PubMed Health⌕ Search

Biomedical subjects

B H Howard

Publications and source records attributed to B H Howard.

At least 37 records · Page 2Linked to original sources
PubMed1996

RAR and RXR selective ligands cooperatively induce apoptosis and neuronal differentiation in P19 embryonal carcinoma cells.

Retinoids cause differentiation in embryonal carcinoma (EC) cells, thus mimicking events in mammalian development. Here, we show that retinoids also cause apoptosis in P19 EC cells. Characteristic DNA fragmentation was observed within 36 h after addition of retinoic acid (RA). Synthetic retinoids that are selective for RA receptors (RAR) were also effective in inducing apoptosis, whereas RXR selective ligands were without effect. The combination of RAR and RXR ligands resulted in a synergistic increase in apoptotic cell death. As with apoptosis, neuronal differentiation of P19 cells was synergistically induced by the combination of RAR and RXR ligands. Data obtained with an RAR antagonist and with P19 cells carrying a dominant negative RXR indicate that the two processes are receptor mediated. Together, our results indicate that retinoid-induced apoptosis and neuronal differentiation are closely coupled, and that both RAR and RXR play a role in these processes as active receptors for their respective ligands.

Apoptosis↗
PubMed1996

Human fibroblast commitment to a senescence-like state in response to histone deacetylase inhibitors is cell cycle dependent.

Human diploid fibroblasts (HDF) complete a limited number of cell divisions before entering a growth arrest state that is termed replicative senescence. Two histone deacetylase inhibitors, sodium butyrate and trichostatin A, dramatically reduce the HDF proliferative life span in a manner that is dependent on one or more cell doublings in the presence of these agents. Cells arrested and subsequently released from histone deacetylase inhibitors display markers of senescence and exhibit a persistent G1 block but remain competent to initiate a round of DNA synthesis in response to simian virus 40 T antigen. Average telomere length in prematurely arrested cells is greater than in senescent cells, reflecting a lower number of population doublings completed by the former. Taken together, these results support the view that one component of HDF senescence mimics a cell cycle-dependent drift in differentiation state and that propagation of HDF in histone deacetylase inhibitors accentuates this component.

Antigens, Viral, Tumor↗
PubMed1996

Specific binding sites for a pol III transcriptional repressor and pol II transcription factor YY1 within the internucleosomal spacer region in primate Alu repetitive elements.

Alu interspersed repetitive elements possess internal RNA polymerase III promoters that are transcribed in vitro and in transfected mouse cells but are nearly silent in human HeLa cells. Transcriptional repression of these elements is to some extent reversible, as pol III-dependent Alu expression can be induced with herpes simplex or adenovirus. To assess whether sequence-specific DNA binding proteins might contribute to Alu transcriptional silencing, we examined the internucleosomal spacer region surrounding the B box of the Alu pol III promoter in HeLa cell nuclei for evidence of proteins bound at specific sites in vivo. We identified a DNase I-hypersensitive site 5' to the B box and a DNase I-resistant region 3' to the B box in nuclei. An Alu-specific repressor binds to a 5-bp inverted repeat motif overlapping the 5' end of the TFIIIC binding site and may inhibit pol III transcription through competitive displacement. The level of Alu-specific pol III repressor activity is significantly reduced in adenovirus-infected HeLa cells, suggesting that the repressor may contribute to Alu transcriptional silencing in vivo. The 3' DNase I-resistant region coincided with a binding site for the pol II transcription factor YY1 in vitro. YY1 is one of the major proteins in HeLa cells having binding specificity for Alu elements. YY1 bound to tandem arrays of genomic Alu elements may play a role in chromatin organization and silencing.

Adenoviridae↗
PubMed1995

Nucleosome positioning by human Alu elements in chromatin.

Alu sequences are interspersed throughout the genomes of primate cells, occurring singly and in clusters around RNA polymerase II-transcribed genes. Because these repeat elements are capable of positioning nucleosomes in in vitro reconstitutes (Englander, E. W., Wolffe, A. P., and Howard, B. H. (1993) J. Biol. Chem. 268, 19565-19573), we investigated whether they also influence in vivo chromatin structure. When assayed collectively using consensus sequence probes and native chromatin as template, Alu family members were found to confer rotational positioning on nucleosomes or nucleosome-like particles. In particular, a 10-base pair pattern of DNase I nicking that spanned the RNA polymerase III box A promoter motif extended upstream to cover diverse 5'-flanking sequences, suggesting that Alu repeats may influence patterns of nucleosome formation over neighboring regions. Computational analysis of a set of naturally occurring Alu sequences indicated that nucleosome positioning information is intrinsic to these elements. Inasmuch as local chromatin organization influences gene expression, the capacity of Alu sequences to affect chromatin structure as demonstrated here may help to clarify some features of these elements.

Base Sequence↗
PubMed1995

Adenovirus type 2 preferentially stimulates polymerase III transcription of Alu elements by relieving repression: a potential role for chromatin.

The number of Alu transcripts that accumulate in HeLa and other human cells is normally very low; however, infection with adenovirus type 5 increases the expression of Alu elements dramatically, indicating that the potential for polymerase III (pol III)-dependent Alu transcription in vivo is far greater than generally observed (B. Panning and J.R. Smiley, Mol. Cell. Biol. 13:3231-3244, 1993). In this study, we employed nuclear run-on in combination with a novel RNase H-based assay to investigate transcription from uninfected and adenovirus type 2-infected nuclei, as well as genomic DNAs from uninfected and infected cells. When performed in the presence of excess uninfected nuclear extract, such assays revealed that (i) the vast majority of transcriptionally competent Alu elements in nuclei are masked from the pol III transcriptional machinery and (ii) the induction of Alu expression upon adenovirus infection can be largely accounted for by an increased availability of these elements to the pol III transcription machinery. We also investigated the role of H1 histone for silencing of Alu genes and, in comparison, mouse B2 repetitive elements. Depletion of H1 led to an approximately 17-fold activation of B2 repetitive elements but did not change Alu transcription relative to that of constitutively expressed 5S rRNA genes. These results are consistent with the view that Alu repeats are efficiently sequestered by chromatin proteins, that such masking cannot be accounted for by nonspecific H1-dependent repression, and that adenovirus infection at least partially overrides the repressive mechanism(s).

3T3 Cells↗
PubMed1994

Methylation- and mutation-dependent stimulation of Alu transcription in vitro.

Alu genes are GC-rich, highly repetitive genetic elements whose functions remain unknown. Members of this family are readily transcribed in vitro by RNA polymerase III, but RNA corresponding to only a small sub-set of Alu elements has been found in vivo. Based on the hypothesis that methylation of Alu elements affects their transcription, the transcriptional activity of unmethylated and methylated template DNA was assessed in vitro. It was found that methylation of a single CG site just 5' to Alu functions to stimulate transcription; the base composition in this region also affects transcriptional activity. These results indicate that the methylation state and sequence of DNA flanking Alu elements influence its transcription rate.

Base Sequence↗
PubMed1994

Lumbar epidural varix as a cause of radiculopathy.

STUDY DESIGN: Venous outflow obstruction or congestion has been implicated in the etiology of some lumbar radicular syndromes. Ten cases of epidural varices or variants manifesting themselves as masses within the lumbar spinal canal or adjacent foramina have been seen. OBJECTIVES: Diagnosis was made by multiplanar cross-sectional magnetic resonance and computed tomography imaging and confirmed in five patients by operative findings. These venous abnormalities differ from cases of arteriovenous malformations described in the literature. METHOD: Surgical treatment consisting of coagulative oblation and/or excision of the lesion was consistently successful in the five patients who underwent operations for recalcitrant symptoms. The other five patients experienced spontaneous resolution of symptoms. Three distinctly different types of these abnormalities have been identified. RESULTS: The described entity may account for some previously unexplained or spontaneously resolving cases of lumbar radiculitis. CONCLUSIONS: Knowledge of the existence of this condition, its possible etiologies, and its imaging characteristics may assist in its recognition and improved management of patients with it.

Epidural Space↗
PubMed1994

Dissociation of retinoblastoma gene protein hyperphosphorylation and commitment to enter S phase.

Mitogenic activities of simian virus 40 large T and small t antigens were studied in serum-deprived human diploid fibroblasts. Wild-type large T and small t cooperated in stimulating DNA synthesis and in inducing hyperphosphorylation of the Rb gene product (pRb). In contrast, a T antigen mutant defective for pRb binding (Rb- T) possessed no detectable mitogenic activity alone and failed to complement small t in stimulating DNA synthesis. Surprisingly, Rb- T and small t cooperated as strongly as wild-type T and small t with respect to pRb hyperphosphorylation. As a consequence, in two closely related conditions (i.e., stimulation by small t plus wild-type T versus small t plus Rb- T), the fraction of pRb in hyperphosphorylated forms dissociated from the fraction of cells in the S phase. These results indicate that pRb hyperphosphorylation is not always tightly coupled with a commitment to initiate DNA replication.

Antigens, Polyomavirus Transforming↗
PubMed1993

Nucleosome interactions with a human Alu element. Transcriptional repression and effects of template methylation.

Alu interspersed repetitive elements possess internal RNA polymerase III promoters which are strongly transcribed in vitro, yet these elements are nearly silent in somatic cells. To examine whether repression by chromatin proteins could contribute to the low level of Alu expression, a conserved Alu element from the fourth intron of the human alpha-fetoprotein gene was reconstituted with purified octamer or tetramer particles. Analysis of reconstitutes revealed that this Alu element directed translational and rotational positioning of octamers as well as tetramers. In vitro transcription experiments with reconstituted templates demonstrated that RNA polymerase III-dependent transcription of the Alu element was profoundly repressed by positioned octamer particles. Furthermore, complete CpG methylation of this template enhanced the capacity of tetramers to repress transcription.

Animals↗
PubMed1992

Expression of Alu and 7SL RNA in Alzheimer's and control brains.

We investigated whether changes in expression of RNA polymerase III (pol III) or heterodisperse RNA polymerase II (pol II) transcripts hybridizing to Alu could be detected in Alzheimer's disease (AD). RNA samples obtained from AD and control brain tissues were examined by Northern analysis for Alu and 7SL RNA expression. All RNA samples contained a prominent band of approximately 300 nucleotides which corresponds to 7SL RNA, the Alu-homologous RNA component of the signal recognition particle. In addition, three small (i.e. less than 300 nucleotide) 7SL/Alu-hybridizing transcripts were detected. The two larger of the low molecular weight transcripts hybridized preferentially to the 7SL RNA probe, while the smallest transcript hybridized to the Alu probe. These transcripts and the heterodisperse RNA were variable in quantity and displayed a lack of correlation with AD.

Alzheimer Disease↗
PubMed1991

Magnetic-affinity cell sorting of human multidrug-resistant cells.

We have developed a method for isolating multidrug-resistant cells from a heterogeneous cell population using the magnetic-affinity cell-sorting system. Human KB carcinoma cell lines expressing different amounts of P-glycoprotein have been selected by use of the monoclonal antibody MRK-16 coupled to magnetic particles. This specific, rapid, and sensitive method allows the selection of viable and clonable drug-resistant cells from various drug-resistant human and murine cell populations. This method may prove useful in isolating drug-resistant cells from tumors with heterogeneous P-glycoprotein expression for further analysis.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1991

Modulation of HeLa cell growth by transfected 7SL RNA and Alu gene sequences.

Alu and 7SL RNA gene sequences were tested for the potential to regulate mammalian cell growth by introducing these sequences into HeLa cells in a coupled DEAE-dextran transfection/affinity cell sorting system. Both Alu and 7SL RNA genes mediated inhibition of [3H]thymidine and [35S]methionine incorporation in recipient cells. In addition, short term growth curves were performed on calcium phosphate/DNA cotransfected, affinity-purified cells. This second assay revealed that transfected Alu and 7SL RNA can also cause suppression of HeLa cell proliferation. To investigate whether transcription or polymerase III (pol III) transcription factor binding was required for inhibitory activity, mutations were introduced into RNA pol III B block promoter elements in each of these genes. Suppression of [3H]thymidine incorporation was dependent on the presence of this pol III element in both genes; likewise, 7SL RNA-mediated growth suppression required the presence of the pol III B block promoter element. The results of this study indicate that Alu and 7SL RNA gene sequences interact with cellular factors that are important for HeLa cell proliferation and suggest that these pol III-transcribed elements may be involved in the regulation of cellular growth.

Base Sequence↗
PubMed1991

Expression of GTP-binding proteins and prostaglandin E2 receptors during human T cell activation.

GTP-binding proteins (G-proteins) are a family of closely related, yet structurally distinct signal transducing proteins. In this study the presence and relative abundance of several G-proteins and of their corresponding mRNAs were measured in resting and activated human T lymphocytes. We found that T lymphocytes contain RNA coding for Gs, Gi2, and Gi3. No Gi1- and Go-specific RNA could be detected. Membrane fractions of resting and activated lymphocytes were studied in immunoblot experiments. Again, Gs, Gi2, and Gi3, but not Gi1 and Go, were detected. Upon mitogenic activation, a relative increase in mRNA for Gs and Gi3, but not for Gi2 could be demonstrated in Northern blot experiments. Immunoblotting indicated an increase in Gs and Gi3 density in membrane fractions of T cells as well. Paralleling the increase in Gs, we found that activated T cells produce five to seven times more cAMP per cell in response to prostaglandin E2 (PGE2) than resting lymphocytes. Finally, PGE2 binding studies showed that the number of receptors for this hormone increased from 435 +/- 322 to 1035 +/- 357 per cell following in vitro stimulation. We propose that in vitro T cell activation is paralleled by an increase in sensitivity to PGE2-induced cAMP generation. This sensitization is accompanied by both an increase in cell surface PGE2 receptor numbers as well as by increased expression of the signal transducing protein Gs and may physiologically be important for limiting an immune response.

Cyclic AMP↗
PubMed1991

Isolation of a population of transiently transfected quiescent and senescent cells by magnetic affinity cell sorting.

Rous sarcoma virus (RSV) and cytomegalovirus (CMV) promoters were tested for activity in proliferating and nonproliferating (quiescent or senescent) human embryo fibroblasts. These promoters were cloned upstream of the coding sequence for the Tac subunit of the interleukin 2 receptor, and activity was calculated from the fraction of Tac antigen positive cells detected in a coupled transient transfection/magnetic affinity cell sorting assay. Differences in promoter activities are substantial in quiescent cells: the efficiency of the RSV promoter is no greater than background whereas the CMV promoter is equally active in serum concentrations ranging from 0.5 to 20%. While both promoters are functional in growing cells (WI-38 and HeLa), the CMV promoter exhibits twofold greater activity. Surprisingly, in senescent cells both promoters exhibit the same degree of activity.

Avian Sarcoma Viruses↗
PubMed1991

Sequence-specific toxicity of transfected retroviral DNA.

Experimental gene transfer and viral infections can result in the accumulation of unintegrated DNA in target cells. The effects of such accumulation on target cell metabolism have not been directly studied. The experiments reported in this paper show that transfection of cloned retroviral long-terminal-repeat (LTR) DNA, or of a variety of eukaryotic promoters, into proliferating HeLa cells results in rapid, sequence-specific, and dose-dependent cell death. Plasmids containing the Rous sarcoma virus LTR or the human immunodeficiency virus LTR cloned in pUC-related plasmids are 5 to 10 times more toxic than pUC19. The demonstrated sensitivity of eukaryotic cells to exogenously introduced DNA has important implications for the interpretation of gene transfer experiments and may be relevant to the pathogenic mechanisms in the course of retroviral infections such as AIDS.

Avian Sarcoma Viruses↗
PubMed1991

A rapid method for recombination and site-specific mutagenesis by placing homologous ends on DNA using polymerase chain reaction.

We have developed a novel polymerase chain reaction (PCR) method that permits the rapid generation of site-specific mutants and recombinant DNA constructs with a minimum number of steps and primers. DNA segments are modified by using amplifying primers that add homologous ends to the polymerase chain reaction product(s). These homologous ends undergo recombination in vivo following transformation of recA-E. coli strains used routinely in cloning. In vivo circularization of PCR products containing plasmid sequences with a selective marker permits the rapid cloning of the desired mutant or recombinant. In the mutagenesis protocol, 7 of the 12 clones contained the product of interest, and 6 of these clones had no detected error (50% of the clones without detected errors). In each of several recombination protocols, at least 50% of the clones tested contained the insert of interest without detected errors.

Base Sequence↗