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Biomedical subjects

E Pfaff

Publications and source records attributed to E Pfaff.

At least 37 records · Page 2Linked to original sources
PubMed1995

Comparative studies of bacterially expressed integrase proteins of caprine arthritis-encephalitis virus, maedi-visna virus and human immunodeficiency virus type 1.

Integrase (IN) proteins mediate an essential step in retroviral life cycles, the integration of reverse-transcribed viral DNA into the host genome. To create tools for direct comparative investigations, hexahistidine-tagged IN proteins of the phylogenetically related lentiviruses caprine arthritis-encephalitis virus (CAEV), maedi-visna virus (MVV) and human immunodeficiency virus type 1 (HIV-1) were expressed in Escherichia coli. After purification by affinity chromatography, the active enzymes were compared in vitro for their site-specific cleavage, integration and disintegration activities on cognate and non-cognate oligonucleotide substrates. It was found that CAEV IN and MVV IN catalyse both site-specific cleavage and disintegration with high efficiencies, reduced substrate specificities and similar reaction patterns. Comparisons with the respective activities of HIV-1 IN revealed basic functional similarities as well as considerable differences such as more restricted substrate requirements for site-specific cleavage. On the other hand, all three enzymes catalyse disintegration almost independent of the substrate origin. Furthermore, MVV IN was shown to join oligonucleotides as efficiently as HIV-1 IN, albeit with reduced substrate specificity. In contrast, no detectable strand transfer activities occurred with CAEV IN.

Amino Acid Sequence↗
PubMed1994

The glomerulosclerosis gene Mpv17 encodes a peroxisomal protein producing reactive oxygen species.

The mutant mouse strain Mpv17 carries a retroviral insert in its genome which inactivates the Mpv17 gene. At a young age these mice develop glomerulosclerosis and nephrotic syndrome which resembles human disease. We show here that the Mpv17 gene product is highly conserved and encodes a peroxisomal protein. Loss of the Mpv17 protein does not impair peroxisome biogenesis but instead leads to a reduced ability to produce reactive oxygen species (ROS). In turn, overproduction of the Mpv17 gene in transfected cells results in dramatically enhanced levels of intracellular ROS indicating a direct involvement of Mpv17 in ROS production. These data reveal a role for the Mpv17 protein in peroxisomal reactive oxygen metabolism and establish a novel link between peroxisomal ROS production and glomerulosclerosis.

Amino Acid Sequence↗
PubMed1994

Characterization of antigenic determinants in the core antigen of the hepatitis C virus.

Antibodies to the hepatitis C virus (HCV) core protein are present in the majority of patients with chronic HCV infection. To characterize the corresponding determinants, synthetic peptides and various deletion clones of the core gene expressed in Escherichia coli were used to test human anti-core positive sera or rabbit anti-peptide antibodies in enzyme-linked immunosorbent assays, immunoblots, and competition assays. Two distinct linear antigenic determinants which are located within aa 1 to 20 and between aa 30 and 47 were found. Further studies using reactive serum after preabsorption of antibodies with N- and C-terminal-deleted HCV core proteins or with peptides directed to the linear epitopes revealed an additional determinant that requires for presentation the participation of the N-terminal 69 amino acids. It is postulated that the HCV core protein forms a three-dimensional structure exposing two linear epitopes and, in addition, presents a conformational determinant within the N-terminal 69 amino acids. The remaining core amino acid sequence spanning from position 69 to 191 does not seem to expose further determinants to induce additional anti-core antibodies.

Animals↗
PubMed1994

B-lymphocytes are predominantly involved in viral propagation of hepatitis C virus (HCV).

Recent reports have shown that HCV infection is not only restricted to hepatocytes. Like hepatitis B virus (HBV), which also was thought to be strictly hepatotropic in early molecular and cellular investigations, infection of lymphoid cells by HCV in vivo has been demonstrated. We showed that total peripheral blood leukocytes of chronically HCV-infected patients are infected by detection of plus- and minus-stranded HCV RNA using strand-specific oligonucleotide primers in the RT-PCR. These cells also represent extrahepatic sites for the viral replication, as demonstrated by incorporation of [3H]-uridine into nascent RNA after stimulation of the cells with a mitogen. Furthermore, total PBML from an uninfected person could be infected in vitro using an HCV-positive serum. It could be shown that replication of HCV RNA takes place in these cells. Examination of different subsets of PBML showed predominant infection of B-lymphocytes during HCV disease. Additionally, infection of T-lymphocytes was detected in about 50% of all chronically HCV-infected patients.

B-Lymphocytes↗
PubMed1994

The major species specific epitope in prion proteins of ruminants.

The species specific nature of an antigenic determinant previously discovered in the scrapie form of prion protein (PrPD) from cattle, sheep and mice, was further investigated in normal prion protein (PrPC) from these and other species. This was carried out with eight different anti-peptide sera raised in rabbits against various synthetic peptides representing segments of the amino acid (aa) sequence 101-122 of ovine, bovine, murine and hamster PrP. Antipeptide serum against a peptide representing aa 107-122 of ovine PrP showed almost specific reaction and crossreacted in immunoblot with caprine and human PrP only. Antisera to the corresponding bovine sequence stained bovine and porcine PrP and to a minor extent PrP of goat, man, cat, and mink, while antiserum to the murine aa sequence reacted with rodent and monkey PrP only. In contrast, antiserum to the corresponding hamster sequence displayed a broader reactivity pattern, just like the four other anti-peptide sera to various ovine and bovine sequences. Antisera were also tested for reactivity with the pathogenic isoforms of PrP of sheep, cow, hamster and mouse and showed generally similar reactivity patterns as by using PrPC. In conclusion, the region close to the actual or putative proteinase K cleavage sites of PrP seems to exhibit high structural variability among mammalian species.

Amino Acid Sequence↗
PubMed1994

Porcine T-cell receptors: molecular and biochemical characterization.

Two subclasses of CD3 associated T-cell receptors (TcR) have been described so far, consisting of either an alpha and beta chain (TcR alpha beta) or a gamma and delta chain (TcR gamma delta). Of the two subclasses, the TcR alpha beta is the one predominantly expressed on peripheral T lymphocytes of humans and rodents. TcR gamma delta T lymphocytes represent only a minor subset in these species. Among all mammalian species studied so far, swine showed the most diversified composition of the T-lymphocyte population characterized by the expression of CD4 and CD8 differentiation antigens. Besides CD4+CD8- and CD4-CD8+ T lymphocytes, CD4+CD8+ and CD4-CD8- T lymphocytes are prominent in the extrathymic T-lymphocyte compartment. Because of the lack of specific monoclonal antibodies (mAb), to date the porcine TcR can only be characterized with biochemical and molecular biological methods. TcR on porcine peripheral blood T lymphocytes with the phenotype CD4+ and/or CD8+ are characterized as 46-48 kDaR heterodimers which were supposed to represent the porcine TcR alpha beta. Biochemical analyses of the CD4-CD8- T lymphocytes revealed three distinct TcR gamma delta; all are characterized by a 40 kDa delta chain but differed in their gamma chains. One gamma chain with a molecular mass of 38 kDaR is preferentially expressed on CD4-CD8- T lymphocytes derived from peripheral blood; another chain with molecular mass of 37 kDaR is evenly distributed between CD4-CD8- T lymphocytes from blood and lymphoid tissues.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1993

Molecular cloning of porcine T cell receptor alpha, beta, gamma and delta chains using polymerase chain reaction fragments of the constant regions.

Fragments of the constant regions of porcine alpha, beta, delta and two types of gamma T cell receptor (TcR) chains were obtained by reverse transcription and polymerase chain reaction (PCR). Screening of a porcine peripheral T cell cDNA library with these PCR fragments led to the isolation of porcine TcR alpha, beta, gamma and delta chain clones. Sequence analysis of these clones and the respective PCR fragments demonstrated the existence of one alpha, one beta, three gamma and one delta chain isotype. Comparisons of the deduced amino acid sequences of the constant region with other species revealed a significant homology. For two of the three identified porcine gamma chain insertions of 38 and 40 amino acids were found within the hinge region. In addition, our sequence data demonstrate a high variability in the cytoplasmic C gamma domain among the three porcine TcR gamma isotypes as well as between species, which might be of structural and/or functional significance. Comparison with biochemical data indicate the existence of four porcine TcR gamma isotypes.

Amino Acid Sequence↗
PubMed1993

Genetic variability of German hepatitis C virus isolates.

Heterogeneity of hepatitis C viral (HCV) genomes of several isolates from different countries has been reported, but there is little information on HCV isolates for the Federal Republic of Germany. Therefore, the nucleotide (nt) and deduced amino acid (aa) sequences of interesting parts of the viral genome derived from different human isolates in Germany were compared with each other and with the nt and predicted aa sequences of recently published isolates. HCV sequences were obtained by reverse transcription of viral RNA extracted from serum followed by polymerase chain reaction (PCR) amplification. Within the 5' nontranslated region we found only 3 single nucleotide exchanges among 2 of our isolates, and in comparison to sequences of Japanese isolates 2 to 3 exchanges, and to U.S. isolates 1 to 5 exchanges (homologies 98% to > 99%). Determination of a 249-bp core sequence from two German isolates exhibited 3% sequence divergence. The sequence of the core region (nt 342-911) showed a homology of about 88-91% on nt level and 96-97% on aa level as compared to U.S. isolates and other German isolates, and a homology of 95-96% (nt) and 96-98% (aa), respectively, to Japanese isolates. Less homologies were noticed for the E1 and E2/NS1 genes, especially in the N-terminal E2/NS1 hypervariable domain. Our isolates HD1 and HD2 showed nt sequence homologies of about 72-81% and aa homologies of 76-88% to U.S., German, and French isolates, and 89-91% (nt) and 88-96% (aa), respectively, to Japanese isolates. These results indicate that various German isolates are more closely related to Japanese isolates and differ from other European isolates as reported so far. Because of a nucleotide sequence heterogeneity of up to 10% among the tested isolates, we conclude that more than one closely related but distinct viral genotype of HCV exists in Germany. Furthermore, heterogeneous sequences of HCV can be detected in a single patient suggesting multiple infection with different genomic variants or, alternatively, a genetic drift forced by mutational events as a consequence of host immune selection.

Amino Acid Sequence↗
PubMed1993

Peripheral blood leukocytes serve as a possible extrahepatic site for hepatitis C virus replication.

To study possible extrahepatic sites for the replication of hepatitis C virus (HCV), we examined fresh and cultured peripheral blood mononuclear leukocytes (PBML), as well as different subpopulations of PBML of HCV-infected patients, for the presence of viral genomic and antigenomic RNA. Sense and antisense oligonucleotide primers derived from HCV sequences were used for reverse transcription (RT) followed by an amplification with the polymerase chain reaction assay (PCR). Using antisense primers for RT, genomic viral RNA could be detected in serum, liver, total PBML and B lymphocytes of chronically infected patients. However, only liver tissue and PBML specimens were positive when a sense primer was used. To demonstrate further the specificity of these findings, total PBML were stimulated using pokeweed mitogen and synthesis of HCV RNA was determined by incorporation of [3H]uridine into nascent viral RNA molecules using a hybrid release assay. Additionally, total PBML from an uninfected person could be infected in vitro using an HCV RNA-positive serum. The PCR products obtained from serum, liver and PBML specimens of an HCV-positive individual were found to have nearly identical sequences. Our findings suggest that PBML could be a site for viral replication of HCV during the natural course of infection and may represent a reservoir for hepatitis C virions.

B-Lymphocytes↗
PubMed1993

Studies on a species-specific epitope in murine, ovine and bovine prion protein.

Transmissible spongiform encephalopathies are fatal neurodegenerative disorders which are linked to abnormal isoforms of the prion protein (PrP), which is expressed in different cells of various mammalian species. Susceptibility to disease and reduced transmission rates upon the first passage to another species are thought to be a result of functional and biochemical differences of the PrP as a consequence of amino acid sequence among species. In 1985 an epidemic of bovine spongiform encephalopathy (BSE) started after accidental transmission of scrapie by feeding infected sheep and goat meat and bone meal products to cattle. In this report we present data demonstrating species-specific epitopes in bovine, ovine and murine PrP that are based on amino acid substitutions at positions 108 and 110. Rabbit antisera to synthetic peptides representing amino acid sequence 108 to 123 of PrP of cattle, sheep and mice reacted strongly with modified PrP of the homologous host but not, or only poorly, with PrP of heterogeneous origin. Cross-reactivity was observed, however, with antisera to bovine and ovine peptide sequences 102 to 117, thus stressing the importance of the location of the amino acid substitution in synthetic peptides used for immunization. Based on these data, BSE PrP and ovine and murine scrapie PrP can be distinguished from each other, and these differences might help elucidate the species barrier effect.

Amino Acid Sequence↗
PubMed1993

Viral resistance to the thiazolo-iso-indolinones, a new class of nonnucleoside inhibitors of human immunodeficiency virus type 1 reverse transcriptase.

Thiazolo-iso-indolinone derivatives with high specificity toward the reverse transcriptase (RT) of human immunodeficiency virus type 1 (HIV-1) were identified. The most potent compound, BM +51.0836, inhibited HIV-1 RT at a 50% inhibitory concentration of 90 nM in vitro. In cell culture assays, similar 50% inhibitory concentrations were obtained with high specificity for HIV-1. These substances were equally active against a zidovudine-resistant isolate. No antiviral effect was observed with an HIV-2 isolate. HIV-1 isolates resistant to the thiazolo-iso-indolinones were generated in cell culture, and the nucleotide sequences of the respective RT genes were analyzed subsequently. Comparison of the deduced amino acid sequences with the wild-type sequence showed an amino acid change at position 181 (Tyr to Cys). Substitutions of amino acid Lys-101 and Lys-103 as well as Tyr-181 and/or Tyr-188 by site-directed mutagenesis led to resistance against the thiazolo-iso-indolinones. A chimeric HIV-2 RT, substituted with amino acids at positions 179 to 190 from HIV-1, acquired only partial susceptibility to BM +51.0836.

Antiviral Agents↗
PubMed1992

Role of hepatitis C virus infection in German patients with fulminant and subacute hepatic failure.

To investigate the possible role of hepatitis C virus (HCV) in fulminant and subacute liver failure, we tested serum and liver of 13 patients undergoing orthotopic liver transplantation for the presence of HCV RNA. HCV RNA was detected in specimens from two out of eight patients negative for all viral markers with suspected hepatitis non-A, non-B infection and in one out of four patients with hepatitis B virus infection. Only in this patient replication of HCV could be demonstrated. We conclude, that fulminant and subacute hepatic failure is induced by hepatitis C virus only in few patients with hepatitis non-A, non-B.

Adult↗
PubMed1991

Reassessment of the v-fms sequence: threonine phosphorylation of the COOH-terminal domain.

The v-fms oncogene product of the McDonough strain of feline sarcoma virus is a member of the receptor tyrosine kinase family. Its cellular counterpart, the c-fms product, is the receptor for colony-stimulating factor 1 (CSF-1) of macrophages. We have reanalyzed the v-fms gene by direct sequencing of a biologically active clone. An additional A nucleotide was detected in position 2810 of the published v-fms sequence. The frameshift changed the COOH-terminal sequence of the v-fms protein from -R-937-G-P-P-L-COOH to -Q-937-R-T-P-P-V-A-R-COOH. Antibodies against a synthetic peptide representing this new sequence precipitated the v-fms proteins from transformed NRK cells as well as from feline sarcoma virus (McDonough)-infected feline fibroblasts. We show by tryptic peptide mapping that threonine 939 present in the new sequence is phosphorylated by a yet unknown serine/threonine kinase in vivo. In chicken fibroblasts expressing the v-fms gene, this phosphorylation clearly depended on the addition of exogenous CSF-1. Furthermore, addition of CSF-1 appeared to activate the serine/threonine kinase, as judged by phosphorylation of the synthetic peptide QRTPPVAR.

Amino Acid Sequence↗
PubMed1990

Use and control of biotechnological methods.

The use of molecular virology is described. Emphasis is placed on molecular biology, especially methods involving gene technology. The respective techniques allow characterisation of viruses by molecular cloning and nucleotide sequencing as well as the development of new diagnostic tools and vaccines. Genetically engineered live viruses serve as examples to study potential risks of vaccines derived from such viruses.

Animals↗
PubMed1989

Isolation and characterization of cytoplasmic and nuclear particles of hepatitis B virus.

The properties of hepatitis B virus (HBV) core particles from liver tissue of two patients with acute HBV infection were investigated. Cores were isolated from cytoplasm and nuclear fractions by centrifugation and two thirds of the cores were located in the cytoplasm. In all properties examined cores isolated from the cytoplasm or nucleus were the same. The cores had a density of 1.38 g/ml and had the same DNA and protein content when analyzed by Southern blotting and by Western blotting with C-specific antisera. Cores from both subcellular fractions had endogenous polymerase activity. We conclude that core particles with identical properties are found in both the cytoplasm and nucleus of cells during acute infection.

Blotting, Southern↗