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T Köhler

Publications and source records attributed to T Köhler.

At least 55 records · Page 3Linked to original sources
PubMed1999

In vivo emergence of multidrug-resistant mutants of Pseudomonas aeruginosa overexpressing the active efflux system MexA-MexB-OprM.

During a 6-month period, 21 pairs of Pseudomonas aeruginosa isolates susceptible (pretherapy) and resistant (posttherapy) to antipseudomonal beta-lactam antibiotics were isolated from hospitalized patients. In vivo emergence of beta-lactam resistance was associated with the overexpression of AmpC beta-lactamase in 10 patients. In the other 11 patients, the posttherapy isolates produced only low, basal levels of beta-lactamase and had increased levels of resistance to a variety of non-beta-lactam antibiotics (e.g., quinolones, tetracyclines, and trimethoprim) compared with the levels of beta-lactamase production and resistance of their pretherapy counterparts. These data suggested the involvement of the MexA-MexB-OprM active efflux system in the multidrug resistance phenotype of the posttherapy strains. Immunoblotting of the outer membrane proteins of these 11 bacterial pairs with a specific polyclonal antibody raised against OprM demonstrated the overexpression of OprM in all the posttherapy isolates. To determine whether mutations in mexR, the regulator gene of the mexA-mexB-oprM efflux operon, could account for the overproduction of the efflux system, sequencing experiments were carried out with the 11 bacterial pairs. Eight posttherapy isolates were found to contain insertions or deletions that led to frameshifts in the coding sequences of mexR. Two resistant strains had point mutations in mexR that yielded single amino acid changes in the protein MexR, while another strain did not show any mutation in mexR or in the promoter region upstream of mexR. Introduction of a plasmid-encoded wild-type mexR gene into five posttherapy isolates partially restored the susceptibility of the bacteria to selected antibiotics. These results indicate that in the course of antimicrobial therapy multidrug-resistant active efflux mutants overexpressing the MexA-MexB-OprM system may emerge as a result of mutations in the mexR gene.

Anti-Bacterial Agents↗
PubMed1999

Carbapenem activities against Pseudomonas aeruginosa: respective contributions of OprD and efflux systems.

While meropenem MICs were strongly influenced by the presence or absence of the MexAB-OprM efflux pump in both OprD-proficient and -deficient strain backgrounds, MICs of imipenem and of ER-35786 remained unchanged, demonstrating that meropenem is a substrate of MexAB-OprM but not imipenem and ER-35786. In vitro, all three carbapenems selected loss of OprD as a first mechanism of resistance. However, in an OprD-deficient background, meropenem was able to select MexAB-OprM overproducers as a secondary resistance mechanism, while ER-35786 selected a mutant cross-resistant to sparfloxacin and cefpirome.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1999

Characterization of MexT, the regulator of the MexE-MexF-OprN multidrug efflux system of Pseudomonas aeruginosa.

We investigated the regulation of the MexEF-OprN multidrug efflux system of Pseudomonas aeruginosa, which is overexpressed in nfxC-type mutants and confers resistance to quinolones, chloramphenicol and trimethoprim. Sequencing of the DNA region upstream of the mexEF-oprN operon revealed the presence of an open reading frame (ORF) of 304 amino acids encoding a LysR-type transcriptional activator, termed MexT. By using T7-polymerase, a 34-kDa protein was expressed in Escherichia coli from a plasmid carrying the mexT gene. Expression of a mexE::lacZ fusion was 10-fold higher in nfxC-type mutants than in the wild-type strain; however, transcription of mexT as well as the mexT DNA region was unchanged. Located adjacent to mexT but transcribed in opposite direction, the beginning of an ORF termed qrh (quinone oxidoreductase homologue) was identified. Expression of a qrh::lacZ fusion was also found to be activated by MexT. Further, we present evidence for coregulation at the transcriptional and the posttranscriptional level between the MexEF-OprN efflux system and the OprD porin responsible for cross-resistance of nfxC-type mutants to carbapenem antibiotics.

4-Quinolones↗
PubMed1999

p53 autoantibodies in patients with primary ovarian cancer are associated with higher age, advanced stage and a higher proportion of p53-positive tumor cells.

Autoantibodies (AAb) directed against the nuclear phosphoprotein p53 can be detected in patients with various forms of cancer. The objective was to determine the prevalence of p53 AAb at the time of diagnosis in ovarian cancer patients and to correlate the presence of p53 AAb with clinicopathological parameters. Sera of 83 patients were analyzed by an ELISA using p53 expressed from a human wild-type cDNA. p53 AAb were detectable at all stages. The overall prevalence was 46%. p53 AAb were more frequent in patients with higher age (p = 0.014), postmenopausal status (p = 0.050), or advanced tumor stage (p = 0.046). p53 AAb positivity was related to the proportion of cells positive in immunohistochemistry but not with the staining intensity. In bivariate analysis, patients with p53 AAb had a 1.96-fold risk for relapse (95% confidence interval 1.02-3.78).

Adult↗
PubMed1999

[Forgetting of word associations in relation to indicators of emotionality. A possibility for evaluating Freud's concept of repression?].

In replication of the well-known study by Levinger & Clark, 30 subjects were presented a list with 60 words under several conditions. In the first part of the experiment they had to tell the first word which came to mind as a response to the stimuli read by the experimenter. Reaction times and skin conductance reactions (SCRs) during associations were recorded. Immediately afterwards the list was read again with the instruction to recall the associations given before. In part 2 of the study, one week later, subjects had to recall again their first associations; in addition, they scaled the emotionality of the word stimuli. Intraindividual correlations were computed for indices of emotionality (reaction time, SCR, perceived emotionality) and forgetting in both short-term and long-term memory. Associations accompanied by larger electrodermal reaction and showing longer reaction time were more likely to be forgotten both for short and long intervals. This is in line with Freuds concept of repression. However, alternative explanations should also be discussed.

Adult↗
PubMed1998

Improving source reconstructions by combining bioelectric and biomagnetic data.

OBJECTIVES: A framework for combining bioelectric and biomagnetic data is presented. The data are transformed to signal-to-noise ratios and reconstruction algorithms utilizing a new regularization approach are introduced. METHODS: Extensive simulations are carried out for 19 different EEG and MEG montages with radial and tangential test dipoles at different eccentricities and noise levels. The methods are verified by real SEP/SEF measurements. A common realistic volume conductor is used and the less well known in vivo conductivities are matched by calibration to the magnetic data. Single equivalent dipole fits as well as spatio-temporal source models are presented for single and combined modality evaluations and overlaid to anatomic MR images. RESULTS: Normalized sensitivity and dipole resolution profiles of the different EEG/MEG acquisition systems are derived from the simulated data. The methods and simulations are verified by simultaneously measured somatosensory data. CONCLUSIONS: Superior spatial resolution of the combined data studies is revealed, which is due to the complementary nature of both modalities and the increased number of sensors. A better understanding of the underlying neuronal processes can be achieved, since an improved differentiation between quasi-tangential and quasi-radial sources is possible.

Brain Mapping↗
PubMed1998

Genotyping of human apolipoprotein E alleles by the new qualitative, microplate-based CASSI-detection assay.

A new qualitative PCR product detection assay called competitive amplified single mutation detection by selective probe hybridization immunoassay (CASSI) was developed for genotyping the most common apolipoprotein E (apoE) polymorphisms. Single target DNA strands immobilized using biotin on streptavidin-coated microplates were hybridized in separate wells with two distinct, 5'-fluorescein isothiocyanate (FITC)-labeled oligonucleotides, complementary to either the 112Arg or 158Arg encoding site. With this assay, only correctly matched hybrids that form between probe and target DNA can be cleaved with the HhaI restriction endonuclease, leading to loss of probe label in corresponding wells. However, allele-specific, probe-target mismatches due to G-->T exchanges in the HhaI recognition sequences are not cleaved. After digestion, the remaining microplate-adsorbed signal is measured colorimetrically by using anti-FITC, Fab-horseradish peroxidase conjugates. Our results show maximum intensity was detected when the respective probe hybridized incompletely to the target (i.e., no cleavage), and minimum signal was obtained when the probe matched the target completely (complete cleavage); whereas, an intermediate signal was recorded at 50% complementarity (i.e., heterozygote alleles). With this assay, we could demonstrate a high prevalence of the apoE2 allele in patients suffering from coronary artery disease even though they displayed normal triglyceride and cholesterol levels. Corresponding results were obtained by CASSI compared with conventional restriction fragment-length polymorphism analysis.

Alleles↗
PubMed1998

[Antibiotic efflux, a mechanism of multiple resistance in Pseudomonas aeruginosa].

Pseudomonas aeruginosa displays three active efflux systems, made of three components: an inner membrane protein acting as a proton motive pump, a periplasmic linking protein and an outer membrane protein acting as an efflux porin. All three proteins are encoded by genes organised as an operon, with a regulator gene in the vicinity. Efflux systems produce phenotypes of multidrug resistance. The constitutively produced system MexAB-OprM generates intrinsic resistance to most beta-lactams, quinolones, tetracycline, chloramphenicol, trimethoprim and sulfamethoxazole; in addition, genetic derepression causes acquired resistance to the same drugs. The non constitutive MexCD-OprJ and MexEF-OprN can be expressed by mutation, producing then slightly different multidrug resistant phenotypes. Efflux systems are responsible for low level resistance, and seem to promote selection of mutations responsible for higher level resistance. The multidrug resistance associated to active efflux systems should be taken into account in our antibiotic policies, both at the individual and at the institutional level.

Anti-Bacterial Agents↗
PubMed1997

[Prevention of thromboembolism in minimal invasive interventions and brief inpatient treatment. Results of a multicenter, prospective, randomized, controlled study with a low molecular weight heparin].

A prospective, randomised, controlled clinical trial was carried out in order to elucidate the incidence of venous thromboembolism in selected patients undergoing laparoscopic cholecystectomy and other types of minimally invasive surgery, as well as to show safety and efficacy of a low-molecular-weight heparin (LMWH) in the prevention of post-operative venous thromboembolism. Seven hundred and eighteen patients were randomly allocated to one of two groups: One group received physical measures for prevention of deep-vein thrombosis, i.e. graduated elastic stockings (n = 359). The second group also received graduated elastic stockings and, additionally, a LMWH (reviparin sodium, Clivarin) s.c. once daily (n = 359). For safety reasons, with respect to the untreated control group, patients with three or more risk factors for venous thromboembolism were not included into the trial. Diagnosis for DVT was systematically done by duplex scan. In this, rather artificial low-risk selection the overall incidence of thromboembolic events was surprisingly low: five cases of suspected pulmonary embolism, confirmed by scintigraphy in one case only, and one patient with phlebographically confirmed calf vein thrombosis. The use of reviparin for prevention of venous thromboembolism was safe and convenient--the rate of post-operative bleeding complications was 2.3% in the LMWH group, even lower than in the control group (3.2%). The real incidence of venous thromboembolism in patients undergoing laparoscopic cholecystectomy remains unclear. Further trials with unselected patients are needed.

Adolescent↗
PubMed1997

The correlation between everyday stress and angina pectoris: a longitudinal study.

The article describes a longitudinal study testing the hypothesis that everyday stress can aggravate angina pectoris (AP). Every weekend for 1 year, 42 patients with coronary heart disease filled out a questionnaire on perceived stress for the preceding week. They also rated AP symptoms, again for the previous week. Within-subject correlation between stress and severity of AP was quite pronounced in some subjects and resulted in a sample mean of 0.38. The distribution of correlation coefficients in the sample was significantly different from a random distribution around 0. Stress values also predicted AP in the week to come. However, this finding was no longer significant when the influence of lag 1 auto-correlations between stress values was eliminated. Our results show that the effect of everyday stress on AP is essentially immediate and that it can be quite important in some subjects.

Aged↗
PubMed1997

Characterization of MexE-MexF-OprN, a positively regulated multidrug efflux system of Pseudomonas aeruginosa.

Antibiotic-resistant mutants of Pseudomonas aeruginosa were generated using chloramphenicol and ciprofloxacin as selective agents. These mutants displayed a multidrug phenotype and overexpressed an outer membrane protein of 50 kDa, which was shown by Western blot analysis to correspond to OprN. A cosmid clone harbouring the oprN gene was isolated by partial complementation of a mutant deficient in OprM, the outer membrane component of the mexAB-oprM efflux operon. Antibiotic-accumulation studies indicated that OprN was part of an energy-dependent antibiotic-efflux system. Sequencing of a 6180bp fragment from the complementing cosmid revealed the presence of three open reading frames (ORFs), which exhibited amino acid similarity to the components of the mexAB-oprM and mexCD-oprJ efflux operons of P. aeruginosa. The ORFs were designated MexE, MexF and OprN. Mutation of the mexE gene eliminated the multidrug-resistance phenotype in an OprN-overexpressing strain, but did not affect the susceptibility profile of the wild-type strain. Expression of the mexEF-oprN operon was shown to be positively regulated by a protein encoded on a 1.5 kb DNA fragment located upstream of mexE and belonging to the LysR family of transcriptional activators. The presence of a plasmid containing this DNA fragment was sufficient to confer a multidrug phenotype onto the wild-type strain but not onto the mexE mutant. Evidence is provided to show that the mexEF-oprN operon may be involved in the excretion of intermediates for the biosynthesis of pyocyanin, a typical secondary metabolite of P. aeruginosa.

Amino Acid Sequence↗
PubMed1997

Human thyroid carcinoma cell lines and normal thyrocytes: expression and regulation of matrix metalloproteinase-1 and tissue matrix metalloproteinase inhibitor-1 messenger-RNA and protein.

Matrix metalloproteinase-1 (MMP-1) and tissue matrix metalloproteinase inhibitor 1 (TIMP-1) play an important role in remodeling the extracellular matrix in normal and pathological processes. The effect of phorbol-myristate acetate (PMA), interleukin-1 (IL-1), and tumor necrosis factor-alpha (TNF-alpha) on MMP-1 and TIMP-1 expression was studied on highly purified thyrocytes and undifferentiated 8505 C, C 643, HTh 74, SW 1736 thyroid carcinoma cells compared with thyroid-derived fibroblasts. Messenger RNA (mRNA) levels were monitored by competitive semiquantitative reverse transcriptase polymerase chain reaction (RT-PCR) after 24 hours. Culture supernatants were assayed for free and/or complexed MMP-1 and TIMP-1 after 48 hours using enzyme-linked immunosorbent assay (ELISA) systems (detection limit: <2 ng/mL). MMP-1 and TIMP-1 mRNA were present in all cell types, although thyrocytes showed MMP-1 mRNA levels near the detection limit. 8505 C expressed MMP-1 mRNA levels of up to 10(6) times those of the other cells analyzed. PMA and IL-1 increased MMP-1 mRNA in most cell types. TIMP-1 mRNA increased after treatment with PMA in all cells except 8505 C, whereas only slight effects were shown after IL-1 stimulation. MMP-1 protein was undetectable in normal thyrocyte cultures, but was secreted spontaneously by all cell lines ([ng/mL]; C 643: 15+/-7; HTh 74: 81+/-1; SW 1736: 13+/-2; 8505 C: 2097+/-320). There was a strong correlation between levels of MMP-1 mRNA and protein (r = 0.99, p < .0001). PMA and IL-1 increased MMP-1 secretion in all cell types after 48 hours. Fibroblasts ([ng/mL] 517+/-55) and the cell lines (C 643: 142+/-48; HTh 74: 115+/-13; SW 1736: 202+/-14; 8505C: 120+/-19) secreted TIMP-1 in unstimulated cultures, whereas only a trace amount was detected in thyrocyte cultures, even after PMA treatment. IL-1 upregulated TIMP-1 secretion after 48 hours in SW 1736, HTh 74, and C 643 cells. Our data suggest that in contrast to normal thyrocytes, dedifferentiated thyroid carcinoma cell lines are potential producers of MMP-1 as well as TIMP-1. High MMP-1 or MMP-1/TIMP-1 expression may play a role in tissue invasion of undifferentiated thyroid cancer cells.

Adult↗
PubMed1997

Differential selection of multidrug efflux systems by quinolones in Pseudomonas aeruginosa.

Resistance mechanisms selected after in vitro exposure to 12 quinolones were analyzed for Pseudomonas aeruginosa. Efflux-type mutants were predominant. Quinolones differed in their ability to select a particular efflux system. While the newer fluoroquinolones favored the MexCD-OprJ system, the older quinolones selected exclusively the MexEF-OprN or MexAB-OprM systems. A protonable C-7 substituent in combination with a C-6 fluorine atom is a structural determinant of quinolones involved in efflux pump substrate specificity.

Anti-Infective Agents↗
PubMed1997

Use of steroids to monitor alterations in the outer membrane of Pseudomonas aeruginosa.

Testosterone (a strongly hydrophobic steroid) and testosterone hemisuccinate (a negatively charged derivative) were used as probes to investigate alterations in the outer membrane of Pseudomonas aeruginosa. Diffusion rates of the steroids across the lipid bilayer were measured by coupling the influx of these compounds to their subsequent oxidation by an intracellular delta1-dehydrogenase enzyme. Wild-type cells of P. aeruginosa (strain PAO1) were found to be 25 times more permeable to testosterone than to testosterone hemisuccinate. The uptake of the latter compound appeared to be partially dependent on the external pH, thus suggesting a preferential diffusion of the uncharged protonated form across the cell envelope. Using various PAO mutants, we showed that the permeation of steroids was not affected by overexpression of active efflux systems but was increased up to 5.5-fold when the outer membrane contained defective lipopolysaccharides or lacked the major porin OprF. Such alterations in the hydrophobic uptake pathway were not, however, associated with an enhanced permeability of the mutants to the small hydrophilic molecule N,N,N',N'-tetramethyl-p-phenylene diamine. Thirty-six agents were also assayed for their ability to damage the cell surface of strain PAO1, using testosterone as a probe. Polymyxins, rBPI23, chlorhexidine, and dibromopropamidine demonstrated the strongest permeabilizing activities on a molar basis in the presence of 1 mM MgCl2. These amphiphilic polycations increased the transmembrane diffusion of testosterone up to 50-fold and sensitized the PAO1 cells to hydrophobic antibiotics. All together, these data indicated that the steroid uptake assay provides a direct and accurate measurement of the hydrophobic uptake pathway in P. aeruginosa.

Anti-Bacterial Agents↗
PubMed1997

Psychophysiological reactivity of borderline hypertensives and their recovery after mental stress.

UNLABELLED: The question of psychophysiological reactivity of borderline hypertensives is still controversial. METHODS: Young males with borderline blood pressure levels and normotensive controls were recruited during a routine examination. Samples of study I comprised 19 subjects, samples of study II 18 subjects. Two stressors were presented (distressing movie, mental arithmetic), each followed by a recovery phase. Systolic and diastolic blood pressure, heart rate, and electrodermal parameters were assessed repeatedly. RESULTS: Borderline hypertensives showed greater reactions to stressors in systolic blood pressure only. Changes in percentage of baseline levels were essentially the same. Recovery after stress did not differ between groups. CONCLUSION: Only moderate support is given to the hypothesis that borderline hypertensives show increased and slowly recovering psychophysiological responses.

Adaptation, Physiological↗
PubMed1997

Calibration and storage of DNA competitors used for contamination-protected competitive PCR.

DNA fragments used as standards in competitive PCR were precisely calibrated using HPLC and commercially available DNA molecular mass markers. The accuracy of calibration was reflected by data that differed by only 2% from the mean when two independently purified and calibrated competitor preparations were compared. Highly dilute competitor solutions were stable at -20 degrees C for up to 1 year in the presence of carrier HindIII-digested lambda DNA, but progressive loss of competitor DNA with increasing storage time was observed when carrier DNA was omitted from the solution. Applying 0.2 U uracil-DNA glycosylase (UDG) per assay of remaining temperature-stable activity did not effect the ratios of synthesized products. This study describes quality management in PCR quantitation that is useful for the measurement of multidrug resistance-associated protein (MRP) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene transcripts.

ATP-Binding Cassette Transporters↗