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Biomedical subjects

S Hayes

Publications and source records attributed to S Hayes.

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

Steroid hormone receptors activate transcription in glial cells of intact retina but not in primary cultures of retinal glial cells.

We have compared the steroid responsiveness of Müller glial cells of intact embryonic chicken retina with that of primary cultures derived from Müller glia. Appropriately constructed fusion genes were found to be highly glucocorticoid inducible after their cotransfection with an expression vector encoding the human glucocorticoid receptor (GR) into intact embryonic d-10 (E10) or E5.5 retina. Dramatically attenuated inductions were obtained after contransfection of Müller cell primary cultures. The progesterone receptor (PR) was also demonstrated to function in intact retina, but not in Müller cell primary cultures. An immunochemical assay was utilized to confirm that a glucocorticoid-responsive, beta-galactosidase-encoding fusion gene was specifically induced in Müller cells after its transfection into intact retina. Thus, in contrast to Müller cells in intact retina, Müller cells in primary culture have lost the capacity to achieve transcriptional activation by steroid receptors. We postulate that coordinate expression of the GR, and other more general factors required for steroid inducibility, is lost by dispersion and primary culture of retinal Müller glial cells.

Animals↗
PubMed1997

p27Kip1 induces an accumulation of the repressor complexes of E2F and inhibits expression of the E2F-regulated genes.

p27Kip1 is an inhibitor of the cyclin-dependent kinases and it plays an inhibitory role in the progression of cell cycle through G1 phase. To investigate the mechanism of cell cycle inhibition by p27Kip1, we constructed a cell line that inducibly expresses p27Kip1 upon addition of isopropyl-1-thio-beta-D-galactopyranoside in the culture medium. Isopropyl-1-thio-beta-D-galactopyranoside-induced expression of p27Kip1 in these cells causes a specific reduction in the expression of the E2F-regulated genes such as cyclin E, cyclin A, and dihydrofolate reductase. The reduction in the expression of these genes correlates with the p27Kip1-induced accumulation of the repressor complexes of the E2F family of factors (E2Fs). Our previous studies indicated that p21WAF1 could disrupt the interaction between cyclin/cyclin-dependent kinase 2 (cdk2) and the E2F repressor complexes E2F-p130 and E2F-p107. We show that p27Kip1, like p21WAF1, disrupts cyclin/cdk2-containing complexes of E2F-p130 leading to the accumulation of the E2F-p130 complexes, which is found in growth-arrested cells. In transient transfection assays, expression of p27Kip1 specifically inhibits transcription of a promoter containing E2F-binding sites. Mutants of p27Kip1 harboring changes in the cyclin- and cdk2-binding motifs are deficient in inhibiting transcription from the E2F sites containing reporter gene. Moreover, these mutants of p27Kip1 are also impaired in disrupting the interaction between cyclin/cdk2 and the repressor complexes of E2Fs. Taken together, these observations suggest that p27Kip1 reduces expression of the E2F-regulated genes by generating repressor complexes of E2Fs. Furthermore, the results also demonstrate that p27Kip1 inhibits expression of cyclin A and cyclin E, which are critical for progression through the G1-S phases.

Animals↗
PubMed1996

The grpD55 locus of Escherichia coli appears to be an allele of dnaB.

Attempts to characterize the grpD55 mutation of Escherichia coli have led us to conclude that the gene had been assigned an incorrect map position. The mutation was found to cotransduce with malF3089:: Tn10 (at approximately 91.5 min) and a dnaB-expressing plasmid was able to complement fully the grpD55 defect in lambda replication. These studies strongly suggest that grpD55 is an allele of dnaB and is localized near 92 min on the E. coli linkage map.

Alleles↗
PubMed1996

Conformational alteration of Oct-1 upon DNA binding dictates selectivity in differential interactions with related transcriptional coactivators.

VP16 (termed VP16-H here) of herpes simplex virus (HSV) belongs to a family of related regulatory proteins which includes VP16-B of bovine herpesvirus (BHV). We show that VP16-B, while also being a powerful transactivator of transcription dependent on Oct-1 binding sites in its target promoters, has virtually no activity on a defined VP16-H-responsive, octamer-containing target promoter. While Oct-1 binds equally well to the VP16-B-responsive and -nonresponsive sites, VP16-B interacts with Oct-1 only when Oct-1 is bound to the BHV octamer site and not when it is bound to the HSV site. We show from the analysis of chimeric proteins that the ability of VP16-B to discriminate between the Oct-1 forms depends on features of its N-terminal region. We also show from an analysis of chimeric DNA motifs that sequences that lie 3' to the POU domain-contacting region of the HSV octamer site play a role in making it unresponsive to VP16-B. Finally, we show by high-resolution hydroxyl radical footprint analysis that the conformation of Oct-l is different on the two sites. These results augment our previous report on an allosteric effect of DNA signals on the conformation of bound proteins and indicate that different conformations of the same DNA binding protein can be recognized selectively by related members of interacting regulatory proteins. The possible implications of our observations for selective gene regulation by Oct-1, a ubiquitous transcription factor, and other multimember transcription families are discussed.

Animals↗
PubMed1995

A transcriptional enhancer of the glutamine synthetase gene that is selective for retinal Müller glial cells.

This article demonstrates that the chicken glutamine synthetase (GS) promoter contains cis-acting elements that direct transcription to retinal Müller glial cells. The transient assay system developed to identify these elements involved electroporation of intact retinal tissue with GS-beta-galactosidase fusion genes followed by preparation of primary cultures and histochemical assay of cells expressing beta-galactosidase. Plasmids containing beta-galactosidase under transcriptional control by two different viral promoters are expressed primarily in neuronal cells after transfection of intact embryonic d 12 retina. In sharp contrast, expression is primarily in Müller glia after transfection with a GS-beta-galactosidase fusion gene. Although GS is glucocorticoid inducible, steroid hormone is not required to achieve Müller cell-selective expression of the GS-beta-galactosidase fusion gene. Deletion studies indicate that multiple cis-acting elements located between nucleotides-436 and -61 relative to the GS transcription start site contribute to produce Müller cell selectivity. Moreover, these upstream elements enhance expression of a heterologous promoter in Müller cells not neurons. These results indicate that an enhancer located between 61 and 436 nucleotides upstream of the transcription start site contributes to Müller cell-selective expression of the GS gene in the retina.

Animals↗
PubMed1995

The bovine herpesvirus alpha gene trans-inducing factor activates transcription by mechanisms different from those of its herpes simplex virus type 1 counterpart VP16.

In herpes simplex virus (HSV)-infected cells, viral gene expression is initiated when the immediate-early, or alpha, genes are transactivated by the alpha gene trans-inducing factor (alpha TIF), a component of the infecting virion. The protein binds to one or more recognition elements (TAATGARAT) in the promoters of alpha genes via interaction with the cellular proteins Oct-1 and CFF. The alpha TIF of HSV (HSV-alpha TIF) is believed to subsequently accelerate the assembly of the transcription complex by direct contact between its carboxyl-terminal acidic activation domain and at least two components of the transcription apparatus, TAFII40 and TFIIB. Like its HSV counterpart, the alpha TIF of bovine herpesvirus (BHV) (designated BHV-alpha TIF) also transactivates alpha gene promoters and for full activity exhibits a requirement for its extended carboxyl-terminal region. Despite this requirement, there is a notable lack of homology to the carboxyl-terminal acidic activation domain of HSV-alpha TIF. We swapped the amino- and carboxyl-terminal domains of HSV-alpha TIF and BHV-alpha TIF to make chimeric proteins. Using these chimeras, we show that the carboxyl terminus of BHV-alpha TIF is insufficient for transactivation, which requires cooperative determinants in both the amino-terminal and carboxyl-terminal regions of the protein. We have previously shown that the amino-terminal determinant in BHV-alpha TIF displays reduced but significant independent transactivation potential. Interestingly, this amino-terminal determinant appears not to reside in the HSV-alpha TIF, which displays no independent amino-terminal activity. Furthermore, we show that the amino-terminal activation domain of BHV-alpha TIF may be able to act synergistically with the carboxyl-terminal activation domain of HSV-alpha TIF, since a chimeric protein containing both domains appeared to be more efficient at activating transcription than either alpha TIF. In addition, the amino terminus of HSV-alpha TIF could not restore activity when linked to the carboxyl terminus of BHV-alpha TIF, while the amino terminus of BHV-alpha TIF reconstituted an intact protein with potent activation potential. We also show that in fusions with the DNA binding domain of GAL4, full activity requires the entire BHV-alpha TIF, although both amino and carboxyl termini display some activity on their own. In contrast, for HSV-alpha TIF, the carboxyl terminus is sufficient and possibly even more potent than the entire protein, while the amino-terminus is devoid of activity.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗
PubMed1995

Managed care in the Twin Cities: what can we learn?

Minneapolis/St. Paul, because of its history of health maintenance organization development and active employer participation in the health care arena, is often cited as a community in which managed competition has been tested to some degree. This paper reviews the historical development of the Twin Cities health care market and summarizes findings from past studies of this market. It also describes the recent consolidation of providers in the Twin Cities, as well as the activities of large purchasing coalitions. Finally, it assesses the elements of the Twin Cities experience that seem most relevant to managed competition-based health care reform proposals.

Group Purchasing↗
PubMed1995

Career motivations of male and female medical students.

Australian medical students (N = 645) were asked at the beginning of their training to rank the importance of a list of motivations relevant to their choice of medicine as a career. Both male and female students ranked the desire to help others as the most important motivation, closely followed by the scientific nature and the intellectual challenge of the profession of medicine. Both genders rated considerations of status and prestige as of low importance. These findings are similar to surveys from other countries that have reported altruism and intellectual challenge as prime motivations for both genders.

Adult↗
PubMed1994

Site-specific conformational alteration of the Oct-1 POU domain-DNA complex as the basis for differential recognition by Vmw65 (VP16).

We show that the presence of a regulatory cis-acting element that flanks the core octamer site and dictates selectivity in the response to Vmw65 (VP16), while dispensable for POU binding per se, induces a conformational alteration in the nature of the POU domain in the DNA complex. A single substitution in the flanking signal distorts the POU complex and without affecting overall POU binding prevents Vmw65 interaction. Alternatively, substitution of a residue in the homeodomain predicted to contact the GARAT region prevents its recognition even on a wild-type motif, causing a reversion to the DNA binding pattern seen on a cellular motif and at the same time inefficient recognition by Vmw65. The results indicate that Vmw65 recognizes a particular POU domain conformation induced by the presence of the flanking GARAT region.

Base Sequence↗
PubMed1994

Transactivation of the 'promoterless' luciferase-encoding vectors pXP1 and pXP2 by C/EBP alpha.

Transactivation of the firefly luciferase-encoding vectors, pXP1 and pXP2, by the alpha isoform of CCAAT/enhancer-binding protein (C/EBP) is reported. Thus, these vectors are not 'promoterless' in every cellular context, and transactivation by C/EBP or a closely related factor should be considered as a possible explanation for the relatively high background levels of luciferase production that are occasionally observed after transfection of certain cells with these or other similar vectors.

Animals↗
PubMed1994

Regional localization of 188 sequence tagged sites on a somatic cell hybrid mapping panel for human chromosome 3.

Chromosome 3 comprises 7% of the genome and contains at least 210 Mb of DNA. To expedite the analysis of this chromosome, we have assembled a somatic cell hybrid mapping panel that subdivides human chromosome 3 into 23 intervals using a total of 19 hybrids. Hybrids were constructed from 16 patients' cells containing chromosome 3 translocations. All of these hybrids selectively retained the derivative 3 chromosome. In addition, we utilized 2 radiation-reduced hybrids and 3 hybrids carrying spontaneous translocations between human chromosome 3 and rodent chromosomes. The entire panel has 9 short arm breakpoints that involve bands p24.2, p22, p21, p14, and p12 plus a total of 11 long arm breakpoints that involve bands q13, q21, q25, q26, and q27. In addition, two cell lines appear to have breakpoints at or near the centromere. To date, we have used this panel to localize 92 sequences regionally on the short arm, 89 sequences on the long arm, and 7 sequences near the centromere. These hybrids are useful tools that allow the rapid localization of markers on chromosome 3 and greatly assist other mapping efforts.

Animals↗
PubMed1994

Plasma clozapine concentrations predict clinical response in treatment-resistant schizophrenia.

Steady-state blood clozapine concentrations in 58 schizophrenic patients varied more than 45-fold (40-1911 ng/mL) after fixed-dose treatment (400 mg/day). Discriminant function analysis determined that a blood clozapine concentration of 420 ng/mL optimally distinguished responders from nonresponders. After 4 weeks of treatment, only 8% of those patients with a blood clozapine concentration < 420 ng/mL responded compared with 60% of those who had a blood clozapine concentration > 420 ng/mL. When plasma concentrations were increased above 420 ng/mL (by a double-blind random assignment procedure), nonresponders increased their response rate to 73% if their plasma concentrations at Week 12 exceeded 420 ng/mL compared with a response rate of 29% if their Week 12 levels remained below 420 ng/mL (chi 2 = 4.2, p < .04).

Adult↗