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Biomedical subjects

M Schwab

Publications and source records attributed to M Schwab.

At least 73 records · Page 4Linked to original sources
PubMed2000

Telomere length in different tissues of elderly patients.

Telomeres are supposed to play a role in cellular aging and might contribute to the genetic background of human aging and longevity. During the past few years telomere length has been measured in various human tissues. However, very little is known about the individual telomere loss in different tissues from the same donor. Therefore we have measured telomere restriction fragment (TRF) length in three unrelated tissues (leukocytes, skin and synovial tissue) of nine elderly patients (age range 73-95 years old). Dependent on the tissue specific proliferation rate we have found significantly shorter telomeres (6546+/-519 bp, mean +/- S.D.) in leukocytes compared to skin (7792+/-596 bp, P<0.01) and synovial tissue (7910+/-420 bp, P<0.001). In general, we have observed an inverse relationship between donor age and TRF length which becomes significant in leukocytes (P=0.04, R(2)=0.49) and skin specimens (P=0.006, R(2)=0.81). Interestingly, linear correlations (P values between 0.017 and 0.038, R(2) values between 0.54 and 0.79) were also obtained on comparison of telomere length in each pair of two different tissues from the same donor without taking donor age into account. This suggests that genetic determination of the regulation of telomere length is tissue-independent. Furthermore, our results indicate that TRF measurement in easily accessible tissues such as blood could serve as a surrogate parameter for the relative telomere length in other tissues.

Aged↗
PubMed2000

Effects of betamethasone administration to the fetal sheep in late gestation on fetal cerebral blood flow.

Glucocorticoid administration to women at risk of preterm delivery to accelerate fetal lung maturation has become standard practice. Antenatal glucocorticoids decrease the incidence of intraventricular haemorrhage as well as accelerating fetal lung maturation. Little is known regarding side effects on fetal cerebral function. Cortisol and synthetic glucocorticoids such as betamethasone increase fetal blood pressure and femoral vascular resistance in sheep. We determined the effects of antenatal glucocorticoid administration on cerebral blood flow (CBF) in fetal sheep. Vehicle (n = 8) or betamethasone (n = 8) was infused over 48 h via the jugular vein of chronically instrumented fetal sheep at 128 days gestation (term 146 days). The betamethasone infusion rate was that previously shown to produce fetal plasma betamethasone concentrations similar to human umbilical vein concentrations during antenatal glucocorticoid therapy. Regional CBF was measured in 10 brain regions, using coloured microspheres, before and 24 and 48 h after onset of treatment, and during hypercapnic challenges performed before and 48 h after onset of betamethasone exposure. Betamethasone exposure decreased CBF in all brain regions measured except the hippocampus after 24 h of infusion (P < 0.05). The CBF decrease was most pronounced in the thalamus and hindbrain (45-50% decrease) and least pronounced in the cortical regions (35-40% decrease). It was mediated by an increase in cerebral vascular resistance (CVR, P < 0.05) and led to a decrease in oxygen delivery to subcortical and hindbrain structures of 30-40%, to 8.6 +/- 1.1 ml x (100 g)(-1) x min(-1), and 40-45 %, to 11.0 +/- 1.6 ml x 100 g(-1) x min(-1), respectively (P < 0.05). After 48 h of betamethasone treatment, the reduction in CBF was diminished to about 25-30 %, but was still significant in comparison to vehicle-treated fetuses in all brain regions except three of the five measured cortical regions (P < 0.05). CVR and oxygen delivery were unchanged in comparison to values at 24 h of treatment. The CBF increase in response to hypercapnia was diminished (P < 0.05). These observations demonstrate for the first time that glucocorticoids exert major vasoconstrictor effects on fetal CBF. This mechanism may protect the fetus against intraventricular haemorrhage both at rest and when the fetus is challenged. Betamethasone exposure decreased the hypercapnia-induced increase in CBF (P < 0.05) due to decreased cerebral vasodilatation (P < 0.05).

Animals↗
PubMed2000

Chromosomal mapping of human genes by radioactive hybridization of cDNAs to Centre d'Etude du Polymorphisme humain high density gridded filter sets.

Chromosomal assignment of human transcribed sequences has been done mainly by high throughput genome analysis in specialized genome centres and, in a more classical fashion, by fluorescence in-site hybridization (FISH) analysis. Not every laboratory has the ability to map cDNAs by FISH analysis. We here report a rapid mapping approach that is based on the hybridization of cDNA probes to high density gridded Centre d'Etude du Polymorphisme Humain filters followed by subsequent computational analysis by database searches in the internet. Not only transcribed sequences but also genomic DNA could be subjected to this mapping approach. The presented approach allows to map human transcribed and genomic DNAs within 1-3 days and with a high level of resolution that will constantly increase in line with the incorporation of data deriving from high throughput genome mapping.

Chromosome Mapping↗
PubMed2000

Identification of the human cytochromes P450 involved in the oxidative metabolism of "Ecstasy"-related designer drugs.

The human cytochrome P450 (CYP) isozymes catalyzing the oxidative metabolism of the widely abused amphetamine derivatives MDMA (N-methyl-3,4-methylenedioxyamphetamine, "Ecstasy"), MDE (N-ethyl-3, 4-methylenedioxyamphetamine, "Eve"), and MDA (3, 4-methylenedioxyamphetamine) were identified. Using a simplified non-extractive reversed-phase HPLC assay with fluorescence detection, biphasic Michaelis-Menten kinetics were obtained for formation of all three dihydroxyamphetamines in liver microsomes from a CYP2D6 extensive metabolizer subject. In contrast, no low K(m) component was detectable in microsomes from a poor metabolizer subject. Additional specific probes for CYP2D6 further confirmed this isozyme as the exclusive low K(m) component for demethylenation. P450-selective inhibitors applied to CYP2D6-inhibited microsomes and activity measurements in a series of recombinant P450s suggested CYP1A2 as the major high K(m) component with contributions by CYP2B6 and CYP3A4. Moreover, the relative CYP1A2 content of a panel of 12 human livers was weakly but significantly correlated to the high K(m) demethylenase activity (Spearman rank correlation coefficient [r(s)] = 0.58; P < 0.05). Microsomal maximal velocities for N-dealkylation were at least 7-fold lower than for demethylenation and were characterized by apparently monophasic kinetics. The most important isozyme for this reaction appeared to be CYP2B6, the microsomal content of which was found to be strongly correlated to N-deethylation of MDE (r(s) = 0.90; P < 0.001). We conclude that, in addition to CP2D6 as the sole high-affinity demethylenase, several other P450 isozymes have the capacity to contribute to microsomal oxidative metabolism of methylenedioxyamphetamines. This may be of particular importance in individuals genetically lacking functional CYP2D6.

Cytochrome P-450 Enzyme System↗
PubMed2000

Retention of imprinting of the human apoptosis-related gene TSSC3 in human brain tumors.

Genomic imprinting is the result of a gamete-specific modification leading to parental origin-specific gene expression in somatic cells of the offspring. Several embryonal tumors show loss of imprinting of genes clustered in human chromosome 11p15.5, an important tumor suppressor gene region, harboring several normally imprinted genes. TSSC3, a gene homologous to mouse TDAG51, implicated in Fas-mediated apoptosis, is also located in this region between hNAP2 and p57 (KIP2). TSSC3 is the first apoptosis-related gene found to be imprinted in placenta, liver and fetal tissues where it is expressed from the maternal allele in normal human development. This study investigated the imprinting status of TSSC3 in human normal, adult brain and in human neuroblastomas, medulloblastomas and glioblastomas. A polymorphism in exon 1 at position 54 was used to analyze the allelic expression of the TSSC3 gene by a primer oligo base extension (PROBE) assay using matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). We found that the TSSC3 gene is not imprinted in human normal, adult brain and blood. In contrast, strong allelic bias resembling imprinting could be detected in most examined tumor specimens. The results demonstrate for the first time that the tumors under investigation are associated with a retention of imprinting of a potential growth inhibitory gene.

Animals↗
PubMed2000

Identification and characterization of novel genes located at the t(1;15)(p36.2;q24) translocation breakpoint in the neuroblastoma cell line NGP.

The distal portion of chromosome 1p is frequently deleted in several human cancers, suggesting the presence of one or more putative tumor suppressor genes on this chromosomal arm. In human neuroblastoma, a consistently deleted region at 1p36.1-p36.2 has been defined by comparison of molecular loss of heterozygosity (LOH) analyses. Recently we described the identification of a yeast artificial chromosome, YAC 927G4, that spans a translocation/duplication breakpoint within the minimally defined LOH region at 1p36.1-p36.2 in the neuroblastoma cell line NGP. Here we describe the identification of two overlapping P1 artificial chromosomes comprising 220 kb at the distal end of YAC 927G4, which we have used as hybridization probes under modified conditions to screen a composite, normalized cDNA library (IMAGE cDNA library). Hybridization screening resulted in the rapid and comprehensive identification of partial cDNAs of which a portion comprised two novel candidate genes, termed DNB1/ARPh and DNB5, which encode putative proteins of 1011 and 447 amino acids, respectively. The DNB1/ARPh gene, which was found to be ubiquitously expressed in human adult and fetal tissues, is highly related to the DRPLA gene, in which expansion of a CAG triplet appears to be causal in the dentatorubral and pallidolysian atrophy disease phenotype. The DNB5 sequence, in contrast, which is predominantly expressed in brain tissues and fetal kidney, failed to show any similarity to sequences in the public domain. A preliminary assessment of transcription and sequence of both genes in several neuroblastoma cell lines does not, thus far, support a causal role in neuroblastoma. However, further analyses are required to confirm these results.

Adult↗
PubMed2000

MycN sensitizes neuroblastoma cells for drug-triggered apoptosis.

BACKGROUND: Amplification of the MYCN gene is found in a large proportion of neuroblastomas and is associated with a poor prognosis. PROCEDURE: To investigate the effect of ectopic MycN expression on the susceptibility of neuroblastoma cells to cytotoxic drugs, we used a human neuroblastoma cell line with tetracycline-controlled expression of MycN. RESULTS: Neither conditional expression of MycN alone nor low drug concentrations induced apoptosis. However, MycN and cytotoxic drugs cooperated to induce cell death. Apoptosis triggered by MycN and doxorubicin was mediated by cleavage of caspases and involved activation of the CD95 system. MycN overexpression and cytotoxic drugs also synergized to induce p53 and Bax protein expression and to trigger mitochondrial permeability transition and cytochrome c release. CONCLUSION: In that amplification of MYCN is considered an adverse prognostic factor, these findings suggest that dysfunctions in apoptosis pathways may be a mechanism by which MycN-induced apoptosis of neuroblastoma cells is inhibited.

Apoptosis↗
PubMed2000

Topology of double minutes (dmins) and homogeneously staining regions (HSRs) in nuclei of human neuroblastoma cell lines.

Amplification of the MYCN gene is a characteristic feature of many neuroblastomas and is correlated with aggressive tumor growth. Amplicons containing this gene form either double minutes (dmins) or homogeneously staining regions (HSRs). To study the nuclear topology of these tumor-specific and transcriptionally active chromatin structures in comparison to chromosome territories, we performed fluorescence in situ hybridization with a MYCN probe and various chromosome paint probes, confocal laser scanning microscopy, and quantitative three-dimensional image analysis. The dmins formed dot-like structures in interphase nuclei and were typically located at the periphery of complexly folded chromosome territories; dmins noted in the chromosome territory interior were often detected within an invagination of the territory surface. Interphase HSRs typically formed extremely expanded structures, which we have never observed for chromosome territories of normal and tumor cell nuclei. Stretches of HSR-chromatin often extended throughout a large part of the cell nucleus, but appeared well separated from neighboring chromosome territories. We hypothesize that dmins are located within the interchromosomal domain (ICD) space and that stretches of HSR-chromatin align along this space. Such a topology could facilitate access of amplified genes to transcription and splicing complexes that are assumed to localize in the ICD space.

Cell Nucleus↗
PubMed2000

Psychotropic drug use, falls and hip fracture in the elderly.

The use of benzodiazepines (BZD) or other psychotropic agents is an established risk factor for falls and hip fractures. The evidence supporting this association has been based solely on history and/or prescription data. In a case-control study we monitored the intake of BZD, tricyclic antidepressants (TCA) and barbiturates in patients with hip fracture by serum analysis, and compared this measurement with drug history and prescription records. The serum was BZD positive in 41% of 82 community-dwelling patients; an equivalent by history and/or prescription records, however, was detected in only 18% (p < 0.0001). In contrast, in 82 age- and gender-matched community-dwelling control patients, there was only a minor discrepancy (3%) in BZD use between anamnestic (N = 19) and analytical (N = 21) data. In 23 patients admitted from nursing homes, a similar trend was visible (39% serum positive for BZD vs 26% by drug history). Barbiturate use (N = 2) could be neglected, and TCA intake was minor (N = 7); in each case both assessments were in accordance. In conclusion, in patients with hip fracture, BZD use is substantially more frequent than reported and previously assumed, pointing to an underestimated risk factor for this injury.

Accidental Falls↗
PubMed2000

Investigation of nonlinear ECoG changes during spontaneous sleep state changes and cortical arousal in fetal sheep.

We examined the processes of cortical activation and deactivation of the fetal brain during spontaneous sleep state transitions and during central nervous processing of vibroacoustic stimulations (VASs) using nonlinear analysis of the electrocorticogram (ECoG). Tests of nonlinearity and a random shuffling routine revealed deterministic and nonlinear portions in the fetal ECoG. As common nonlinear measures are not applicable to nonstationary time series, we developed an algorithm to estimate the predictability of the ECoG in its time course by means of a point prediction error (PPE). The ECoG was recorded before and during VAS from the maternal abdominal surface in seven chronically instrumented fetal sheep at 0.8 of gestation. The PPE during REM sleep was significantly higher than during NREM sleep. VAS in NREM sleep resulted in an abrupt increase of the PPE not reaching the level of REM sleep. The steep increase of the PPE at onset and its slow decrease after cessation of the stimulus were very similar to the dynamics of spontaneous sleep state transitions, suggesting the involvement of the same cortical activating mechanisms. In conclusion, the stage and the time course of fetal brain activation and deactivation patterns can be clearly shown by PPE techniques. The PPE is a useful complement to spectral analysis. Both techniques describe different properties of the ECoG.

Alpha Rhythm↗
PubMed2000

Elucidation of the genetic basis of the common 'intermediate metabolizer' phenotype for drug oxidation by CYP2D6.

A subgroup of 10-15% of Caucasians are termed phenotypical 'intermediate metabolizers' of drug substrates of CYP2D6 because they have severely impaired yet residual in-vivo function of this cytochrome P450. Genotyping based on the currently known CYP2D6 alleles does not predict this phenotype satisfactorily. A systematic sequencing strategy through 1.6 kb of the CYP2D6 5'-flanking sequence revealed six mutations of which three were exclusively associated with the functional CYP2D6*2 allele (-1496 C to G; -652 C to T; and -590 G to A), two were associated with the nonfunctional *4 and with the functional *10-alleles (-1338 C to T and -912 G to A) and one (-1147 A to G) was seen in all *2, *4 and *10-alleles investigated. The -1496 C to G mutation was found to be polymorphic within CYP2D6*2 alleles. In a family study, the wild-type CYP2D6 *2[-1496 C] and the novel variant [-1496 G] allele co-segregated with lower and higher CYP2D6 in-vivo function, respectively, as shown by phenotyping using sparteine as probe drug. In a representative population sample selected for genotypes comprising one CYP2D6*2 and one non-functional allele, the median urinary metabolic ratio (MRs) for sparteine oxidation was 4.4-fold reduced in individuals with the variant allele (*2[-1496 G], MRs = 0.53, n = 27) compared with individuals lacking the mutation (*2[-1496 C], MRs = 2.33, n = 12; P < 0.0001). The mutation -1496 C to G has an estimated frequency of approximately 20% in the general population and allows establishment of a genotype for the identification of over 60% of intermediate metabolizers in Caucasian populations.

Base Sequence↗
PubMed2000

Epstein-Barr virus in pediatric patients after renal transplantation.

BACKGROUND: Pediatric allograft recipients are at an increased risk for lymphotropic virus-associated disorders, particularly in association with primary EBV infection. PATIENTS AND METHODS: Twenty-nine children, adolescents and young adults after renal transplantation were studied in comparison with a healthy young adult control group for evidence of primary, reactivated or chronic active EBV infection at two different time points. RESULTS: Prevalence of antibodies against viral capsid antigen (VCA) was > or = 90% in both groups, whereas anti-Epstein-Barr nuclear antigen (EBNA) was detected only in 19 of 26 seropositive patients compared with seropositive controls (p = 0.01). Persistence of EBV DNA in leukocytes for > or = 6 months was observed in 11 seropositive patients (38%) and one control patient (p < 0.007) using nested polymerase chain reaction. In the transplant recipients, 3 cases of primary EBV infection and 3 cases of chronic active EBV infection were identified. One of these cases developed a non-Hodgkin lymphoma one year later. CONCLUSION: The results suggest that determination of pretransplant antibody status in recipients, rapid detection of EBV infection in seronegative symptomatic recipients, and regular screening for persistent EBV DNA in patients at risk to develop post-transplantation lymphoproliferative disease should be performed.

Adolescent↗
PubMed1999

Human rhinovirus HRV14 uncoats from early endosomes in the presence of bafilomycin.

Determination of infectious progeny virus and in vivo labelling with [(35)S]methionine followed by immunoprecipitation demonstrates that the major receptor group human rhinovirus HRV14 is able to infect HeLa cells in the presence of the V-ATPase inhibitor bafilomycin A1. However, host cell shut off is delayed and viral yield is decreased in the presence of the drug. Uncoating can thus take place under conditions that prevent endosomal acidification indicating that it is catalysed by the viral receptor alone. Since transport is arrested in early endosomes upon inhibition of vesicle acidification, the data also suggest that productive uncoating takes place from early endocytic compartments.

Anti-Bacterial Agents↗
PubMed1999

Nmi protein interacts with regions that differ between MycN and Myc and is localized in the cytoplasm of neuroblastoma cells in contrast to nuclear MycN.

Myc family proteins play an important role in cellular processes such as proliferation, differentiation, apoptosis and transformation. A number of interaction partners of Myc have been identified, such as Max, p107, TBP, YY1, Miz-1, AP-2 and Nmi. Both Max and Nmi also bind to MycN. In contrast to the well defined binding of Max to Myc family proteins the interaction of Nmi with Myc or MycN is only poorly characterized. By employing the yeast two-hybrid system we have mapped the regions of MycN and Myc responsible for binding to Nmi. For MycN exclusively a central region mediates binding to Nmi. In contrast, for Myc a C-terminal portion of the protein, and possibly also a central part, is involved in Nmi interaction. Nmi does not interact with Max and has no transactivation capabilities in yeast, suggesting that Nmi alone is not a transcriptional activator in mammalian cells. Immunofluorescence demonstrates that both in 293 embryonic kidney cells and in Kelly neuroblastoma cells all detectable ectopically expressed Nmi is localized in the cytoplasm, in part in a punctate, granular pattern. MycN, which is highly expressed in Kelly cells consequent to amplification, appears to be localized exclusively in the nuclei. This directly demonstrates that in the same cell at least the major proportion of MycN and Nmi is localized in different cellular compartments. This result is confirmed by the finding that endogenous Nmi, which is expressed in Kelly cells only after stimulation with interferon gamma, is detected exclusively in the cytoplasm of these cells. Therefore only a very small amount of MycN and Nmi is likely to be involved in MycN/Nmi interaction in vivo.

Carrier Proteins↗
PubMed1999

Two regions of deletion in 9p23-24 in sporadic breast cancer.

Allelic deletions of 9p including band 21-22 are common in various types of human carcinomas including breast cancer. Our previous cytogenetic studies had identified constitutional chromosomal changes in 9p23-24 in patients of a male-breast-cancer family and 9p23-24 alterations in a cell line established from a sporadic female breast cancer. To find out whether this genomic region is involved more frequently in alterations in sporadic breast cancers, we have surveyed 80 microdissected tumor samples for both loss of heterozygosity (LOH) and homozygous deletion at 22 microsatellite loci spanning 9p22 to 9p24 using fluorescent multiplex PCR. LOH at one or more loci was observed in 32 (40%) of these tumors. Homozygous deletion was detected in four cases. Eleven tumors had LOH at all of the informative loci analyzed, whereas 21 tumors showed partial-terminal or interstitial allelic loss of 9p. Deletion mapping identified two common regions of deletion: (a) 4 cM including D9S281 to D9S286; and (b) 1 cM including D9S1808 to D9S268.

Breast Neoplasms↗
PubMed1999

MycN sensitizes neuroblastoma cells for drug-induced apoptosis.

Amplification of the MYCN gene is found in a large proportion of neuroblastoma and considered as an adverse prognostic factor. To investigate the effect of ectopic MycN expression on the susceptibility of neuroblastoma cells to cytotoxic drugs we used a human neuroblastoma cell line harboring tetracycline-controlled expression of MycN. Neither conditional expression of MycN alone nor low drug concentrations triggered apoptosis. However, when acting in concert, MycN and cytotoxic drugs efficiently induced cell death. Apoptosis depended on mitochondrial permeability transition and activation of caspases, since the mitochondrion-specific inhibitor bongkrekic acid and the caspase inhibitor zVAD-fmk almost completely abrogated apoptosis. Loss of mitochondrial transmembrane potential and release of cytochrome c from mitochondria preceded activation of caspase-8 and caspase-3 and cleavage of PARP. CD95 expression was upregulated by treatment with cytotoxic drugs, while MycN cooperated with cytotoxic drugs to increase sensitivity to CD95-induced apoptosis and enhancing CD95-L expression. MycN overexpression and cytotoxic drugs also synergized to induce p53 and Bax protein expression, while Bcl-2 and Bcl-X(L) protein levels remained unchanged. Since amplification of MYCN is usually associated with a poor prognosis, these findings suggest that dysfunctions in apoptosis pathways may be a mechanism by which MycN-induced apoptosis of neuroblastoma cells is inhibited.

Apoptosis↗