Biomedical subjects

G Scherer

Publications and source records attributed to G Scherer.

At least 55 records · Page 3Linked to original sources

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

Novel and recurrent tyrosine aminotransferase gene mutations in tyrosinemia type II.

Tyrosinemia type II (Richner-Hanhart syndrome, RHS) is a disorder of autosomal recessive inheritance characterized by keratitis, palmoplantar hyperkeratosis, mental retardation, and elevated blood tyrosine levels. The disease results from deficiency in hepatic tyrosine aminotransferase (TAT). We have previously described one deletion and six different point mutations in four RHS patients. We have now analyzed the TAT genes in a further seven unrelated RHS families from Italy, France, the United Kingdom, and the United States. We have established PCR conditions for the amplification of all twelve TAT exons and have screened the products for mutations by direct sequence analysis or by first performing single-strand conformation polymorphism analysis. We have thus identified the presumably pathological mutations in eight RHS alleles, including two nonsense mutations (R57X, E411X) and four amino acid substitutions (R119W, L201R, R433Q, R433W). Only the R57X mutation, which was found in one Scottish and two Italian families, has been previously reported in another Italian family. Haplotype analysis indicates that this mutation, which involves a CpG dinucleotide hot spot, has a common origin in the three Italian families but arose independently in the Scottish family. Two polymorphisms have also been detected, viz., a protein polymorphism, P15S, and a silent substitution S103S (TCG-->TCA). Expression of R433Q and R433W demonstrate reduced activity of the mutant proteins. In all, twelve different TAT gene mutations have now been identified in tyrosinemia type II.

Adult↗

PubMed1998

The SOX10/Sox10 gene from human and mouse: sequence, expression, and transactivation by the encoded HMG domain transcription factor.

The SOX genes form a gene family related by homology to the high-mobility group (HMG) box region of the testis-determining gene SRY. We have cloned and sequenced the SOX10 and Sox10 genes from human and mouse, respectively. Both genes encode proteins of 466 amino acids with 98% sequence identity. Significant expression of the 2.9-kb human SOX10 mRNA is observed in fetal brain and in adult brain, heart, small intestine and colon. Strong expression of Sox10 occurs throughout the peripheral nervous system during mouse embryonic development. SOX10 shows an overall amino acid sequence identity of 59% to SOX9. Like SOX9, SOX10 has a potent transcription activation domain at its C-terminus and is therefore likely to function as a transcription factor. Whereas SOX9 maps to 17q, a SOX10 cosmid has previously been mapped by us to the region 22q13.1. Mutations in SOX10 have recently been identified as one cause of Waardenburg-Hirschsprung disease in humans, while a Sox10 mutation underlies the mouse mutant Dom, a murine Hirschsprung model.

Adult↗

PubMed1998

Misclassification of smoking in a follow-up population study in southern Germany.

Smoking prevalence in southern Germany was studied in 1984-1985 using a representative cohort of 4022 subjects aged 25 to 64 years, with 3753 reinterviewed in 1987-1988. Data were available for analysis from interviews on self-reported smoking behavior and from serum cotinine measurements in both investigations. More men than women reported current smoking, and particularly heavy smoking. Serum cotinine levels increased steadily with the daily number and nicotine yield of cigarettes smoked. Mean cotinine levels in ex-smokers were higher than those in never smokers, suggesting that a higher percentage of current smokers are misclassified as ex-smokers than never smokers. Using cotinine rather than self-reported smoking data increased the proportion of true smokers in the subgroup of self-reported smokers by about 3% in males and by about 1% in females. Data from the reinterviews revealed that reported smoking status confirmed by cotinine measurement in 1987-1988 conflicted in a number of cases with the data obtained in 1984-1985 using the same procedure. For example, 0.1% of those who stated they were current regular smokers, 4.3% of those who stated they were current occasional smokers, and 17.6% of those who stated they were ex-smokers in 1984-1985 claimed in 1987-1988 to have never smoked. This misclassification of ex-smokers was higher in women. Altogether the true proportion of ex-smokers among self-reported never smokers was about 9.7% (17.8% in men and 6.7% in women). The widely variable uptake of tobacco smoke by smokers, as well as the misclassification of true smokers and ex-smokers as never smokers, needs to be considered in epidemiological studies evaluating the health risks from both active and passive smoking.

Adult↗

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

Direct interaction of SRY-related protein SOX9 and steroidogenic factor 1 regulates transcription of the human anti-Müllerian hormone gene.

For proper male sexual differentiation, anti-Müllerian hormone (AMH) must be tightly regulated during embryonic development to promote regression of the Müllerian duct. However, the molecular mechanisms specifying the onset of AMH in male mammals are not yet clearly defined. A DNA-binding element for the steroidogenic factor 1 (SF-1), a member of the orphan nuclear receptor family, located in the AMH proximal promoter has recently been characterized and demonstrated as being essential for AMH gene activation. However, the requirement for a specific promoter environment for SF-1 activation as well as the presence of conserved cis DNA-binding elements in the AMH promoter suggest that SF-1 is a member of a combinatorial protein-protein and protein-DNA complex. In this study, we demonstrate that the canonical SOX-binding site within the human AMH proximal promoter can bind the transcription factor SOX9, a Sertoli cell factor closely associated with Sertoli cell differentiation and AMH expression. Transfection studies with COS-7 cells revealed that SOX9 can cooperate with SF-1 in this activation process. In vitro and in vivo protein-binding studies indicate that SOX9 and SF-1 interact directly via the SOX9 DNA-binding domain and the SF-1 C-terminal region, respectively. We propose that the two transcription factors SOX9 and SF-1 could both be involved in the expression of the AMH gene, in part as a result of their respective binding to the AMH promoter and in part because of their ability to interact with each other. Our work thus identifies SOX9 as an interaction partner of SF-1 that could be involved in the Sertoli cell-specific expression of AMH during embryogenesis.

Animals↗

PubMed1998

Three novel SRY mutations in XY gonadal dysgenesis and the enigma of XY gonadal dysgenesis cases without SRY mutations.

Mutations in the Y-located testis-determining gene SRY are one cause for XY sex reversal. We have previously identified four SRY mutations in a total of 45 sex-reversed females with XY gonadal dysgenesis (XY GD). In a new sample of 16 XY GD cases, three previously undescribed SRY mutations were identified. Two are point mutations that lead to amino acid substitutions in the HMG domain of SRY, M64R, and F67V. The third SRY mutation is a single base insertion 5' to the HMG box within codon 43, converting this lysine codon to a stop codon (K43X). A total of 33 SRY mutations have so far been described that account for only 10-15% of XY GD females. A further 10-15% of these cases result from deletion of SRY due to aberrant X/Y interchange. The etiology of the remaining 70-80% of XY GD cases is still enigmatic. Possible explanations for these XY sex-reversal cases are discussed.

Adolescent↗

PubMed1998

Prolyl isomerases do not catalyze isomerization of non-prolyl peptide bonds.

Prolyl isomerases accelerate the cis <--> trans isomerization of prolyl peptide bonds during protein folding and probably also in folded proteins. We asked whether this catalytic function is in fact restricted to prolyl bonds or whether the isomerizations of 'normal' non-prolyl peptide bonds are catalyzed as well. By using the P39A variant of ribonuclease T1 as a substrate we find that the trans --> cis isomerization of the Tyr38-Ala39 bond in the refolding of this protein is not catalyzed by prolyl isomerases of the cyclophilin, FKBP and parvulin families. These enzymes are neither able to catalyze amide bond isomerizations in the proline-free model peptide Ala-Ala-Tyr-Ala-Ala.

Amides↗

PubMed1997

Assay of S-ethyl-N-acetyl-l-cysteine in urine by high-performance liquid chromatography using post-column reaction detection.

The assay of the ethyl chloride metabolite S-ethyl-N-acetyl-L-cysteine in human urine by HPLC is described. The compound is enriched by adsorption on a non-polar adsorbent of graphitized non-porous carbon, and then stripped from positively charged compounds by application onto a strong acid cation-exchanger. Subsequently, an enzymatic deacetylation is carried out and the acylase is removed by centrifugal ultrafiltration. Separation of the sample is performed by cation-exchange chromatography applying an eluent of a very low elution strength (diluted formic acid). In the column effluent S-ethyl-L-cysteine is derivatized by o-phthaldialdehyde and the reaction product is detected by fluorescence measurement. In human urine a detection limit in the low ppb range is achieved.

Acetylation↗

PubMed1997

Two independent nuclear localization signals are present in the DNA-binding high-mobility group domains of SRY and SOX9.

SRY and SOX9, members of the family of high-mobility group (HMG) domain transcription factors, are both essential for testis formation during human embryonic development. The HMG domain is a DNA-binding and DNA-bending motif comprising about 80 amino acid residues. It has been shown that SRY and SOX9 are nuclear proteins. Using normal or mutant SRY-beta-galactosidase and SOX9-beta-galactosidase fusion proteins in transfection studies involving COS-7 cells, we have identified two nuclear localization signals (NLSs) within the HMG domains of both proteins that can independently direct the fusion proteins into the nucleus. Only mutational inactivation of both NLS motifs resulted in complete exclusion of the fusion proteins from the nucleus. The NLS sequences are located at the N and C termini of the HMG domain and are a bipartite NLS motif and a basic cluster NLS motif, respectively. Both NLS motifs are conserved in the HMG domains of other transcription factors. The implications of the present results are discussed regarding (a) the apparent dual function of certain basic amino acid residues in the HMG domain of SRY in both DNA binding and in nuclear localization and (b) the possible control of SOX9 in early gonadal differentiation at the level of nuclear translocation.

Amino Acid Sequence↗

PubMed1997

DNA adducts in human placenta in relation to tobacco smoke exposure and plasma antioxidant status.

The DNA adduct 8-hydroxy-2'-deoxyguanosine (8-OHdG) has been widely used as a biomarker for oxidative stress. Bulky DNA adducts, which are detectable by the 32P-postlabelling method, provide evidence for exposure to and metabolic activation of large, mainly apolar compounds, e.g. polycyclic aromatic hydrocarbons. We determined both types of adducts in placental tissues of 30 term pregnancies and related the adduct levels to the exposure to tobacco smoke and the plasma antioxidant status. Urine and plasma continine concentrations were used to select 10 nonsmokers, 9 nonsmokers exposed to environmental tobacco smoke (ETS) and 11 smoking women. Placental levels of 8-OHdG were 0.84 +/- 0.11, 0.90 +/- 0.21 and 0.83 +/- 0.20/10(5) deoxyguanosine bases (dG) for nonsmokers, nonsmokers exposed to ETS and smokers, respectively. The differences between the groups were not significant. Smoking women had significantly lower plasma vitamin C and beta-carotene concentrations than nonsmoking women or nonsmoking women exposed to environmental tobacco smoke. The 8-OHdG adduct level in placental DNA was inversely correlated with the plasma vitamin E concentration (r = -0.47, P < 0.05). There was no association between placental 8-OHdG adducts and vitamin A, C and beta-carotene in plasma. In total, 15 different adducts could be identified in the 30 placenta samples by the 32P-postlabelling method. There was a strong inter-individual variation in both the number of adducts and adduct intensities. No smoking-related or vitamin-related effects on adduct patterns or intensities were found. Our findings suggests that, within the limits of the methods used, tobacco smoke exposure during pregnancy does not lead to a measurable increase in placental DNA adduct levels and that vitamin E appears to have a protective effect on placental 8-OHdG formation.

8-Hydroxy-2'-Deoxyguanosine↗

PubMed1997

High-resolution mapping of a linkage group on mouse chromosome 8 conserved on human chromosome 16Q.

We have performed a high-resolution linkage analysis for the conserved segment on distal mouse Chromosome (Chr) 8 that is homologous to human Chr 16q. The interspecific backcross used involved M. m. molossinus and an M. m. domesticus line congenic for an M. spretus segment from Chr 8 flanked by phenotypic markers Os (oligosyndactyly) and e, a coat colormarker. From a total of 682 N2 progeny, the 191 animals revealing a recombination event between these phenotypic markers were typed for 23 internal loci. The following locus order with distances in cM was obtained: (centromere)-Os-4.1-Mmp2-0.2-Ces1,Es1, Es22-1.2-Mt1,D8Mit15-2.2-Got2, D8Mit11-3.7-Es30-0.3-Es2, Es7-0.9-Ctra1,Lcat-0.3-Cdh1, Cadp, Nmor1, D8Mit12-0.2-Mov34-2.5-Hp,Tat-0.2-Zfp4-1.6-Zfp1,+ ++Ctrb-10.9-e. In a separate interspecific cross involving 62 meioses, Dpep1 was mapped together with Aprt and Cdh3 at 12.9 cM distal to Hp, Tat, to the vicinity of e. Our data give locus order for markers not previously resolved, add Mmp2 and Dpep1 as new markers on mouse Chr 8, and indicate that Ctra1 is the mouse homolog for human CTRL. Comparison of the order of 17 mouse loci with that of their human homologs reveals that locus order is well conserved and that the conserved segment in the human apparently spans the whole long arm of Chr 16.

Animals↗

PubMed1997

trans,trans-muconic acid as a biomarker of non-occupational environmental exposure to benzene.

Excretion of trans,trans-muconic acid (2,4-hexadienedioic acid; t,t-MA), a potential biomarker of low-level exposure to benzene, was determined in 32 smokers and 82 nonsmokers. In smokers the median background excretion of t,t-MA was 0.13 (0.06-0.39) mg/g creatinine and was significantly higher (P < 0.05) than the value of 0.065 (0.02-0.59) mg g creatinine in nonsmokers. For nonsmokers, the correlation between t,t-MA excretion and environmental exposure to benzene in ambient air, which was determined during the 8-day study period by personal diffusion samplers, was not significant (r = 0.164, P = 0.18). Nonsmokers living in the city tended to have higher t,t-MA excretion rates than nonsmokers living in the suburbs. However, the difference was only significant for nonsmokers from nonsmoking homes. For the same location (suburb or city), smoking at home leads to a marginal increase in t,t-MA excretion of the nonsmoking members of the household. In a further study with eight nonsmokers we found that dietary supplementation with 500 mg sorbic acid significantly increased (P < 0.001) the mean urinary t,t-MA excretion from 0.08 (0.04-0.12) to 0.88 (0.57-1.48) mg/24 h. Under study conditions 0.12% of the sorbic acid dose was excreted in urine as t,t-MA, thereby indicating that a typical dietary intake of 6-30 mg/day sorbic acid accounts for 10-50% of the background t,t-MA excretion in nonsmokers, and for 5-25% in smokers. As a consequence, sorbic acid in the diet is a significant confounding factor in assessing low-level benzene exposure if t,t-MA excretion in urine is used as a biomarker.

Benzene↗

PubMed1997

Mutational analysis of the SOX9 gene in campomelic dysplasia and autosomal sex reversal: lack of genotype/phenotype correlations.

It has previously been shown that, in the heterozygous state, mutations in the SOX9 gene cause campomelic dysplasia (CD) and the often associated autosomal XY sex reversal. In 12 CD patients, 10 novel mutations and one recurrent mutation were characterized in one SOX9 allele each, and in one case, no mutation was found. Four missense mutations are all located within the high mobility group (HMG) domain. They either reduce or abolish the DNA-binding ability of the mutant SOX9 proteins. Among the five nonsense and three frameshift mutations identified, two leave the C-terminal transactivation (TA) domain encompassing residues 402-509 of SOX9 partly or almost completely intact. When tested in cell transfection experiments, the recurrent nonsense mutation Y440X, found in two patients who survived for four and more than 9 years, respectively, exhibits some residual transactivation ability. In contrast, a frameshift mutation extending the protein by 70 residues at codon 507, found in a patient who died shortly after birth, showed no transactivation. This is apparently due to instability of the mutant SOX9 protein as demonstrated by Western blotting. Amino acid substitutions and nonsense mutations are found in patients with and without XY sex reversal, indicating that sex reversal in CD is subject to variable penetrance. Finally, none of 18 female patients with XY gonadal dysgenesis (Swyer syndrome) showed an altered SOX9 banding pattern in SSCP assays, providing evidence that SOX9 mutations do not usually result in XY sex reversal without skeletal malformations.

Child↗

PubMed1997

A somatic cell hybrid panel for distal 17q: GDIA1 maps to 17q25.3.

A somatic cell hybrid panel was constructed consisting of seven hybrids with translocation breakpoints spanning the region 17q23-->q25. Hybrid clones carrying the longarm derivative of chromosome 17 in the absence of the normal chromosome 17 and of the derivative 17 were initially identified by PCR typing for a proximal and distal 17q marker. The translocation breakpoints of the hybrids were then mapped in more detail by PCR analysis for a number of microsatellite markers from chromosome 17q as well as for five gene loci (CACNLG, GH1, SOX9, TIMP2, TK1) previously mapped to the region 17q23-->q25. In addition, the locus for GDIA1 was mapped by FISH to 17q25.3 and fine mapped with the help of the hybrid panel. These seven new hybrids complement the existing somatic cell hybrid panel for the long arm of chromosome 17q.

Animals↗

PubMed1997

Biomonitoring exposure to environmental tobacco smoke (ETS): a critical reappraisal.

1 The most frequently used biomarkers for exposure to environmental tobacco smoke (ETS) are cotinine and thiocyanate in body fluids, carboxyhaemoglobin in red blood cells (COHb) and carbon monoxide in the expired air. Although not ideal, cotinine in blood, saliva or urine is an established biomarker for ETS exposure within the past 1-3 days. Comparison with cotinine concentrations in cigarette smokers reveals that passive smokers take up less than 1/100 of the nicotine dose of smokers. 2 Biomonitoring data available for the ETS-related exposure to genotoxic substances comprise uptake of benzene, polycyclic aromatic hydrocarbons (PAH), aromatic amines, tobacco-specific nifrosamines (TSNA), electrophilic compounds giving rise to urinary thioethers, mutagens causing urinary mutagenic activity and the formation of various DNA adducts. With the exception of TSNA, these biomarkers are related to chemicals occurring ubiquitously in the environment and in the food. As a consequence, the background levels in unexposed nonsmokers are high compared to the observed increases (if any) associated with ETS exposure. 3 Some markers of biological effects, which, by definition, are non-specific with regard to the underlying exposure, have also been investigated in relation to ETS exposure. These markers comprise cytogenetic effects, aryl hydrocarbon hydroxylase (AHH) induction, urinary hydroxyproline excretion and various factors indicative of cardiovascular risks. The available data suggest that passive smoking is associated with a small induction of placental AHH and also with effects on cardiovascular risk markers. The latter findings in particular may be confounded by other risk factors, which have been observed to be more frequent in passive smokers than in unexposed nonsmokers.

Benzene↗

PubMed1996

Extended binding sites of cyclophilin as revealed by the interaction with HIV-1 Gag polyprotein derived oligopeptides.

Oligopeptides derived from the gag polyprotein (Pr55gag) of human immunodeficiency virus type 1 (HIV-1) segment were used to evaluate the extension of the putative binding region for the complex of Pr55gag and the human cytosolic peptidyl prolyl cis/trans isomerase (PPIase) 18 kDa cyclophilin (Cyp18). Five N-terminally acetylated, C-terminally amidated oligopeptides containing one (HIV-1 Gag218-224; 1), two (HIV-1 Gag218-226 and HIV-1 Gag217-224; 2 and 3, respectively), three (HIV-1 Gag217-226; 4) or four (HIV-1 Gag213-237; 5) proline residues were synthesized. Using competition experiments with a standard substrate the binding affinities to Cypl8 of the synthesized peptides were determined. The IC50 value of 184 microM for the 25-mer peptide 5 was fivefold or more lower than those of the peptides 1-4 lacking one or more prolines. Failure of competition in assays containing enzymes of other PPIase families by millimolar concentrations of 5 revealed a Cyp18 specific interaction involving the active site of the enzyme. In its far UV circular dichroism, aqueous solutions of 5 display properties of random coil conformation, but spectra were also consistent with a small contribution of proline specific secondary structures. However, a proline-rich peptide typical of forming left-handed polyproline II helices did not compete for the active site of Cypl8. The results demonstrate that the putative binding region of HIV-1 gag polyprotein has a certain degree of binding affinity to the PPIase site of Cyp18, and may add a previously unrecognized topological component to the known subsite specificity of cyclophilins.

Amino Acid Isomerases↗

PubMed1996

Urinary mutagenicity and thioethers in nonsmokers: role of environmental tobacco smoke (ETS) and diet.

The urinary excretion of mutagens and thioethers was investigated in a controlled diet study and in two field studies. A diet containing charcoal-broiled meat and other items rich in mutagenic compounds increased the urinary mutagenicity as assessed in Salmonella typhimurium strain TA98 with metabolic activation approximately 46-fold compared to a diet low in mutagens. The excretion of thioethers after ingestion of the diet rich in mutagens also increased significantly when compared to the diet low in mutagens. The increase was associated with the content of preformed thioethers in the diet. In the first field study with 21 nonsmokers, urinary mutagenicity as assessed in Salmonella typhimurium strain TA98 and excretion of thioethers showed no relation to either the self-reported exposure to environmental tobacco smoke (ETS) or to serum cotinine concentrations used as an objective marker for ETS exposure. In the second field study, urinary mutagenicity was determined with a tobacco-smoke sensitive Salmonella typhimurium strain YG1024 with metabolic activation. No correlation was found between the mutagenic activity in urine and ETS exposure duration, nicotine on the personal sampler, cotinine in saliva and cotinine in urine. Our results suggest that real-life ETS exposure does not measurably increase either urinary mutagen or urinary thioether excretion. Furthermore, diet seems to be the most important source for both urinary mutagen and thioether excretion in nonsmokers.

Adolescent↗