Only half of GPs in study knew that advance directives could carry legal force in UK.
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Advance Directives↗
Biomedical subjects
S Hayes
Publications and source records attributed to S Hayes.
Acquired mutations in phage lambda genes O or P that enable constitutive expression of a cryptic lambdaN+cI[Ts]cro- prophage in E. coli cells shifted from 30 degreesC to 42 degreesC, accompanied by loss of immlambda and Rex+ phenotypes and emergence of a non-immune exclusion-state.
The majority of bacteria, which carry the N+-cI857[Ts]-cro--O+-P+ fragment of lambda genome, are killed when derepressed by shifting from 30 degreesC to 42 degreesC. Among rare survivors, we observed a proportion of colony-forming units (cfu) that retained the typical immlambda-immunity phenotype when grown at 30 degreesC; however, when shifted from 30 degreesC to 42 degreesC, they lost lambda immunity and acquired a non-immune exclusion-state (Nie phenotype). We also found that the immlambda survivor cfu quickly lost their Rex+ exclusion phenotype (as measured by T4rII plating inhibition) when shifted from 30 degreesC to 42 degreesC, even though they produced CII, which stimulates pE-cI-rexA-rexB transcription. The Nie phenotype was characterized by an inhibition of plating of the homoimmune phage, lambdawt, and the heteroimmune phage, lambdaimm434. However, lambdavir and spontaneous mutants of lambdawt (lambdase mutations localized within oR) escaped the Nie exclusion-state and plated efficiently on lawns of Nie cfu at 42 degreesC. Thus, we examined the scope of the Nie exclusion-state toward lambda mutants blocked for lysogeny, and lambda hybrids substituted for immunity or replication genes. Phage like lambdawt, competent for lysogeny, were severely excluded compared to some mutants of lambda defective for lysogeny. Among this latter type, there was high variance in the Nie exclusion of various cI mutants; some of which were not excluded. The Nie exclusion-state was attributed to the constitutive expression of the defective lambda fragment in the survivor cfu, made possible by the acquired replication defect(s). We characterized, both genetically and physically, the mutations in the defective integrated lambda prophage that permitted growth of the survivor cfu at 42 degreesC. In five of seven survivor cfu, we identified IS2 insertions within lambda genes O and P that can block replication initiation from the lambda fragment. The remaining survivor cfu had multiple base substitutions within the C-terminal end of O and N-terminal half of P, the majority of which were silent. In some of these mutants, either an ochre nonsense mutation or a single-base frameshift deletion inactivated P.
Amino Acid Sequence↗
Images in cardiovascular medicine. Cardiac tuberculosis.
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Cardiomegaly↗
Analysis of SCA1, DRPLA, MJD, SCA2, and SCA6 CAG repeats in 48 Portuguese ataxia families.
The spinocerebellar ataxias (SCAs) are clinically and genetically a heterogeneous group of neurodegenerative disorders. To date, eight different loci causing SCA have been identified: SCA1, SCA2, Machado-Joseph disease (MJD)/SCA3, SCA4, SCA5, SCA6, SCA7, and dentatorubropallidoluysian atrophy (DRPLA). Expansion of a CAG repeat in the disease genes has been found in five of these disorders. To estimate the relative frequencies of the SCA1, DRPLA, MJD, SCA2, and SCA6 mutations among Portuguese ataxia patients, we collected DNA samples from 48 ataxia families and performed polymerase chain reaction (PCR) amplification of the CAG repeat mutations on chromosomes 6p, 12p, 14q, 12q, and 19p, respectively. Fifty-five individuals belonging to 34 dominant families (74%) had an expanded CAG repeat at the MJD gene. In five individuals from two kindreds with a dominant pattern of inheritance (4%), an expanded CAG repeat at the SCA2 gene was found. In MJD patients, the normal allele size ranged from 13 to 41, whereas the mutant alleles contained 65 to 80 repeats. For the SCA2 patients, normal alleles had 22 or 23, while expanded alleles had between 36 and 47 CAG units. We did not find the SCA1, DRPLA, or SCA6 mutations in our group of families. The MJD mutation remains the most common cause of SCA in Portugal, while a small number of cases are caused by mutations at the SCA2 gene, and 22% are due to still unidentified genes.
Adolescent↗
DDB, a putative DNA repair protein, can function as a transcriptional partner of E2F1.
The transcription factor E2F1 is believed to be involved in the regulated expression of the DNA replication genes. To gain insights into the transcriptional activation function of E2F1, we looked for proteins in HeLa nuclear extracts that bind to the activation domain of E2F1. Here we show that DDB, a putative DNA repair protein, associates with the activation domain of E2F1. DDB was identified as a heterodimeric protein (48 and 127 kDa) that binds to UV-damaged DNA. We show that the UV-damaged-DNA binding activity from HeLa nuclear extracts can associate with the activation domain of E2F1. Moreover, the 48-kDa subunit of DDB, synthesized in vitro, binds to a fusion protein of E2F1 depending on the C-terminal activation domain. The interaction between DDB and E2F1 can also be detected by coimmunoprecipitation experiments. Immunoprecipitation of an epitope-tagged DDB from cell extracts resulted in the coprecipitation of E2F1. In a reciprocal experiment, immunoprecipitates of E2F1 were found to contain DDB. Fractionation of HeLa nuclear extracts also revealed a significant overlap in the elution profiles of E2F1 and DDB. For instance, DDB, which does not bind to the E2F sites, was enriched in the high-salt fractions containing E2F1 during chromatography through an E2F-specific DNA affinity column. We also observed evidence for a functional interaction between DDB and E2F1 in living cells. For instance, expression of DDB specifically stimulated E2F1-activated transcription. In addition, the transcriptional activation function of a heterologous transcription factor containing the activation domain of E2F1 was stimulated by coexpression of DDB. Moreover, DDB expression could overcome the retinoblastoma protein (Rb)-mediated inhibition of E2F1-activated transcription. The results suggest that this damaged-DNA binding protein can function as a transcriptional partner of E2F1. We speculate that the damaged-DNA binding function of DDB, besides repair, might serve as a negative regulator of E2F1-activated transcription, as damaged DNA will sequester DDB and make it unavailable for E2F1. Furthermore, the binding of DDB to damaged DNA might be involved in downregulating the replication genes during growth arrest induced by damaged DNA.
Base Sequence↗
DNA analysis of Duffy genes in American blacks.
BACKGROUND: Because limited studies have shown that the Duffy genotype is not accurately reflected by the erythrocyte phenotype, we used PCR-RFLP to determine the true FY1 and FY2 gene frequencies in blacks. METHODS: Genomic DNA was extracted from 81 randomly selected black individuals, amplified using PCR, digested with BanI and electrophoresed in 1.8% gels. Direct DNA sequencing of the FY regulatory region was done on a sample which had weak Fyb antigens. Standard serological techniques were used to type the erythrocytes for Fya and Fyb. RESULTS: The most common genotype (83%) in blacks was Fy(a-b+) but almost all of the samples serologically typed as Fy(a-b-). The new gene frequencies are 0.08 for FY1 and 0.92 for FY2. Two donors whose erythrocytes typed weakly positive for Fyb and appeared to be Fyx were homozygous for FY2 by DNA testing. DNA sequencing of the regulatory region for Duffy found a mutation in an Sp1 binding site in the Fyx donor. CONCLUSIONS: We conclude that (1) the FY2 gene occurs in a much higher frequency in blacks than previously reported which may explain the lack of anti-Fyb in this ethnic group and (2) Fyx appears to be a the product of a mutant FY2 gene giving a quantitative variation in expression.
Black People↗
Avirulence gene D of Pseudomonas syringae pv. tomato may have undergone horizontal gene transfer.
Avirulence gene D (avrD) is carried on the B-plasmid of the plant pathogen Pseudomonas syringae pv. tomato with plasmid-borne avrD homologs widely distributed among the Pseudomonads. We now report sequences in the soft rot pathogen Erwinia carotovora that cross-hybridize to avrD suggesting a conserved function beyond avirulence. Alternatively, avrD may have been transferred horizontally among species: (i) DNA linked to avrD shows evidence of class II transpositions and contains a novel IS3-related insertion sequence, and (ii) short sequences linked to avrD are similar to pathogenicity genes from a variety of unrelated pathogens. We have also identified the gene cluster that controls B-plasmid stability.
Bacterial Proteins↗
The Rex phenotype of altruistic cell death following infection of a lambda lysogen by T4rII mutants is suppressed by plasmids expressing OOP RNA.
The coordinate expression from induced lambda prophages of pLit-rexB-tImm (late immunity transcription, LIT RNA) and po-oop-t(o) (OOP RNA) has remained unexplained. The initial assigned sequence for pLit bore no relationship to po. We have identified two promoter sites for independent rexB transcription, denoted here pLit2 and pLit1, which are separated by about 330 bp. The upstream pLit1 site shares with po a common 9 bp sequence between the -10 and -35 regions, with strong homology to aspects of the SOS box or LexA operator site. This sequence is also found within OOP RNA, suggesting that OOP RNA, or another regulatory factor recognizing the common sequence, was involved in the regulation of rexB expression and hence Rex exclusion. We measured the influence of OOP synthesis from plasmids on the Rex phenotype, finding that plasmids producing OOP can suppress Rex exclusion by a lambda prophage. The possibility was suggested that low level constitutive rexB transcription occurs from pLit2. Potential binding sites were identified for DnaA, for the LexA, CI and Cro repressors and for lambda O protein in the 80 nt DNA interval upstream from and including pLit1, suggesting a complex regulatory pattern for rexB expression from this promoter.
Bacteriophage lambda↗
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↗
Review of recent trials of chemotherapy for advanced breast cancer: studies excluding taxanes.
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Antineoplastic Agents↗
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↗
Sexuality in women receiving chemotherapy for breast cancer.
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Breast Neoplasms↗
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↗
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↗
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↗
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↗
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↗
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↗