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Biomedical subjects

G Scherer

Publications and source records attributed to G Scherer.

At least 19 recordsLinked to original sources
PubMed2007

Two novel translocation breakpoints upstream of SOX9 define borders of the proximal and distal breakpoint cluster region in campomelic dysplasia.

The semilethal skeletal malformation syndrome campomelic dysplasia (CD) with or without XY sex reversal is caused by mutations within the SOX9 gene on 17q24.3 or by chromosomal aberrations (translocations, inversions or deletions) with breakpoints outside the SOX9 coding region. The previously published CD translocation breakpoints upstream of SOX9 fall into two clusters: a proximal cluster with breakpoints between 50-300 kb and a distal cluster with breakpoints between 899-932 kb. Here, we present clinical, cytogenetic and molecular data from two novel CD translocation cases. Case 1 with karyotype 46,XY,t(1;17)(q42.1;q24.3) has characteristic symptoms of CD, including mild tibial bowing, cryptorchidism and hypospadias. By standard fluorescence in situ hybridization (FISH) and by high-resolution fiber FISH, the 17q breakpoint was mapped 375 kb from SOX9, defining the centromeric border of the proximal breakpoint cluster region. Case 2 with karyotype 46,X,t(Y;17)(q11.2;q24.3) has the acampomelic form of CD and complete XY sex reversal. By FISH and somatic cell hybrid analysis, the 17q breakpoint was mapped 789 kb from SOX9, defining the telomeric border of the distal breakpoint cluster region. We discuss the structure of the 1 Mb cis-control region upstream of SOX9 and the correlation between the position of the 14 mapped translocation breakpoints with respect to disease severity and XY sex reversal.

Abnormalities, Multiple↗
PubMed2005

Biological activity and physicochemical parameters of marine halogenated natural products 2,3,3',4,4',5,5'-heptachloro-1'-methyl-1,2'-bipyrrole and 2,4,6-tribromoanisole.

Physicochemical parameters (vapor pressure, water solubility, Henry's law constant) and biological activities of two halogenated natural products frequently detected in marine samples and food were determined. Synthetic 2,3,3',4,4',5,5'-heptachloro-1'-methyl-1,2'-bipyrrole (Q1) and 2,4,6-tribromoanisole (TBA) were available in pure form. The physicochemical parameters were in the range of anthropogenic chlorinated compounds of concern. The aqueous solubilities at 25 degrees C (S(w,25)) of Q1 and TBA were 4.6 microg/L and 12,200 microg/L, respectively, whereas subcooled liquid vapor pressures were 0.00168 Pa (Q1) and 0.06562 Pa (TBA) as measured by the gas chromatographic-retention time technique. Q1 was negative by established test systems for the determination of ethoxyresorufin-O-deethylase (EROD) induction and by sulforhodamine B assay. EROD induction potency was at least 10(-7) times lower than that of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). At a relatively high concentration (20 microM), Q1 inhibited specific binding of 2 nM [(3)H]TCDD to the in vitro-expressed human aryl hydrocarbon receptor (AHR) by 18%; lower concentrations showed no effect. Molecular modeling showed that Q1 is nonplanar, consistent with its relatively modest affinity as an AHR ligand. When tested for cell-growth inhibitory/cytocidal activity in human tumor cells, Q1 was only marginally, if at all, active with an IC(50) value >50 microM compared with five to ten times lower IC(50) values for potent cytotoxins tested in the test system used. Furthermore, standard pesticide tests on insecticidal, herbicidal, and fungicidal activity did not provide any significant activity at highest concentrations. For TBA, the results in all tests were comparable with Q1. The SRB assay was also applied to the halogenated natural product 4,6-dibromo-2-(2',4'-dibromo)phenoxyanisole, but no toxic response was found. Although it was apparent that Q1 and TBA had been proven to have relatively low biological activity in all tests performed, further research is necessary to clarify whether metabolites of the compounds eventually may possess a risk to humans or other living organisms. Nevertheless, the role of Q1 in nature remains uncertain.

Animals↗
PubMed2004

The effect of environmental tobacco smoke on eczema and allergic sensitization in children.

BACKGROUND: The negative impact of environmental tobacco smoke (ETS) on airway diseases in children is well known. Whether there is an effect on atopic eczema is not clear. OBJECTIVES: To determine the impact of ETS on atopic eczema, allergic sensitization and allergic airway diseases in 1669 school beginners. METHODS: The prevalence of atopy-related health outcomes was assessed by questionnaire, dermatological examination, skin prick testing and specific immunoglobulin E measurement. Exposure assessments were based on measurement of cotinine [expressed as cotinine to creatine ratio (CCR)] in spot urine samples (n = 1220) together with questionnaire and interview data on smoking behaviour of the parents. RESULTS: In the total study group, prevalence of atopic eczema diagnosed on examination was significantly associated with urinary CCR values. The odds ratio (OR) and 95% confidence interval (CI), calculated for an increase of 100 ng mg-1 CCR was 1.97 (95% CI 1.23-3.16). The prevalence of skin manifestations according to questionnaire data as well as a history of asthma, wheezing, and hay fever were positively although not significantly associated with ETS exposure. When genetically predisposed children (defined by the presence of parental atopy) were compared with children whose parents had no atopy, the ORs of allergic outcome variables were generally higher in the first group. In the group of predisposed children, significant associations with urinary CCR were found for allergic sensitization against house dust mites as measured by skin prick test (OR 3.10, 95% CI 1.63-5.90). CONCLUSIONS: Children are at a higher risk of developing an atopic eczema when exposed to ETS and genetically predisposed children are at higher risk of developing a sensitization against house dust mites.

Child↗
PubMed2002

In vitro genotoxicity assay of sidestream smoke using a human bronchial epithelial cell line.

Genotoxic effects of air contaminants, such as gaseous or particulate compounds, have been difficult to investigate due to inefficient methods for exposing cell cultures directly to these substances. New cultivation and exposure techniques enable treatment of epithelial cells with sample atmospheres with subsequent in vitro assays, as demonstrated by a new system called CULTEX (CULTEX: patent No. DE 19801763; PCT/EP99/00295), which uses a transwell membrane technique for direct exposure of complex mixtures, for example sidestream cigarette smoke, at the air/liquid interface. The sensitivity and susceptibility of human bronchial epithelial cells to this complex mixture have already been shown for cytotoxic endpoints. In this study, genotoxic effects of sidestream cigarette smoke at different concentrations were assessed using the alkaline comet assay. HFBE 21 cells were exposed for 1 h to clean air, nitrogen dioxide or sidestream smoke. Exposure of the cells to sidestream cigarette smoke induced DNA strand breaks in a dose-dependent manner. The combination of gas phase exposure and the comet assay provides a realistic and efficient model for sensitive detection of DNA strand breaks induced by airborne and inhalable compounds.

Bronchi↗
PubMed2001

Acampomelic campomelic syndrome.

Campomelic syndrome (or campomelic dysostosis, CD; MIM *114290) is an autosomal dominant skeletal malformation syndrome characterized by shortness and bowing of long bones, especially of the lower limbs. Additional radiological and clinical findings are 11 pairs of ribs and a bell-shaped thorax, hypoplastic scapulae, narrow iliac wings, non-mineralized thoracic pedicles, clubbed feet, Robin sequence, typical facial anomalies and tracheomalacia. The disorder is frequently lethal due to respiratory distress. Sex reversal occurs in most patients with an XY karyotype. CD is caused by heterozygous mutations in the SOX9 gene, an SRY-related gene at 17q24.3-q25.1 with pleiotropic effects on the skeletal and genital systems. In addition, cases with chromosomal rearrangements involving 17q have been described that are most likely caused by disturbing one or more cis-regulatory elements from an extended control region. Campomelia (bowed limbs) is seen in most but not all patients, defining a so-called acampomelic campomelic dysostosis (ACD). Half of the CD cases with 17q rearrangements have no or mild campomelia. Furthermore, campomelia is absent or only mildly present in a small subgroup of cases with a normal karyotype. We present a chromosomally normal boy with ACD and his clinical follow-up up to the age of 2 years, in whom a heterozygous SOX9 missense mutation (H165Y) was identified. A SOX9 missense mutation was published in two other patients with ACD. Although up to now a general genotype-phenotype correlation could not be established for CD, a correlation emerges for the ACD variant that needs further confirmation.

Bone Diseases, Developmental↗
PubMed2001

Comparison of cytochrome P450 2A6 polymorphism frequencies in Caucasians and African-Americans using a new one-step PCR-RFLP genotyping method.

CYP2A6 (cytochrome P450 2A6), which was first identified as the human coumarin 7-hydroxylase, is the most important enzyme in nicotine C-oxidation. The enzyme also metabolically activates the tobacco specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) in vitro. Polymorphisms in the CYP2A6 gene may thus impact on both smoking behavior and lung cancer susceptibility. Several different genotyping methods have been reported with conflicting results in the frequencies of CYP2A6 polymorphic variants. Thus we decided to perform a sequence analysis of the entire CYP2A6 gene. Sequencing confirmed the published CYP2A6 cDNA sequence. However, intron sequences differed considerably from the reported sequence of the CYP2A6*3 (v2) variant. Our analyses revealed that parts of introns shared homologies with the published sequence of CYP2A13. Based on our sequence data we developed a one step protocol for specific amplification of exon 3 of CYP2A6. The resulting PCR product can be used directly for restriction endonuclease digestion with XcmI and DdeI to determine the frequencies of the reported variant alleles CYP2A6*2 and CYP2A6*3. In a population of 305 African-Americans and 145 Caucasians, we found allele frequencies of 0.003 (2/610) for CYP2A6*2 and 0 (0/610) for CYP2A6*3 in African-Americans and allele frequencies of 0.014 (4/290) and 0 (0/290) in Caucasians. We conclude that both alleles are considerably less frequent in populations than previously reported.

Alleles↗
PubMed2001

High-performance liquid chromatographic-tandem mass spectrometric determination of 3-hydroxypropylmercapturic acid in human urine.

A sensitive and specific high-performance liquid chromatographic-tandem mass spectrometric (HPLC-MS-MS) method was developed for the determination of 3-hydroxypropylmercapturic acid (3-HPMA) in human urine. Samples were extracted using ENV+ cartridges and then injected onto a C8 Superspher Select B column with acetonitrile and formic acid as eluent (5:95, v/v). N-Acetylcysteine was used as internal standard for HPLC-MS-MS. Linearity was given in the tested range of 50-5000 ng/ml urine. The limit of quantification was 50 ng/ml. Precision, as C.V., in the tested range of 50-5000 ng/ml was 1.47-6.04%. Accuracy ranged from 87 to 114%. 3-HPMA was stable in human urine at 37 degrees C for 24 h. The method was able to quantify 3-HPMA in urine of non-smokers and smokers.

Acetylcysteine↗
PubMed2001

Comparative genomics of the SOX9 region in human and Fugu rubripes: conservation of short regulatory sequence elements within large intergenic regions.

Campomelic dysplasia (CD), a human skeletal malformation syndrome with XY sex reversal, is caused by heterozygous mutations in and around the gene SOX9. SOX9 has an extended 5' control region, as indicated by CD translocation breakpoints scattered over 1 Mb proximal to SOX9 and by expression data from mice transgenic for human SOX9-spanning yeast artificial chromosomes. To identify long-range regulatory elements within the SOX9 5' control region, we compared approximately 3.7 Mb and 195 kb of sequence around human and Fugu rubripes SOX9, respectively. We identified only seven and five protein-coding genes in the human and F. rubripes sequences, respectively. Four of the F. rubripes genes have been mapped in humans; all reside on chromosome 17 but show extensive intrachromosomal gene shuffling compared with the gene order in F. rubripes. In both species, very large intergenic distances separate SOX9 from its directly flanking genes: 2 Mb and 500 kb on either side of SOX9 in humans, and 68 and 97 kb on either side of SOX9 in F. rubripes. Comparative sequence analysis of the intergenic regions revealed five conserved elements, E1-E5, up to 290 kb 5' to human SOX9 and up to 18 kb 5' to F. rubripes SOX9, and three such elements, E6-E8, 3' to SOX9. Where available, mouse sequences confirm conservation of the elements. From the yeast artificial chromosome transgenic data, elements E3-E5 are candidate enhancers for SOX9 expression in limb and vertebral column, and 8 of 10 CD translocation breakpoints separate these elements from SOX9.

5' Flanking Region↗
PubMed2001

Haemoglobin adducts from aromatic amines in children in relation to area of residence and exposure to environmental tobacco smoke.

OBJECTIVE: The influence of area of residence on haemoglobin (Hb) adducts of 4-aminobiphenyl (4-ABP), o-, m-, p-toluidine and o-anisidine was investigated in children from three different-sized Bavarian cities - Munich, Augsburg and Eichstätt, with 1,300,000, 250,000 and 13,000 inhabitants, respectively--and was compared with that of exposure to environmental tobacco smoke (ETS). METHODS: Blood samples from Munich (n = 34) and Eichstätt (n = 64) were from children attending the Paediatric Clinic of the Technical University of Munich (TUM) or a practice in Eichstätt, respectively. Blood samples (n = 126) together with urine samples (n = 88) were collected from Augsburg children during school medical examination. Personal data including possible sources of ETS exposure were obtained at the interview. Hb adduct levels were analysed by a gas chromatographic method, using mass spectrometry with selected-ion monitoring. Urinary cotinine was determined by radioimmunoassay. RESULTS: 4-ABP Hb adduct levels in children from Munich were 1.5 and 1.2 times higher than those in children from Eichstätt and Augsburg (P < 0.001). Children from Munich also had significantly higher Hb adduct levels of monocyclic aromatic amines than did children from Eichstätt and, except for o-toluidine, children from Augsburg (P < 0.005). Compared with children from Eichstätt, children from Augsburg had higher Hb adduct levels of 4-ABP, o- and m-toluidine (P < 0.01) but not p-toluidine and o-anisidine. In a multivariate analysis, gender, age and body mass index had no consistent influence on Hb adducts. ETS exposure resulted in a slight, nonsignificant increase in 4-ABP Hb adduct levels. In contrast, adduct levels from monocyclic aromatic amines were consistently decreased in ETS-exposed children (significant for o- and m-toluidine, P<0.05). CONCLUSIONS: Hb adducts from aromatic amines in children were strongly influenced by site of residence, whereas ETS exposure did not significantly increase the adduct levels.JECT

Amines↗
PubMed2001

A method for in vitro analysis of the biological activity of complex mixtures such as sidestream cigarette smoke.

Studies of the cytotoxicity of air contaminants such as gaseous or particulate compounds and complex mixtures have traditionally used in animal experiments because of the difficulties in exposing cell cultures directly to these substances. New cultivation and exposure techniques enhance the efficiency of in vitro methods, as demonstrated by a new system called CULTEX* which uses a transwell membrane technique for direct exposure of complex mixtures like sidestream cigarette smoke at the air/liquid interface. The factors influencing the susceptibility of human bronchial epithelial cells (e.g. gas flow rate or duration of exposure) were studied and the cells were finally exposed for one hour to clean air or different concentrations of sidestream smoke. The biological parameters estimated were number of cells, metabolic activity and glutathione concentration. After exposure of the cells to sidestream cigarette smoke, dose-dependent effects were measured. Thus, the introduction of these cultivation and exposure techniques offers new testing strategies for the toxicological evaluation of a broad range of airborne and inhalable compounds.

Cell Culture Techniques↗
PubMed2001

Longitudinal study of urinary 8-hydroxy-2'-deoxyguanosine excretion in healthy adults.

Numerous studies have investigated the urinary excretion of 8-hydroxy-2'-deoxyguanosine (8-OHdG) as a biomarker for the assessment of oxidative DNA damage in humans. In this study, we performed six consecutive series of measurement of urinary levels of 8-OHdG in 68 healthy probands, in order to provide information on the intra- and inter-individual variability of 8-OHdG and to estimate the influence of smoking, age, sex, body weight and body mass index (BMI) on the excretion of 8-OHdG. The intra-individual coefficient of variation (CV) of urinary 8-OHdG/24 h ranged from 0.18 to 1.06 (mean CV = 0.48). Women excreted significantly lower amounts of 8-OHdG/24 h than men, but the difference lost its significance when the body weight or urinary creatinine were used as covariates. By multiple linear regression analysis significant correlations between the mean individual levels of 8-OHdG/24 h excretion and urinary creatinine (rp = 0.61), and cotinine (rp = 0.27) have been observed, whereas no statistically significant effect of age, body weight and BMI was found. The 8-OHdG/creatinine ratio was found to be significantly increased in 23 smokers (1.95 +/- 0.40 mumol/mol) opposed to 45 non-smoking probands (1.62 +/- 0.50 mumol/mol), which is in good agreement with previously published data. No effect of passive smoking on the excretion of 8-OHdG was found. From our data we conclude that the intra-individual variability of urinary 8-OHdG excretion has been underestimated so far, indicating that values of 8-OHdG measured by single spot monitoring are not representative for individual base levels.

8-Hydroxy-2'-Deoxyguanosine↗
PubMed2000

Acampomelic campomelic dysplasia with SOX9 mutation.

Acampomelic campomelic dysplasia is a rare clinical variant of the more commonly encountered campomelic dysplasia (CMD1), characterized by absence of long bone curvature (acampomelia). We present a patient with acampomelic CMD1 with a de novo SOX9 missense mutation and report his clinical course to age one year, thereby contributing to genotype-phenotype correlation in CMD1. 2000.

High Mobility Group Proteins↗
PubMed2000

Sensitization to death receptor cytotoxicity by inhibition of fas-associated death domain protein (FADD)/caspase signaling. Requirement of cell cycle progression.

Upon binding of their ligands, death receptors belonging to the tumor necrosis factor (TNF) receptor family initiate a signaling pathway leading to the activation of caspases and ultimately apoptosis. TNF, however, in parallel elicits survival signals, protecting many cell types from cell death that can only be induced by combined treatment with TNF and inhibitors of protein synthesis. Here, we report that in NIH3T3 cells, apoptosis in response TNF and cycloheximide is not inhibited by the broad spectrum caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone (zVAD. fmk). Moreover, treatment with zVAD.fmk sensitizes the cells to the cytotoxic action of TNF. Sensitization was also achieved by overexpression of a dominant-negative mutant of Fas-associated death domain protein and, to a lesser extent, by specific inhibition of caspase-8. A similar, but weaker sensitization of zVAD.fmk to treatment with the TNF-related apoptosis-inducing ligand (TRAIL) or anti-CD95 antibody was demonstrated. The unexpected cell death in response to TNF and caspase inhibition occurs despite the activation of nuclear factor kappaB and c-Jun N-terminal kinases. The mode of cell death shows several signs of apoptosis including DNA fragmentation, although activation of caspase-3 was excluded. TNF/zVAD.fmk-induced cell death is preceded by an accumulation of cells in the G(2)/M phase of the cell cycle, indicating an important role of cell cycle progression. This hypothesis is further strengthened by the observation that arresting the cells in the G(1) phase of the cell cycle inhibited TNF/zVAD.fmk-induced cell death, whereas blocking them in the G(2)/M phase augmented it.

3T3 Cells↗
PubMed2000

Male specific expression suggests role of DMRT1 in human sex determination.

Sex determination in mammals is controlled by various transcription factors. Following the identification of SRY on the Y chromosome, several other factors have been identified. They can normally be identified as being involved in sex determination by the identification of sex reversal mutations or deletions, functional studies, and also by male-specific expression patterns in embryos. Here, it is shown that DMRT1, recently demonstrated to be deleted in 9p monosomies associated with sex reversal, is specifically expressed during sex determination in the genital ridge of human male, but not female, embryos, similar to SRY.

Female↗
PubMed2000

Fast quantification of the urinary marker of oxidative stress 8-hydroxy-2'-deoxyguanosine using solid-phase extraction and high-performance liquid chromatography with triple-stage quadrupole mass detection.

Exogenous and endogenous oxidants constantly cause oxidative damage to DNA. Since the reactive oxidants itself are not suitable for analysis, oxidized bases like 8-hydroxy-2'-deoxyguanosine (8OHdG) are used as biomarkers for oxidative stress, either in cellular DNA or as elimination product in urine. A simple, fast and robust analytical procedure is described for urinary 8OHdG as an indicator of oxidative damage in humans. The adduct was purified from human urine by applying a single solid-phase extraction step on LiChrolut EN. After evaporation of the eluate, the residue was resolved and an aliquote was injected into a HPLC system with a triple quadrupole mass spectrometer. The limit of detection was 0.2 ng ml(-1) (7 fmol absolute) when using one product ion as quantifier and two further product ions as qualifier. The coefficient of variation was 10.1% (n=5 at 2.8 ng ml(-1) urine). The sample throughput was about 50 samples a day. Thus, this method is more sensitive and much faster than the common method using HPLC with electrochemical detection. The results of a study with nine volunteers investigated at six time-points each over 5 days are presented. The mean excretion of 8OHdG was 2.1 ng mg(-1) creatinine (range 0.17-5.9 ng mg(-1) creatinine; 4 of 53 samples were below the LOD). A relatively large intra- (relative SD 66%) and inter-individual (relative SD 71%) variation in urinary 8OHdG excretion rates was found.

8-Hydroxy-2'-Deoxyguanosine↗
PubMed2000

The SOX8 gene is located within 700 kb of the tip of chromosome 16p and is deleted in a patient with ATR-16 syndrome.

SOX proteins are transcription factors that are characterized by a common DNA-binding motif known as the HMG domain. We describe the 5. 4-kb human SOX8 gene that codes for a 446-amino-acid protein and that is expressed strongly in brain and less abundantly in other tissues. SOX8 shows an overall identity of 47% to SOX9 and SOX10. The latter two possess a C-terminal transactivation domain, whereas in SOX8, this domain is located in the central part of the protein. We have mapped SOX8 within 700 kb of the telomeric repeats of band 16p13.3. Hemizygosity for 1 Mb from this region causes the ATR-16 syndrome characterized by alpha-thalassemia and mental retardation. We show that SOX8 is deleted in an ATR-16 patient, and from its location, we deduce that it should be deleted in all previously described cases. Thus, SOX8 is a good candidate gene contributing to the mental retardation phenotype seen in ATR-16 patients.

Amino Acid Sequence↗