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K Köhrer

Publications and source records attributed to K Köhrer.

At least 37 records · Page 2Linked to original sources
PubMed1999

Molecular epidemiology of quinolone resistance and comparative in vitro activities of new quinolones against European Staphylococcus aureus isolates.

UNLABELLED: New fluoroquinolones (FQ) may possibly be used as alternative therapeutic options for Staphylococcus aureus infections. Our objectives were: (1) to define the in vitro activities of seven FQs in a collection of 434 methicillin-susceptible and 457 methicillin-resistant S. aureus from 23 European university hospitals; (2) to characterise the prevalence of mutations in the grlA and gyrA genes in all ciprofloxacin-resistant (n=433) isolates of S. aureus; (3) to determine the percentage of ciprofloxacin-resistant S. aureus strains with measurable quinolone efflux. METHODS: (1) The in vitro activities of different FQs were determined by microdilution tests. (2) PCR-amplified DNA was sequenced. (3) Ciprofloxacin minimum inhibitory concentrations (MIC) were determined in the presence and absence of reserpine, which inhibits efflux pumps. RESULTS: (1) Irrespective of the methicillin resistance of the isolates, sitafloxacin and clinafloxacin showed the best in vitro activities. (2) All ciprofloxacin-resistant isolates exhibited GrlA alterations, namely Ser-80-->Phe or Tyr or Glu-84-->Lys or Ala-116-->Glu or Pro or a combination of Ser-80-->Phe and Glu-84-->Val. These alterations in GrlA were combined with alterations in GyrA, namely Ser-84-->Leu or Lys or Glu-88-->Lys or Val. (3) Reserpine reduced ciprofloxacin MIC values in ca. 30% of the clinical isolates tested. CONCLUSIONS: (1) This current European overview of mutations involved in FQ resistance demonstrates that only a limited number of classical mutations in grlA and gyrA contributed to resistance in clinical isolates. (2) An efflux pump is involved in ca. 30% of ciprofloxacin-resistant S. aureus isolates. (3) Sitafloxacin and clinafloxacin are two very promising new FQs with good anti-staphylococcal activity. New FQs, perhaps in combination with efflux pump inhibitors, might play a role in the treatment of S. aureus infections.

Anti-Infective Agents↗
PubMed1999

Association of alterations in ParC and GyrA proteins with resistance of clinical isolates of Enterococcus faecium to nine different fluoroquinolones.

The parC and gyrA genes of 73 ciprofloxacin-resistant and 6 ciprofloxacin-susceptible Enterococcus faecium clinical isolates were partly sequenced. Alterations in ParC and GyrA, possibly in combination with other resistance mechanisms, severely restricted the in vitro activities of the nine quinolones tested. For all isolates, clinafloxacin and sitafloxacin showed the best activities.

Anti-Infective Agents↗
PubMed1999

Comparative in vitro activities of ciprofloxacin, clinafloxacin, gatifloxacin, levofloxacin, moxifloxacin, and trovafloxacin against Klebsiella pneumoniae, Klebsiella oxytoca, Enterobacter cloacae, and Enterobacter aerogenes clinical isolates with alterations in GyrA and ParC proteins.

The in vitro activities of ciprofloxacin, clinafloxacin, gatifloxacin, levofloxacin, moxifloxacin, and trovafloxacin were tested against 72 ciprofloxacin-resistant and 28 ciprofloxacin-susceptible isolates of Klebsiella pneumoniae, Klebsiella oxytoca, Enterobacter cloacae, and Enterobacter aerogenes. Irrespective of the alterations in GyrA and ParC proteins, clinafloxacin exhibited greater activity than all other fluoroquinolones tested against K. pneumoniae and E. aerogenes.

Anti-Infective Agents↗
PubMed1998

Differential modulation of pro- and anti-inflammatory cytokine receptors by N-(4-trifluoromethylphenyl)-2-cyano-3-hydroxy-crotonic acid amide (A77 1726), the physiologically active metabolite of the novel immunomodulator leflunomide.

N-(trifluoromethylphenyl)-2-cyano-3-hydroxy-crotonic acid amide (A77 1726), the physiologically active metabolite of leflunomide, has been described to exert antiproliferative effects in vitro and anti-inflammatory actions in several animal models. Currently, its use is being evaluated in clinical trials in psoriasis, which is characterized by epidermal hyperproliferation and infiltration of inflammatory cells. We studied the effects of A77 1726 on growth and gene expression in cultured epidermal cells by 5-bromo-2'-deoxy-uridine (BrdU) incorporation, reverse transcriptase-polymerase chain reaction (RT-PCR), Northern blot hybridizations and flow cytometry. A77 1726 inhibited epidermal proliferation at concentrations above 5 microM after 24 hr. However, the cells were still fully viable at a concentration of 100 microM. The drug caused a dose-dependent reduction in the mRNA level of the type A receptor for the proinflammatory cytokine interleukin-8 (IL-8-RA) and, in contrast, induced gene expression of the receptor for the anti-inflammatory cytokine IL-10 (IL-10R) at the mRNA and protein levels. In addition, the mRNA and protein levels of the p53 gene, which is a negative cell cycle regulator, were up-regulated by A77 1726. These data suggest that A77 1726 exerts its anti-inflammatory action via the modulation of epidermal gene expression.

Aniline Compounds↗
PubMed1998

A female-specific cDNA sequence of Schistosoma mansoni encoding an amidase that is expressed in the gastrodermis.

A female-specifically expressed cDNA clone was obtained by screening of a subtractive cDNA library enriched for RNA from Schistosoma mansoni females. The deduced protein shows significant homology to a class of enzymes functioning as amidases. Northern blots revealed a transcript of 4.0 kb which is absent in larval stages, but is expressed in adult female worms. By in situ hybridization, the expression site of the gene was exclusively localized in the gastrodermis of female schistosomes. This is the first report of a female-specifically transcribed sequence of S. mansoni that is not expressed in the reproductive organs.

Animals↗
PubMed1998

Regulation of the HGF-receptor c-met in the thyroid gland.

HGF (hepatocyte growth factor) has been characterised as an important mitogen and motogen in many epithelial cells. The biological and clinical significance of HGF and its receptor c-met in the thyroid is currently under study. Overexpression of c-met is an important feature of papillary thyroid cancer. We developed a quantitative differential RT-PCR method in order to examine HGF-receptor regulation using RNA of about 50 cells per analysis. Experiments were performed in three spontaneously transformed follicular thyroid cancer (FTC) cell lines FTC-133, 236, and 238, 7 primary cultures derived from multinodular goitres, one from Graves disease and 1 from papillary thyroid cancer (PTC). TGF-alpha and to a minor degree HGF were shown to induce a marked up-regulation of the receptor whereas bovine TSH, NaI, dbcAMP or basic FGF had no apparent effect. We conclude that expression of the HGF-receptor is under control of paracrine growth factors activating tyrosine-kinase-dependent pathways. We could demonstrate a minor stimulation of thyroid cell proliferation by HGF in presence but not in absence of 10% fetal calf serum.

Adult↗
PubMed1998

Rare loss of heterozygosity of the MTS1 and MTS2 tumor suppressor genes in differentiated human thyroid cancer.

Loss of heterozygosity (LOH) of the MTS1 (p16) tumor suppressor gene has been reported to occur frequently in thyroid cancer cell lines. In order to determine the frequency of LOH for these multiple tumor suppressor genes, we used microsatellite markers IFNA and D9S171 to perform differential quantitative polymerase chain reaction. Tumor DNA was isolated from native sections of tumor tissue. Control DNA was isolated from blood. PCR products were separated on 6% polyacrylamide sequencing gels and quantified according to peak height and area. Analysis was informative in 70% of cases for both markers, and in 88% for at least one out of both. LOH was found in 3 out of 35 informative patients (8.6%) with papillary thyroid cancer, in 1 out of 7 patients with follicular thyroid cancer (14.2%), and in 0 out of 18 medullary cancers (0%). No LOH was found in 11 informative patients with multinodular goitre, 7 with follicular adenoma, 4 with Graves' disease, and 6 with other thyroid disease. 75% of LOH was found in T1 and T2 stages, it was not more frequent in patients with lymphonodular metastasis. The low frequency of LOH in these types of thyroid cancer argues against a role of loss of heterozygosity at the MTS 1 and 2 gene locus in the development of differentiated thyroid neoplasia.

Adenoma↗
PubMed1998

Relationship between ciprofloxacin, ofloxacin, levofloxacin, sparfloxacin and moxifloxacin (BAY 12-8039) MICs and mutations in grlA, grlB, gyrA and gyrB in 116 unrelated clinical isolates of Staphylococcus aureus.

The in-vitro activities of five fluoroquinolones were tested against 70 ciprofloxacin-resistant and 46 ciprofloxacin-susceptible unrelated isolates of Staphylococcus aureus. All 116 S. aureus isolates were studied for the presence of mutations in the grl and gyr loci. The order of efficacy of the fluoroquinolones tested, from least to most active, was: ciprofloxacin, ofloxacin, levofloxacin, sparfloxacin and moxifloxacin (BAY 12-8039), in response to all characterized mutations in grlA, grlB, gyrA and gyrB. Moxifloxacin was active against most S. aureus isolates tested (MIC90 = 1 mg/L for ciprofloxacin-resistant isolates) and was less influenced by known mutations.

Anti-Infective Agents↗
PubMed1998

Development of a multiplex-PCR for direct detection of the genes for enterotoxin B and C, and toxic shock syndrome toxin-1 in Staphylococcus aureus isolates.

As well as conventional methods such as immunodiffusion, ELISA, or agglutination for the detection of toxin production in Staphylococcus aureus, amplification techniques like PCR allow a very sensitive and specific identification of the genes responsible for enterotoxin B and C, and TSST-1 production. These toxins might be a cause of the toxic shock syndrome (TSS). For that reason an easy and quick test system for determining the toxin production pattern of S. aureus isolates is desirable so that strains suspected to be toxin producers may be identified much faster and easier. In the present investigation, a new multiplex-PCR method was used that allowed single bacterial colonies grown on agar plates to be used directly in the PCR assay without preceding preparation. This procedure generated information concerning the presence of seb, sec-1 and tst genes within 4 h in a single test. To analyse the sensitivity and the specificity of this procedure, 100 methicillin-resistant S. aureus (MRSA), 50 coagulase-negative staphylococci and 50 other eubacterial isolates were tested initially with sets of single primer pairs followed by a combined multiplex-PCR. Results of this amplification technique were compared to a conventional and widely used method for toxin detection, reversed passive latex agglutination (RPLA). With the RPLA assay results as the basis, sensitivity and specificity of the seb and tst primer sets were 100%, whereas sensitivity and specificity of the sec-1 primer set were 100% and 82%, respectively. With the sec-1 primer set, two isolates were identified as carrying the corresponding toxin gene although the RPLA test did not show any detectable toxin. The multiplex-PCR rapidly generated reliable information concerning the toxin-producing capacity of staphylococcal strains and could be easily integrated into a multiplex procedure described previously. The latter enabled the identification of specific PCR products for eubacteria and staphylococci as well as the detection of the coa and mecA genes.

Bacterial Toxins↗
PubMed1998

Typing of methicillin-resistant Staphylococcus aureus isolates from Düsseldorf by six genotypic methods.

Nosocomial infections caused by methicillin-resistant Staphylococcus aureus (MRSA) represent an increasing problem in hospitals. Quick and reliable typing methods are required to obtain information about the relatedness of MRSA isolates and to allow faster implementation of appropriate infection control measures. This investigation describes the distribution of MRSA isolates from 11 hospitals in the Düsseldorf region of Germany, and the ability of six different genotypic typing techniques -- pulsed-field gel electrophoresis (PFGE), random amplification of polymorphic DNA (RAPD), 16S-23S rDNA spacer amplification, protein A-gene PCR, PCR characterisation of the hypervariable region (HVR) adjacent to mecA, and coagulase gene-PCR -- to detect different unrelated types. Of 7814 S. aureus isolates tested, 489 (6.3%) were MRSA, of which 183 were selected for subsequent molecular analyses on the basis of being the first MRSA isolated from colonised or infected patients. Larger hospitals had a higher incidence of MRSA and a greater variability in genotypes than smaller hospitals. All methods confirmed the presence of two main clonal types. The ability of techniques to detect different unrelated types was found to be as follows: PFGE, 28 types; 16S-23S rDNA spacer-amplification, 10 types; RAPD, nine types; protein A-gene PCR, five types; HVR-PCR, five types; and coa gene-PCR, two types. Combination of PFGE and one other PCR-based method (spacer-amplification, RAPD or protein-A gene PCR) provided the best resolution of types and allowed the identification of subtypes. Similar molecular types were identified with international MRSA isolates. Although PCR-based techniques have the advantage of rapid performance and easy handling, their discriminatory capacity is inferior compared to the more labour intensive PFGE.

Bacterial Typing Techniques↗
PubMed1998

Characterization of grlA, grlB, gyrA, and gyrB mutations in 116 unrelated isolates of Staphylococcus aureus and effects of mutations on ciprofloxacin MIC.

One hundred sixteen unrelated clinical isolates of Staphylococcus aureus (70 ciprofloxacin resistant and 46 ciprofloxacin susceptible) from eight countries were studied for the presence of mutations in the grlA, grlB, gyrA, and gyrB gene loci. Two mutations within grlA (located at codons 80 and 84) and two mutations within gyrA (located at codons 84 and 88) were clearly associated with ciprofloxacin resistance, although other mutations detected within the four genes studied may also contribute to decreased susceptibility.

Anti-Infective Agents↗
PubMed1998

Experience with cardiac troponin T in difficult cases.

Recent years have seen the advent of new cardiac markers. These have been shown to be valuable in large controlled clinical trials. The breakthrough of the troponins world-wide for detection of myocardial lesions has meant that the Cardiac Troponin T parameter has been available in our hospital since August 1996. Analysis of the samples is carried out on the Elecsys, an automated immunology analyzer from Roche Diagnostics, Austria. The purpose of the present paper is to investigate whether study results on the diagnostic utility, sensitivity and specificity of troponin T can be confirmed in multimorbid patients in routine clinical practice, who are otherwise excluded from such trials. Troponin T was investigated in five cases including patients presenting with myocarditis and parallel damage of the skeletal muscles, as well as in haemodialysis patients. Cardiac troponin T has proved to be a very reliable parameter in all of these patients due to its high sensitivity and specificity, where other conventional diagnostic procedures were not helpful due to co-morbidity and confounding factors. New clinically relevant information can be obtained by both individual as well as serial measurements. Cardiac troponin T determination is a powerful new tool and should be considered seriously, regardless of other underlying diseases: it is, therefore, not only a diagnostic but also a management tool.

Aged↗
PubMed1997

The VPS4 gene is involved in protein transport out of a yeast pre-vacuolar endosome-like compartment.

Four yeast mutants were isolated in a screen for dominant-negative vacuolar protein-sorting mutants, secreting a carboxypeptidase Y-invertase hybrid protein. In addition to defects in the sorting/transport of soluble vacuolar hydrolases, the mutants accumulated a pre-vacuolar endosome-like compartment. The mutant alleles causing the defects were identified as the members of the VPS4 gene locus, each harbouring single-point mutations leading to amino-acid exchanges at positions 233 (E233Q), 211 (E211 K), and 178 (G178D). These mutations all reside within a 200 amino-acid-long ATPase module, common to members of the AAA-protein family. The VPS4 gene product shows homology to the yeast Sec18p (50% similarity and 25% identity), which is involved in several vesicle-mediated protein transport steps and homotypic membrane fusion events. Disruption of the VPS4 gene leads to a recessive vacuolar protein-sorting phenotype. About 40% of newly synthesized CPY is secreted as the Golgi-modified p2CPY precursor form. Transport of secretory proteins to the plasma membrane is normal as demonstrated by the secretion of invertase and alpha-factor. The alpha-factor, however, is secreted as a partially processed precursor, caused by defects in late Golgi function. The vps4 mutants also exhibit defects in fluid-phase endocytosis, as demonstrated by the accumulation of Lucifer Yellow in a pre-vacuolar endosome-like compartment. Based on the pleiotropic phenotype of the vps4 mutants and on the sequence homology to NSF/Sec18p, we propose that the VPS4 gene product is required for efficient transport out of the pre-vacuolar endosome-like compartment.

Adenosine Triphosphatases↗
PubMed1997

Specific information concerning taxonomy, pathogenicity and methicillin resistance of staphylococci obtained by a multiplex PCR.

The use of DNA amplification techniques such as the polymerase chain reaction (PCR) in modern diagnostic microbiology not only allows the sensitive and specific identification of micro-organisms but also the detection of specific antibiotic resistance genes. This study describes a multiplex PCR on bacterial colonies picked directly from agar plates without preceding DNA preparation. Eubacteria and staphylococci were identified by, 16S rRNA specific PCR products. In parallel, specific primers were used for the detection of staphylococcal coa and mecA genes. This 4-h multiplex PCR, consisting of four sets of primers, was evaluated for rapid and specific differential diagnosis of methicillin-resistant and methicillin-susceptible strains of Staphylococcus aureus and coagulase-negative staphylococci. To analyse specificity of the amplification products, 100 non-staphylococcal, eubacterial isolates and 20 Candida albicans strains were tested. In a first step, specificity of all four single sets of primers was evaluated before the co-amplification within the multiplex PCR procedure was performed. The results were compared with those of conventional susceptibility and typing methods. The specific 16S rRNA PCR product for eubacterial isolates (n = 786) and staphylococci (686) was found in all strains tested. The coa gene was detected only in S. aureus (488) strains with a specificity of 100%, and was not detected in any of the coagulase-negative staphylococci (198). The mecA gene was detected in 98% of methicillin-resistant staphylococci (393) and in 2% of all methicillin-susceptible staphylococci (293). The multiplex PCR with co-amplification of different determinants provides rapid reliable information on staphylococcal identification and methicillin susceptibility supporting the diagnosis, treatment and control of staphylococcal infections.

Bacterial Proteins↗
PubMed1997

Analysis of UVB-modulated gene expression in human keratinocytes by mRNA differential display polymerase chain reaction.

Ultraviolet (UV) light is the most important environmental insult to skin. Even a single exposure to UVB radiation can result in inflammation and may also lead to DNA damage and apoptosis in the acute response of the cutaneous tissue. To elucidate the complex alterations of gene expression in human keratinocytes underlying these UV responses we took advantage of differential display polymerase chain reaction (DD-PCR) technology's ability to detect qualitative and quantitative changes in gene expression in more than two cell populations simultaneously. We demonstrate that low-dose UVB (100 Jm-2) leads to both induction and downregulation of different genes during the 24 h after irradiation in a time-dependent manner. In addition to the identification of known genes as possible effectors or targets in the UV response of human keratinocytes, we here identify a new sequence that is negatively regulated by UVB irradiation and was termed HUR 7 (HaCaT UV repressed). In general our results showed that DD-PCR is a useful tool in the analysis of quantitative changes of mRNA levels in human keratinocytes after UV irradiation. The identification of new UVB-repressed genes offers the opportunity to identify unrecognized molecular mechanisms in the UV response of human cells.

Blotting, Northern↗
PubMed1997

Cloning and characterization of a dominant-negative vps1 allele of the yeast Saccharomyces cerevisiae.

The gene product of the yeast VPS1 gene is a member of a family of high-molecular-weight GTP-binding proteins that are involved in diverse cellular processes. The Vps1 protein (Vps1p) was shown to perform an essential function in the yeast secretory pathway. Here, we report the isolation and characterization of a mutant allele of the VPS1 gene, causing a dominant-negative vacuolar protein sorting (vps) defect, as demonstrated by the mislocalization of the vacuolar hydrolase carboxypeptidase Y (CPY). DNA sequence analysis of the mutant vps1 allele (vps1d-293) revealed a single point mutation, resulting in an amino acid exchange at position 293 from Ala to Asp. The mutation is located downstream of the tripartite GTP-binding motif found in the amino-terminal half of the protein. The observation that expression of wild-type Vps1p partially suppressed the dominant-negative CPY sorting phenotype indicates competition of a non-functional mutant Vps1 protein and a functional wild-type VPS1p for a Vps1p-binding site of an as yet unknown vacuolar protein sorting factor.

Alanine↗
PubMed1996

The ribosomal protein gene RPS3 is an essential single copy gene of the yeast Saccharomyces cerevisiae.

The communication reports the cloning, sequencing, and analysis of the RPS3 gene from yeast, which codes for the ribosomal protein YS3. Sequence analyses of a 2.45 kb DNA fragment revealed an open reading frame with the potential to code for a 240 amino-acid long protein. The first 20 amino acids display a 90% identity to a 20 amino-acid long protein sequence of yeast ribosomal protein S3, that was obtained by protein sequencing of purified yeast ribosomal proteins. The promoter region of the RPS3 gene contains several upstream conserved sequence elements (UASrpg, T-rich region) that usually regulate transcription of ribosomal protein genes. Northern blot experiments demonstrate that this ORF is transcribed into an approximately 900 nt long mRNA. The major start site of transcription is located near position -20. The RPS3 gene is a single copy gene in yeast. Its disruption yields non viable haploid spores of Saccharomyces cerevisiae.

Amino Acid Sequence↗