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Biomedical subjects

R Laufs

Publications and source records attributed to R Laufs.

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

The influence of age on the prevalence of hepatitis C virus subtypes 1a and 1b.

The distribution of hepatitis C virus (HCV) genotypes was determined in isolates of 447 chronically HCV-infected German patients by nucleotide sequencing. Of these, 206 (46.1%) were infected with the subtype 1a, 215 (48.1%) with subtype 1b, 2 (0.4%) with subtype 1c, 9 (2.0%) with subtype 3a, and 15 (3.4%) with subtype 4a. Subtype 1a was predominant in those < 40 years old (62.6%) and was associated with the risk factor of intravenous drug addiction and with shorter duration of disease. Conversely, subtype 1b was more frequent in patients > 50 years old (84.7%; P < .001) and was associated with the risk factor of blood transfusions and with longer duration of disease. These data suggest that a shift from subtype 1b to subtype 1a occurred in the population studied. An increase in HCV infection with subtype 1a and a diminution of subtype 1b in the future can be expected.

Adult↗
PubMed1997

Minimal effect of delayed sample processing on results of quantitative PCR for cytomegalovirus DNA in leukocytes compared to results of an antigenemia assay.

Quantitative cytomegalovirus antigenemia and DNAemia were determined in peripheral leukocytes of 25 patients stored for up to 72 h at room temperature (RT) and 4 degrees C before processing. Numbers of antigen-positive cells significantly decreased with time. The decline was greater at RT than at 4 degrees C. In contrast, no significant alterations in DNAemia occurred.

Antigens, Viral↗
PubMed1997

Nonstructural protein 3 of hepatitis C virus blocks the distribution of the free catalytic subunit of cyclic AMP-dependent protein kinase.

Chronic hepatitis resulting from hepatitis C virus (HCV) infection develops into cirrhosis in at least half of infected patients and increases the risk of hepatocellular carcinoma. The pathogenic effects of a number of viruses result from the disturbance of intracellular signal cascades caused by viral antigens. Therefore, we investigated the interaction of nonstructural protein 3 (NS3) of HCV with the cyclic AMP-dependent signal pathway. We found a similarity between the HCV sequence Arg-Arg-Gly-Arg-Thr-Gly-Arg-Gly-Arg-Arg-Gly-Ile-Tyr-Arg localized in NS3 and the general consensus sequence of protein kinase A (PKA). Consequently, the catalytic (C) subunit of PKA bound to a bacterially expressed fragment of HCV polyprotein containing amino acid residues 1189 to 1525. When this fragment was introduced into cells, it inhibited the translocation of the C subunit into the nucleus after stimulation with forskolin. The result of this inhibition was significantly reduced histone phosphorylation. Therefore, the presence of NS3 in the cytoplasm of infected cells may affect a wide range of PKA functions and contribute to the pathogenesis of the diseases caused by HCV.

Biological Transport↗
PubMed1997

High percentage of seronegative HCV infections in hemodialysis patients: the need for PCR.

Among patients undergoing maintenance hemodialysis, a high prevalence of hepatitis C virus (HCV) infection can be observed. In a prospective study, sera of 273 patients were examined for the presence of HCV infection by serological tests and by PCR. Thirty-five patients (12.8%) were HCV antibody positive, and in 31 of them HCV RNA could be detected by PCR. Among the 238 seronegative patients HCV infection was detected in 12 cases (5.0%) exclusively by PCR. Only in 1 of these patients seroconversion could be observed within the 18-month follow-up period. These findings demonstrate that in hemodialysis patients PCR is necessary for the diagnosis of HCV infection.

Follow-Up Studies↗
PubMed1996

Non-structural protein 3 of hepatitis C virus inhibits phosphorylation mediated by cAMP-dependent protein kinase.

Inspection of the amino acid sequence of the non-structural region of the hepatitis C virus (HCV) gene product reveals a sequence of 14 amino acids, Arg1487-Arg-Gly-Arg-Thr-Gly-Arg-Gly-Arg-Arg-Gly-Ile-Tyr-Arg1500 , located in the non-structural protein, NS3. This sequence is highly similar to the inhibitory site of the heat-stable inhibitor of cAMP-dependent protein kinase (PKA) and to the autophosphorylation site in the hinge region of the PKA type II regulatory domain. A synthetic peptide that corresponds to the HCV sequence above and a set of shorter analogues act as competitive inhibitors of PKA. A 43.5-kDa fragment of NS3 that consists of residues 1189-1525 of the HCV polyprotein inhibits PKA in a similar range to the investigated synthetic peptides. In contrast to the short peptides, which show competitive inhibition, HCV-polyprotein-(1189-1525) influences PKA in a mixed-inhibition-type manner. A possible mechanism explaining these differences is the formation of complexes that consist of the protein substrate, the enzyme and the HCV-polyprotein-(1189-1525). Binding studies with PKA and the non-hydrolysable ATP analogue [14C]fluorosulfonylbenzoyladenosine and [3H]cAMP do not reveal any influence of the short HCV-derived peptides or HCV-polyprotein-(1189-1525) upon the affinity of PKA for these nucleotides. The complex interactions of the NS3 fragments could influence one of the most important signal pathways of the cell and, therefore, could possibly provide new pathological mechanisms for HCV infections of liver.

Amino Acid Sequence↗
PubMed1996

Detection of hepatitis B virus DNA in serum samples via nested PCR and MALDI-TOF mass spectrometry.

In a blind study, nested polymerase chain reaction (PCR) was performed with control DNA and DNA preparations from serum samples of six patients. The detection limit was determined to be 100 molecules of template in 1 ml of serum. Hepatitis B virus (HBV) related products of nested PCR were purified by ultrafiltration and immobilisation on streptavidin coated magnetic beads. The immobilized PCR products were denatured from the beads and analyzed via matrix assisted laser desorption/ionisation time-of-flight (MALDI-TOF) mass spectrometry. The results of MALDI-TOF MS analysis were in agreement with the results obtained by polyacrylamide gel electrophoresis (PAGE) and with the data obtained by serological analysis. The detection strategy introduced here has a high potential for automation and represents a fast and reliable method of detection for HBV DNA in serum without the need for time consuming gel electrophoresis and labeling or hybridization procedures.

DNA, Viral↗
PubMed1996

Characterization of the C-terminal domain of ras-GTPase-activating protein (ras-GAP) as substrate for epidermal growth factor receptor and p60c-src kinase.

We describe in vitro tyrosine phosphorylation of the C-terminal 334 amino acids of ras-GTPase-activating protein (ras-GAP)1 that contains the activity domain for ras interaction. To date, there have been no other phosphorylation sites determined than the reported in N-terminal domain of ras-GAP Tyr-460, which is considered to be the major phosphorylation site of ras-GAP. In our assays some differences of the kinetic parameters were observed when the reaction was catalyzed by EGF-R compared to p60c-src. Enzyme specific regulation of activity is associated with autophosphorylation which leads to reduced (in case of EGF-R) or increased (in case of p60c-src) phosphorylation of the C-terminal 334 amino acids of ras-GAP (GAP334). Because of the characteristics of these investigated reactions the phosphorylation of GAP334 seems to be-independent from the presence of SH2 or SH3 domains-triggered off by complex mechanisms different from those regulating the phosphorylation at Tyr-460.

Electrophoresis, Polyacrylamide Gel↗
PubMed1996

Association of biofilm production of coagulase-negative staphylococci with expression of a specific polysaccharide intercellular adhesin.

An association between adherent biofilm production on tissue culture plates and expression of a specific polysaccharide intercellular adhesion (PIA), which is functionally involved in cell clustering, was investigated for 179 Staphylococcus epidermidis isolates. Of the S. epidermidis strains, 50.8% were biofilm producers (A570 of > 0.1). There was a significant positive association between biofilm production and PIA expression: 86.8% of biofilm-producing S. epidermidis strains produced PIA as detected with a specific coagglutination assay. In contrast, 88.6% of the biofilm-negative isolates did not express PIA (P < .001). A linear association between the amount of PIA produced as detected by inhibition ELISA and the amount of biofilm produced was established for 49 S. epidermidis strains, representing a continuum from biofilm-negative to strongly biofilm-producing (r = .81, P < .001). Apparently, PIA is important for biofilm accumulation in the majority of clinical S. epidermidis isolates.

Adhesins, Bacterial↗
PubMed1996

The intercellular adhesin involved in biofilm accumulation of Staphylococcus epidermidis is a linear beta-1,6-linked glucosaminoglycan: purification and structural analysis.

The primary attachment to polymer surfaces followed by accumulation in multilayered cell clusters leads to biofilm production of Staphylococcus epidermidis, which is thought to contribute to virulence in biomaterial-related infections. We purified a specific polysaccharide antigen of biofilm-producing S. epidermidis 1457 and RP62A, which was recently shown to have a function in the accumulative phase of biofilm production by mediating intercellular adhesion (D. Mack, M. Nedelmann, A. Krokotsch, A. Schwarzkopf, J. Heesemann, and R. Laufs, Infect. Immun. 62:3244-3253, 1994). Following Sephadex G-200 gel filtration, this antigen was separated by Q-Sepharose chromatography into a major polysaccharide, polysaccharide I (> 80%), which did not bind to Q-Sepharose, and a minor polysaccharide, polysaccharide II (< 20%), which was moderately anionic. As shown by chemical analyses and nuclear magnetic resonance spectroscopy, polysaccharide I is a linear homoglycan of at least 130 beta-1,6-linked 2-deoxy-2-amino-D-glucopyranosyl residues. On average, 80 to 85% of them are N acetylated; the rest are non-N-acetylating and positively charged. Chain cleavage by deamination with HNO2 revealed a more or less random distribution of the non-N-acetylated glucosaminyl residues, with some prevalence of glucosaminyl-rich sequences. Cation-exchange chromatography separated molecular species whose content of non-N-acetylated glucosaminyl residues varied between 2 and 26%. Polysaccharide II is structurally related to polysaccharide I but has a lower content of non-N-acetylated D-glucosaminyl residues and contains phosphate and ester-linked succinate, rendering it anionic. Enzyme-linked immunosorbent assay inhibition with various monosaccharides revealed the beta-anomeric form and the acetylated amino group of the D-glucosaminyl residues as important for reactivity with the specific antiserum. The unbranched polysaccharide structure favors long-range contacts and interactions between polysaccharide strands and the cell wall and/or lectin-like proteins, leading to intercellular adhesion and biofilm accumulation. The structure of the polysaccharide is, so far, considered to be unique and, according to its function, is referred to as S. epidermidis polysaccharide intercellular adhesin (PIA).

Acetylglucosamine↗
PubMed1996

Experience with quantitative PCR for the management of HCMV disease.

Human cytomegalovirus (HCMV) is a major pathogen in newborn children and immunosuppressed individuals. There are various diagnostic methods for HCMV, but all test systems either suffer from limited sensitivity and specificity or are prone to sampling errors. Due to its unequaled sensitivity, strong hopes were pinned on the polymerase chain reaction (PCR) for the early diagnosis of active HCMV infections. However, when monitoring immunocompromised patients, unwanted positive results are often obtained without further evidence of active HCMV infection. To better discriminate between latent and active infections, different methods for the quantitation of viral genomes in clinical samples by PCR have been devised in the past years. Here we discuss the current standing of quantitative PCR in HCMV diagnostics.

Cytomegalovirus Infections↗