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Biomedical subjects

D Schindler

Publications and source records attributed to D Schindler.

At least 37 records · Page 2Linked to original sources
PubMed1998

Fanconi's anaemia cells have normal steady-state levels and repair of oxidative DNA base modifications sensitive to Fpg protein.

Cells from Fanconi's anaemia (FA) patients are abnormally sensitive to oxygen. However, a distinct genetic defect in either the cellular defence against reactive oxygen species (ROS) or in their metabolic generation has not been identified to date. Recently, the gene for the human 8-hydroxyguanine (8-oxoG) glycosylase, which removes this oxidative base modification from the genome, has been localized on chromosome 3p25, i.e., in the same region as the FA complementation group D (FAD) gene. We therefore studied the removal of photosensitization-induced 8-oxoG residues from the DNA of FA cells, using Fpg protein, the bacterial 8-oxoG glycosylase, to quantify the lesions by alkaline elution. Similar repair kinetics (approx. 50% removal within 2 h) were observed in Epstein-Barr virus (EBV) immortalized lymphoid cells from FA complementation groups A, B, C and D and in control cells from normal donors, as well as in primary fetal lung fibroblasts not yet assigned to a specific complementation group. The susceptibility for the induction of oxidative DNA modifications by photosensitization was similar in all cells. In addition, the background (steady-state) levels of Fpg-sensitive oxidative DNA base modifications, which reflect the balance between generation and removal of the lesions, were similar in control and FA cells. It is concluded that both the generation and the overall removal of 8-oxoG residues in nuclear DNA is not impaired in FA cells.

Cell Division↗
PubMed1998

Molecular cloning, structural organization, sequence, chromosomal assignment, and expression of the mouse alpha-N-acetylgalactosaminidase gene.

Alpha-N-acetylgalactosaminidase (2-acetamido-2-deoxy-alpha-d-galactoside acetamidodeoxy-galactohydrolase, NAGA; EC 3.2.1.49) deficiency is a recently recognized autosomal recessive lysosomal disease. As a prerequisite for the generation of an animal model, the mouse NAGA gene was cloned and characterized. The NAGA gene was assigned to mouse chromosome 15 band E3, syntenic to the region encompassing the human gene, and NAGA-deficient mutant human cells transfected with the cosmid clone containing the mouse NAGA gene expressed NAGA activity. Comparison of the mouse NAGA nucleotide sequence with the human NAGA sequence predicted that the mouse NAGA gene contains an open reading frame of 1245bp, comprising nine coding exons and spanning a genomic region of 8258bp, and a 3' untranslated region of 0.5kb. The 5' untranslated region was determined in primer extension studies to be 235bp in length. Nucleotide identity between the human and mouse NAGA exons ranged from 67.4 to 89.5%, with better matches for exons 1-7 than for 8 and 9. The overall amino acid identity between the mouse and human deduced NAGA polypeptides was 82.0%, between those of mouse and chicken 72.9%. Homology was found to only one other mouse gene, i.e. the alpha-galactosidase A (GALA; EC 3.2.1. 22) gene. The amino acid identity ranged from 51.6 to 62.1% in the polypeptide regions corresponding to NAGA exons 2-7 and GALA exons 1-6, but little, if any, in the remainder. These analyses gave emphasis to the strong conservation of the NAGA gene and its origin from an ancestor common with the GALA gene, with NAGA exons 8 and 9 and GALA exon 7 being the most divergent regions in the evolution of the two genes.

Amino Acid Sequence↗
PubMed1998

Small deletions in the regulatory 3' UTR of the human alpha-tropomyosin gene identified by differential display.

The differential display technique (DDT) was used to compare Fanconi anaemia (FA) fibroblasts with those of normal controls in a screen for genes involved in DNA repair, recognizing and handling damage or indicating cell cycle abnormalities as a result of genetic changes. The DDT revealed two different deletions of 5 and 11 bp at a single locus in the 3' untranslated region (UTR) of a gene known to encode human alpha-tropomyosin (TPM1) in FA cells. These small deletions were detected by analysis of shifted 900-bp long cDNA fragments on polyacrylamide gels. They were characterized as loss of GTTTT or TGTTTTGTTTT, respectively, in a region with five GTTTT tandem repeats. Since it was postulated that the 3' UTR of the TPM1 gene plays a regulatory role in cell differentiation and tumour suppression, the existence and possible patterns of deletions in a variety of normal donors was investigated. The heterogenous distribution of non-deleted, 5- and 11-bp deleted 3' UTR regions indicate a polymorphism of the TPM1 gene in this tandem repeat motif. Therefore the expression pattern of these mutations among FA and non-FA cells rendered any direct relationship to the putative DNA repair defect in FA unlikely. Of note, however, the fact remains that such deletions reportedly facilitate mRNA degradation and may bear significance in the TPM1 gene action. Finally, of further interest is the finding that even small deletions can be identified by DDT in addition to the identification of the differential expression patterns of genes.

Amino Acid Sequence↗
PubMed1998

The Fanconi anaemia group G gene FANCG is identical with XRCC9.

Fanconi anemia (FA) is an autosomal recessive disease with diverse clinical symptoms including developmental anomalies, bone marrow failure and early occurrence of malignancies. In addition to spontaneous chromosome instability, FA cells exhibit cell cycle disturbances and hypersensitivity to cross-linking agents. Eight complementation groups (A-H) have been distinguished, each group possibly representing a distinct FA gene. The genes mutated in patients of complementation groups A (FANCA; refs 4,5) and C (FANCC; ref. 6) have been identified, and FANCD has been mapped to chromosome band 3p22-26 (ref. 7). An additional FA gene has recently been mapped to chromosome 9p (ref. 8). Here we report the identification of the gene mutated in group G, FANCG, on the basis of complementation of an FA-G cell line and the presence of pathogenic mutations in four FA-G patients. We identified the gene as human XRCC9, a gene which has been shown to complement the MMC-sensitive Chinese hamster mutant UV40, and is suspected to be involved in DNA post-replication repair or cell cycle checkpoint control. The gene is localized to chromosome band 9p13 (ref. 9), corresponding with a known localization of an FA gene.

5' Untranslated Regions↗
PubMed1998

Cytotoxic and genotoxic effects of paclitaxel (Taxol) and radiation in a squamous cell carcinoma cell line of the larynx.

Paclitaxel (Taxol) is an antimicrotubular agent which blocks the cells in the G2/M phase of the cell cycle. Because of this mechanism it is presumed that this drug could function as a radiation sensitizer. The cytotoxic and genotoxic effects of paclitaxel and a combination of paclitaxel and radiation were studied in the human laryngeal carcinoma cell line HLac 79. The growth of the cells was significantly reduced at concentrations of paclitaxel as low as 10 nM. Flow cytometry data showed a G2/M block after exposure to paclitaxel. Radiation at 12 and 24 h after drug treatment exerted an additive but no radiation sensitizing effect. As genotoxic effect paclitaxel induced multinucleated cells, possibly in a synergistic manner, at low concentrations (10 nM) and radiation doses up to 3 Gy.

Antineoplastic Agents, Phytogenic↗
PubMed1998

Unusual mutations in high functioning fragile X males: apparent instability of expanded unmethylated CGG repeats.

We report on further cases of high functioning fragile X males showing decreased expression of FMR1 protein, absence of detectable methylation at the EagI site in the FMR1 gene promoter, and highly unusual patterns of fragile X mutations defined as smear of expansions extending from premutation to full mutation range. Very diffuse and therefore not easily detectable patterns of full mutations were also observed on prenatal testing using DNA from chorionic villi sampled at a time of development when full mutations were still unmethylated in this particular tissue. In the search for possible determinants of such unusual patterns, repeat expansions in the premutation and in the lower full mutation range were identified on genomic PstI blots previously prepared for fragile X DNA testing. Cases with 130 or more triplets, and a number of shorter repeats, were reinvestigated on EcoRI plus EagI digests. Among the 119 expansions, there were 22 in our sample showing either blurred bands or smears on PstI blots. This particular characteristic was strongly associated with the coincidence of a repeat size of more than 130 triplets and absence of EagI site methylation. Our data set also includes cases of mosaic patterns consisting of smears of unmethylated expansions to more than 130 CGGs and of clear bands of methylated expansions. We therefore suggest that in fragile X syndrome unusual smeared patterns of mutations result from somatic instability of larger repeats under circumstantial absence of repeat methylation.

Blotting, Southern↗
PubMed1998

Degradation of blood group A glycolipid A-6-2 by normal and mutant human skin fibroblasts.

The degradation of blood group glycolipid A-6-2 (GalNAc(alpha1-->3)[Fuc alpha1-->2]Gal(beta1-->4)GlcNAc(beta1-->3)Gal(beta1-->4)Glc(beta1-->1')C er, IV2-alpha-fucosyl-IV3-alpha-N-acetylgalactosaminylneolact otetraosylceramide), tritium-labeled in its ceramide moiety, was studied in situ, in skin fibroblast cultures from normal controls, from patients with defects of lysosomal alpha-N-acetylgalactosaminidase, and from patients with other lysosomal storage diseases. Uptake of the glycolipid with apolipoprotein E-coated liposomes was linear with time and with the amount of glycolipid added. In normal cells, the expected array of less polar products and some lipids resulting from re-using the liberated sphingosine, mainly sphingomyelin and phosphatidylcholine, were formed. In alpha-N-acetylgalactosaminidase-deficient cells, the glycolipid was virtually not degraded; product formation was less than 2% of the normal control rate, suggesting that blood group A-active glycolipids contribute as storage compounds to the pathogenesis of this disease. The expected accumulation of degradation intermediates was seen in fucosidosis, and in Sandhoff, Gaucher, and Farber disease cells, whereas normal turnover rates were found in Tay-Sachs disease cells, G(M2) activator-deficient (variant AB of G(M2) gangliosidosis) and in sulfatide activator- (sap-B-) deficient cells. In G(M1) gangliosidosis and in sap precursor-deficient cells, the lysosomal glycolipid catabolism was found to be strongly retarded; accumulation of individual products could not be seen. Skin fibroblasts from patients with alpha-N-acetylgalactosaminidase deficiency (Schindler disease) cannot degrade the major blood group A glycolipid.

ABO Blood-Group System↗
PubMed1996

New mutations in the ataxia telangiectasia gene.

We report the detection of four new mutations in the ataxia telangiectasia gene (ATM). Reverse-transcribed RNA extracted from cultured cells was analysed for mutations by polymerase chain reaction amplifications and restriction endonuclease fingerprinting. Three deletions and a base substitution are described. The deletions reported here would result in severe disruptions of the ATM gene product by leading either to a protein truncation (a 4-bp deletion) or the loss of stretches of 53 and 58 amino acids (a 159-bp deletion and a 174-bp deletion, respectively); whereas the base substitution would lead to an amino acid change from a highly conserved glycine to an arginine residue.

Adolescent↗
PubMed1996

Human alpha-N-acetylgalactosaminidase (alpha-NAGA) deficiency: new mutations and the paradox between genotype and phenotype.

Up to now eight patients with alpha-NAGA deficiency have been described. This includes the newly identified patient reported here who died unexpectedly aged 1 1/2 years of hypoxia during convulsions; necropsy was not performed. Three patients have been genotyped previously and here we report the mutations in the other five patients, including two new mutations (S160C and E193X). The newly identified patient is consanguineous with the first patients reported with alpha-NAGA deficiency and neuroaxonal dystrophy and they all had the alpha-NAGA genotype E325K/E325K. Clinical heterogeneity among patients with alpha-NAGA deficiency is extreme. Two affected sibs, homozygotes for E325K, are severely affected and have the signs and symptoms of infantile neuroaxonal dystrophy, but prominent vacuolisation is lacking. The mildly affected patients (two families, three patients) at the opposite end of the clinical spectrum have clear vacuolisation and angiokeratoma but no overt neurological manifestations. Two of them are homozygous for the stop mutation E193X, leading to complete loss of alpha-NAGA protein. These observations are difficult to reconcile with a simple genotype-phenotype correlation and we suggest that factors or genes other than alpha-NAGA contribute to the clinical heterogeneity of the eight patients with alpha-NAGA deficiency. At the metabolic level, the patients with alpha-NAGA deficiency are similar. The major abnormal urinary oligosaccharides are sialylglycopeptides of the O linked type. Our enzymatic studies indicated that these compounds are not the primary lysosomal storage products.

Animals↗
PubMed1995

Comparative evaluation of diepoxybutane sensitivity and cell cycle blockage in the diagnosis of Fanconi anemia.

Fanconi anemia (FA) is a clinically and genetically heterogenous disease that is usually diagnosed on the basis of chromosomal instability reflecting the hypersensitivity towards the DNA cross-linking agents diepoxybutane (DEB) and/or mitomycin C. A less well-known cellular feature that characterizes FA patients is an intrinsic cell cycle disturbance consisting of prolonged progression through, and arrest within, the G2 phase compartment of the cell cycle. In a collaborative blind study, we have evaluated 72-hour lymphocyte cultures from 66 patients with clinical suspicion of FA both for DEB sensitivity and cell cycle disturbance. A concordant result was obtained in 63 of 66 cases. Each of the 3 discordant, but only 1 of the concordant cases presented with overt leukemia. Seventeen cases were identified as classical FA because of their increased DEB sensitivity and G2 phase blockage. Five cases showed a cell cycle disturbance but only borderline DEB sensitivity. These cases might represent atypical or nonclassical forms of FA. They would have been missed by cell cycle studies without concomitant DEB testing. Used in conjunction, cytogenetic and flow cytometric testing provide for the currently optimal diagnosis of FA in nonleukemic patients.

Cells, Cultured↗
PubMed1995

Neuroaxonal dystrophy in infantile alpha-N-acetylgalactosaminidase deficiency.

Morphologic alterations in biopsies of central and peripheral nervous tissue were investigated at the light-and electron-microscopic level in the first cases of lysosomal alpha-N-acetylgalactosaminidase deficiency. Widespread spheroid formation was observed in terminal and preterminal axons. Neocortical and peripheral autonomic axons contained tubulovesicular and lamelliform membranous arrays, prominent acicular clefts, and electron-dense axoplasmic matrix, the typical ultrastructural abnormalities corresponding to axonal spheroids in many inherited and acquired axonopathies. Central and peripheral membranous distal axonal spheroids were the only neuropathologic abnormality identified; other alterations resembling those in various neuronopathic lysosomal storage diseases were not observed. The morphologic findings and the distribution of the lesion in the present disorder are remarkably similar to those reported in the inherited infantile form of neuroaxonal dystrophy with normal alpha-N-acetylgalactosaminidase activity (Seitelberger disease).

Axons↗
PubMed1994

Modulation of the spontaneous G2 phase blockage in Fanconi anemia cells by caffeine: differences from cells arrested by X-irradiation.

The effect of caffeine on the endogenous G2 phase cell cycle blockage of Fanconi anemia (FA) cells was compared with the effect of caffeine on the G2 phase blockage induced in control cells by X-irradiation. The G2 phase accumulations in FA cells could be completely resolved by exposure to 1.5 mM caffeine. This was also observed in three brothers with endogenous G2 phase blockage due to unusual BrdU sensitivity. In contrast, G2 phase blockage induced by X-irradiation was only partially resolved by exposure to caffeine. The rescued G2 phase cells from FA patients were arrested within the following G1 phase compartments. This was not seen in X-irradiated cells from control donors. These results point towards a different nature and/or repair mechanism of the endogenous G2 phase lesion in FA cells compared to that induced by X-irradiation in control cells.

Caffeine↗
PubMed1994

Factors that contribute to the reduced weight gain following chronic ventromedial hypothalamic stimulation.

This report follows up our earlier finding that chronic ventromedial hypothalamic stimulation caused an inhibition of weight gain. In this study we examined the contribution of stimulation-induced activity and brown adipose tissue thermogenesis to the reduced weight gain following three sessions of low-level stimulation delivered every other day to the ventromedial hypothalamus and adjacent areas. During stimulation trials, activity level was ranked on a dichotomous scale. Weight gain and food intake were subsequently monitored for an additional 4 weeks, after which the effects of a 60-s stimulation trial on the temperature of core and interscapular brown adipose tissue were evaluated. The highest activity was associated with the ventromedial hypothalamic sites and this factor contributed significantly to the difference in weight gain and food intake resulting from stimulation of the ventromedial hypothalamus and other areas. These differences largely disappeared during the follow-up period. With little exception, none of the sites elicited temperature changes in brown adipose tissue. As demonstrated in acute work, the contribution of stimulation-induced activity must be dissociated from the metabolic changes that occur in response to ventromedial hypothalamic stimulation.

Adipose Tissue, Brown↗
PubMed1994

alpha-N-acetylgalactosaminidase deficiency with mild clinical manifestations and difficult biochemical diagnosis.

Two additional patients with alpha-N-acetylgalactosaminidase (alpha-NAGA) deficiency are described. An 11-month-old girl with nonconsanguineous parents had generalized seizures and no angiokeratoma. Biochemical investigation showed persistent slight oligosacchariduria; enzymatic analysis of plasma, leukocytes, and fibroblasts revealed profound alpha-NAGA deficiency. Heterozygote enzyme levels were found in both parents. The mother has epilepsy, and epilepsy is present in the father's family. A younger, clinically healthy brother also had the enzyme deficiency. Electron microscopy of lymphocytes from the index patient showed no vacuolization. Incubation of cultured fibroblasts with Helix pomatia lectin showed the presence of intracellular N-acetylgalactosamine-containing storage material, not present in a series of 12 normal fibroblast lines. Our cases cannot be classified definitely as infantile cases. Biochemically the diagnosis could easily have been missed. Urinary oligosaccharide pattern after resorcinol staining was identical to those previously described, but excretion was significantly lower than in the reported infantile cases and the bands disappeared after the urine was desalted. The enzyme defect in leukocytes would have been missed with one of the commercial substrates used. For this mild variant of alpha-NAGA deficiency, the clinical pattern is not yet clear; a longer follow-up period is needed.

Diagnosis, Differential↗
PubMed1993

G2 phase cell cycle disturbance as a manifestation of genetic cell damage.

The predominant cell cycle change induced by X-rays and clastogens in peripheral blood mononuclear cells is the accumulation of cells in the G2 phase of the cell cycle. We show that this accumulation consists of cells that are either delayed or arrested within the G2 phase. Since both X-rays and DNA crosslinking chemicals are known to damage DNA, the G2 phase inhibition caused by these agents is thought to be one of the primary manifestations of (unrepaired) DNA damage. This interpretation is supported by two additional findings. (1) Older individuals have elevated baseline levels of mononuclear blood cells that are delayed and/or arrested in the G2 phase of the cell cycle. This coincides with the increased chromosomal breakage rates reported for older individuals. (2) Irrespective of their age, individuals with inherited genetic instability syndromes (such as Fanconi anemia and Bloom syndrome) exhibit elevated G2 phase cell fractions. We show that the method used to detect such induced or spontaneous cell cycle changes, viz. BrdU-Hoechst flow cytometry, is a rapid and highly sensitive technique for the assessment of genetic cell damage.

Cells, Cultured↗
PubMed1993

A strategy for the characterization of minute chromosome rearrangements using multiple color fluorescence in situ hybridization with chromosome-specific DNA libraries and YAC clones.

The identification of marker chromosomes in clinical and tumor cytogenetics by chromosome banding analysis can create problems. In this study, we present a strategy to define minute chromosomal rearrangements by multicolor fluorescence in situ hybridization (FISH) with "whole chromosome painting" probes derived from chromosome-specific DNA libraries and Alu-polymerase chain reaction (PCR) products of various region-specific yeast artificial chromosome (YAC) clones. To demonstrate the usefulness of this strategy for the characterization of chromosome rearrangements unidentifiable by banding techniques, an 8p+ marker chromosome with two extra bands present in the karyotype of a child with multiple anomalies, malformations, and severe mental retardation was investigated. A series of seven-color FISH experiments with sets of fluorochrome-labeled DNA library probes from flow-sorted chromosomes demonstrated that the additional segment on 8p+ was derived from chromosome 6. For a more detailed characterization of the marker chromosome, three-color FISH experiments with library probes specific to chromosomes 6 and 8 were performed in combination with newly established telomeric and subtelomeric YAC clones from 6q25, 6p23, and 8p23. These experiments demonstrated a trisomy 6pter-->6p22 and a monosomy 8pter-->8p23 in the patient. The present limitations for a broad application of this strategy and its possible improvements are discussed.

Abnormalities, Multiple↗
PubMed1993

Chromosomal breakage, endomitosis, endoreduplication, and hypersensitivity toward radiomimetric and alkylating agents: a possible new autosomal recessive mutation in a girl with craniosynostosis and microcephaly.

A high frequency of spontaneous chromosomal breakage, endomitosis, endoreduplication and hypersensitivity toward both the alkylating agent Trenimon and the radiomimetric drug bleomycin was observed in phytohemagglutinin-stimulated peripheral lymphocytes from a girl with craniosynostosis, microcephaly, ptosis, bird-like facies, and moderate mental retardation. We also observed abnormal chromosomal spiralization and some aspects of abnormal cellular division. Several fruitless attempts were made to establish a cell line. The parents were consanguineous, supporting the existence of a new, rare, autosomal, recessive condition in man. The mutation might involve a gene involved in DNA repair and/or regulation of the mitotic cycle.

Adult↗