Biomedical subjects
P Frank
Publications and source records attributed to P Frank.
Purification of Saccharomyces cerevisiae RNase H(70) and identification of the corresponding gene.
We purified Saccharomyces cerevisiae RNase H(70) to homogeneity, using an optimized chromatographic purification procedure. Renaturation gel assay assigned RNase H activity to a 70 kDa polypeptide. Sequencing of tryptic peptides identified the open reading frame YGR276c on chromosome VII of the S. cerevisiae genome as the corresponding gene, which encodes a putative polypeptide of molecular mass of 62849. We therefore renamed this gene RNH70. Immunofluorescence microscopy using a RNH70-EGFP fusion construct indicates nuclear localization of RNase H(70). Deletion of RNH70 from the yeast genome did not result in any serious phenotype under the conditions tested. Homology searches revealed striking similarity with a number of eukaryotic proteins and open reading frames, among them the chimpanzee GOR protein, a homolog of a human autoimmune antigen, found to elicit autoimmune response in patients infected with hepatitis C virus.
Saccharomyces cerevisiae RNase H(35) functions in RNA primer removal during lagging-strand DNA synthesis, most efficiently in cooperation with Rad27 nuclease.
Correct removal of RNA primers of Okazaki fragments during lagging-strand DNA synthesis is a critical process for the maintenance of genome integrity. Disturbance of this process has severe mutagenic consequences and could contribute to the development of cancer. The role of the mammalian nucleases RNase HI and FEN-1 in RNA primer removal has been substantiated by several studies. Recently, RNase H(35), the Saccharomyces cerevisiae homologue of mammalian RNase HI, was identified and its possible role in DNA replication was proposed (P. Frank, C. Braunshofer-Reiter, and U. Wintersberger, FEBS Lett. 421:23-26, 1998). This led to the possibility of moving to the genetically powerful yeast system for studying the homologues of RNase HI and FEN-1, i.e., RNase H(35) and Rad27p, respectively. In this study, we have biochemically defined the substrate specificities and the cooperative as well as independent cleavage mechanisms of S. cerevisiae RNase H(35) and Rad27 nuclease by using Okazaki fragment model substrates. We have also determined the additive and compensatory pathological effects of gene deletion and overexpression of these two enzymes. Furthermore, the mutagenic consequences of the nuclease deficiencies have been analyzed. Based on our findings, we suggest that three alternative RNA primer removal pathways of different efficiencies involve RNase H(35) and Rad27 nucleases in yeast.
Cloning of the cDNA encoding the large subunit of human RNase HI, a homologue of the prokaryotic RNase HII.
Two RNases H of mammalian tissues have been described: RNase HI, the activity of which was found to rise during DNA replication, and RNase HII, which may be involved in transcription. RNase HI is the major mammalian enzyme representing around 85% of the total RNase H activity in the cell. By using highly purified calf thymus RNase HI we identified the sequences of several tryptic peptides. This information enabled us to determine the sequence of the cDNA coding for the large subunit of human RNase HI. The corresponding ORF of 897 nt defines a polypeptide of relative molecular mass of 33,367, which is in agreement with the molecular mass obtained earlier by SDS/PAGE. Expression of the cloned ORF in Escherichia coli leads to a polypeptide, which is specifically recognized by an antiserum raised against calf thymus RNase HI. Interestingly, the deduced amino acid sequence of this subunit of human RNase HI displays significant homology to RNase HII from E. coli, an enzyme of unknown function and previously judged as a minor activity. This finding suggests an evolutionary link between the mammalian RNases HI and the prokaryotic RNases HII. The idea of a mammalian RNase HI large subunit being a strongly conserved protein is substantiated by the existence of homologous ORFs in the genomes of other eukaryotes and of all eubacteria and archaebacteria that have been completely sequenced.
Vanadium K-edge x-ray absorption spectroscopy reveals species differences within the same ascidian genera. A comparison of whole blood from Ascidia nigra and Ascidia ceratodes.
Vanadium K-edge x-ray absorption spectroscopy (XAS) was used to examine whole blood preparations from the tunicates Ascidia nigra and Ascidia ceratodes. Each XAS spectrum exhibits a rising edge inflection near 5480 eV characteristic of vanadium(III) and an intensity maximum at 5484.0 eV. In A. ceratodes blood cells, intrinsic aquo-VSO4+ complex ion is indicated by an inflection feature at 5476 eV in the first derivative of the vanadium K-edge XAS spectrum, but this feature is notably absent from the first derivative of the vanadium K-edge spectrum of blood cells from A. nigra. A strong pre-edge feature at 5468.6 eV also uniquely distinguishes the vanadium K-edge XAS spectrum of A. nigra blood cells, implying that vanadyl ion represents approximately 25% of the endogenous vanadium. However, the energy position of the rising edge inflection of the vanadium K-edge XAS spectrum of A. nigra (5479.5 eV) is 1 eV lower than that of A. ceratodes (5480.5 eV), the reverse of any expected shift arising from the endogenous vanadyl ion. Thus, in contrast to A. ceratodes, a significant fraction of the blood cell vanadium(III) in A. nigra is apparently in a ligation environment substantially different from that provided by water. These novel species-related differences may have taxonomic significance.
Yeast RNase H(35) is the counterpart of the mammalian RNase HI, and is evolutionarily related to prokaryotic RNase HII.
We cloned the Saccharomyces cerevisiae homologue of mammalian RNase HI, which itself is related to the prokaryotic RNase HII, an enzyme of unknown function and previously described as having minor activity in Escherichia coli. Expression of the corresponding yeast 35 kDa protein (named by us RNase H(35)) in E. coli and immunological analysis proves a close evolutionary relationship to mammalian RNase HI. Deletion of the gene (called RNH35) from the yeast genome leads to an about 75% decrease of RNase H activity in preparations from the mutated, still viable cells. Sequence comparison discriminates this new yeast RNase H from earlier described yeast enzymes, RNase H(70) and RNase HI.
Cloning, subcellular localization and functional expression of human RNase HII.
Recently we showed that the major mammalian RNase H, RNase HI, is evolutionarily related to prokaryotic RNase HII (Frank et al., FEBS-Lett. 421, 23-26, 1998), an enzyme described to be a minor activity in E. coli. As a consequence we addressed the question of whether a human RNase H exists, sharing homology with the main E. coli enzyme, RNase HI. Employing sequence analysis of expressed sequence tags, followed by specific PCR amplification of human cDNA, we cloned, sequenced and expressed a human open reading frame, coding for a 32 kDa protein. Purification of the recombinant His(6)-tagged protein from E. coli extracts using Ni(2+)-chelating chromatography and subsequent renaturation gel assay proved that it is an active RNase H. The properties of this enzyme suggest that it is identical with the human RNase HII, previously purified by one of us (Frank et al., Nucleic Acids Res. 22, 5247-5254, 1994). Studies using a green fluorescent protein-fusion construct reveal that this protein is located in the nucleus.
Purification and nucleic-acid-binding properties of a Saccharomyces cerevisiae protein involved in the control of ploidy.
Scp160p (Saccharomyces cerevisiae protein involved in the control of ploidy), a polypeptide with a molecular mass of around 160 kDa, is associated with the nuclear envelope and the endoplasmic reticulum. The most noteworthy phenotype of SCP160 deletion mutants is a decrease in viability and an increased number of chromosomes in the surviving cells [Wintersberger, U., Kühne, C. & Karwan, A. (1995) Yeast 11, 929-944]. Scp160p contains 14 KH domains, conserved motifs that have lately been identified in a variety of RNA-binding proteins. In this report, we demonstrate that the Scp160p sequence shows nearly perfect colinearity with the putative gene product of C08H9.2 from the nematode Caenorhabditis elegans as well as with the vigilins, vertebrate RNA-binding proteins with a cellular location similar to that of Scp160p. Moreover, we found that Scp160p contains a potential nuclear-export signal (NES) near its N-terminus and a potential nuclear-localization signal (NLS) between KH domains 3 and 4. To determine whether the protein is able to bind to RNA, we purified Scp160p from yeast cell extract by DNA-cellulose and anti-Scp160p affinity chromatography. In northwestern blotting experiments, the electrophoretically homogeneous protein bound to ribohomopolymers and ribosomal RNA as well as to single-stranded and double-stranded DNA. Subcellular fractionation studies revealed that the major part of Scp160p is membrane associated via ionic interactions and can be released from the membrane fraction under conditions that lead to a dissociation of ribosomes. Together, our findings suggest that Scp160p is the yeast homologue of the vigilins, and point to a role for Scp160p in nuclear RNA export or in RNA transport within the cytoplasm.
Experimental investigation of the effects of frequency-altered auditory feedback on the speech of adults who stutter.
A series of single-subject experiments evaluated the effects of frequency-altered auditory feedback (FAF) on the speech performance of four adult males who stutter. Using alterations of plus or minus one octave, FAF was compared with normal auditory feedback (NAF) in oral reading and spontaneous speech with measurements made of stuttered intervals, stutter-free speech rate, and speech naturalness. The effects of extended FAF conditions on spontaneous speech were also evaluated for two subjects who demonstrated a positive response to FAF. Results showed no consistencies across subjects in responses to FAF: One subject showed no response, another produced an initial temporary response, a third showed a deterioration in speech quality with minimal reductions in stuttering, and a fourth displayed substantial and sustained improvements in speech performance. Some implications of these findings for current research and theory about the relationship between stuttering and FAF are discussed.
Reactions between vanadium ions and biogenic reductants of tunicates: spectroscopic probing for complexation and redox products in vitro.
Several species of marine tunicates store oxygen-sensitive VIII in blood cells. A sensitive colorimetric VIII assay was used to survey the leading candidates for the native reducing agent of vanadate in tunicates (i.e., An-type tunichromes, glutathione, NADPH, and H2S) in reactions with VV or VIV ions under anaerobic, aqueous conditions at acidic or neutral pH. Except for the case of An-1 and VV ions in pH 7 buffer, the assay results for the biogenic reducing agents clearly showed that appreciable quantities of VIII products were not generated under the conditions tested. Therefore, the assay results place new limits on hypothetical mechanisms of VIII formation in vivo. For reactions between An-1 and VV ions in pH 7 buffer, low levels of VIII products could not be ruled out because of an interfering peak in the colorimetric assays. For similar reactions between VV ions and An-1, or an An-1,2 mixture, in mildly to moderately basic media, the product mixtures precipitated as greenish black solids. Analyses of the precipitated V/An mixtures using vanadium K-edge X-ray absorption spectroscopy (XAS) showed that the major products were tris(catecholate)-type VIV complexes (65 +/- 6%) and bis(catecholate)-type VIVO complexes (20 +/- 4%). XAS analysis of the V/An-1 product mixture also provided evidence of a minor VIII component (9 +/- 5% of total V), notable for possible relevance to tunicate biochemistry. The combined results of XAS studies, spectrophotometric studies [Ryan, D. E., et al. (1996) Biochemistry 35, 8640-8650], and EPR studies [Grant, K. B., et al. (1996) J. Inorg. Biochem. (manuscript in preparation)] consistently establish that reactions between tunichromes (Mm-1 or An-1) and VV ions generate predominantly VIV-tunichrome complexes in neutral to moderately basic aqueous media.
Endotoxin and cytokines induce direct cardiodepressive effects in mammalian cardiomyocytes via induction of nitric oxide synthase.
In patients with septic shock or inflammatory cardiac diseases like myocarditis myocardial contractility is depressed. These patients have elevated circulating levels of bacterial endotoxins (lipopolysaccharides, LPS) and pro-inflammatory cytokines like interleukin-1 beta (IL-alpha 1 beta) or tumor necrosis factor-alpha (TNF-alpha). It is not clear, whether LPS and/or cytokines have direct inotropic effects on cardiomyocytes and whether these effects are mediated via the L-arginine-nitric oxide synthase (NOS) pathway as demonstrated in vascular smooth muscle cells. Therefore, we examined the direct effects of LPS. IL-1 beta and TNF-alpha on contractility and cGMP content in isolated guinea-pig ventricular cardiomyocytes. Furthermore, the influence of the NOS inhibitor NG-nitro-L-arginine (L-NNA) and dexamethasone on these effects was studied as well as inducible NOS (iNOS) protein expression. LPS (1000 ng/ml), IL-1 beta (25 ng/ml) and TNF-alpha (100 ng/ml) decreased contractility by 48%, 55% and 65% and augmented cGMP content by 135%, 88% or 70% after long-term treatment (18 h) in cardiomyocytes, without altering contractility or cGMP content after short-term treatment (30 min). These effects were blocked by L-NNA (100 microM) and dexamethasone (3 microM). Furthermore iNOS protein was expressed in LPS- and cytokine-treated cardiomyocytes. These findings demonstrate that LPS. IL-1 beta and TNF-alpha have direct negative inotropic effects on cardiomyocytes, which are accompanied by an increase in cGMP content. These effects are mediated via de novo synthesis of a myocardial iNOS. The direct negative inotropic effects of endotoxins and cytokines on cardiomyocytes may in part contribute to the contractile dysfunction observed in patients with septic shock or inflammatory cardiac diseases.
The synthesis of fluorine-18 lomefloxacin and its preliminary use in human studies.
Lomefloxacin is a new fluorine-containing antibiotic that has recently been approved for general use. Fluorine-18 lomefloxacin has been prepared by fluoride exchange between fluorine-18 fluoride and lomefloxacin in DMSO. Both time and temperature of the reaction have been optimized and conditions developed for the isolation and purification of the labeled product in a form suitable for oral administration. The exchange reaction provides sufficient labeled material for human studies with pharmacologically relevant quantities of the drug. We have performed preliminary human studies with this compound using positron emission tomography to estimate the tissue distribution of the compound and show the distribution of the compound into the liver and lungs.
Use of a postal questionnaire to estimate the likely under-diagnosis of asthma-like illness in adults.
BACKGROUND: Asthma is thought to be under-diagnosed. AIM: To estimate how many adults in two practices may have unknown asthma-like illness. METHOD: Results from a postal respiratory questionnaire, sent to 11,206 adults registered with two practices, were linked with practice records to identify patients who may have unknown asthma-like illness. RESULTS: The questionnaire replies were almost identical in both practices; 30.4% of respondents [95% confidence interval (CI) 29.4-31.4] reported wheezing and 14.1% (95% CI 13.3-14.9) had been woken by breathlessness during the previous year. Using a simple scoring system, 1112 (13.8%) of the respondents were judged possibly to have asthma-like illness; 529 (6.6%) had not previously been diagnosed as having asthma and had not received asthma treatments. CONCLUSION: The frequency of asthma-like symptoms was high. A large proportion of adults in the practices may have an unknown asthma-like illness which warrants further evaluation.
Termination of pregnancy and psychiatric morbidity.
BACKGROUND: We investigated whether reported psychiatric morbidity was increased after termination of pregnancy compared with other outcomes of an unplanned pregnancy. METHOD: This was a prospective cohort study of 13,261 women with an unplanned pregnancy. Psychiatric morbidity reported by GPs after the conclusion of the pregnancy was compared in four groups: women who had a termination of pregnancy (6410), women who did not request a termination (6151), women who were refused a termination (379), and women who changed their minds before the termination was performed (321). RESULTS: Rates of total reported psychiatric disorder were no higher after termination of pregnancy than after childbirth. Women with a previous history of psychiatric illness were most at risk of disorder after the end of their pregnancy, whatever its outcome. Women without a previous history of psychosis had an apparently lower risk of psychosis after termination than postpartum (relative risk RR = 0.4, 95% confidence interval CI = 0.3-0.7), but rates of psychosis leading to hospital admission were similar. In women with no previous history of psychiatric illness, deliberate self-harm (DSH) was more common in those who had a termination (RR 1.7, 95% CI 1.1-2.6), or who were refused a termination (RR 2.9, 95% CI 1.3-6.3). CONCLUSIONS: The findings on DSH are probably explicable by confounding variables, such as adverse social factors, associated both with the request for termination and with subsequent self-harm. No overall increase in reported psychiatric morbidity was found.
Vanadium in ascidians and the chemistry of tunichromes.
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Purification and characterization of human ribonuclease HII.
A ribonuclease H activity from human placenta has been separated by ion exchange chromatography from the major RNase HI enzyme. Additional chromatographic steps allowed further purification, more than 3,000 fold compared to the crude extract in which it represents about 15% of the total RNase H activity. The enzyme requires Mg2+ ions for its activity, is strongly inhibited by the addition of Mn2+ ions or other divalent transition metal ions, and exhibits a pH optimum between 8.5 and 9. It shows a strong sensitivity to the SH-blocking agent N-ethylmaleimide. It has a strict specificity for double-stranded RNA-DNA duplexes and exhibits neither single-stranded nor double-stranded RNase (or DNase) activities. Therefore, this enzyme displays the characteristics of class II RNase H and is now termed RNase HII. Renaturation gel assays and gel filtration experiments proved a monomeric structure for the active enzyme with a native molecular weight of about 33 kDa. The human RNase HII acts as an endonuclease and releases oligoribonucleotides with 3'-OH and 5'-phosphate ends. It is therefore a candidate for the RNase H-mediated effect of antisense oligodeoxynucleotides.
Mutational analysis of the fingers and palm subdomains of human immunodeficiency virus type-1 (HIV-1) reverse transcriptase.
We have analyzed the human immunodeficiency virus type-1 reverse transcriptase (HIV-1 RT) polymerase domain between amino acids 91 and 157 by site-directed mutagenesis. We have constructed a series of amino acid substitutions using BspMI cassettes, and have assayed the RNA-dependent DNA polymerase, DNA-dependent DNA polymerase, and RNase H activities of the mutant HIV-1 RTs. The regions of HIV-1 RT between amino acids 91 and 119 and between amino acids 151 and 157 lie within the palm subdomain and include part of the polymerase active site. A number of amino acids within these regions have been identified as being directly or indirectly involved with polymerization, since amino acid substitutions at these residues decrease the polymerase activity without affecting RNase H activity. The region of HIV-1 RT between amino acids 120 and 150 lies within the fingers subdomain of the HIV-1 polymerase. We believe that the fingers subdomain plays a role in positioning the template. Many amino acid substitutions in this region decrease or abolish both the polymerase and the RNase H functions.