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Biomedical subjects

E Richter

Publications and source records attributed to E Richter.

At least 73 records · Page 4Linked to original sources
PubMed2000

Differentiation of clinical Mycobacterium tuberculosis complex isolates by gyrB DNA sequence polymorphism analysis.

The discriminatory power of gyrB DNA sequence polymorphisms for differentiation of the species of the Mycobacterium tuberculosis complex (MTBC) was evaluated by sequencing and restriction fragment length polymorphism (RFLP) analysis of a 1,020-bp fragment amplified from clinical isolates of M. tuberculosis, Mycobacterium bovis (pyrazinamide [PZA] resistant as well as PZA susceptible), Mycobacterium africanum subtypes I and II, and Mycobacterium microti types vole and llama. We found sequence polymorphisms in four regions described previously and at one additional position. These differences in the gyrB sequences allow an accurate discrimination of M. bovis, M. microti, and M. africanum subtype I. The PZA-susceptible subtypes of M. bovis shared the M. bovis-specific substitution at position 756 with the PZA-resistant strains, but can be unambiguously differentiated by a characteristic substitution at position 1311. As a drawback, M. tuberculosis and M. africanum subtype II showed an identical gyrB sequence that facilitates discrimination from the other species, but not from each other. A PCR-RFLP technique applying three restriction enzymes could be shown to be a rapid and easy-to-perform tool for the differentiation of the members of the MTBC. Based on these results, we present a clear diagnostic algorithm for the differentiation of species of the MTBC.

Animals↗
PubMed2000

Internalized SP-A and lipid are differentially resecreted by type II pneumocytes.

Biochemical and morphological assays were developed to study surfactant protein A (SP-A) and lipid resecretion kinetics by isolated type II cells in vitro. After a 10-min uptake period with SP-A (3 microg/10(6) cells) in combination with liposomes (60 microg/10(6) cells), the cells were allowed to resecrete. After 5 min of resecretion, only 21.7 +/- 4.6% of the internalized SP-A remained intracellularly compared with 54 +/- 2.9% of the lipids. Extracellular SP-A present during the resecretion period partially inhibited resecretion (SP-A, 36% at 5 min; lipid, approximately 16% at 5 min). Lipid resecretion was also dependent on the SP-A concentration present during the uptake period. Although, as shown by confocal laser scanning microscopy, after a 10-min uptake period at 37 degrees C, most of the fluorescein isothiocyanate-labeled SP-A and rhodamine-phosphatidylethanolamine-labeled lipids colocalized within the cells, after an additional 10 min of resecretion, both the strength of the fluorescence signals and the extent of colocalization had markedly decreased. These data indicate that internalized lipid and SP-A can be resecreted rapidly by type II cells, likely via different pathways.

Animals↗
PubMed2000

Death and injury from motor vehicle crashes in Colombia.

We report data on the distribution and determinants of road deaths and injuries for all victims in Colombia, with the aim of defining targets and priorities for highway death prevention in that country and other rapidly urbanizing nations. Using information from Colombia's Fund for the Prevention of Road Injury and the national death registry, we studied data on deaths and injuries from 1991 to 1995 for the nation as a whole and for the country's two largest cities, Santa Fe de Bogotá and Medellín. Deaths and injuries are rising in the nation as a whole. Of the deaths, 75% occur in urban areas, and 80% are in males. Pedestrians aged 15-34 are a peak subgroup. Thirty-four percent of deaths are attributable to speeding and/or alcohol consumption. Death tolls are highest at night and on weekends. Specific priority targets for intervention are indicated by the fact that 75% of road deaths in Colombia occur in urban areas and that 80% of all victims are males.

Accidents, Traffic↗
PubMed2000

PET with a dual-head coincidence camera: spatial resolution, scatter fraction, and sensitivity.

UNLABELLED: Scintillation cameras with options for detecting positron annihilation quanta in the coincidence acquisition mode may be the most innovative diagnostic devices introduced in nuclear medicine during the last few years. Besides conventional low-energy imaging in the collimated single-photon mode, these options offer a relatively inexpensive opportunity to perform uncollimated PET by switching into the coincidence acquisition mode. Instead of collimators, scatter frames (with 2 optional configurations: axial or open scatter frame) can be mounted to reduce the amount of quanta reaching the detectors from parts of the patient's body outside the field of view. This study investigates the coincidence imaging properties of the scintillation camera by measuring spatial resolution, scatter fraction, sensitivity, and count-rate response for 18F. METHODS: A needle in air and a plastic tube in water, each filled with 18F, were oriented axially and transversally to measure the transverse and axial spatial resolutions, respectively. Using either the axial or the open scatter frame, a standard cylinder filled homogeneously with activity was studied over several half-life periods to deduce the respective scatter and random fractions of the system by means of a sinogram technique. The activity of the cylinder was kept low to determine the sensitivity to coincidence events for both scatter frames. RESULTS: Depending on the distance between the line source and the axis of rotation and on the choice of the axial acceptance angle used to reconstruct the coincidence events (single-slice rebinning algorithm), the axial resolution was found to be between 6 and 10 mm (full width at half maximum) with the axial scatter frame mounted. The transversal resolution was 6-6.5 mm on the axis of rotation, independent of the scatter frame used. The scatter fraction amounted to roughly 25% for the axial and 38% for the open scatter frames. The sensitivity when measuring true coincidence pairs ran to nearly 650 Hz/kBq/mL, when acquisition was performed with the axial scatter frame using a 30%-wide photopeak energy window. When acquiring with the open scatter frame, the sensitivity increased to nearly 3000 Hz/kBq/mL. Using the axial scatter frame, the homogeneously filled cylinder could be scanned with a maximum true coincidence rate of 2000 Hz for an activity of 55-60 MBq. Although this maximum true coincidence counting rate did not change significantly when the acquisition was performed with the open scatter frame, the respective activity in the standard cylinder was decreased to 10-15 MBq. CONCLUSION: The spatial resolution of the scintillation camera is sufficient for high-resolution coincidence imaging. Compared with a dedicated PET scanner, the scatter fraction is relatively high and should therefore be corrected adequately. The relatively low sensitivity and the rather low maximum true coincidence counting rate are considerably inferior compared with a conventional PET scanner. However, these drawbacks can be partially compensated for, facilitating its clinical use.

Gamma Cameras↗
PubMed1999

The effect of physical activity on absorption and metabolism of an orally applied drug (pentoxifylline).

STUDY OBJECTIVE: In this study the effect of physical activity on absorption and metabolism of a therapeutic drug was investigated by comparing plasma concentrations and metabolism of pentoxifylline during rest and after standardized physical activity. METHODS AND PATIENTS: After an oral dose of 600 mg pentoxifylline plasma concentrations of the drug and its metabolite 1 were monitored in nine healthy volunteers during five hours with the persons at rest and in a second experiment after ability-adapted standardized bicycle ergonometry. MAIN RESULTS: In six of nine test persons exercise led to significant higher plasma concentrations of the drug at 2 hours after application (+77%; p <0.01) compared to the concentrations in the same persons at rest. Reductive metabolism of pentoxifylline decreased in all test persons after physical activity giving 19% lower maximal concentrations of metabolite 1 (p = 0.06, not significant) and a 16% lower area under the concentration time curve (p <0.02). CONCLUSIONS: Our results indicate that physical activity can influence absorption and metabolism of a drug.

Administration, Oral↗
PubMed1999

High-performance liquid chromatographic method for the simultaneous determination of HIV-1 protease inhibitors indinavir, saquinavir and ritonavir in plasma of patients during highly active antiretroviral therapy.

A new high-performance liquid chromatographic method for the simultaneous determination of indinavir, saquinavir and ritonavir in human plasma is described. Quantitative recovery following liquid-liquid extraction with diethyl ether from 500 microl of human plasma was achieved. Subsequently, the assay was performed with a linear gradient starting at 67 mM potassium dihydrogenphosphate-acetonitrile (65:35 to 40:60, v/v) as a mobile phase, a Phenomenex C18 column and UV detection at 240 and 258 nm, respectively. Linear standard curves were obtained for concentrations ranging from 75 to 20,000 ng/ml for indinavir, from 10 to 6000 ng/ml for saquinavir, and from 45 to 30,000 ng/ml for ritonavir. The calculated intra- and inter-day coefficients of variation were below 6%.

Chromatography, High Pressure Liquid↗
PubMed1999

Rat sensory neurons contain cytochrome b558 large subunit immunoreactivity.

Cytochrome b558 is part of the NADPH oxidase complex of phagocytes, but it has also been proposed to function as a cellular oxygen sensor, e.g. in the carotid body. Thus, we investigated whether cytochrome b558 is present in rat primary afferent neurons. Immunohistochemistry and Western blotting using the monoclonal antibody 54.1 directed towards the large subunit of cytochrome b558, gp91phox, revealed a ubiquituous occurrence of cytochrome b558-immunoreactivity in neurons of the petrosal ganglion that innervates the carotid body, and also in dorsal root ganglia. This ubiquituous occurrence in sensory neurons of various locations and functional modalities points to a general role of cytochrome b558 in primary afferent neurons rather than involvement in a specialized function such as arterial chemoreception.

Animals↗
PubMed1999

Doxorubicin-resistant LoVo adenocarcinoma cells display resistance to apoptosis induction by some but not all inhibitors of ser/thr phosphatases 1 and 2A.

LoVo adenocarcinoma cells are fairly sensitive to cytostatic drugs, e.g. doxorubicin, but can develop drug resistance by expression of a P-glycoprotein-mediated MDR1 phenotype. LoVo cells respond with apoptosis to nanomolar concentrations of okadaic acid and micromolar concentrations of cantharidic acid. Interestingly, LoVoDx cells which had become about 10-fold less sensitive to doxorubicin by incubation in increasing concentrations of this cytostatic drug were also less sensitive to the toxicity of okadaic acid. Resistance to both agents was lost or significantly reduced by incubation in drug-free medium for about 4 months. On the other hand, LoVoDx cells did not lose responsiveness to the structurally different phosphatase inhibitor cantharidic acid but were about twofold more sensitive to the cytotoxic effect of this agent. Thus, MDR expression protects LoVo cells from the toxicity of phosphatase inhibitors that presumably are substrates of the P-glycoprotein, e.g. okadaic acid and its derivatives but not cantharidic acid, despite the fact that both agents are potent inducers of apoptotic cell death via ser/thr phosphatase inhibition.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1999

Synthesis and in vitro multidrug resistance modulating activity of a series of dihydrobenzopyrans and tetrahydroquinolines.

A series of dihydrobenzopyrans and tetrahydroquinolines was synthesized and pharmacologically tested for their ability to inhibit P-glycoprotein mediated daunomycin efflux in multidrug resistant CCRF-CEM vcr1000 cells. Several compounds exhibit activities in the range of the reference compounds verapamil and propafenone. Preliminary structure-activity relationship studies propose the importance of high molar refractivity values of the compounds and the presence of an additional basic nitrogen atom.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1999

Topography of cell traces studied by atomic force microscopy.

Migrating adherent cells release material onto artificial substrates like glass and silicon while moving. Traces of mouse fibroblasts (L929) have been visualised by atomic force microscopy (AFM). "Non-contact" mode AFM in a liquid environment can extract topographic information from these traces. This dynamic mode allows the study of these soft structures without damage or compression. The AFM images show crossing and branching networks (with specific angles of branching), structured patches, nodular elements, linear elements with irregular height and other features. Fourier analysis of segment spacing in the strands is presented. These spatial features of fibroblast traces are strong indications that actin linked to structural proteins is involved in the formation of cell traces. We also give methods for trace preparation and undistorted imaging and discuss further perspectives.

Animals↗
PubMed1999

Culture-independent prediction of isoniazid resistance in Mycobacterium tuberculosis by katG gene analysis directly from sputum samples.

BACKGROUND: The molecular prediction of isoniazid (INH) resistance in Mycobacterium tuberculosis is hampered by the need for specialized equipment, expertise, high costs, a limited range of detectable mutations, or several of these factors. The rationale for the study was to find a practical alternative and to demonstrate generally valid problems. METHODS AND RESULTS: DNA extracted from decontaminated sputum pellets was used to amplify a 0.26 kb target sequence within the katG gene. Mutations of codon 315, frequently found in isoniazid-resistant isolates, could be discriminated in a simple agarose minigel format following an AciI digest of the nested polymerase chain reaction (PCR) product. Within a panel of 22 sputum samples, INH resistance could be predicted in 5 of 10 samples containing isoniazid-resistant M. tuberculosis. The protocol is robust, requires little expertise and no specialized equipment, and provides the test results within 2 days. CONCLUSION: The results show the feasibility to rapidly and easily detect mutations highly predictive of isoniazid resistance. Nevertheless, this, like any other molecular resistance prediction test, is affected by often neglected factors, including mutation prevalences, the phenomenon of heteroresistance, and a possible bias toward one's own method.

Antitubercular Agents↗
PubMed1999

Green fluorescent protein as a marker to investigate targeting of organellar RNA polymerases of higher plants in vivo.

The recent identification of phage-type RNA polymerases encoded in the nuclear genome of higher plants has provided circumstantial evidence for functioning of these polymerases in the transcription of the mitochondrial and plastid genomes, as demonstrated by sequence analysis and in vitro import experiments. To determine the subcellular localization of the phage-type organellar RNA polymerases in plants, the putative transit peptides of the RNA polymerases RpoT;1 and RpoT;3 from Arabidopsis thaliana and RpoT from Chenopodium album were fused to the coding sequence of a green fluorescent protein (GFP). The constructs were used to stably transform A. thaliana. Transgenic plants were examined for green fluorescence with epifluorescence and confocal laser scanning microscopy. Plants expressing the GFP fusions under control of the CaMV35S promoter exhibited a distinct subcellular localization of the GFP fluorescence for each of the fusion constructs. In plants expressing GFP fusions with the putative transit peptides of ARAth;RpoT;1 and CHEal;RpoT, fluorescence was found exclusively in mitochondria, both in root and leaf cells. In contrast, GFP fluorescence in plants expressing the ARAth;RpoT;3-GFP construct accumulated in chloroplasts of leaf cells and nongreen plastids (leucoplasts) of root cells. By demonstrating targeting in plants, the data add substantial evidence for the phage-type RNA polymerases from C. album and A. thaliana to function in the transcriptional machinery of mitochondria and plastids.

Arabidopsis↗
PubMed1999

Stability of Mycobacterium tuberculosis IS6110 restriction fragment length polymorphism patterns and spoligotypes determined by analyzing serial isolates from patients with drug-resistant tuberculosis.

The stability of Mycobacterium tuberculosis IS6110 fingerprint patterns and spoligotypes has been assessed by analyzing serial isolates from patients with drug-resistant tuberculosis. Altogether, 165 M. tuberculosis isolates obtained from 56 patients have been analyzed. The time spans between the first and the last or a changed isolate from one patient ranged from 1 to 772 days. Among the 56 patients, 5 (9%) were infected with isolates with changes in their IS6110 fingerprint patterns. According to the total number of strains analyzed, 5% of the subsequent isolates showed variations in their IS6110 restriction fragment length polymorphism patterns compared to the pattern of the first isolates. Up to 10 isolates from one patient sampled at time intervals of up to 772 days with no changes in their IS6110 patterns have been analyzed. A statistically significant correlation could be found between changes in insertion sequence (IS) patterns and the increased time intervals over which the isolates were obtained, whereas changes in IS patterns are not correlated to changes in the drug resistance of the isolates. In contrast to the observed variations in IS6110 fingerprint patterns, no changes in the spoligotypes of the isolates analyzed could be found. In conclusion, our results confirm that the IS6110 fingerprint patterns of M. tuberculosis isolates have high degrees of stability. Compared to IS6110, the direct repeat (DR) region, which is the basis for spoligotyping, has a lower rate of change. Partial deletions, e.g., deletions induced by homologous recombination between the repetitive DR elements, could not be detected in this study.

Adult↗
PubMed1999

Identification of Mycobacterium kansasii by using a DNA probe (AccuProbe) and molecular techniques.

The newly formulated Mycobacterium kansasii AccuProbe was evaluated, and the results obtained with the new version were compared to the results obtained with the old version of this test by using 116 M. kansasii strains, 1 Mycobacterium gastri strain, and 19 strains of several mycobacterial species. The sensitivity of this new formulation was 97.4% and the specificity was 100%. Still, three M. kansasii strains were missed by this probe. To evaluate the variability within the species, genetic analyses of the hsp65 gene, the spacer sequence between the 16S and 23S rRNA genes, and the 16S rRNA gene of several M. kansasii AccuProbe-positive strains as well as all AccuProbe-negative strains were performed. Genetic analyses of the one M. gastri strain from the comparative assay and of two further M. gastri strains were included because of the identity of the 16S rRNA gene in M. gastri to that in M. kansasii. The data confirmed the genetic heterogeneity of M. kansasii. Furthermore, a subspecies with an unpublished hsp65 restriction pattern and spacer sequence was described. The genetic data indicate that all M. kansasii strains missed by the AccuProbe test belong to one subspecies, the newly described subspecies VI, as determined by the hsp65 restriction pattern and the spacer sequence. Since the M. kansasii strains that are missed are rare and all M. gastri strains are correctly negative, the new formulated AccuProbe provides a useful tool for the identification of M. kansasii.

Bacterial Proteins↗
PubMed1999

Micro-column affinity test and gel test: comparative study of two techniques for red cell antibody screening.

BACKGROUND AND OBJECTIVES: We describe the results of a comparative evaluation of a gel test (ID Micro Typing) and a micro-column affinity test (MCAT, Cellbind Screen) for red cell antibody screening and identification under routine conditions. MATERIALS AND METHODS: 3,000 serum samples of patients from the Mannheim University Hospital were tested in parallel by means of the gel test and the MCAT, using the low-ionic-strength-saline indirect antiglobulin test and the protein G affinity technique, respectively. Test cells used were the same in all tests. In addition, we performed titration studies with all detected antibodies as well as with 59 frozen sera containing antibodies of known specificity. RESULTS: A total of 154 antibodies (5.1%) were detected, 149 by gel test and 147 by MCAT. The overall sensitivity and specificity of the gel test was 96.8 and 96.5% and of the MCAT 95.5 and 97.2%. No significant differences between the gel test and MCAT were found when the titer scores of all 213 (fresh and frozen) antibodies were used to check the results. The mean scores for the gel test and the MCAT were 26.8 and 28.5, respectively. For anti-Fy(a) and anti-Kell, a significantly higher titration score could be obtained in the MCAT, whereas anti-Lu(a) showed a significantly higher score with the gel test. CONCLUSION: For the screening of unexpected red blood cell antibodies, the MCAT is as sensitive as the gel indirect antiglobulin test. The sensitivity and specificity of the two systems are more or less the same although it seems that IgM antibodies are better detected by the gel test.

Antibodies, Blocking↗
PubMed1999

Analysis of the Kveim-Siltzbach test reagent for bacterial DNA.

The sarcoid spleen-derived reagent for the Kveim-Siltzbach test (KST) elicits a sarcoid-specific, granulomatous, cutaneous response used to establish the diagnosis of sarcoidosis. In the context of the ongoing discussion of a bacterial cause of sarcoidosis we asked the question whether bacterial DNA could be found in the KST reagent. For this purpose two different KST reagents, an identical preparation from a normal spleen, and a native sarcoid spleen were analyzed by polymerase chain reaction (PCR) employing universal primers detecting conserved DNA sequences coding for bacterial ribosomal 16S RNA. Neither KST reagents, the control preparation, nor the spleen yielded a positive signal, indicating that the preparations are free of bacterial contamination. Because the KST reagent elicits granuloma, these results do not support the hypothesis of a bacterial cause of sarcoid granuloma.

Conserved Sequence↗