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Biomedical subjects

W Schaffner

Publications and source records attributed to W Schaffner.

At least 73 records · Page 4Linked to original sources
PubMed1997

A serine/arginine-rich nuclear matrix cyclophilin interacts with the C-terminal domain of RNA polymerase II.

The largest subunit of RNA polymerase II shows a striking difference in the degree of phosphorylation, depending on its functional state: initiating and elongating polymerases are unphosphorylated and highly phosphorylated respectively. Phosphorylation mostly occurs at the C-terminal domain (CTD), which consists of a repetitive heptapeptide structure. Using the yeast two-hybrid system, we have selected for mammalian proteins that interact with the phosphorylated CTD of mammalian RNA polymerase II. A prominent isolate, designated SRcyp/CASP10, specifically interacts with the CTD not only in vivo but also in vitro . It contains a serine/arginine-rich (SR) domain, similar to that found in the SR protein family of pre-mRNA splicing factors, which is required for interaction with the CTD. Most remarkably, the N-terminal region of SRcyp includes a peptidyl-prolyl cis - trans isomerase domain characteristic of immunophilins/cyclophilins (Cyp), a protein family implicated in protein folding, assembly and transport. SRcyp is a nuclear protein with a characteristic distribution in large irregularly shaped nuclear speckles and co-localizes perfectly with the SR domain-containing splicing factor SC35. Recent independent investigations have provided complementary data, such as an association of the phosphorylated form of RNA polymerase II with the nuclear speckles, impaired splicing in a CTD deletion background and inhibition of in vitro splicing by CTD peptides. Taken together, these data indicate that factors directly or indirectly involved in splicing are associated with the elongating RNA polymerases, from where they might translocate to the nascent transcripts to ensure efficient splicing, concomitant with transcription.

Amino Acid Isomerases↗
PubMed1997

Gene activation at a distance and telomeric silencing are not affected by yeast histone H1.

Until recently, it was believed that the budding yeast Saccharomyces cerevisiae has no histone H1 gene. However, a search of the yeast genome database revealed a possible H1 homologue of 258 amino acids, termed yeast histone H1 (HHO1). The protein shows 36% identity to the human H1 core domain over a stretch of 93 amino acids. Unlike other H1 proteins, Hho1p has a second possible core domain which shows 43% identity to the first core domain. Since vertebrate H1 histone had been implied in gene repression as well as gene activation at a distance, we tested the effect of deleting the yeast H1-like gene on remote activation of a modified GAL1 promoter, which contains a synthetic GAL4 binding site close to the TATA box, and the natural UASG, consisting of four GAL4 binding sites. Different spacing up to 1.8 kb between the proximal binding site and the distal UASG enhancer revealed no differences in gene activation between wild-type and knockout strains. Overexpression of a heterologous histone H1 from sea urchin showed an overall inhibition of gene activation by the GAL1 promoter, whereas overexpression of the yeast histone H1 had no effect. Also, the expression of A1, ALPHA2 or SUC2 genes, all of which are known to be responsive to an altered chromatin structure, was unchanged in HHO1 knockout or HHO1-overexpressing strains when compared to wild-type cells. We also considered the possibility that HHO1 was involved in forming the heterochromatin at telomeres. On testing for telomeric silencing of a URA reporter gene introduced 1.3 kb away from the chromosomal end, we again observed no differences between wild-type and knockout strains. Thus, the yeast histone H1-like gene appears to have no role in gene activation at a distance or in silencing under the conditions tested. It remains to be seen whether the yeast H1 histone is a gene-specific regulator rather than a general chromatin-associated protein.

Animals↗
PubMed1997

Inhibition of benzodiazepine binding in vitro by amentoflavone, a constituent of various species of Hypericum.

Flower extracts of Hypericum perforatum, Hypericum hirsutum, Hypericum patulum and Hypericum olympicum efficiently inhibited binding of [3H]flumazenil to rat brain benzodiazepine binding sites of the GABAA-receptor in vitro with IC50 values of 6.83, 6.97, 13.2 and 6.14 micrograms/ml, respectively. Single constituents of the extracts like hypericin, the flavones quercetin and luteolin, the glycosylated flavonoides rutin, hyperoside and quercitrin and the biflavone 13, II8-biapigenin did not inhibit binding up to concentrations of 1 microM. In contrast, amentoflavone revealed an IC50 = 14.9 +/- 1.9 nM on benzodiazepine binding in vitro. Comparative HPLC analyses of hypericin and amentoflavone in extracts of different Hypericum species revealed a possible correlation between the amentoflavone concentration and the inhibition of flumazenil binding. For hypericin no such correlation was observed. Our experimental data demonstrate that amentoflavone, in contrast to hypericin, presents a very active compound with regard to the inhibition of [3H]-flumazenil binding in vitro and thus might be involved in the antidepressant effects of Hypericum perforatum extracts.

Animals↗
PubMed1997

Neonatal tetanus in the United States: a sentinel event in the foreign-born.

BACKGROUND: Neonatal tetanus occurred in a 7-day-old infant born to Mexican immigrant parents in Tennessee in February, 1995. This was the first patient with neonatal tetanus reported in the United States since 1989. METHODS: We interviewed the infant's mother and physicians and reviewed the medical record. We conducted a telephone survey of 103 (17%) of the 609 licensed obstetrician/gynecologists practicing in Tennessee to assess vaccination history-taking practices during prenatal care. RESULTS: The mother was a 30-year-old gravida 4 para 3 woman who grew up in rural Mexico. After moving to the United States in 1987, she had delivered two children before this delivery. The hospital-based delivery and nursery stay in February, 1995, were uncomplicated. On the sixth day of life the infant became irritable and developed muscle stiffness. The next day he was examined by a pediatrician who diagnosed neonatal tetanus. The infant recovered fully after a 2-month hospitalization. The survey of obstetrical practices revealed that 61 (59%) of 103 respondents asked about the patient's vaccination status during prenatal care. However, of all respondents, only 14 (14%) confirmed that they specifically asked about prior tetanus vaccinations. Tetanus toxoid was available in 47% of offices on the day of the survey. CONCLUSIONS: Neonatal tetanus can still occur in the United States. This infant's immigrant mother had multiple missed opportunities to be vaccinated against tetanus during her three pregnancies in this country. Health care providers should ask patients about their vaccination status, particularly those patients who are foreign-born or who grew up outside the United States.

Emigration and Immigration↗
PubMed1997

The VP16 paradox: herpes simplex virus VP16 contains a long-range activation domain but within the natural multiprotein complex activates only from promoter-proximal positions.

Removal of core promoter elements like the TATA box converts several regulatory upstream regions of viral and cellular genes into classical enhancers, i.e., cis-regulatory elements capable of activating transcription over long distances in an orientation-independent manner. This is not the case with herpes simplex virus (HSV) immediate-early gene promoters, which are strongly induced by the viral transactivator VP16 (Vmw65, alphaTIF, ICP25) complexed with the cellular factors Oct-1 and HCF. Here we report that the VP16 complex can readily bring about strong activation from a promoter-proximal position but fails to induce transcription from a distal downstream enhancer position. This is in striking contrast to results obtained with GAL fusion proteins: in this context, the C-terminal "general" activation domain of VP16 activates transcription to high levels over long distances. Thus, this paradoxical behavior suggests that the VP16 activation domain is not accessible to the transcription machinery when the VP16-Oct-1-HCF complex is bound in a remote position. Only upon specific interactions in a promoter-proximal position, perhaps with the basal transcription factors, can transcription be strongly induced. In agreement with such a proposed mechanism, VP16 proteins to which a heterologous general activation domain has been added strongly activate transcription from a downstream position. The biological role of this unexpected and sophisticated mechanism is most probably a limitation of the VP16 activity to the associated immediate-early genes, without undesired long-range effects on other viral promoters within the tightly packed HSV genome.

Enhancer Elements, Genetic↗
PubMed1997

Long non-stop reading frames on the antisense strand of heat shock protein 70 genes and prion protein (PrP) genes are conserved between species.

Several mammalian genes, including heat shock protein (Hsp70) and prion protein (PrP) genes, have been reported to have long open reading frames (ORFs) or non-stop reading frames (NRFs) in the antisense direction. A simple explanation would be that these long antisense reading frames, which are usually in the same triplet frame as the coding strand, are the fortuitous byproduct of a high overall [G+C] content with concomitant preference for G/C over A/T in the third codon position, a preference for RNY type codons (purine/any nucleotide/pyrimidine), and/or a bias against serine and leucine, the only amino acids with codons that can be read as stop codons in the antisense direction. The PrP genes and most heat shock genes with long antisense NRFs (aNRFs) are indeed relatively [G+C] rich but do not show a bias against serine and leucine. In several vertebrates investigated, at least one of the Hsp70 genes has a long antisense reading frame, and we found that some, though not all, putative stop codons in long Hsp70 antisense reading frames were due to sequencing errors. The PrP gene contains an extended antisense open reading frame in all 45 eutherian mammals tested, but not in a marsupial and in a bird. In the PrP gene, the long, protein-coding exon also harbors the antisense nonstop reading frame. In both Hsp70 and PrP genes, the putative antisense protein sequence is well conserved. Even though there is no clear evidence in Hsp70 or PrP genes for the existence of the respective antisense proteins, we speculate that such antisense proteins serve to regulate the genuine Hsp and PrP proteins under special circumstances. Alternatively, regulation might occur at the RNA level, and the antisense RNA would merely lack stop codons to prevent its rapid degradation by an mRNA quality control mechanism that is triggered by premature stop codons. We note that both Hsp and PrP are involved in physiological or pathological protein aggregation phenomena, that scrapie prions have been reported to modify the expression or localization of heat shock proteins, and that in yeast, propagation of a prion-like state (PSI+) depends on a heat shock (Hsp104) protein.

Amino Acid Sequence↗
PubMed1997

A novel SR-related protein specifically interacts with the carboxy-terminal domain (CTD) of RNA polymerase II through a conserved interaction domain.

The largest subunit of the RNA polymerase II (pol II) contains at the carboxy-terminus a peculiar repetitive sequence that consists of 52 tandem repeats of the consensus motif Tyr-Ser-Pro-Thr-Ser-Pro-Ser, referred to as the C-terminal domain (CTD). Upon transcriptional initiation/promoter clearance, the CTD becomes extensively phosphorylated and apparently remains so during elongation. While the underphosphorylated CTD plays a role in transcriptional initiation, recent evidence couples the highly phosphorylated CTD to RNA processing, namely polyadenylation and splicing. Using a yeast two-hybrid screen, we have selected for human proteins that interact with the CTD of RNA polymerase II. The CTD-GAL fusion protein used as a bait is highly phosphorylated in yeast and, accordingly, we did not isolate proteins implicated in transcriptional regulation but rather proteins with possible roles in RNA splicing. One major cDNA clone isolated this way encodes SRrp129/CASP11, a protein that contains a conserved CTD-interaction domain at the C-terminus and an internal serine-arginine rich domain (SR domain). Proteins of the SR family have been implicated in RNA splicing, notably in the regulation of alternative splicing. Thus we consider it likely that SRrp129 is an auxiliary splice factor. We also improved our method to quickly map domains involved in protein-protein interaction (Stagljar et al., 1996, BioTechniques 21, 430-432). Instead of using sonication for the production of a random DNA fragment library, we took advantage of the fact that DNAse I in the presence of manganese (II) produces double strand rather than single strand DNA breaks. The DNA fragment library of the SRrp129 clone was then used in the yeast two-hybrid system to identify the 100-amino acid domain that interacts with the CTD of RNA polymerase II.

Alternative Splicing↗
PubMed1997

Silencing of RNA polymerases II and III-dependent transcription by the KRAB protein domain of KOX1, a Krüppel-type zinc finger factor.

The so-called KRAB domain, which is present in about one third of the vertebrate Kruppel-type zinc finger factors, has previously been shown to inhibit transcription in cis when tethered to promoter regions. Here we analyze this effect with fusions of the KRAB domain derived from KOX1/ZNF10 zinc finger protein to the heterologous DNA binding domains of both LexA and GAL4 factors. In transfected human cells, repression of reporter gene transcription is observed not only from proximal promoter positions, but also when KRAB is tethered to DNA at a remote position more than 1.8 kb downstream of the initiation site of transcription. Furthermore, KRAB-mediated silencing over short and long distances is not restricted to RNA polymerase II, since transcription by RNA polymerase III is also repressed. However, transcription by RNA polymerase I and by phage T7 RNA polymerase in mammalian cells are not significantly influenced by the KRAB domain. These latter results may indicate that repression by the KRAB domain, at least under our assay conditions, involves specific inhibition of some component(s) of RNA polymerase II and III transcription, rather than inducing some gross physical alteration of template chromatin structure.

Binding Sites↗
PubMed1997

Q fever pneumonia.

Pneumonia is one of the principal manifestations of Q fever, a disease caused by Coxiella burnetii. This bacterium can replicate only within cells, yet it is capable of surviving in the environment because it can withstand drying and substantial temperature variations. Livestock, especially sheep, goats and cattle, are a major reservoir of C burnetii. The organism is transmitted to humans by direct contact with animal products, especially during parturition. Aerosols have transmitted infection over considerable distances. The illness is characterized by an influenza-like syndrome with patchy pulmonary infiltrates. The pneumonia may be accompanied by biochemical evidence of mild hepatitis. The diagnosis is established serologically. Tetracycline or doxycycline provide effective therapy.

Animals↗
PubMed1997

Psittacosis.

Pneumonia caused by Chlamydia psittaci likely occurs considerably more frequently than is suggested by the mere 100 to 200 cases reported annually to public health authorities. Parrots and other psittacine birds still are regarded as the major reservoir of the infectious agent and most recognized cases are associated with owning pet birds or working in a pet store. It was once thought that birds imported from abroad, often illegally, were a principal source, but many domestic breeder flocks of pet birds now have become infected. There is little that is distinctive in the clinical presentation of psittacosis. Therefore, a careful history of bird contact is often the first clue to the diagnosis. The diagnosis frequently is first considered in evaluating a patient whose pneumonia has not responded to therapy with a beta-lactam antibiotic. The diagnosis usually is established serologically. Tetracycline is the preferred therapy and is administered for three weeks to prevent relapses.

Animals↗
PubMed1997

Autopsy risk and acquisition of human immunodeficiency virus infection: a case report and reappraisal.

Autopsy findings have contributed greatly to our understanding of acquired immunodeficiency syndrome. To our knowledge, documented autopsy-acquired infection with human immunodeficiency virus type 1 has not been reported, suggesting autopsy performance is of limited risk. We present a well-documented case of autopsy-acquired human immunodeficiency virus infection in a pathologist who sustained a scalpel wound to the hand.

Accidents, Occupational↗