Biomedical subjects

R Wirth

Publications and source records attributed to R Wirth.

At least 37 records · Page 2Linked to original sources

PubMed1993

Biochemical, immunological and ultrastructural characterization of aggregation substances encoded by Enterococcus faecalis sex-pheromone plasmids.

The sex-pheromone system of Enterococcus faecalis can be viewed as a unique and highly efficient plasmid-collection mechanism. The contact needed for transfer of the conjugative sex-pheromone plasmids is mediated by an adhesin, called aggregation substance, which is encoded by these plasmids. We show here that for 17 of the 18 sex-pheromone plasmids (pAM373 being the exception) described to date, their adhesins are immunologically related to each other. In each case, we observed the presence of an N-terminal fragment of about 78 kDa in addition to the 137-kDa form of mature aggregation substance. The cross-reactions were different for the various plasmids. In the case of pPD1 the 78-kDa fragment reacted only weakly. The aggregation substance encoded by sex-pheromone plasmid pAD1 (Asa1) was characterized in detail. The conditions used for SDS/PAGE had a drastic influence on the migration behavior of mature aggregation substance and differently migrating, interconvertible forms were identified. Preliminary data indicate that Asa1 might be a glycoprotein. Antibodies were isolated which are directed against the N- and C-terminal parts of aggregation substance. They showed about the same reactivity on Western blots; however, only antibodies directed against the N-terminal part of the aggregation substance could inhibit the bacterial cell/cell contact. The reactions of the two antibody preparations with induced cells of E. faecalis was analyzed by transmission electron microscopy. The results indicated that especially the N-terminal part of aggregation substance is exposed on the cell surface of E. faecalis; the C-terminal part seems to be much less exposed.

Antibodies↗

PubMed1992

Identification of new sex pheromone plasmids in Enterococcus faecalis.

We describe the identification of the following new sex pheromone plasmids in Enterococcus faecalis: a haemolysin-bacteriocin plasmid, pIP964; three R plasmids, pIP1017, pIP1438 and pIP1440; and two cryptic conjugative plasmids, pIP1141 and pMV120. The identification was based on the formation of cell aggregates on filter membranes during conjugation, on efficient transfer in broth matings, and on a positive clumping reaction of cells carrying these plasmids. In addition these plasmids hybridized with DNA probes specific for sex pheromone-induced structural genes encoding surface proteins required for conjugative transfer of the plasmids.

DNA Probes↗

PubMed1992

Sex pheromone plasmid pAD1-encoded surface exclusion protein of Enterococcus faecalis.

During conjugative transfer of sex pheromone plasmids of Enterococcus faecalis a so-called surface exclusion protein reduces the frequency with which these plasmids are transferred to cells already possessing the same plasmid. We report here the DNA sequence of a 3.8 kb fragment of the sex pheromone plasmid pAD1 containing the structural gene sea1 for surface exclusion protein and a small open reading frame (ORF) upstream of sea1. Surface exclusion protein Sea1 was found to be highly homologous to the surface exclusion protein Sec10 encoded by the sex pheromone plasmid pCF10. Hybridization studies with DNA probes derived from the structural gene sea1 demonstrated that, with the exception of pAM373, all known sex pheromone plasmids carry a homologous gene. These studies also indicated that the genetic organization is similar in these plasmids, with the structural gene for surface exclusion protein being located 5' to that for aggregation substance.

Amino Acid Sequence↗

PubMed1992

Transcriptional control of sex-pheromone-inducible genes on plasmid pAD1 of Enterococcus faecalis and sequence analysis of a third structural gene for (pPD1-encoded) aggregation substance.

The expression of several neighbouring genes on plasmid pAD1 that are necessary for conjugation depend on induction with sex pheromone cAD1. Analyses of transcripts by Northern blot hybridization demonstrated that the genes sea1 (encoding surface exclusion protein) and asa1 (encoding aggregation substance) are transcribed independently. Both genes are organized in different operons together with neighbouring open reading frames of unknown function. Several transcripts could be identified for sea1 and asa1. Their transcriptional start sites were determined by primer extension experiments, confirming the results of the Northern blot experiments. We also could identify sea1- and iad- (encoding an inhibitory peptide counteracting sex pheromone cAD1) specific transcripts which are expressed constitutively, but to a lower extent relative to induced conditions. In addition, we localized the asp1 gene coding for aggregation substance of sex pheromone plasmid pPD1 and determined its DNA sequence, which was found to be highly homologous to asa1 (aggregation substance gene of pAD1) and prgB (aggregation substance gene of pCF10). The structural genes were found to be organized more or less identically on the three sex-pheromone plasmids pAD1, pCF10, and pPD1, and to be highly conserved. Regions supposed to be of crucial importance for regulatory functions, however, were found to differ. We also could identify some conserved DNA motifs which might be potential target sites for transcriptional regulators. In combination these data allowed us to formulate a model for the regulation of sex-pheromone-inducible genes of plasmid pAD1. Its main statement is that only in the presence of cAD1 can the gene traE1 be transcribed. The positive regulatory factor TraE1 then can trigger expression of the structural genes sea1 and asa1.

Amino Acid Sequence↗

PubMed1992

Efficacy of different irrigation methods and concentrations of root canal irrigation solutions on bacteria in the root canal.

The effectiveness of two different root canal irrigating solutions, each in two different concentrations or formulations, with two different irrigation methods was compared in vitro by means of bacterial survival determinations. 75 human root canals were enlarged, sterilized and inoculated with a mixed culture of Escherichia coli and Streptococcus mutans. After inoculation, the root canals were irrigated either manually or with an ultrasonic device for equal times (20s) with the same amount (5 ml) of sodium hypochlorite (1% and 2%), Fokalhydran I and Fokalhydran II. Sodium hypochlorite (1% and 2%) was used in a 1:100 dilution. Fokalhydran I and II were used in a 1:10 dilution. In the sodium hypochlorite group, the 1% concentration applied with a syringe proved to be most effective against Escherichia coli and Streptococcus mutans. The least effective concentration and application method against both bacteria species was obtained with 2% NaOCL and ultrasonics. Against Escherichia coli and Streptococcus mutans, a significantly lower effectiveness was found with 2% NaOCL applied with ultrasonics with respect to the rest of the sodium hypochlorite group. Fokalhydran I was significantly better than Fokalhydran II against Escherichia coli. However, no significant differences could be seen against Streptococcus mutans within this group.

Analysis of Variance↗

PubMed1992

Aggregation substance of Enterococcus faecalis mediates adhesion to cultured renal tubular cells.

The sex pheromone system of Enterococcus faecalis is a unique, highly efficient plasmid collection mechanism for this species. A crucial role in this system is played by an adhesin called aggregation substance which enables the cell-cell contact between donor and recipient strains. The existence of the amino acid motif Arg-Gly-Asp-Ser in the adhesin prompted us to look for a possible binding of E. faecalis cells expressing aggregation substance to eucaryotic cells. We were able to show that the adhesin mediated binding to cultured renal tubular cells (porcine cell line LLC-PK1) via light microscopic, electron microscopic, and enzyme-linked immunosorbent assay-based studies. Synthesis of the adhesin was induced by some component(s) of serum. These data are interpreted to mean that aggregation substance is an adhesin mediating not only cell-cell contact between different E. faecalis strains but also binding of E. faecalis to eucaryotic cells, and therefore it might contribute to virulence.

Amino Acid Sequence↗

PubMed1992

Habitual physical activity in adult IDDM patients. A study with portable motion meters.

The objective of this study was to assess the habitual physical activity of insulin-dependent diabetes mellitus (IDDM) patients on intensive insulin therapy. This case-control study consisted of 34 IDDM patients (14 on multiple injection therapy, 20 on continuous subcutaneous insulin infusion [CSII], and control subjects matched for sex, age, weight, height, body mass index, smoking behavior, and occupational status). Seven IDDM patients were studied before and after changing from multiple injection to CSII therapy. All patients were well controlled according to HbA1c. Portable motion meters were used to assess habitual physical activity during 7 consecutive days with appreciable reproducibility (coefficient of variation 1.24%) and agreement to standardized activity protocols (r = 0.96, P < 0.001). Habitual physical activity was similar in IDDM patients on injection treatment and in controls, respectively. CSII-treated patients exhibited on the average 17% less habitual physical activity than control subjects (P < 0.05). Changing from multiple injection therapy to CSII lowered habitual physical activity insignificantly in seven patients. There was no indication of decreasing physical exercise (e.g., sports) by CSII in this patient group. No correlations were found between habitual physical activity and HbA1c or body mass index, respectively. Habitual physical activity is similar in IDDM patients on multiple injection therapy and control subjects, but may decrease by CSII therapy.

Adult↗

PubMed1991

Genetic transformation of various species of Enterococcus by electroporation.

A transformation system for Enterococcus faecalis was developed which uses untreated (i.e., non-protoplasted) cells and the electroporation technique. The optimized protocol resulted in transformation efficiencies of up to 4 x 10(6) transformants per microgram of plasmid DNA. All strains of E. faecalis tested could be transformed by this method, albeit with differing transformation efficiencies. Using the protocol optimized for E. faecalis we successfully transformed Enterococcus faecium, E. hirae, E. malodoratus and E. mundtii.

Cell Division↗

PubMed1991

Computed tomography as an adjunct to ultrasound in the diagnosis of acute acalculous cholecystitis.

The sonographic and computed tomographic (CT) findings were reviewed in 17 patients with acute acalculous cholecystitis (AAC) over a 6-year period from 1984 to 1989. Of the six patients in whom both ultrasound and CT were performed, CT revealed marked gallbladder (GB) wall abnormalities, including perforation, and pericholecystic fluid collections in five patients not demonstrated by sonography. Of the total group, five patients had GB wall thicknesses of less than or equal to 3 mm (normal) at pathologic examination, which demonstrated a spectrum of disease ranging from acute hemorrhagic/necrotizing, to gangrenous acalculous cholecystitis with perforation. Sonography was falsely negative or significantly underestimated the severity of AAC in seven of the 13 patients examined by sonography. CT because of its superior ability to assess pericholecystic inflammation may provide additional diagnostic information even after a thorough sonographic study in cases of AAC.

Acute Disease↗

PubMed1991

Nucleotide sequence of the gelatinase gene (gelE) from Enterococcus faecalis subsp. liquefaciens.

The gene coding for gelatinase (also called metalloendopeptidase II; microbial proteinase, EC 3.4.24.4) of Enterococcus faecalis subsp. liquefaciens strain OG1-10 was cloned in an Escherichia coli-Enterococcus shuttle vector, and its nucleotide sequence was determined. The DNA sequence encodes one large open reading frame (ORF) with 509 amino acid residues. The ORF contains a signal sequence in its N-terminal region, whereas the N-terminal amino acid sequence determined from the purified extracellular proteinase starts at residue 192 deduced from the ORF. This implies that the gelatinase is synthesized as a prepropolypeptide or prezymogen. The mature gelatinase contains 318 amino acid residues (molecular weight, 34,582) and has significant homology with neutral proteinases from Bacillus species and elastase from Pseudomonas aeruginosa.

Amino Acid Sequence↗

PubMed1991

Comparative analysis of Enterococcus faecalis sex pheromone plasmids identifies a single homologous DNA region which codes for aggregation substance.

An analysis of the 11 known sex pheromone plasmids of Enterococcus faecalis was performed by DNA-DNA hybridization. Plasmids pAD1, pJH2, and pBEM10 turned out to be closely related, whereas pAM373 showed only weak homology with pAD1. A comparison of the hemolysin/bacteriocin determinants of pAD1, pJH2, and pOB1 revealed strong similarities at the DNA level. Our main finding was that one DNA region is conserved among all sex pheromone plasmids, with pAM373 again being an exception; for pAD1 this region was shown earlier to code for aggreagation substance. Detailed hybridization studies of the genes for this plasmid-coded adhesin, which is responsible for cell-cell contact during conjugative transfer via the so-called sex pheromone system of E. faecalis, support the idea of their common origin.

Adhesins, Escherichia coli↗

PubMed1990

Sequence analysis of Enterococcus faecalis aggregation substance encoded by the sex pheromone plasmid pAD1.

The location of the structural gene for aggregation substance on the sex pheromone plasmid pAD1 of Enterococcus faecalis was determined using an oligonucleotide deduced from the N-terminal amino acid sequence of the purified protein. The nucleotide sequence was determined for the corresponding region and two open reading frames (ORFs) could be identified. ORF1 codes for a small (Mr 13,160) acidic protein of unknown function. The gene for aggregation substance (named asa1) was found to code for a protein of 1296 amino acids (Mr 142,248). The protein has a signal peptide of 43 amino acids (the resulting Mr for mature aggregation substance is 137,429) and contains in its C-terminal region a proline-rich sequence, previously characterized as being involved in cell wall association, which is followed by a membrane anchor. The membrane anchor showed significant similarity to that of other Gram-positive organisms, but no other similarities to surface proteins from Gram-positive bacteria were found. In particular, no repeats on the DNA or protein level could be detected for pAD1-specific aggregation substance. The protein contains the amino acid motifs Arg-Gly-Asp-Ser and Arg-Gly-Asp-Val (once each), which, it is proposed, play a crucial role in adherence to eukaryotic cells.

Amino Acid Sequence↗

PubMed1989

Transformation of various species of gram-negative bacteria belonging to 11 different genera by electroporation.

We have undertaken a systematic study to test the transformation of various species of gram-negative bacteria using the electroporation method. The data obtained show very clearly that a great variety of gram-negative bacteria--15 different species belonging to 11 different genera--including freshly isolated wild-type strains can be transformed efficiently by use of the electric-field mediated transformation technique. These include species of the families Enterobacteriaceae, Pseudomonadaceae, Rhizobiaceae, photosynthetic bacteria and strains for which transformation could not be achieved, up to now, by other methods.

Electricity↗

PubMed1989

Identification of aggregation substances of Enterococcus faecalis cells after induction by sex pheromones. An immunological and ultrastructural investigation.

The sex pheromone system of Enterococcus faecalis is responsible for the clumping response of a plasmid carrying donor strain with a corresponding plasmid free recipient strain due to the production of sex pheromones by the recipient strain. The clumping response is mediated by a surface material (called aggregation substance) which is synthesized upon addition of sex pheromones to the cultures. Here we show that after induction a dense layer of "hairlike" structures is formed on the cell wall of the bacteria. These hairlike structures are responsible for the cell-cell contact which leads to the aggregation of cells. Formation of these structures was specific, only occurring after the addition of homologous sex pheromone.

Biological Factors↗

PubMed1989

Localization of aggregation substances of Enterococcus faecalis after induction by sex pheromones. An ultrastructural comparison using immuno labelling, transmission and high resolution scanning electron microscopic techniques.

The distribution of sex pheromone induced aggregation substance was studied on the cell surface of various Enterococcus faecalis strains. In the accompanying paper we have shown that the aggregation substance appears as a layer of hairlike structures. Using direct and indirect immunogold technique, transmission electron microscopy and high resolution scanning electron microscopy we investigated the appearance and distribution of the aggregation substance. The "hairs" increase in number with increasing exposure to sex pheromones (maximum density: 1300/microns2). We show that these structures are unequally distributed over the cell surface, even if the cells were induced by sex pheromones for a long period of time. Statistical analysis of the unequal distribution indicates that aggregation substance is incorporated into pre-existing "old" cell-walls and that this incorporation shows a saturation ca. 40 min after addition of sex pheromones.

Biological Factors↗

PubMed1988

Transformation of bacteria with plasmid DNA by electroporation.

The possibility of electric field-mediated transformation ("electroporation") of a gram-positive bacterium (Enterococcus faecalis) and two gram-negative bacteria (Escherichia coli and Pseudomonas putida) with plasmid DNA was investigated. E. faecalis protoplasts could be transformed by electroporation with a transformation frequency of 10(4) to 10(5) transformants/micrograms plasmid. Untreated--i.e., washed--cells of E. coli could be transformed with rates of 1 X 10(5) transformants/micrograms plasmid DNA. Transformation rates for P. putida cells were up to 3 X 10(4) if the method developed for E. coli was used. Detailed protocols for these systems, including the results of various optimization experiments, are given.

DNA, Bacterial↗

PubMed1988

Transient heart block associated with head trauma.

Atrioventricular nodal block is a rare consequence of head trauma. We report a case of a 63-year-old male with coronary artery disease who sustained a skull fracture. Twelve hours later the cardiac monitor recorded an episode of high-grade atrioventricular block with a ventricular escape rhythm. Subsequent electrophysiologic evaluation revealed no abnormality of atrioventricular nodal conduction nor of intraventricular conduction. Atrioventricular block after head trauma may be caused by enhanced vagal tone.

Creatine Kinase↗

PubMed1986

Studies on the lysosomal binding of 141Ce, 239Np, 239Pu and 241Am in rat and Syrian hamster liver using carrier-free electrophoresis.

The binding of 141Ce, 239Np, 239Pu and 241Am in the liver of rats and Syrian hamsters, following injection in essentially monomeric form, was analyzed by carrier-free electrophoresis at 4-9 days and several months after radionuclide injection. In contrast to density gradient methods lysosomes can be clearly separated from other cell organelles by carrier-free electrophoresis. These, and previous results from this and other laboratories confirm that lysosomes are the main initial binding site for these four radionuclides in the livers of rats and Syrian or Chinese hamster. Light microscopic autoradiography showed that at all the time intervals studied 241Am was more or less uniformly distributed in the liver of all three species. Thus, the changes in the electrophoretic pattern of the nuclides, observed at later time periods in hamsters, cannot be explained by gross redistribution phenomena such as accumulation in macrophages.

Americium↗