patients in the control group were supplied with orthoses and three months later, in June 1994, 60% of the ulcers in the experimental group and 62% of ulcers in the control group had healed. The results of this study demonstrate that orthotic intervention can significantly reduce the duration of ulceration.

Adult↗
PubMed1995

A water-vapour giga-maser in the active galaxy TXFS2226-184.

Active galactic nuclei are thought to be powered by gas falling into a massive black hole; the different types of active galaxy may arise because we view them through a thick torus of molecular gas at varying angles of inclination. One way to determine whether the black hole is surrounded by a torus, which would obscure the accretion disk around the black hole along certain lines of sight, is to search for water masers, as these exist only in regions with plentiful molecular gas. Since the first detection of an extra-galactic water maser in 1979, they have come to be associated primarily with active galaxies, and have even been used to probe the mass of the central engine. Here we report the detection of a water giga-maser in the radio galaxy TXFS2226-184. The strength of the emission supports a recently proposed theory of maser pumping that allows for even more powerful masers, which might be detectable at cosmological distances. Water masers may accordingly provide a way to determine distances to galaxies outside the usual distance ladder, providing an independent calibration of the Hubble constant.

Astronomical Phenomena↗
PubMed1995

Displacement of sequence-specific transcription factors from mitotic chromatin.

The general inhibition in transcriptional activity during mitosis abolishes the stress-inducible expression of the human hsp70 gene. Among the four transcription factors that bind to the human hsp70 promoter, the DNA-binding activities of three (C/EBP, GBP, and HSF1) were normal, while Sp1 showed reduced binding activity in mitotic cell extracts. In vivo footprinting and immunocytochemical analyses revealed that all of the sequence-specific transcription factors were displaced from promoter sequences as well as from bulk chromatin during mitosis. The correlation of transcription factor displacement with chromatin condensation suggests an involvement of chromatin structure in mitotic repression. However, retention of DNase I hypersensitivity suggests that the hsp70 promoter was not organized in a canonical nucleosome structure in mitotic chromatin. Displacement of transcription factors from mitotic chromosomes could present another window in the cell cycle for resetting transcriptional programs.

Base Sequence↗
PubMed1995

Nuclear disintegration in chicken peritoneal macrophages exposed to fumonisin B1 from Indian maize.

The effects of fumonisin B1 (FB1) on the morphology of chicken peritoneal macrophages were studied. It was found that FB1-exposure caused nuclear disintegrations in peritoneal macrophages. The number of macrophages experiencing nuclear disintegration increased with the rise in the dose of FB1 from 6 to 18 micrograms ml-1. The finding implies that dietary intake of FB1 may damage the macrophages in consumers rendering the latter susceptible to infection.

Animals↗
PubMed1995

Patterns of cocaine use among individuals in substance abuse treatment.

Recognition of the heterogeneity among substance abusers has led to increased attention to the issue of client-treatment matching. One approach involves identifying different patterns of use within samples of substance abusers. The present preliminary report employed a cluster analysis of various aspects of cocaine use to examine subgroups of cocaine users within a sample of individuals in treatment for substance abuse. Three clusters emerged, differentiated primarily by quantity/frequency of use and route of administration. The resulting subgroups were compared on measures of external validity. Results suggest that subgroups of cocaine use patterns do exist within a sample of substance abusers. In addition, these subgroups differ in age of cocaine use onset and the severity of negative consequences. Future research is needed to establish the clinical utility of these clusters.

Adolescent↗
PubMed1995

The efficacy of podiatric orthoses as an adjunct to the treatment of plantar ulceration in leprosy.

This study examines the outcome of a management approach to plantar ulceration secondary to leprotic neuropathy. Locally-available resources were used to produce podiatric orthoses which were supplied to an experimental group. The effects on healing time and quality of healing were compared with a control group. Both groups were ambulant (the programme sought not to interfere with socioeconomic independence). Using standard nonparametric methods of analysis, it was demonstrated that the effects on healing rate, attributed to the experimental intervention, were highly significant. Over a 7-month period, 57% of the experimental group ulcers healed, while only 12.5% of the control group ulcers healed. The experimental intervention also demonstrated a positive effect on the quality of ulcer healing. This effect was not reflected in the control group.

Foot Ulcer↗
PubMed1994

Dominant negative retinoid X receptor beta inhibits retinoic acid-responsive gene regulation in embryonal carcinoma cells.

Retinoid X receptors (RXRs) heterodimerize with multiple nuclear hormone receptors and are thought to exert pleiotropic functions. To address the role of RXRs in retinoic acid- (RA) mediated gene regulation, we designed a dominant negative RXR beta. This mutated receptor, termed DBD-, lacked the DNA binding domain but retained the ability to dimerize with partner receptors, resulting in formation of nonfunctional dimers. DBD- was transfected into P19 murine embryonal carcinoma (EC) cells, in which reporters containing the RA-responsive elements (RAREs) were activated by RA through the activity of endogenous RXR-RA receptor (RAR) heterodimers. We found that DBD- had a dominant negative activity on the RARE reporter activity in these cells. P19 clones stably expressing DBD- were established; these clones also failed to activate RARE-driven reporters in response to RA. Further, these cells were defective in RA-induced mRNA expression of Hox-1.3 and RAR beta, as well as in RA-induced down-regulation of Oct3 mRNA. Gel mobility shift assays demonstrated that RA treatment of control P19 cells induces RARE-binding activity, of which RXR beta is a major component. However, the RA-induced binding activity was greatly reduced in cells expressing DBD-. By genomic footprinting, we show that RA treatment induces in vivo occupancy of the RARE in the endogenous RAR beta gene in control P19 cells but that this occupancy is not observed with the DBD- cells. These data provide evidence that the dominant negative activity of DBD- is caused by the lack of receptor binding to target DNA. Finally, we show that in F9 EC cells expression of DBD- leads to inhibition of the growth arrest that accompanies RA-induced differentiation. Taken together, these results demonstrate that RXR beta and partner receptors play a central role in RA-mediated gene regulation and in the control of growth and differentiation in EC cells.

Animals↗
PubMed1994

Ligand-dependent occupancy of the retinoic acid receptor beta 2 promoter in vivo.

Retinoic acid (RA) activates transcription of the RA receptor beta 2 (RAR beta 2) gene in embryonal carcinoma (EC) cells. This activation involves binding of the RAR/retinoid X receptor (RAR/RXR) heterodimer to the RA-responsive element (beta RARE). Dimethyl sulfate-based genomic footprinting was performed to examine occupancy of this promoter in P19 EC cells. No footprint was detected at the beta RARE prior to RA treatment, but a footprint was detected within the first hour of RA treatment. Concomitantly, other elements in the promoter, the cyclic AMP-responsive element and tetradecanoyl phorbol acetate-like-responsive element became footprinted. Footprints at these elements were induced by RA without requiring new protein synthesis and remained for the entire duration of RA treatment but rapidly reversed upon withdrawal of RA. A delayed protection observed at the initiator site was also reversed upon RA withdrawal. The RA-inducible footprint was not due to induction of factors that bind to these element, since in vitro assays showed that these factors are present in P19 cell extracts before RA treatment. Significantly, no RA-induced footprint was observed at any of these elements in P19 cells expressing a dominant negative RXR beta, in which RXR heterodimers are unable to bind to the beta RARE. Results indicate that binding of a liganded heterodimer receptor to the beta RARE is the initial event that allows other elements to gain access to the factors. In accordance, reporter analyses showed that a mutation in the beta RARE, but not those in other elements, abrogates RA activation of the promoter. It is likely that the RAR beta 2 promoter opens in a hierarchically ordered manner, signalled by the occupancy of liganded heterodimers.

Animals↗
PubMed1993

A regulatory element in the beta 2-microglobulin promoter identified by in vivo footprinting.

Expression of the beta 2-microglobulin (beta 2-m) and major histocompatibility complex (MHC) class I genes is coordinately regulated. By ligation-mediated polymerase chain reaction, we have analyzed in vivo factor binding to the promoter region of the murine beta 2-m gene. In adult spleen, in which beta 2-m is expressed, strong protection was found in three elements. Two of these elements, the beta 2-m NF-kappa B binding site and the interferon consensus sequence, are homologous to the regulatory elements of the MHC class I genes and were also found to be protected in spleen. A third protected element, PAM, identified in this work, is unique to the beta 2-m gene. None of the elements showed protection in brain tissue, in which neither the beta 2-m nor the MHC class I gene is expressed. In vivo footprinting was also performed with F9 embryonal carcinoma cells, in which expression of the beta 2-m and MHC class I genes is induced at a low level only upon stimulation with retinoic acid (RA). No in vivo protection was detected before and after RA treatment of F9 cells, indicating that RA induction of beta 2-m (and MHC class I) expression occurs without detectable in vivo factor occupancy, whereas EL4 T lymphocytes expressing beta 2-m at a high level exhibited strong protection similar to that in spleen. Despite the lack of in vivo occupancy, the nuclear factors specific for each of the three elements were present in brain tissue and F9 cells as well as in spleen tissue and EL4 cells. We show that PAM, an element identified by its in vivo protection, binds nuclear factors ranging from 40 to 50 kDa in size and is capable of enhancing transcription of a reporter in F9 and other cells. Taken together, these results indicate that in vivo factor occupancy for the beta 2-m and MHC class I promoters is coordinated and occurs through a mechanism other than mere expression of relevant factors.

Animals↗
PubMed1992

Occupancy of upstream regulatory sites in vivo coincides with major histocompatibility complex class I gene expression in mouse tissues.

The major histocompatibility complex (MHC) class I HLA-B7 transgene carrying a 660-bp upstream sequence is expressed in the mouse with tissue specificity that parallels that of the expression of endogenous mouse MHC class I (H-2) genes. We have performed in vivo genomic footprinting for the HLA-B7 transgene and the endogenous H-2Kb gene. We show that the upstream region of both the transgene and the endogenous gene was extensively occupied in spleen tissue, where these genes are expressed at high levels. In contrast, no occupancy was detected in brain tissue, where expression of these genes is virtually absent. Sites exhibiting in vivo protection correspond to cis elements previously shown to bind to nuclear factors in vitro, including the constitutive enhancer region I and the interferon response element. The strongest tissue-specific protection was detected at site alpha, located downstream from the interferon response element. Site alpha bound a constitutively expressed nuclear factor(s) in vitro that exhibited an overlapping specificity which may involve a nuclear hormone receptor, RXR, and an AP-1-related factor. Site alpha was functional in vivo, as it enhanced MHC class I transcription in lymphocytes. These results show that the tissue-specific occupancy of the MHC class I regulatory sequences in vivo correlates with their expression and suggest that in vivo occupancy is controlled by a mechanism other than the mere presence of factors capable of binding to these sites. Our results suggest that a sequence present in the 660-bp upstream region in a human leukocyte antigen gene directs tissue-specific occupancy of MHC class I genes in vivo, independently of their position and copy number, illustrating a potential advantage of using a transgene for delimitation of the sequence requirement for in vivo occupancy.

Animals↗
PubMed1991

The AP-1 site and the cAMP- and serum response elements of the c-fos gene are constitutively occupied in vivo.

The c-fos proto-oncogene is inducible by cAMP, phorbol esters, serum, and growth factors. The induction by cAMP is mediated by the conserved cAMP response element (CRE), while induction by phorbol esters, serum, and growth factors requires a distal element called the serum response element (SRE). In addition to these elements, a consensus AP-1 transcription factor binding site is located next to SRE. Upstream regions of the mouse and human c-fos genes were footprinted in vivo by the ligation-mediated polymerase chain (PCR). Our results show that all three elements are constitutively protected in mouse liver and lung and in cultured human A431 cells. No major change in the protection profile was detected in A431 cells following stimulation with epidermal growth factor or in mice at birth, when c-fos is known to be induced. These results suggest that the inducible cis elements of the c-fos gene are poised, ready to respond immediately to external signals.

Animals↗