Analysis of the canine EDAR gene and exclusion as a candidate for the hairless phenotype in the Chinese Crested dog.
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Animals↗
Biomedical subjects
P Sander
Publications and source records attributed to P Sander.
Impact of amphotericin B on the cytochrome P450 system in HIV-infected patients.
OBJECTIVE: To investigate whether cytochrome P450-dependent enzymes are influenced by amphotericin B (Am-B) during the treatment of Candida oesophagitis in HIV-infected patients. METHODS: Twelve HIV-infected, antiretroviral-naive patients (CDC/WHO stage C3) with Candida oesophagitits were enrolled into a prospective clinical trial. The patients were treated with Am-B (0.4 mg/kg body weight) for two weeks. At baseline and after Am-B therapy the clearance of antipyrine and its metabolites were investigated by high-performance liquid chromatography. In addition, the urinary excretion of 6-beta-hydroxycortisol and 17-hydroxycorticosteroids was assessed by means of a radioimmunoassay. RESULTS: The following significant changes were observed after Am-B treatment (P < 0.01): increase of antipyrine half-life (12.4 h vs 16.8 h) and the area under the plasma concentration-time curve (27.9 mg min/ml vs 38.1 mg min/ml); decrease of the total body clearance (61.2 ml/min vs 43.7 ml/min); decrease of the renal clearance of antipyrine metabolites - norantipyrine (7.45 ml/min vs 5.31 ml/min), 4-hydroxyantipyrine (15.4 ml/min vs 10.3 ml/min), hydroxymethylantipyrine (4.31 ml/min vs 3.65 ml/min); decrease of urinary 6-beta-hydroxycortisol excretion (453 microg/24h vs 298 microg/24h) and the ratio of 6-beta-hydroycortisol to 17-hydroxycorticosteroids (8.8% vs 6.4%). CONCLUSIONS: Our data indicate that Am-B therapy has an inhibitory effect on cytochrome P450-dependent enzymes in HIV-infected patients. These results are of particular significance for HIV-infected patients who are concomitantly treated with drugs that are predominantly metabolised in the liver. A careful drug monitoring system seems advisable, especially for proteinase inhibitor experienced HIV-1-infected subjects.
17-Hydroxycorticosteroids↗
Lipoprotein processing is required for virulence of Mycobacterium tuberculosis.
Lipoproteins are a subgroup of secreted bacterial proteins characterized by a lipidated N-terminus, processing of which is mediated by the consecutive activity of prolipoprotein diacylglyceryl transferase (Lgt) and lipoprotein signal peptidase (LspA). The study of LspA function has been limited mainly to non-pathogenic microorganisms. To study a potential role for LspA in the pathogenesis of bacterial infections, we have disrupted lspA by allelic replacement in Mycobacterium tuberculosis, one of the world's most devastating pathogens. Despite the presence of an impermeable lipid outer layer, it was found that LspA was dispensable for growth under in vitro culture conditions. In contrast, the mutant was markedly attenuated in virulence models of tuberculosis. Our findings establish lipoprotein metabolism as a major virulence determinant of tuberculosis and define a role for lipoprotein processing in bacterial pathogenesis. In addition, these results hint at a promising new target for therapeutic intervention, as a highly specific inhibitor of bacterial lipoprotein signal peptidases is available.
Animals↗
The role of cortisol suppression on craving for and satisfaction from nicotine in high and low impulsive subjects.
The rewarding properties of drug self-administration have been shown to depend on mesolimbic dopamine release but also on the availability of corticosterone, as shown in rats. Since this became particularly evident when tested in rat strains bred for high (HR) and low (LR) reactivity to novel stimuli, the role of cortisol and personality in drug craving was investigated in 60 male nicotine-deprived smokers divided according to questionnaire scores into high and low impulsives, a dimension considered to represent HR/LR rats. They either received the peripheral cortisol blocker metyrapone (MET) or the centrally blocking substance dexamethasone (DEX) or placebo (n=20 each) and were then allowed to smoke. MET more than DEX reduced craving for cigarettes during deprivation. This was only observed in low impulsives while high impulsives developed low craving unmodified by blockade of cortisol. Satisfaction from smoking was less achieved with DEX than with MET and not modified by impulsivity. Differences between MET and DEX in influencing craving during deprivation and satisfaction from smoking were discussed on the basis of different glucocorticoid receptors. The mineralocorticoid receptor (MR) seems to be the 'turn on switch' and the glucocorticoid receptor (GR) the 'turn off switch' in mediating the rewarding properties of dopamine in drug self-administration.
Adult↗
Ribosomal and non-ribosomal resistance to oxazolidinones: species-specific idiosyncrasy of ribosomal alterations.
A derivative of Mycobacterium smegmatis, which carries only one functional rRNA (rrn) operon, was used to isolate mutants resistant to the ribosome-targeted antibiotic linezolid. Isolation and characterization of linezolid-resistant clones revealed two classes of mutants. Ribosomes from class I mutants are resistant to oxazolidinones in an in vitro peptidyl transferase assay, indicating that resistance maps to the ribosome component. In contrast, ribosomes from class II mutants show wild-type susceptibility to a linezolid derivative in vitro, pointing to a non-ribosomal mechanism of resistance. Introduction of a wild-type ribosomal RNA operon into linezolid-resistant strains restored linezolid sensitivity in class I mutants, indicating that resistance (i) maps to the rRNA and (ii) is recessive. Sequencing of the entire rrn operon identified a single nucleotide alteration in 23S rRNA of class I mutant strains, 2447G --> T (Escherichia coli numbering). Introduction of mutant rrl2447T into M. smegmatis rrn- resulted in a linezolid-resistant phenotype, demonstrating a cause-effect relationship of the 2447G --> T alteration. The 2447G --> T mutation, which renders M. smegmatis linezolid resistant, confers lethality in E. coli. This finding is strong evidence of structural and pos-sibly functional differences between the ribosomes of Gram-positive and Gram-negative bacteria. In agreement with the results of the in vitro assay, class II mutants show a wild-type sequence of the complete rRNA operon. The lack of cross-resistance of the class II mutants to other antibiotics suggests a resistance mechanism other than activation of a broad-spectrum multidrug transporter.
Acetamides↗
Oxidative stress response genes in Mycobacterium tuberculosis: role of ahpC in resistance to peroxynitrite and stage-specific survival in macrophages.
The Mycobacterium tuberculosis ahpC gene, encoding the mycobacterial orthologue of alkylhydroperoxide reductase, undergoes an unusual regulatory cycle. The levels of AhpC alternate between stages of expression silencing in virulent strains grown as aerated cultures, secondary to a natural loss of the regulatory oxyR function in all strains of the tubercle bacillus, and expression activation in static bacilli by a yet undefined mechanism. The reasons for this unorthodox regulatory cycle controlling expression of an antioxidant factor are currently not known. In this work, M. tuberculosis H37Rv and Mycobacterium smegmatis mc(2)155 ahpC knockout mutants were tested for sensitivity to reactive nitrogen intermediates, in particular peroxynitrite, a highly reactive combinatorial product of reactive nitrogen and oxygen species, and sensitivity to bactericidal mechanisms in resting and activated macrophages. Both M. tuberculosis ahpC::Km(r) and M. smegmatis ahpC::Km(r) showed increased susceptibility to peroxynitrite. In contrast, inactivation of ahpC in M. tuberculosis did not cause increased sensitivity to donors of NO alone. M. tuberculosis ahpC::Km(r) also showed decreased survival in unstimulated macrophages, but the effect was no longer detectable upon IFNgamma activation. These studies establish a specific role for ahpC in antioxidant defences involving peroxynitrite and most likely additional cidal mechanisms in macrophages, with the regulatory cycle likely contributing to survival upon coming out of the stationary phase during dormancy (latent infection) or upon transmission to a new host.
Cell Line↗
Instability and site-specific excision of integration-proficient mycobacteriophage L5 plasmids: development of stably maintained integrative vectors.
Integrative vectors expressing foreign genes are used as tools for the development of recombinant vaccines in mycobacteria since it is assumed that these vectors are stably maintained even without antibiotic selection. We here demonstrate that integration-proficient vectors are lost from the mycobacterial genome in high frequency. Loss of integrated vectors occurred in recA+ and in recA-strains, indicating a RecA-independent mechanism. Loss of the integrated vector was prevented when integrase gene function was carried on a separate plasmid that is unable to replicate in mycobacteria, indicating that excision is a function of integrase. By providing attP in cis and integrase function in trans, vectors integrating at the attB site are stably maintained, even when carrying genes that deleteriously affect the host.
Attachment Sites, Microbiological↗
Structural basis for selectivity and toxicity of ribosomal antibiotics.
Ribosomal antibiotics must discriminate between bacterial and eukaryotic ribosomes to various extents. Despite major differences in bacterial and eukaryotic ribosome structure, a single nucleotide or amino acid determines the selectivity of drugs affecting protein synthesis. Analysis of resistance mutations in bacteria allows the prediction of whether cytoplasmic or mitochondrial ribosomes in eukaryotic cells will be sensitive to the drug. This has important implications for drug specificity and toxicity. Together with recent data on the structure of ribosomal subunits these data provide the basis for development of new ribosomal antibiotics by rationale drug design.
Alleles↗
Mechanisms of streptomycin resistance: selection of mutations in the 16S rRNA gene conferring resistance.
Chromosomally acquired streptomycin resistance is frequently due to mutations in the gene encoding the ribosomal protein S12, rpsL. The presence of several rRNA operons (rrn) and a single rpsL gene in most bacterial genomes prohibits the isolation of streptomycin-resistant mutants in which resistance is mediated by mutations in the 16S rRNA gene (rrs). Three strains were constructed in this investigation: Mycobacterium smegmatis rrnB, M. smegmatis rpsL(3+), and M. smegmatis rrnB rpsL(3+). M. smegmatis rrnB carries a single functional rrn operon, i.e., rrnA (comprised of 16S, 23S, and 5S rRNA genes) and a single rpsL+ gene; M. smegmatis rpsL(3+) is characterized by the presence of two rrn operons (rrnA and rrnB) and three rpsL+ genes; and M. smegmatis rrnB rpsL(3+) carries a single functional rrn operon (rrnA) and three rpsL+ genes. By genetically altering the number of rpsL and rrs alleles in the bacterial genome, mutations in rrs conferring streptomycin resistance could be selected, as revealed by analysis of streptomycin-resistant derivatives of M. smegmatis rrnB rpsL(3+). Besides mutations well known to confer streptomycin resistance, novel streptomycin resistance conferring mutations were isolated. Most of the mutations were found to map to a functional pseudoknot structure within the 530 loop region of the 16S rRNA. One of the mutations observed, i.e., 524G-->C, severely distorts the interaction between nucleotides 524G and 507C, a Watson-Crick interaction which has been thought to be essential for ribosome function. The use of the single rRNA allelic M. smegmatis strain should help to elucidate the principles of ribosome-drug interactions.
Alleles↗
Silencing of oxidative stress response in Mycobacterium tuberculosis: expression patterns of ahpC in virulent and avirulent strains and effect of ahpC inactivation.
Intracellular pathogens such as Mycobacterium tuberculosis are able to survive in the face of antimicrobial products generated by the host cell in response to infection. The product of the alkyl hydroperoxide reductase gene (ahpC) of M. tuberculosis is thought to be involved in protecting the organism against both oxidative and nitrosative stress encountered within the infected macrophage. Here we report that, contrary to expectations, ahpC expression in virulent strains of M. tuberculosis and Mycobacterium bovis grown in vitro is repressed, often below the level of detection, whereas expression in the avirulent vaccine strain M. bovis BCG is constitutively high. The repression of the ahpC gene of the virulent strains is independent of the naturally occurring lesions of central regulator oxyR. Using a green fluorescence protein vector (gfp)-ahpC reporter construct we present data showing that repression of ahpC of virulent M. tuberculosis also occurred during growth inside macrophages, whereas derepression in BCG was again seen under identical conditions. Inactivation of ahpC on the chromosome of M. tuberculosis by homologous recombination had no effect on its growth during acute infection in mice and did not affect in vitro sensitivity to H2O2. However, consistent with AhpC function in detoxifying organic peroxides, sensitivity to cumene hydroperoxide exposure was increased in the ahpC::Km(r) mutant strain. The preservation of a functional ahpC gene in M. tuberculosis in spite of its repression under normal growth conditions suggests that, while AhpC does not play a significant role in establishing infection, it is likely to be important under certain, as yet undefined conditions. This is supported by the observation that repression of ahpC expression in vitro was lifted under conditions of static growth.
Animals↗
Mycobacterium bovis BCG recA deletion mutant shows increased susceptibility to DNA-damaging agents but wild-type survival in a mouse infection model.
Pathogenic microorganisms possess antioxidant defense mechanisms for protection from reactive oxygen metabolites which are generated during the respiratory burst of phagocytic cells. These defense mechanisms include enzymes such as catalase, which detoxifies reactive oxygen species, and DNA repair systems, which repair damage resulting from oxidative stress. To (i) determine the relative importance of the DNA repair system when oxidative stress is encountered by the Mycobacterium tuberculosis complex during infection of the host and to (ii) provide improved mycobacterial hosts as live carriers to express foreign antigens, the recA locus was inactivated by allelic exchange in Mycobacterium bovis BCG. The recA mutants are sensitive to DNA-damaging agents and show increased susceptibility to metronidazole, the first lead compound active against the dormant M. tuberculosis complex. Surprisingly, the recA genotype does not affect the in vitro dormancy response, nor does the defect in the DNA repair system lead to attenuation as determined in a mouse infection model. The recA mutants will be a valuable tool for further development of BCG as an antigen delivery system to express foreign antigens and as a source of a genetically stable vaccine against tuberculosis.
Alkylating Agents↗
pKa of adenine 2451 in the ribosomal peptidyl transferase center remains elusive.
The universally conserved A2451 of 23S rRNA has been proposed to participate directly in the catalysis of peptide bond formation in the ribosomal peptidyl transferase center. An unusually high, near neutral, pKa of A2451 is a prerequisite for its action as a general acid-base catalyst. Increased reactivity of A2451 to dimethylsulfate (DMS) at pH 8.5 compared to pH 6.5 was taken as evidence that the pKa of this nucleotide falls within this pH range. Structural data suggested that the interaction between A2451 and G2447 in the ribosome is responsible for A2451 pKa perturbation. In contrast to expectation, our studies did not show pH dependence of A2451 dimethylsulfate modification in ribosomes of Thermus aquaticus and Mycobacterium smegmatis. Other rRNA regions, however, showed major alterations in DMS reactivity at pH 8.5 compared to pH 6.5, suggesting that conformational rearrangements in the structure of the large ribosomal subunit may occur upon the pH shift. The G2447U mutant of M. smegmatis was viable, indicating that the G2447-A2451 interaction is not critical for the ribosome function. We concluded that the proposed unusual pKa of A2451, if existing, may not be crucial for the ribosome activity and that the previously reported pH-dependent alterations in the DMS modification of A2451 do not necessarily reveal an unusual pKa of this nucleotide.
Adenine↗
Contribution of the multidrug efflux pump LfrA to innate mycobacterial drug resistance.
Multidrug resistance (MDR) in bacteria has been associated with efflux pumps that export structurally unrelated compounds and decrease cytoplasmic drug accumulation. To investigate MDR in mycobacteria, we studied the Mycobacterium smegmatis mutant mc(2)11, which is resistant to doxorubicin, tetracycline, rhodamine, ethidium bromide and the hydrophilic fluoroquinolones. A genomic library constructed from this mutant was used to select clones conferring resistance to doxorubicin. Surprisingly, the clone selected encodes the efflux pump LfrA, which has been reported to confer resistance to hydrophilic fluoroquinolones, ethidium bromide, rhodamine, and acriflavine. To define the contribution of LfrA to the innate mycobacterial drug resistance and to the MDR phenotype in mc(2)11, the lfrA gene was disrupted in both the mc(2)11 mutant and the mc(2)155 wild-type parent. LfrA disruption of the wild-type strain decreased resistance to ethidium bromide and acriflavine, and increased accumulation of ethidium bromide. However, disruption of lfrA gene results only in a 2-fold decrease in minimal inhibitory concentrations (MICs) for ciprofloxacin, doxorubicin, rhodamine, and accumulation of [(14)C]ciprofloxacin was unchanged. LfrA disruption of the MDR strain mc(2)11 produced a similar phenotype. Thus, LfrA contributes significantly to the intrinsic MICs of M. smegmatis for ethidium bromide and acriflavine, but not for ciprofloxacin, doxorubicin or rhodamine.
Acriflavine↗
[The effect of a vaginal device on urinary leakage and quality of life of women with stress urinary incontinence].
The aim of this study was to assess the effect of a vaginal device (Continence Guard) on urine leakage and its impact on quality of life (QoL). Fifty-five women participated in a three month study using the Continence Guard. QoL was assessed by an incontinence specific questionnaire (IIQ), two incontinence specific questions and the generic SF-36 health questionnaire. A total of 41 (74.5%) women completed the study. Use of the vaginal device was associated with subjective cure in 11 women (27%) and improvement in 27 (66%). The mean 24-hour pad test leakage decreased significantly. QoL measured by the IIQ and the two incontinence specific questions showed highly significant improvements. The SF-36 questionnaire showed no significant changes. In conclusion treatment with the Continence Guard significantly decreases leakage and improves QoL in women with the symptom of urinary stress incontinence. The SF-36 questionnaire was not sensitive enough to detect alterations in QoL in patients with stress urinary incontinence.
Adult↗
The visual G protein of fly photoreceptors interacts with the PDZ domain assembled INAD signaling complex via direct binding of activated Galpha(q) to phospholipase cbeta.
Visual transduction in the compound eye of flies is a well-established model system for the study of G protein-coupled transduction pathways. Pivotal components of this signaling pathway, including the principal light-activated Ca(2+) channel transient receptor potential, an eye-specific protein kinase C, and the norpA-encoded phospholipase Cbeta, are assembled into a supramolecular signaling complex by the modular PDZ domain protein INAD. We have used immunoprecipitation assays to study the interaction of the heterotrimeric visual G protein with this INAD signaling complex. Light-activated Galpha(q)- guanosine 5'-O-(thiotriphosphate) and AlF(4)(-)-activated Galpha(q), but not Gbetagamma, form a stable complex with the INAD signaling complex. This interaction requires the presence of norpA-encoded phospholipase Cbeta, indicating that phospholipase Cbeta is the target of activated Galpha(q). Our data establish that the INAD signaling complex is a light-activated target of the phototransduction pathway, with Galpha(q) forming a molecular on-off switch that shuttles the visual signal from activated rhodopsin to INAD-linked phospholipase Cbeta.
Animals↗
Evaluation of a simple, non-surgical concept for management of urinary incontinence (minimal care) in an open-access, interdisciplinary incontinence clinic.
Our objective was to evaluate a new concept for assessment and treatment of urinary incontinence in an open-access, interdisciplinary incontinence clinic. A standardized program for investigation and treatment of incontinence was based on minimal relevant investigations, primarily non-surgical treatment with a limited consumption of resources ("minimal care"). This was a prospective observational study of 408 consecutive women examined and treated in the clinic. The main characteristics of the women were a high median age and a high prevalence of severe concomitant diseases with possible influence on lower urinary tract function. More than half of the patients had urge or mixed incontinence. Most of the patients were managed with conservative treatment. Fifteen percent were referred to in-hospital treatment, with 5% to incontinence surgery. In total 44% felt cured or very much improved. Before and after treatment one third of the women completed quality-of-life questions and voiding charts, while 43% completed the pad tests. Quality of life improved significantly. Objectively leakage on pad test and voiding charts was significantly improved. The patients were in general very satisfied with clinic's program. Almost one fourth of the women were followed up for 6 months after discharge. No significant deterioration in the subjective results were found compared to status at discharge. In conclusion, the results highlight the need for advice and treatment of patients with incontinence. The minimal care program and interdisciplinary structure in the incontinence clinic offer effective and low cost treatment for urinary incontinence. The open-access, interdisciplinary incontinence clinic model is recommended. Neurourol. Urodynam. 18:9-17, 2000.
Adolescent↗
Age-related differences in twitch contractile properties of plantarflexor muscles in women.
This study compared maximal voluntary isometric strength and electrically evoked twitch characteristics of the plantarflexor muscles among the groups of women of the 3rd (n=14), 4th (n=13), 5th (n=11), 6th (n=12) and 8th (n=13) decade. A significant decrease (P < 0.05-0.001) has been found in isometric maximal voluntary contraction (MVC) force from the 5th decade and in twitch maximal force from the 6th decade. The 3rd decade group produced 72% greater MVC force and 43% greater twitch maximal force than the 8th decade group (P < 0.001). A prolongation (P < 0.01) in twitch contraction time was observed from the 5th decade. Twitch contraction time in the 3rd decade group was 16% shorter (P < 0.001) compared with the 8th decade group (P < 0.01). Twitch half-relaxation time did not differ significantly (P > 0.05) among the groups. A decrease (P < 0. 05-0.001) has been found in twitch maximal rate of force development from the 5th decade and in twitch maximal rate of relaxation from the 6th decade. The 3rd decade group produced 63% greater (P < 0. 001) twitch maximal rate of force development than the 8th decade group. It was concluded that in women a marked age-related reduction in maximal voluntary force-generating capacity of the plantarflexor muscles and speed of contraction of the electrically evoked twitch takes place after 40 years of age, while reduction in maximal force-generating capacity and speed of relaxation of the twitch occurs after 50 years of age.
Adult↗
In vivo splicing and functional characterization of Mycobacterium leprae RecA.
The RecA proteins from Mycobacterium tuberculosis and Mycobacterium leprae contain inteins. In contrast to the M. tuberculosis RecA, the M. leprae RecA is not spliced in Escherichia coli. We demonstrate here that M. leprae RecA is functionally spliced in Mycobacterium smegmatis and produces resistance toward DNA-damaging agents and homologous recombination.
Mycobacterium leprae↗