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Biomedical subjects

T Richter

Publications and source records attributed to T Richter.

At least 19 recordsLinked to original sources
PubMed2001

Identification and biochemical characterization of Arabidopsis thaliana sulfite oxidase. A new player in plant sulfur metabolism.

In mammals and birds, sulfite oxidase (SO) is a homodimeric molybdenum enzyme consisting of an N-terminal heme domain and a C-terminal molybdenum domain (EC ). In plants, the existence of SO has not yet been demonstrated, while sulfite reductase as part of sulfur assimilation is well characterized. Here we report the cloning of a plant sulfite oxidase gene from Arabidopsis thaliana and the biochemical characterization of the encoded protein (At-SO). At-SO is a molybdenum enzyme with molybdopterin as an organic component of the molybdenum cofactor. In contrast to homologous animal enzymes, At-SO lacks the heme domain, which is evident both from the amino acid sequence and from its enzymological and spectral properties. Thus, among eukaryotes, At-SO is the only molybdenum enzyme yet described possessing no redox-active centers other than the molybdenum. UV-visible and EPR spectra as well as apparent K(m) values are presented and compared with the hepatic enzyme. Subcellular analysis of crude cell extracts showed that SO was mostly found in the peroxisomal fraction. In molybdenum cofactor mutants, the activity of SO was strongly reduced. Using antibodies directed against At-SO, we show that a cross-reacting protein of similar size occurs in a wide range of plant species, including both herbacious and woody plants.

Amino Acid Sequence↗
PubMed2001

Gene expression profiling of human stent-induced neointima by cDNA array analysis of microscopic specimens retrieved by helix cutter atherectomy: Detection of FK506-binding protein 12 upregulation.

BACKGROUND: Restenosis due to neointima formation is the major limitation of stent-supported balloon angioplasty. Despite abundant animal data, molecular mechanisms of neointima formation have been investigated on only a limited basis in patients. This study sought to establish a method for profiling gene expression in human in-stent neointima and to identify differentially expressed genes that may serve as novel therapeutic targets. METHODS AND RESULTS: We retrieved tissue specimens from patients with symptomatic in-stent restenosis using a novel helix cutter atherectomy device. cDNA samples prepared from neointima (n=10) and, as a control, from the media of normal arteries (n=14) were amplified using a novel polymerase chain reaction protocol and hybridized to cDNA arrays. Immunohistochemistry characterized the atherectomy material as neointima. cDNA arrays readily identified differentially expressed genes. Some of the differentially expressed genes complied with expected gene expression patterns of neointima, including downregulation of desmin and upregulation of thrombospondin-1, cyclooxygenase-1, and the 70-kDa heat shock protein B. Additionally, we discovered previously unknown gene expression patterns, such as downregulation of mammary-derived growth inhibitor and upregulation of FK506-binding protein 12 (FKBP12). Upregulation of FKBP12 was confirmed at the protein level in neointimal smooth muscle cells. CONCLUSIONS: Gene expression patterns of human neointima retrieved by helix-cutter atherectomy can be reliably analyzed by cDNA array technology. This technique can identify therapeutic targets in patients, as exemplified by the findings regarding FKBP12. FKBP12 is the receptor for Rapamycin (sirolimus), which in animal models reduced neointima formation. Our study thus yields a rationale for the use of Rapamycin to prevent restenosis in patients.

Aged↗
PubMed2001

Circular stationary solutions in two-dimensional neural fields.

Stationary solutions are studied in two-dimensional homogeneous neural fields of the lateral-inhibition type. It is shown that in extending the one-dimensional theory to two dimensions, new phenomena arise. We discuss the conditions for the existence of localized solutions analogous to the one-dimensional theory and show that they are no longer sufficient in two dimensions. We give indications for the existence of mono- and bistable dynamics as known from the one-dimensional theory and, additionally, a tri-stable type of dynamic in two-dimensional neural fields, where, depending on the input, excitation dies out, spreads without limit, or causes a stable localized excitation.

Brain↗
PubMed2001

Quantitative gene expression analysis in microdissected archival formalin-fixed and paraffin-embedded tumor tissue.

Formalin-fixed, paraffin-embedded tissue is the most widely available material for retrospective clinical studies. In combination with the potential of genomics, these tissues represent an invaluable resource for the elucidation of disease mechanisms and validation of differentially expressed genes as novel therapeutic targets or prognostic indicators. We describe here an approach that, in combination with laser-assisted microdissection allows quantitative gene expression analysis in formalin-fixed, paraffin-embedded archival tissue. Using an optimized RNA microscale extraction procedure in conjunction with real-time quantitative reverse transcriptase-polymerase chain reaction based on fluorogenic TaqMan methodology, we analyzed the expression of a panel of cancer-relevant genes, EGF-R, HER-2/neu, FGF-R4, p21/WAF1/Cip1, MDM2, and HPRT and PGK as controls. We demonstrate that expression level determinations from formalin-fixed, paraffin-embedded tissues are accurate and reproducible. Measurements were comparable to those obtained with matching fresh-frozen tissue and neither fixation grade nor time significantly affected the results. Laser microdissection studies with 5-microm thick sections and defined numbers of tumor cells demonstrated that reproducible quantitation of specific mRNAs can be achieved with only 50 cells. We applied our approach to HER-2/neu quantitative gene expression analysis in 54 microdissected tumor and nonneoplastic archival samples from patients with Barrett's esophageal adenocarcinoma and showed that the results matched those obtained in parallel by fluorescence in situ hybridization and immunohistochemistry. Thus, the combination of laser-assisted microdissection and real-time TaqMan reverse transcriptase-polymerase chain reaction opens new avenues for the investigation and clinical validation of gene expression changes in archival tissue specimens.

Animals↗
PubMed2001

Transcriptome analysis reveals a role of interferon-gamma in human neointima formation.

The most effective immediate cure for coronary stenosis is stent-supported angioplasty. Restenosis due to neointima proliferation represents a major limitation. We investigated the expression of 2435 genes in atherectomy specimens and blood cells of patients with restenosis, normal coronary artery specimens, and cultured human smooth muscle cells (SMCs). Of the 223 differentially expressed genes, 37 genes indicated activation of interferon-gamma (IFN-gamma) signaling in neointimal SMCs. In cultured SMCs, IFN-gamma inhibited apoptosis. Genetic disruption of IFN-gamma signaling in a mouse model of restenosis significantly reduced the vascular proliferative response. Our data suggest an important role of IFN-gamma in the control of neointima proliferation.

Animals↗
PubMed2001

The effect of increasing topotecan infusion from 30 minutes to 4 hours on the duration of exposure in cerebrospinal fluid.

BACKGROUND: The development of metastatic disease throughout the neuroaxis from primary central nervous system (CNS) tumors and non-CNS tumors suggests the cerebrospinal fluid (CSF) is an important source of exposure for chemotherapeutic agents. In non-human primates, a 4-hour, as compared to a 30-minute, topotecan (TPT) infusion prolonged TPT exposure in the CSF. PATIENT AND METHODS: We evaluated this approach in a 51-year-old woman with breast cancer metastatic to the CNS. TPT was administered at 1.5 mg/m2/day (cycle 1) and 1.0 mg/m2/day (cycles 2 and 3) as a 30-minute infusion on days 0-4, and as a 4-hour infusion on day 5. Cycles were repeated every 21 days. Plasma, lateral ventricular CSF, and lumbar CSF samples were obtained after 30-minute and 4-hour infusions, and assayed for TPT lactone and total by HPLC. A three-compartment model was used to calculate area under the plasma (AUCplasma) and lateral ventricular CSF (AUCCSF) concentation-time curves. TPT CSF penetration was calculated as the ratio of AUCCSF to AUCplasma. RESULTS: Mean +/- SD values for TPT total CSF penetration in lateral CSF after 30-minute and 4-hour infusions were 0.25 +/- 0.15 and 0.29 +/- 0.02, respectively. TPT total lumbar CSF concentration was 3-fold greater after a 4-hour as compared to a 30-minute infusion. For TPT lactone and TPT total, time > 1 ng/ml in lateral CSF was 1.8- and 1.7-fold greater, respectively, for a 4-hour as compared to a 30-minute infusion. CONCLUSIONS: Prolonging TPT infusion from 30 minute to 4 hours increases the duration of exposure in the CSF. This study demonstrates the ability to develop treatment strategies of systemically administered chemotherapy to enhance cytotoxic exposure in the CSF.

Antineoplastic Agents↗
PubMed2001

A new rapid immunohistochemical staining technique using the EnVision antibody complex.

Rapid immunohistochemical investigation, in addition to staining with hematoxylin and eosin, would be useful during intraoperative frozen section diagnosis in some cases. This study was undertaken to investigate whether the recently described EnVision system, a highly sensitive two-step immunohistochemical technique, could be modified for rapid immunostaining of frozen sections. Forty-five primary antibodies were tested on frozen sections from various different tissues. After fixation in acetone for 1 min and air-drying, the sections were incubated for 3 min each with the primary antibody, the EnVision complex (a large number of secondary antibodies and horseradish peroxidase coupled to a dextran backbone), and the chromogen (3,3'diaminobenzidine or 3-amino-9-ethylcarbazole). All reactions were carried out at 37C. Specific staining was seen with 38 antibodies (including HMB-45 and antibodies against keratin, vimentin, leukocyte common antigen, smooth muscle actin, synaptophysin, CD34, CD3, CD20, and prostate-specific antigen). A modification of the EnVision method allows the detection of a broad spectrum of antigens in frozen sections in less than 13 min. This method could be a useful new tool in frozen section diagnosis and research. (J Histochem Cytochem 49:623-630, 2001)

Antibodies↗
PubMed2001

Helicobacter pylori prevalences and risk factors among school beginners in a German urban center and its rural county.

In 1998, the Helicobacter pylori [(13)C]urea breath test was offered to all school beginners (birth cohort 1991/1992) in the city of Leipzig and in Leipzig County, Germany, to determine the colonization prevalence and potential transmission pathways of the bacterium. A total of 3,347 school beginners participated in the test, and 2,888 parents completed the detailed, self-administered questionnaire. The H. pylori prevalence was 6.5% [95% confidence interval (CI), 5.3-7.6] in the city and 5.7% (CI, 4.2-7.0) in the county. Using cluster analysis (WARD's method, Euclidean distances), we identified different sets of variables (confirmed by multivariate logistic regression analyses [odds ratios (ORs)] that are signficantly associated with H. pylori positivity. Among city children, the risk is significantly increased with contact to a pet hamster (OR = 2.4; 95% CI, 1.2-4.7; p < 0.015) and travels to Asian countries (OR = 3.7; 95% CI, 1.6-8.7; p < 0.002). Among county children, H. pylori positivity increased significantly with drinking of water from nonmunicipal sources (OR = 16.4; 95% CI, 3.1-88.5; p < 0.001), more than 3 children living in a household (OR = 4.2; 95% CI, 1.2-14.6; p < 0.02), and contact with pet hamsters (OR = 2.4; 95% CI, 1.0-5.7; p < 0.04). These data suggest that, in a general population sample, indirect fecal-oral transmission and living conditions are important risk factors in the spread of H. pylori infection. However, clinical symptoms do not necessarily indicate H. pylori positivity.

Breath Tests↗
PubMed2000

Importance of cysteine residues for the stability and catalytic activity of human pancreatic beta cell glucokinase.

The low-affinity glucose phosphorylating enzyme glucokinase has the function of a physiological glucose sensor in pancreatic beta cells and in liver. In contrast to the high-affinity hexokinase types I-III glucokinase shows extraordinary sensitivity toward SH group oxidizing compounds. To characterize the function of sulfhydryl groups cysteine residues in the vicinity of the sugar binding site (Cys 213, Cys 220, Cys 230, Cys 233, and Cys 252) as well as cysteine residues a distance from the active site (Cys 364, Cys 371, and Cys 382), they were replaced in human beta cell glucokinase by serine through site-directed mutagenesis. Controlled proteolysis of wild-type glucokinase by proteinase K revealed that the SH group oxidizing agent alloxan can induce the formation of multiple intramolecular disulfide bridges corresponding to a double-band pattern of glucokinase protein in nonreducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The formation of intramolecular disulfide bridges altered the mobility of the protein. None of the cysteine mutations could prevent the formation of the 49-kDa glucokinase conformation after alloxan treatment. The cysteine mutants Cys 233, Cys 252, and Cys 382 showed nearly complete loss of catalytic activity, whereas the V(max) values of the Cys 213, Cys 220, Cys 364, and Cys 371 mutants were decreased by 30-60%. Only the Cys 230 mutant showed kinetic characteristics comparable to those of wild-type glucokinase. The sensitivity of the Cys 213, Cys 230, Cys 364, and Cys 371 mutants toward alloxan-induced inhibition of enzyme activity was up to 10-fold lower compared with wild-type glucokinase. d-Glucose and dithiotreitol provided protection against alloxan-induced inhibition of wild-type glucokinase and all catalytically active cysteine mutants. Conclusively our data demonstrate the functional significance of the cysteine residues of beta cell glucokinase for both structural instability of the enzyme and catalytic function. Knowledge of sensitive cysteine targets may help to develop strategies that improve glucokinase enzyme function under conditions of oxidative stress.

Alloxan↗
PubMed2000

[Prospective detection of important bacterial pathogens in pyoderma and their in vitro antibiotic susceptibility].

BACKGROUND AND OBJECTIVE: For rational therapeutic recommendations the spectrum and resistance of bacteria in skin diseases were investigated. PATIENTS/METHODS: Within 4 months 911 swabs of dematoses possibly caused by bacteria were taken prospectively (481 outpatients, 430 inpatients) and the material cultured on standard media. The positive cultures including resistance screening of 210 swabs of 168 outpatients and of 175 swabs of 85 inpatients could be evaluated, the remaining cultures were sterile. RESULTS: Staphylococcus aureus was the most frequent pathogen (outpatient 67%, inpatient 61% of all positive cultures), followed by streptococci (groups A and B; 25%). In patients Pseudomonas aeruginosa was the most common pathogen in leg ulcers and between the toes (45% and 70% respectively). 13% of the Staphylococcus aureus isolates were resistant to tetracyclines and erythromycin; one strain proved to be methicillin resistant. CONCLUSIONS: Cephalosporins (I. generation), penicillins with beta-lactamase-inhibitors, and to a lesser extent isoxazoyl-penicillin and clindamycin can be recommended for the treatment of skin infections. Oral quinolones are suited for infections with gram-negative bacteria (such as bewteen the toes). The indications for systemic antibiotic therapy of leg ulcers should be restricted.

Bacterial Infections↗
PubMed2000

Genome-wide, large-scale production of mutant mice by ENU mutagenesis.

In the post-genome era, the mouse will have a major role as a model system for functional genome analysis. This requires a large number of mutants similar to the collections available from other model organisms such as Drosophila melanogaster and Caenorhabditis elegans. Here we report on a systematic, genome-wide, mutagenesis screen in mice. As part of the German Human Genome Project, we have undertaken a large-scale ENU-mutagenesis screen for dominant mutations and a limited screen for recessive mutations. In screening over 14,000 mice for a large number of clinically relevant parameters, we recovered 182 mouse mutants for a variety of phenotypes. In addition, 247 variant mouse mutants are currently in genetic confirmation testing and will result in additional new mutant lines. This mutagenesis screen, along with the screen described in the accompanying paper, leads to a significant increase in the number of mouse models available to the scientific community. Our mutant lines are freely accessible to non-commercial users (for information, see http://www.gsf.de/ieg/groups/enu-mouse.html).

Animals↗
PubMed2000

B cell epitopes of gliadin.

A phage displayed dodecapeptide library and synthetic octapeptides spanning the complete sequence of alpha- and gamma-type gliadin and overlapping in six amino acids (pepscan) were screened for binding to human gliadin antibodies (AGA). Phage display experiments led to four sequences recognized with significantly higher frequency by sera with raised IgA-AGA titres than by control sera. All these peptides contained the core sequence PEQ. Pepscan experiments revealed binding of AGA to five prominent regions: (i) QXQPFP (binding to IgG and IgA, X representing P, Q, and L); (ii) IPEQ (IgG) and WQIPEQ (IgA); (iii) FFQP (IgG) and QGXFQP (IgA, X representing F and S); (iv) PQQLPQ (IgG and IgA), all in alpha-type gliadin; and (v) QPQQPF (IgG and IgA) in gamma-type gliadin. In two of the sequences (QPQQPF and QQQPFP), substitution of Q by E resulting in QPEQPF and QEQPFP, respectively, increased significantly binding of AGA from sera of patients with biopsy-proven or suspected coeliac disease (CoD), all positive for endomysium antibodies (EmA). In contrast, binding of sera with high AGA titre from EmA-negative patients (CoD and dermatitis herpetiformis excluded) was not enhanced by this substitution. Thus, AGA directed against these modified epitopes can be regarded as specific for CoD. This is the first study demonstrating that deamidation of gliadin improves reactivity of AGA of CoD patients.

Amino Acid Sequence↗
PubMed2000

Helicobacter pylori prevalence in Leipzig's 1998 school entries: methodology and first results.

The non-invasive, stable-isotope-aided Helicobacter pylori (H. pylori) tests--breath and equivalent urine tests--were offered on a voluntary basis as part of the mandatory school entry medical examination to the 1998 school entry cohort of the City of Leipzig (480,000 residents). Parents of participating subjects were asked to fill out a detailed epidemiologic questionnaire. The response rate was 94% (n = 2228 of 2369 school starters born in 1991/92). Parent-completed questionnaires were returned by 1890 (80%) children. The overall H. pylori positive prevalence was 7.2%. The prevalence among children with a test and a parent-completed questionnaire was 6.5%. Prevalences among subsequently tested family members of the positive tested children was 65, 60 and 39% for mothers, fathers and siblings respectively. Though studies have shown that the direct transmission of the bacterium (oral-oral and fecal-oral) is a dominant pathway of infection, the questionnaire analyses indicate associations between H. pylori colonisation and living as well as environmental conditions.

Breath Tests↗
PubMed2000

Diagnosis of Helicobacter pylori infection in children: is the 15N urine test more reliable than the 13C breath test?

BACKGROUND: The [13C]urea breath test is a convenient method to estimate Helicobacter pylori colonization non-invasively. As an alternative method, the [15N]urea urine test has been established. It is hypothesized that the urine test could be advantageous under some circumstances. The diagnostic value of the breath test might, for example, be jeopardized owing to fluctuating 13C isotope dilution by endogenous carbon dioxide. METHODS: To compare the reliability and practicability of the two tests, 13C breath tests and 15N urine tests were performed simultaneously in three groups of patients: A) 36 adults under standardized conditions, B) 67 children who were allowed to move around during the test, and C) 18 children once without and once during standardized physical activity. RESULTS: In the 36 adults there was a close correlation of the test results of the two methods (r = 0.88). In contrast, the correlation in the group of 67 children and infants was poor (r = 0.10). In the children with controlled activity the 13C results fluctuated, whereas the 15N results remained unchanged. CONCLUSIONS: Owing to their identical biochemical basis the breath test and the urine test produce the same diagnostic results in the same individuals if standardized pre-test and test conditions are followed. However, factors such as physical activity during the test may provoke fluctuations with a tendency to decreasing 13C values. This could be due to additional isotope dilution by increases in the endogenous production of 12C-carbon dioxide. We conclude that in very active children the 15N urine test might be advantageous and more reliable than the 13C breath test.

Adult↗
PubMed2000

Evidence for existence of coeliac disease autoantigens apart from tissue transglutaminase.

BACKGROUND: The pathogenesis of coeliac disease (CD) and of dermatitis herpetiformis (DH) is strongly associated with production of autoantibodies, defined by indirect immunohistology. Recently, tissue transglutaminase (tTG) was identified as a prominent autoantigen. It would be important to investigate if further molecules apart from tTG are involved in autoimmunity. METHODS: Tissue sections of human foetal intestine were used to compare the distribution of tTG with the autoantibody binding patterns of 14 sera samples from patients with CD or DH. Double label experiments were performed using monoclonal as well as polyclonal tTG antibodies (anti-tTG) and patient sera. The staining was investigated by using conventional light and confocal laser scanning microscopy. RESULTS: Most autoantibody binding sites were matched by tTG. Further, the binding of autoantibodies could be inhibited by preincubation with monoclonal anti-tTG. However, in nine serum samples (64%) autoantibody staining suggested a few distinct binding sites apart from tTG. In three sera (21 %) autoantibody binding fibres were detected which definitely did not match monoclonal anti-tTG signals. Distinctly stained fibres were confirmed by applying polyclonal anti-tTG. This indicates the existence of autoantigenic epitopes not related to tTG.

Animals↗
PubMed2000

Response times in attitudinal items as indicators of the continuous accessibility of knowledge related to attitudes.

In the paradigm of social cognition, the measurement of response latencies is important not only for priming procedures directed at the cognitive representation, but also for the assessment of strength-related properties of attitudes. The present paper explored the possibility of using response times as indicators of the continuous accessibility of attitude-relevant knowledge. For two different sets of attitudinal items concerning social welfare (idiographic and nomothetic selection) response times were obtained from a sample of 20 university students. In addition to different reliability coefficients for aggregated response times, information about individual subjects' consistencies and stabilities was obtained. Further, evidence for the validity of response times as indicators of the continuous accessibility of attitude-related knowledge is reported. The aggregated response times had significant correlations with self-report data and interview-related variables (such as number of propositions mentioned) which were obtained by a content analysis of semistructured interviews.

Adult↗
PubMed2000

[Porcine stress syndrome and PSE meat: clinical symptoms, pathogenesis, etiology and animal rights aspects].

A review is given about the clinical symptoms, pathogenesis and aetiology of the porcine stress syndrome, furthermore aspects of animal welfare are discussed. The current breeding programmes of pig industry in Germany in many cases include animals with a mutation of the ryanodine-receptor (RYR-1)-gene--homozygous or heterozygous. This situation is the result of an intensive breeding of pigs during the last decades with the intention of increased lean carcass content and corresponding proceeds. The homozygous pigs are more stress susceptible (porcine stress syndrome) and produce meat of poor quality (PSE), which is also the case to some extend in heterozygous animals. The clinical symptoms of this muscle disease are characterised by a deficit of oxygen and a rapid glycolysis accompanied by a production of lactic acid and acidosis primarily in II B white muscle fibres. There is no doubt that a very close causal relation exists between the mutation of the RYR-1 and the porcine stress syndrome as well as the poor meat quality. The present knowledge of this disease, the genetic background, the physiology and pathophysiology of the mutation of the RYR-1 leads to the imperative conclusion to eliminate this mutated RYR-1 by selection of healthy pigs, which has been done successfully in other countries with important pig production. This conclusion is also supported by simple economic reasons because fertility, reproduction and daily weight gain are significantly reduced in stress susceptible pigs. Furthermore, it should be emphasised that regular breeding with the mutated RYR-1 is also a matter of animal welfare. The evident correlation between the mutated RYR-1 and the porcine stress syndrome, which includes degeneration of the muscle, pain and even life threatening malignant hyperthermia, can easily lead to the accusation in the public that diseased animals are used for pig meat production. Consequently, the authors would like to urge the breeding companies and the responsible authorities to discuss the problem with the intention to finish the current breeding programmes using animals with the mutated RYR-1 within a reasonable period of time.

Animal Husbandry↗