Biomedical subjects
Publications and source records attributed to G Scherer.
An SOX9 mutation in a previously published case of campomelic dysplasia is described. This case did not have overt campomelia of the femora or tibiae although campomelic dysplasia has subsequently been confirmed by gene analysis.
In this report we present the analysis of two overlapping mouse cosmid clones that contain the entire Kcnk6, Map3k11 and Pcnxl3 genes, as well as part of the Sipa1 gene. The sequence and genomic organisation of the Kcnk6 and Map3k11 genes are described in detail. Sipa1 and Map3k11, which have independently been mapped with low resolution to the centromeric region of mouse chromosome 19, are shown here to lie close to each other and to the Kcnk6 gene, which has not previously been mapped. This gene cluster maps to the vicinity of the Dancer (Dc) mutation, which involves inner ear abnormalities and circling phenotypes. Since potassium channels have been implicated in deafness disorders, we have analysed the Kcnk6 gene, which encodes a two-P domain potassium channel, in the Dc mutant. No Dc-causing mutation in the Kcnk6 coding region could be identified. However, we detected a polymorphism in the Kcnk6 gene that leads to a C-terminal extension of the encoded protein by eight amino acids.
Females with XY gonadal dysgenesis are sterile, due to degeneration of the initially present ovaries into nonfunctional streak gonads. Some of these sex-reversal cases can be attributed to mutation or deletion of the SRY gene. We now describe an SRY-deleted 47,XXY female who has one son and two daughters, and one of her daughters has the same 47,XXY karyotype. PCR and FISH analysis revealed that the mother carries a structurally altered Y chromosome that most likely resulted from an aberrant X-Y interchange between the closely related genomic regions surrounding the gene pair PRKX and PRKY on Xp22.3 and Yp11.2, respectively. As a consequence, Yp material, including SRY, has been replaced by terminal Xp sequences up to the PRKX gene. The fertility of the XXY mother can be attributed to the presence of the additional X chromosome that is missing in XY gonadal dysgenesis females. To our knowledge, this is the first human XXY female described who is fertile.
In a field study with 69 subjects, we investigated the influence of smoking, exposure to environmental tobacco smoke (ETS), diet, and location of residence on biomarkers for polycyclic aromatic hydrocarbons (PAH), including urinary excretion of 1-hydroxypyrene and benzo[a]pyrene (BaP) adducts of hemoglobin and albumin. The self-reported smoking status and the extent of ETS exposure were verified by urinary cotinine measurements. ETS exposure was quantified by nicotine and 3-ethenylpyridine measurements on personal samplers worn by the nonsmokers over 5 or 7 days before blood and urine samples were collected. Smokers (n = 27), on average, excreted 0.346 microg/24 h 1-hydroxypyrene, whereas the corresponding value for nonsmokers (n = 42) was 0.157 microg/24 h. Average BaP adduct levels with hemoglobin and albumin were 0.105 fmol/mg and 0.042 fmol/mg, respectively, for smokers, and 0.068 fmol/mg and 0.020 fmol/mg, respectively, for nonsmokers. The differences, except for the hemoglobin adducts, were statistically significant. Of the 42 nonsmokers, 19 were classified as passive smokers. There was no significant difference in the PAH biomarkers between nonsmokers exposed to ETS and those not or rarely exposed to ETS. Total dietary BaP intake, as calculated from questionnaire data, did not correlate with any of the PAH biomarkers (r < 0.1). Subjects living in the suburbs tended to have higher BaP-protein adduct levels than subjects living in the city. Our findings suggest that diet and smoking are major sources for PAH exposure of persons not occupationally exposed to PAH, whereas the influence of ETS exposure is negligible. The lack of correlation between the dietary PAH intake and the PAH biomarkers may be due to the inaccuracy of the estimate for the dietary PAH intake.
Thioguanine-resistant clones of the human lymphoblastoid cell, MCL-5, which carries two recombinant plasmids expressing xenobiotic metabolizing enzymes, were obtained spontaneously and after treatment with 0.1 microgram/ml benzo[a]pyrene (BaP), 1.0 microgram/ml 4-(N-nitrosomethylamino)-1-(3-pyridyl)-1-butanone (NNK), and 10 micrograms/ml cigarette smoke condensate (CSC). Treatment with the chemicals reduced the cloning efficiency (CE) of MCL-5 cells from about 30% in untreated cultures to about 10% after treatment with NNK and to about 1% or to less than 1% after treatment with CSC or BaP, respectively. At the same time, the mutant frequencies were increased about sevenfold above those of untreated cultures. Among a total of 138 independent mutant clones that had resulted from 55 separate cultures, 60 point mutations were identified within the hypoxanthine guanine phosphoribosyltransferase (HPRT) reading frame by sequencing full-size reverse transcription polymerase chain reaction (RT-PCR) products from thioguanine-resistant clones. The identified 53 coding errors were distributed among 33 locations and types. Among the 30 types of single basepair substitutions leading to coding errors, 12 had not been described before. In the present set of point mutations, the distribution of base substitution types as well as of mutated sites appeared to be influenced by the treatment with the chemicals. Thus, the ratio of G to T transversions increased from 3 among 19 spontaneous point mutations in the HPRT coding region to 9 among 21 BaP-induced point mutations. The G119T and G208T transversions were found three times each, exclusively after treatment with BaP, while the accumulation of two to eight incidences of the G97T, CG142/3TA, C508T, T583A and G599A mutations was split among different treatments. All eight identified point mutations identified after NNK treatment were at G or T residues on either strand that were followed by additional G or T residues.
The average daily uptake of the common food preservative sorbic acid is estimated to range from 0.01 to 1.1 mg kg-1. Sorbic acid mainly is metabolised to carbon dioxide. Minor amounts are converted to trans,trans-muconic acid (ttMA) as well as excreted unchanged into the urine. Since urinary ttMA is a biomarker for the occupational and environmental exposure to benzene, there is an additional need for monitoring the uptake of sorbic acid, particularly at low, environmental benzene exposure levels. For this purpose, a simple, robust and rapid method for the determination of sorbic acid in urine at trace levels was developed. After addition of 10 ml of water and 5 ml of 8 M hydrochloric acid to 10 ml of the thawed urine, the sample was water steam distilled using an automated distillation device. A total of 100 ml of the distillate were solid-phase extracted. After washing, the sorbic acid was eluted with 4 ml methanol. The eluate was reduced under a stream of nitrogen to a volume of 300 microliters. After addition of 500 microliters boron trifluoride in methanol and incubation for 1 h at 60 degrees C, the resulting sorbic acid methyl ester was extracted three times with 1 ml heptane. To the combined heptane layers, sorbic acid ethyl ester was added as an internal standard. After reducing to a volume of 100 microliters in a stream of nitrogen, the final analysis was performed by GC-MS using the fragment ions m/z 126 for the analyte and m/z 140 for the internal standard. The limit of detection was 0.7 ng ml-1 urine and the R.S.D. of 69 duplicate determinations was 7.5%. In a controlled, experimental study and in a field study, we were able to show that urinary sorbic acid is a marker for the dietary uptake of sorbic acid and that sorbic acid is converted to ttMA. On average, 0.1% of the dietary sorbic acid is excreted unchanged into the urine. Excretion is complete within 24 h. We found that, on average, 0.23% of the oral dose of sorbic acid is excreted as urinary ttMA. There was a significant correlation between urinary excretions of sorbic acid and ttMA (r = 0.74, n = 69). We conclude that urinary sorbic acid can be used to correct the urinary ttMA level in order to determine the portion related to benzene exposure. This appears to be necessary particularly at low, environmental benzene levels.
The WD-40 repeat protein FAN binds to a distinct domain of the p55 receptor for tumor necrosis factor (TNF) and signals the activation of neutral sphingomyelinase (N-SMase). To analyze the physiological role of FAN in vivo, we generated FAN-deficient mice by targeted gene disruption. Mice lacking a functional FAN protein do not show any overt phenotypic abnormalities; in particular, the architecture and cellular composition of lymphoid organs appeared to be unaltered. An essential role of FAN in the TNF-induced activation of N-SMase was demonstrated using thymocytes from FAN knockout mice. Activation of extracellular signal-regulated kinases in response to TNF treatment, however, was not impaired by the absence of the FAN protein. FAN-deficient mice show delayed kinetics of recovery after cutaneous barrier disruption suggesting a physiological role of FAN in epidermal barrier repair. Although FAN exhibits striking structural homologies with the CHS/Beige proteins, FAN-deficient mice did not reproduce the phenotype of beige mice.
RATIONALE: Compensation or compensatory smoking, accurately defined, deals with the question of whether switching to cigarette brands with different smoke yields is associated with a change in smoke uptake proportional to the change in machine-derived yields. The issue of compensation is important because it bears on whether switching to "lighter" brands means lower overall smoke intake or not. OBJECTIVES: The present review investigated whether and to what extend low yield cigarettes are smoked more intensively. In addition, published data on whether nicotine, "tar", or any other smoke constituent or property influence compensational smoking are summarized. METHODS: The studies on compensation were classified as follows: (1) studies on smoking behaviour in relation to cigarette yields (with and without brand switching); (2) studies on compensation for nicotine (switching between cigarettes which differ "only" in their nicotine yield, nicotine supplementation, manipulation of renal nicotine excretion, administration of nicotine agonists or antagonists); (3) studies on compensation for other factors (influence of tar, taste, irritation, draw resistance). In order to quantify the degree of compensation, an index is defined and applied to selected brand switching studies. This compensation index determines, in relative units, the degree to which a smoker responds to a change in smoke yields with a change in smoke uptake measured by suitable biomarkers. The role of vent blocking is also briefly discussed. RESULTS: Most of the studies which compare the smoking behaviour when smoking cigarettes with different smoke yields supply evidence for "partial" compensation, suggesting that cigarettes with lower yields are smoked more intensively than those with higher yields. These studies also show that a change in the daily number of cigarettes is not a common mechanism of compensation. Effective vent blocking during smoking is a rare event and can therefore also be regarded as an uncommon mechanism of compensation. Evaluation of a suitable subset of brand-switching studies revealed an average compensation of 50-60% of the nicotine yield. Compensation tended to be more complete when changing to cigarettes with higher yields than when changing to cigarettes with lower yields. In general, brand-switching studies do not supply information on the underlying causal factors responsible for compensatory smoking. Results of the nicotine supplementation studies are not conclusive: some report evidence of nicotine titration, others do not. A general problem with this type of investigation is that continuous nicotine application does not mimic the spike-wise application with cigarette smoking, and may lead to nicotine tolerance. There is limited evidence that cigarettes were smoked more intensively when the urinary clearance of nicotine was increased. A small number of studies provide some evidence that smoking intensity increased after smokers were administered a nicotine antagonist. Several reports indicate that tar, taste and sensory properties of the smoke as well as the draw resistance of the cigarette may play a role in compensatory smoking. Low-yield cigarettes usually have reduced pressure drops which smoke researchers have suggested leads to increased puff volume. This effect seems to be independent of the smoke yield of the cigarette. There is also some evidence that some smokers maintain a consistent pattern of smoking which works independent of any changes in nicotine or tar yields, taste or design features of the cigarette ("functional autonomy"). CONCLUSIONS: The available data suggest that smokers partially compensate for a different smoke yield. While the factors and their interaction responsible for compensational smoking are not fully understood, there are data suggesting that a subgroup of smokers may partially compensate for nicotine. Even in this subgroup of smokers, however, the relative importance of the pharmacological versus
UNLABELLED: We report on a male infant with ambiguous genitalia (scrotal hypospadias, sinus urogenitalis) trisomic for 8q23-ter and monosomic for 9p23-ter, who shared craniofacial and other abnormalities with either phenotype. Gonadal histology was nearly normal for age. Normal endocrinological findings and exclusion of mutations in SRY, androgen receptor and alpha-reductase genes point to supplementary gene(s) located in 9p2305-ter, haplo-insufficiency (by deletion) of which is expected to cause defective male morphogenesis. CONCLUSION: This observation lends further support to the hypothesis that genetic factors are located at 9p23-ter which are involved in normal sex determination.
Campomelic dysplasia (CD), a skeletal malformation syndrome with or without XY sex reversal, is usually caused by mutations within the SOX9 gene on distal 17q. Several CD translocation and inversion cases have been described with breakpoints outside the coding region, mapping to locations >130 kb proximal to SOX9. Such cases are generally less severely affected than cases with SOX9 coding-region mutations, as is borne out by three new translocation cases that we present. We have cloned the region extending 1.2 Mb upstream of the SOX9 gene in overlapping bacterial-artificial-chromosome and P1-artificial-chromosome clones and have established a restriction map with rare-cutter enzymes. With sequence-tagged-site-content mapping in somatic-cell hybrids, as well as with FISH, we have precisely mapped the breakpoints of the three new and of three previously described CD cases. The six CD breakpoints map to an interval that is 140-950 kb proximal to the SOX9 gene. With exon trapping, we could isolate five potential exons from the YAC 946E12 that spans the region, four of which could be placed in the contig in the vicinity of the breakpoints. They show the same transcriptional orientation, but only two have an open reading frame (ORF). We failed to detect expression of these fragments in several human and mouse cDNA libraries, as well as on northern blots. Genomic sequence totaling 1,063 kb from the SOX9 5'-flanking region was determined and was analyzed by the gene-prediction program GENSCAN and by a search of dbEST and other databases. No genes or transcripts could be identified. Together, these data suggest that the chromosomal rearrangements most likely remove one or more cis-regulatory elements from an extended SOX9 control region.
Explore the source record for details and available documents.
1. In order to elucidate the role of exposure to environmental tobacco smoke (ETS) in various acute and chronic illnesses in children, it is important to assess the degree of exposure by suitable methods. For this purpose, we determined the exposure to ETS in 39 children (4-15 years) and 43 adults (16+ years) by questionnaires, personal diffusion samplers for nicotine, and cotinine measurements in saliva and urine. In addition, the influence of the smoking status and the location of the home (urban or suburban) on the benzene exposure of the children was investigated. 2. On average, the 24 children living in homes with at least one smoker were exposed to ETS for 3.1 h/d. This is significantly longer (P<0.001) than the daily exposure time of the 15 children from nonsmoking homes (0.3 h/d). The nicotine concentrations on the personal samplers worn over 7 days were 0.615 and 0.046 microg/m3 for children from smoking and nonsmoking homes, respectively (P<0.001). Average salivary cotinine levels were 1.95 ng/ml in children from smoking homes and 0.11 ng/ml in children from nonsmoking homes (P< 0.01). The corresponding urinary cotinine levels were 29.4 and 4.5 ng/mg creatinine (P< 0.001). There was no difference in the extent of ETS exposure between children and adults from smoking households. Adults from nonsmoking homes tended to have higher ETS exposure than children from nonsmoking homes. 3. Exposure to benzene, which was determined by means of personal samplers, measurements of benzene in exhaled air and of the urinary benzene metabolite trans, trans-muconic acid, was not significantly related to the smoking status of the home but primarily dependent on the location of the home.
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.
Benzene is an important industrial chemical and, due to its occurrence in mineral oil and its formation in many combustion processes, a widespread environmental pollutant. Since benzene is hematoxic and has been classified as a human carcinogen, monitoring and control of benzene exposure is of importance. Although trans,trans-muconic acid (ttMA) was identified as a urinary metabolite of benzene at the beginning of this century, only recently has its application as a biomarker for occupational and environmental benzene exposure been investigated. The range of metabolic conversion of benzene to ttMA is about 2-25% and dependent on the benzene exposure level, simultaneous exposure to toluene, and probably also to genetic factors. For the quantitation of ttMA in urine, HPLC methods using UV and diode array detection as well as GC methods combined with MS or FID detection have been described. Sample pretreatment for both HPLC and GC analysis comprises centrifugation and enrichment by solid-phase extraction on anion-exchange sorbents. Described derivatization procedures prior to GC analysis include reaction with N,O-bis(trimethysilyl)acetamide, N,O-bis(trimethylsilyl)trifluoroacetamide, pentafluorobenzyl bromide and borontrifluoride-methanol. Reported limits of detection for HPLC methods range from 0.1 to 0.003 mg l(-1), whereas those reported for GC methods are 0.03-0.01 mg l(-1). Due to its higher specificity, GC methods appear to be more suitable for determination of low urinary ttMA levels caused by environmental exposure to benzene. In studies with occupational exposure to benzene (>0.1 ppm), good correlations between urinary ttMA excretion and benzene levels in breathing air are observed. From the reported regressions for these variables, mean excretion rates of ttMA of 1.9 mg g(-1) creatinine or 2.5 mg l(-1) at an exposure dose of 1 ppm over 8 h can be calculated. The smoking-related increase in urinary ttMA excretion reported in twelve studies ranged from 0.022 to 0.2 mg g(-1) creatinine. Only a few studies have investigated the effect of exposure to environmental levels of benzene (<0.01 ppm) on urinary ttMA excretion. A trend for slightly increased ttMA levels in subjects living in areas with high automobile traffic density was observed, whereas exposure to environmental tobacco smoke did not significantly increase the urinary ttMA excretion. It is concluded that urinary ttMA is a suitable biomarker for benzene exposure at occupational levels as low as 0.1 ppm. Biomonitoring of exposure to environmental benzene levels (<0.01 ppm) using urinary ttMA appears to be possible only if the ingestion of dietary sorbic acid, another precursor to urinary ttMA, is taken into account.
Kinectin has been characterized as the first known receptor for the molecular motor kinesin, which is critically involved in microtubule-based vesicle transport and membrane trafficking. Here we identify kinectin as a target for caspase-mediated proteolysis during apoptosis. Treatment of cells with diverse apoptotic stimuli including TNF, anti-Fas, anticancer drugs, gamma-radiation or ceramide leads to rapid proteolytic cleavage of the 160-kDa form of kinectin to a 120-kDa fragment. Evidence is provided that kinectin cleavage is mediated by caspase 7.
A novel method has been developed for the determination of (+)-anti-benzo[a]pyrene-diol-epoxide [(+)-anti-BPDE] albumin adducts in the low-picogram range. Blood from rats and humans was investigated for the validation of the method. Instead of the usual acid hydrolysis we used alkaline conditions for the cleavage of the esters formed with asparagic or glutamic acid residues of albumin. Alkaline hydrolysis gave rise to benzo[a]pyrene-r-7,t-8,t-9,c-10-tetrahydrotetrol (BT I-1) which was separated from the matrix by HPLC with a column switching technique. The analytes were collected by an automated fraction collector and after silylation determined with GC-MS using negative chemical ionization. Adduct concentrations were calculated by the internal standard method. Benzo[a]pyrene-r-7,t-8,c-9,c-10-tetrahydrotetrol (BT-II-2) was used as an internal standard because of its similar physicochemical properties and its absence from human samples. To determine the recovery of the analytical procedure benzo[a]pyrene-r-7,t-8,t-9,t-10-tetrahydrotetrol (BT I-2) was added at the end of the sample clean-up. Single ion recording mode was applied for the detection of the analyte and the standards using the abundant fragment ion m/z 284 for quantitation of the three tetrols. The mean recovery of the internal standard BT II-2 was about 50%. The limit of detection was 0.15 pg per injection corresponding to 0.01 fmol/mg albumin. Regression coefficients of the calibration curves were r2=0.99 and r2=0.98 for BT I-1 concentration ranges of 4-400 ng/l and 4-40 ng/l, respectively. The mean coefficient of variation for duplicate analyses of human albumin samples was found to be 22%.
Explore the source record for details and available documents.
Peptidyl-prolyl cis/trans isomerisation has been frequently found as a rate limiting step in the folding of proteins. In order to determine whether the nature of the amino acid preceding proline controls the probability of cis prolyl bonds in native proteins, systematic studies on the thermodynamics and kinetics of the prolyl isomerisation in the pentapeptide series Ac-Ala-Xaa-Pro-Ala-Lys-NH2 were performed. All proteinogenic amino acids were substituted in the position preceding proline. When measured by 1H-NMR and CD spectroscopy both isomers proved to be devoid of ordered structure in the whole series of the oligopeptides in aqueous solution. Thus, isomerization rates and cis/trans ratios calculated from solvent jump and 1H-NMR magnetisation transfer experiments exclusively reflect the side-chain effects of the Xaa position in the peptide series. There is a rough correlation between the cis content in the oligopeptides and the propensity of Xaa-Pro cis prolyl bonds in proteins. This correlation suggests that the prolyl bond conformation is mainly determined by local effects in proteins. The rate constants kc-->t of pentapeptides containing unionised amino acids preceding proline range from 3.2 x 10(-3) s-1 (Xaa = Ala) to 0.5 x 10(-3) s-1 (Xaa = Trp) at 4 degrees C. Proline clustering led to an isomerisation cycle indicating considerable influence on the isomerisation rates of the peptide bond conformations flanking the rotating bond. Both tyrosine and histidine specifically reduce isomerisation rates severalfold by deprotonation of their respective side-chains.