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Biomedical subjects

G Krause

Publications and source records attributed to G Krause.

At least 19 recordsLinked to original sources
PubMed1999

Regulation of CD97 protein in thyroid carcinoma.

CD97 is a dimeric glycoprotein belonging to the secretin receptor superfamily and is abundantly expressed in cells of hematopoietic origin. The aim of this study was to analyze the expression of the CD97 protein in thyroid carcinomas and the role of all-trans-retinoic acid (RA) in the regulation of CD97 protein in monolayer culture of the human follicular thyroid carcinoma cell line FTC-133. In normal thyroid tissue, no immunoreactivity of CD97 could be found, whereas in differentiated thyroid carcinomas, CD97 expression was either lacking or low. Undifferentiated anaplastic thyroid carcinomas revealed high CD97 expression. The expression of CD97 protein seems to be correlated to the postoperative histopathological classification staging. Approximately 50% of FTC-133 cells expressed the CD97 protein under basal culture conditions. No differences were found in the number of CD97-positive cells after TSH, forskolin, and insulin treatment compared to control values. Epidermal growth factor treatment led to an increase in CD97 immunostaining (up to 90%), whereas phorbol 12-myristate 13-acetate slightly decreased the immunoreactivity of CD97 (from 50% to 30%). Under basal conditions, RA treatment for 72 h led to a decrease in total cell number by 33% and in CD97-positive cells from 50% to 30%. TSH, forskolin, phorbol 12-myristate 13-acetate, and insulin showed no effect after 72-h pretreatment with RA, whereas epidermal growth factor treatment led to a slight increase in the number of the CD97-positive cells (from 30% to 40%) compared to the control value. These data suggest that CD97 expression may play an important role in the dedifferentiation of thyroid tumors, and RA might interfere with this process in thyroid carcinoma by suppressing the dedifferentiation marker CD97.

Adult↗
PubMed1999

Base-substitution profiles of externally activated polycyclic aromatic hydrocarbons and aromatic amines determined in a lacZ reversion assay.

Using an improved set of lactose-auxotrophic Escherichia coli tester strains, the proportion of the six possible transitions and transversions after mutagen exposure was assessed. Mutagenic specificity was determined in plate-incorporation assays using lactose-containing minimal medium for the selection of revertants, either after application of directly acting mutagens or by including a metabolic activation system with rat liver S9-extract. The differential and dose-dependent response of the six tester strains was shown by treating the bacteria with described diagnostic mutagens and other directly DNA damaging substances, e.g., N-methyl-N-nitrosoguanidine (MNNG) and benzo[a]pyrene-diolepoxide (BPDE). Polycyclic aromatic hydrocarbons and aromatic amines were investigated in the presence of an external metabolic activation system. Benzo[a]pyrene (BaP) yielded similar mutation profiles as its ultimate mutagen BPDE, if 100-fold increased doses were applied. In contrast to the mutation profile of BaP, which was dominated by G:C-T:A transversions, mutagenesis with benzo[c]phenanthrene (BcPh) produced predominantly A:T-T:A transversions. The same base change was observed with 5-methylchrysene and found to be missing with 5,6-dimethylchrysene, while both compounds caused G:C-A:T transitions. The aromatic amines 4-aminobiphenyl (4-ABP), 2-aminoanthracene (2-AA) and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhlP) yielded similar yet distinguishable mutation profiles. Base-substitution reversion profiles of the chemical mutagens were in agreement with those obtained in other systems and with molecular analysis of mutants induced by these agents.

Amines↗
PubMed1998

Genotyping metabolic polymorphisms in a cohort of Caucasians and single strand conformation polymorphism analysis of point mutations in human hprt exons 7 and 8.

In genetic toxicology, the main fields of applications of the polymerase chain reaction (PCR) with subsequent electrophoretic characterization of amplificates include genotyping polymorphisms in the xenobiotic metabolism and mutant analysis. To assess the role of the individual sets of biotransformation enzymes for the internal dose resulting from xenobiotic exposure, we investigated blood samples from 69 healthy donors for the occurrence of known genetic polymorphisms in the xenobiotic metabolizing enzymes N-acetylaminotransfrase II (NAT2), glutathione-S-transferase (GST) mu and theta, and several cytochromes P450 (CYP), namely CYP1A1, CYP2E1 and CYP2A6. Using single strand conformation polymorphism (SSCP) analysis, five known single base substitutions located in the middle portion of 144 bp amplificates comprising exons 7 and 8 of the human hypoxanthine guanine phosphoribosyl transferase (hprt) cDNA, were clearly distinguished from wild type and from each other. Biomagnetic strand separation assigned the slower migrating single strand bands to the biotinylated sense strands.

Aryl Hydrocarbon Hydroxylases↗
PubMed1998

An experimental study of the esthetic effect of facial profiles.

In this study good-looking "male" and "female" as well as ugly facial profiles were shaped by 104 lay persons using an especially constructed device according to specific instructions. These profiles were photographed and subsequently evaluated using a series of parameters from soft tissue profile analyses. Although some significant mean value differences were found between the good-looking and ugly profile variants, they were not substantial. In contrast, markedly significant differences were revealed between the variances of all variables. In some instances the variance of the ugly profiles was more than 3 to 4 times higher than that of the good-looking profiles. These findings were convincingly confirmed when statistical distribution of the data was established and compared. This implies that perception of beauty is associated with regularity of facial features and is conveyed by measurement values which are located close to the mean. Ugliness is associated with extreme deviations from the latter in either direction. Apart from the facial proportions, the degree of convexity or concavity of facial profile and their sequence seem to be important for the esthetic effects. "Male" profiles in contrast to "female" profiles exhibited more conspicuous facial features such as pronounced convexity and concavity.

Adolescent↗
PubMed1998

Rapid characterization of mutations in amplified human hprt cDNA by polyacrylamide gel electrophoresis.

Compiling hprt mutation spectra involves the isolation and analysis of numerous 6-thioguanine-resistant clones for identifying characteristic point mutations. Since cDNA amplificates are compulsory intermediates in most mutant classification protocols, we suggest their preliminary characterization by polyacrylamide gel electrophoresis for the rapid distinction of clonal and independent mutants and for streamlining mutant analysis procedures. Based on the human hprt cDNA sequence a strategy was developed for mapping missing exons by analytical digests with a small panel of restriction enzymes. In mutant classification schemes, polyacrylamide gel electrophoresis of AluI-digested cDNA amplificates increased the sensitivity for detecting RT-PCR products of reduced size, e.g., in the case of missing exon 5. Restriction analysis of cDNA amplificates from 109 independent mutant clones showed a significant increase of exon loss after NNK induction as compared to spontaneous or BaP-induced mutants. The determination of exon loss from cDNA amplificates, as carried out for 39 independent mutant PCR products, might direct towards the genomic target sequences carrying the point mutations, that caused the aberrant splicing, thus eliminating the need of laborious multiplex PCR comprising all exons. For single-strand conformation polymorphism (SSCP) analysis of five known point mutations, sub-amplificates comprising exons 7 and 8 of hprt cDNA were obtained. After a combined heat and alkali denaturation of the double-stranded PCR products, the samples were separated in pre-cast polyacrylamide gels under non-denaturing conditions. Five known nucleotide substitutions within the amplified region, including the C508T hot spot mutation, resulted in mobility shifts of single-strand bands relative to the wild type pattern.

Cell Line↗
PubMed1998

Specific interactions between the syntrophin PDZ domain and voltage-gated sodium channels.

Syntrophins are modular proteins belonging to the dystrophin associated glycoprotein complex and are thought to be involved in the regulation of the muscular system. Screening of peptide libraries revealed selectivity of the synotrophin PDZ domain toward the motif R/K/Q-E-S/T-X-V-COO- found to be highly conserved in the alpha-subunit C-terminus of vertebrate voltage gated sodium channels (VGSCs). The solution structure of the domain in complex with the peptide G-V-K-E-S-L-V shows specific interactions between the conserved residues in the peptide and syntrophin-characteristic residues in the domain. We propose that syntrophins localize VGSCs to the dystrophin network through its PDZ domain.

Amino Acid Sequence↗
PubMed1998

Diagnostic quality in rural health centres in Burkina Faso.

OBJECTIVE: To study the quality of diagnostic practice in rural Burkina Faso. METHOD: In 9 health centres of 3 districts, 313 outpatient consultations were observed, and 417 diagnoses by 15 nurses were analysed. Criteria for evaluation of patient history and clinical examination were based on the diagnostic guidelines distributed by the Ministry of Health. RESULTS: In only 20% of the diagnoses the nurses took a sufficient history and in only 40% they conducted a sufficient clinical examination. In 21% patients underwent no clinical examination at all. Only 12% of all diagnoses were based on sufficient history-taking and adequate clinical examinations. The individual elements of clinical examination were performed correctly in 82% of cases. The variation between nurses was immense, but no correlation could be found with regard to their basic training. However, nurses who had received the diagnostic guidelines examined patients more carefully than those who had not. Larger numbers of patients per day are not associated with shorter nurse-patient contact, and neither is sufficiency of patient history associated with duration of the consultation. CONCLUSION: The low diagnostic quality of the outpatient consultations in the studied area indicates that this issue has been neglected in national public health initiatives. But examination skills are good and diagnostic guidelines may have had a positive effect on the diagnostic quality.

Adolescent↗
PubMed1998

Performance of village pharmacies and patient compliance after implementation of essential drug programme in rural Burkina Faso.

After implementation of a nation-wide essential drug programme in Burkina Faso a prospective study was undertaken consisting of non-participant observation in the health centre and in the village pharmacy, and of household interviews with the patients. The study covered all general consultations in nine health centres in three districts over a two-week period as well as all client-vendor contacts in the corresponding village pharmacies; comprising 313 patients in consultations and 498 clients in eight village pharmacies with 12 vendors involved in dispensing 908 drugs. Additionally patients were interviewed in their households. Performance and utilization of the village pharmacy: 82.0% of the drugs prescribed in the health centres were actually dispensed at the village pharmacy, 5.9% of the drugs were not available at the village pharmacy. Wrong drugs were dispensed in 2.1% of cases. 41.3% of the drugs dispensed in the village pharmacy were bought without a prescription. Differences are seen between the district and are put in relation to different onset of the essential drug programme. Patient compliance: Patients could recall the correct dosage for 68.3% of the drugs. Drug taking compliance was 63.1%, derived from the pills remaining in the households. 11.5% of the drugs had obviously been taken incorrectly to such an extent that the occurrence of undesired drug effects was likely. The study demonstrates the success of the essential drug programme not only in performance but also in acceptability and utilization by the population.

Burkina Faso↗
PubMed1998

A conserved tyrosine residue (Y601) in transmembrane domain 5 of the human thyrotropin receptor serves as a molecular switch to determine G-protein coupling.

In the human thyroid, the wild-type thyrotropin receptor (TSHR) couples to adenylyl cyclase and phospholipase C and constitutively increases intracellular cAMP levels. The first human TSHR sequence submitted differs from subsequently cloned wild-type receptors by an exchange of a conserved Y residue within transmembrane domain 5 (TM5) for an H residue. We did not detect the Y601H mutant in 263 European individuals, but confirmed the homozygous occurrence of TSHR-Y601. Expression of TSHR-Y601H in COS-7 cells revealed a loss of constitutive cAMP production and selective lack of TSH-induced phosphoinositide hydrolysis, whereas agonist-induced cAMP formation remained unaltered. Analysis of several mutant receptors (Y601A, Y601D, Y601F, Y601K, Y601P, Y601S, Y601W, Y601Delta) did not show restoration of constitutive activity and dual signaling, thus suggesting a functional role of a properly spaced hydroxyl group at position 601. Molecular modeling revealed that the formation of a hydrogen bond between the hydroxyl group of Y601 in TM5 and the carbonyl oxygen of A623 in the peptide backbone of TM6 is critical for the receptor to adopt active conformations that impart wild-type signaling properties. Our findings indicate that multiple active receptor states underlie coupling of a G-protein-coupled receptor to different G-proteins.

Animals↗
PubMed1998

A dileucine sequence and an upstream glutamate residue in the intracellular carboxyl terminus of the vasopressin V2 receptor are essential for cell surface transport in COS.M6 cells.

Little is known concerning the intracellular transport of the G protein-coupled receptors (GPCRs). Previous studies suggested a functional role for those residues immediately preceding the conserved palmitoylated cysteine residues in the intracellular carboxyl termini of some GPCRs in cell surface transport. For the human vasopressin V2 receptor, we assessed the significance of a dileucine sequence with an upstream glutamate residue (ELRSLLCC) in mediating cell surface delivery. A series of deletion and point mutants in this region were constructed, and the mutant receptors were expressed in transiently transfected COS.M6 cells. By using [3H]arginine vasopressin binding assays to intact cells and immunofluorescence studies with intact and permeabilized cells, we show that residues E335 (mutant E335Q) and L339 (mutant L339T) are obligatory for receptor transport to the plasma membrane. Residue L340 has a minor but significant influence. [3H]Arginine vasopressin binding experiments on membranes of lysed cells failed to detect any intracellular binding sites for the transport-deficient mutant receptors, suggesting that residues E335 and L339 participate in receptor folding. Studies with green fluorescent protein-tagged receptors demonstrate that the bulk of the mutant receptors E335Q and L339T are trapped in the endoplasmic reticulum. Complex glycosylation was absent in these mutant receptors, supporting this conclusion. These data demonstrate that the glutamate/dileucine motif of the vasopressin V2 receptor is critical for the escape of the receptor from the endoplasmic reticulum, most presumably by establishing a functional and transport-competent folding state. A databank analysis revealed that these residues are part of a conserved region in the GPCR family.

Amino Acid Sequence↗
PubMed1998

The interaction of class B G protein-coupled receptors with their hormones.

In common with many G protein-coupled receptors, dysfunction in members of the Class B or glucagon-like receptors can elicit a wide spectrum of disease related activities. Consequently, they are potential targets in many different areas of pharmacological research. Unlike the class A or rhodopsin-like receptors, for which at least some structural similarity to bacteriorhodopsin has been detected, absolutely no structural information is available for the Class B G protein-coupled receptors. We present a computational study that exploits the experimental work performed by evolution to indicate residues that are potentially involved in ligand binding in the Class B G protein-coupled receptors. We perform an analysis of mutations that occurred in a correlated fashion between the receptors and their peptidic ligands. The inference that the residues detected in this manner are involved in a direct interaction between the receptor and the ligand is in good agreement with the mutation studies that have already been published.

Amino Acid Sequence↗
PubMed1997

Absence of p53 mutations in squamous carcinomas of the tongue in nonsmoking and nondrinking patients younger than 40 years.

BACKGROUND: Although carcinoma of the tongue usually occurs in patients older than 60 years, up to 4% of these tumors may occur in patients younger than 40 years. Many of the younger patients with this tumor have had no exposure or brief exposure to tobacco smoke or alcohol consumption, to which oral carcinoma is usually attributed. The molecular mechanism responsible for carcinogenesis in this group of patients is not known. OBJECTIVE: To assess the role of p53 gene mutation in oral carcinogenesis in a group of patients younger than 40 years with squamous carcinoma of the tongue. DESIGN: Squamous carcinoma cells were isolated from paraffin blocks by microdissection. DNA extracted from these cells was tested for the presence of p53 mutations by polymerase chain reaction and single-stranded conformational polymorphism analysis. Mutations identified by this procedure were directly sequenced. Sections of the tumors were also stained using an immunoperoxidase immunohistochemical technique for expression of p53 protein. SUBJECTS: Eleven patients were selected on the basis of 2 criteria: presence of squamous cell carcinoma and age younger than 40 years. Six of the 11 patients had no history of measurable tobacco or alcohol exposure. RESULTS: Two mutations were detected among 11 tumors by single-stranded conformational polymorphism analysis, one in exon 4 and a second in exon 7. The former mutation consisted of G:C to C:G (guanine:cytosine to cytosine:guanine) transition in codon 72 (CGC to CCC), which would have resulted in the substitution of a proline residue for arginine. With the immunoperoxidase immunohistochemical technique for p53 protein, strong, diffuse nuclear staining was observed only in this tumor. The second mutation was a G:C to A:T (guanine:cytosine to adenine:thymine) transition in codon 248 (CGG to CGA), which would have resulted in no amino acid change since both mutant and wildtype codon sequences encode arginine. Weaker and more variable anti-p53 immunostaining was noted in this and 4 other tumors. Five tumors were negative for p53 protein by the immunoperoxidase immunohistochemical technique. CONCLUSIONS: Our results suggest that p53 gene mutations are less frequent in squamous carcinomas occurring in nonsmoking young patients who do not drink alcohol than in young smokers or in the general population. Paucity of p53 mutations may be explained by the absence of exposure to tobacco smoke or alcohol. These data leave unanswered the question of the molecular mechanism responsible for oral carcinogenesis in this group of patients and suggest that this group may be a suitable population in which to study genetic susceptibility to aerodigestive carcinoma isolated from the confounding factors of tobacco and alcohol exposure.

Adult↗
PubMed1997

Applications of consensus polymerase chain reaction with subsequent electrophoretic distinction of amplificates.

Conserved sequences within gene families permit the design of consensus primers that match several members of a given class of homologous genes. Polymerase chain reaction (PCR) products obtained with such consensus primers were characterized by restriction mapping or single-strand conformation polymorphism (SSCP) analysis, using precast polyacrylamide minigels and automated silver staining. Examples for the electrophoretic distinction of consensus amplificates are presented in the fields of guanylyl cyclase expression studies and in the determination of B-cell clonality in human blood samples. Guanylyl cyclase expression in inner ear tissues of guinea pigs was investigated by reverse transcription PCR using consensus primers with specificity for the subclass of particulate guanylyl cyclases. The resulting PCR products were assigned to three representatives of this group by restriction mapping. The consensus PCR approach enabled the detection of an unexpected receptor type, namely guanylyl cyclase C, in the inner ear. The distinction by SSCP analysis of denatured consensus amplificates was appropriate for the identification of clone-specifically rearranged immunoglobulin heavy chain genes of B-lymphocytes. Genomic DNA isolated from blood samples of leukemia patients served as the template for the consensus amplification of clone-specific VDJ rearrangements. Rapid distinction and re-identification of consensus PCR products was achieved by SSCP analysis for regular antigen receptor rearrangements and for t(14; 18) translocations. The potential of these procedures for detecting leukemia or lymphoma clones when monitoring minimal residual disease was assessed.

Animals↗
PubMed1997

Transcripts encoding three types of guanylyl-cyclase-coupled trans-membrane receptors in inner ear tissues of guinea pigs.

The distribution of membrane-bound guanylyl cyclase (GC) transcription in inner ear tissues of the guinea pig was addressed by a reverse transcription-PCR approach using consensus primers flanking a region of about 630 bp in the intracellular domains in the target sequences. Restriction mapping of such amplificates obtained from cochlear and vestibular specimens permitted us to demonstrate GC-A, GC-B, and GC-C expression by differentiating overall PCR signals. This assay indicated that GC-A was expressed in the cochlea and vestibular organ. PCR products resulting from transcripts of the GC-B gene were obtained at considerably lower abundance than amplificates typical of the GC-A gene. The consensus primer approach with subsequent restriction mapping provided the opportunity to examine at the same time expression of GC-C in the inner ear and revealed the occurrence of GC-C transcripts in both inner ear compartments under investigation. The distribution pattern found by analysing the intracellular domains of membrane-bound guanylyl cyclases was confirmed by demonstrating transcription of the corresponding extracellular receptor domains. In addition, single-strand conformation polymorphism analysis of cDNA amplificates comprising the catalytic domain of guanylyl cyclases also indicated the presence of GC-C expression in the inner ear tissues examined. The GC-C transcripts detected in inner ear tissues appeared to correlate with functional receptor expression, since the production of cyclic GMP catalyzed by cochlear and vestibular specimens was stimulated by 1 microM of heat-stable enterotoxin to 18 and 80% above basal levels, respectively. Thus, GC-C may be involved in the fluid regulation by typical ligands (e.g., the peptide hormone guanylin or the toxins causing travellers' diarrhea), not only in the intestine but also in the organs responsible for hearing and gravitational orientation.

Animals↗
PubMed1997

Atherogenesis and the homocysteine-folate-cobalamin triad: do we need standardized analyses?

BACKGROUND: Bioscientists, physicians and nutritionists are newly interested in the homocysteine-folate-cobalamin triad, in part because homocysteine may be important both in atherogenesis and thrombogenesis. Homocysteine imbalance may be an early marker for cobalamin disorders because cobalamin is a cofactor in remethylation of homocysteine to methionine. METHODS: In 139 men and 32 women of similar mean age of 65 years, we measured markers which have been cited as risk for atherosclerosis: serum homocysteine, folate, total cobalamin, holotranscobalamin I and II, (TCI and TCII), total serum cholesterol (SCHOL), high density lipoprotein cholesterol (HDLC), triglycerides (STG) as well as red blood cell (RBC) folate, food records and body composition by whole body counting of potassium-forty (40K). RESULTS: Statistical relationships among the data showed healthy women had lower mean serum homocysteine and their mean RBC folate and TCI and TCII were higher than men. Eighty-three subjects had TCII much lower than 60 pg/ml (subnormal), yet only 11 of these men and two women had total cobalamin < 200 pg/ml (abnormal). Fifty-two subjects with serum homocysteine greater than 17.5 nmol/ml had TCII less than 60 pg/ml, suggesting serum homocysteine may be a marker for early cobalamin negative balance. None of the subjects in the study had serum folate below abnormal values, i.e., less than 1.6 mg/ml. All subjects had RBC folate within normal range. Serum homocysteine showed inverse relationship with RBC folate and serum total cobalamin, TCI and TCII. CONCLUSIONS: 1) importance of using serum holotranscobalamin TCI and TCII as markers of cobalamin deficiency, 2) necessity to use documented quantitative components of dietary intake if strong comparisons are to be made among quantitative values of serum or plasma homocysteine, folate, cobalamin, and nutrients in food intake.

Adult↗
PubMed1997

Complementary anchor PCR of rearranged variable T-cell receptor beta-chain cDNA regions.

Sequencible amplificates comprising the variable cDNA sequences of the rearranged T-cell receptor (TCR) beta-chain were obtained from the T-leukemia cell line Jurkat using a single-sided PCR approach based on five synthetic oligonucleotides derived from the flanking constant sequence. Double-stranded cDNA was cleaved by a restriction enzyme creating cohesive ends, to which an anchor oligonucleotide was ligated. Since this anchor was complementary to the antisense strand of the known constant region, exclusively the desired ligation product folded into a stem-loop-structure that was enzymatically extended to yield a PCR template, now flanked at both ends by primer binding sites appropriate for nested PCR.

Binding Sites↗