Biomedical subjects

A Rolfs

Publications and source records attributed to A Rolfs.

At least 55 records · Page 3Linked to original sources

PubMed1997

Allelic association of a dopamine transporter gene polymorphism in alcohol dependence with withdrawal seizures or delirium.

Hereditary factors confer susceptibility to alcohol dependence. Alcohol mediates its reinforcing effects by enhancing dopamine activity in the mesolimbic dopamine system. The role of the dopamine transporter in terminating dopaminergic activity in synaptic neurotransmission suggests that variants of the dopamine transporter gene (DAT1) might contribute to individual differences in vulnerability to addictive behavior. Our population-based association study investigated whether variants of DAT1 confer susceptibility to alcohol dependence in 293 alcoholics and clinically more homogeneous subgroups formed by: positive family history, early age-at-onset, delirium, withdrawal seizures, antisocial tendencies, type 1 and 2 alcoholics. Analyzing a VNTR polymorphism in the 3' untranslated region of DAT1, we found a significantly increased prevalence of the nine-repeat allele in 93 alcoholics displaying withdrawal seizures or delirium, compared with 93 ethnically matched nonalcoholic controls (p = 0.003; OR = 2.44; 95% confidence interval: 1.35-4.43). Our data provide evidence that a major genetic determinant of DAT1 influences vulnerability to severe alcohol withdrawal symptoms.

Adult↗

PubMed1997

Hypoxia-inducible factor-1 alpha is regulated at the post-mRNA level.

The hypoxia-inducible factor-1 (HIF-1) is involved in the induction of oxygen regulated genes such as erythropoietin and vascular endothelial growth factor (VEGF). HIF-1 is a heterodimeric transcription factor consisting of an alpha and a beta subunit. The question of how HIF-1 itself is regulated remains to be elucidated. Studies performed in human Hep3B hepatoma cells suggested that the prevalent mode of HIF-1 action is an increase in HIF-1 alpha steady-state mRNA and protein levels after hypoxic exposure. In contrast to the reported very low basal HIF-1 alpha mRNA levels, however, we detected HIF-1 alpha mRNA in several cell lines cultured under normoxic conditions, although no HIF-1 DNA binding activity was observed. Following hypoxic induction, VEGF mRNA levels and HIF-1 DNA binding activity increased, but HIF-1 alpha mRNA levels remained largely unchanged. One possible explanation for this discrepancy might be that HIF-1 DNA binding activity does not follow HIF-1 alpha mRNA kinetics. We therefore incubated HeLaS3 cells in tonometers for 7.5 minutes up to four hours at either 20% O2 or 0.5% O2. Although there was some variation in HIF-1 alpha mRNA levels, we did not find significant changes over this time frame, suggesting that HIF-1 alpha is not transcriptionally regulated.

Animals↗

PubMed1997

Oxygen- and dioxin-regulated gene expression in mouse hepatoma cells.

The discovery that the oxygen-regulated transcription factor HIF-1 alpha and the dioxin receptor AhR share the common heterodimerization partner ARNT (HIF-1 beta) raised the question whether a cross-talk between oxygen and dioxin signal transduction pathways exists. To answer this question we investigated an ARNT-deficient mutant cell line (Hepa1C4), which has lost its capability of responding to dioxin. The results demonstrate that the presence of ARNT is indispensable for hypoxia-inducible HIF-1 DNA binding as well as for oxygen-regulated reporter gene activity mediated by the EPO 3' hypoxia response element (HRE). Hypoxic induction of the vascular endothelial growth factor (VEGF) gene, however, was only partially abrogated in Hepa1C4 cells, suggesting that HIF-1-independent oxygen signaling pathways might exist. We further studied HIF-1 and AhR/ARNT DNA binding activity as well as the regulation of oxygen- and xenobiotic-responsive genes by treating mouse Hepa1 hepatoma cells with hypoxia and/or the dioxin analogue ICZ. Hypoxia-inducible VEGF expression was found to be independent of ICZ-treatment, whereas ICZ-inducible cytochrome P-450IA1 expression was slightly reduced by hypoxic treatment of the cells. Interestingly, the enhancer function of a xenobiotic response element (XRE) linked to a reporter gene was induced by hypoxia, but expression of a HRE-containing reporter gene was not affected by ICZ treatment.

Animals↗

PubMed1997

Influence of the dopamine D2 receptor (DRD2) genotype on neuroadaptive effects of alcohol and the clinical outcome of alcoholism.

The present study was performed to test the hypotheses that allelic variants at the human dopamine D2 receptor gene locus (DRD2) confer susceptibility to alcoholism or are associated with clinical subtypes of alcoholism. We investigated an A --> G substitution polymorphism in the 3'-untranslated region of exon 8 (E8) of DRD2 with allele frequencies of f(G) = 0.295 - 0.329. No significant association of the DRD2 genotype or allele frequencies with alcoholism was found in an association study including 283 alcoholics and 146 non-alcoholic controls. However, the frequent homozygous E8 A/A genotype with f(AA) = 0.47 - 0.48 was associated with increased anxiety and depression scores in alcoholics during the follow up after clinical detoxification treatment. In addition, E8 A/A was associated with increased suicide attempts and showed a tendency towards more severe withdrawal symptoms, early relapse and reduced responsiveness to the dopaminergic agonist apomorphine. Regression analysis revealed the DRD2 E8 genotype as the only significant factor determining withdrawal severity in female alcoholics. The findings suggest an influence of the DRD2 genotype on the neuropharmacological effects of chronic alcohol exposure and the clinical course of alcoholism.

Adaptation, Physiological↗

PubMed1997

Absence of angiotensin II type 1 receptor gene mutations in human adrenal tumors.

Regulatory actions of angiotensin II (AngII), which is involved in the pathophysiology of hypertension and also participates in cell proliferation and cell differentiation, are mainly mediated by AngII type 1 (AT1) receptor. Recently, activating mutations of receptors causing hyperfunctioning endocrine diseases have been described in the case of the TSH and LH receptors, implicating that such mutations might occur in other G-protein-coupled receptors. Furthermore it seems to be possible that genetic variations of AT1 receptor have an influence upon the action of AngII. Therefore, we searched by sequence analysis of the coding region of AT1 receptor gene for activating mutations and genetic polymorphisms in 56 human adrenal tumors (16 aldosterone-producing adenomas, 10 cortisol-producing adenomas, 1 aldosterone-producing carcinoma, and 29 incidentalomas). We were not able to identify any activating mutation in the coding region of AT1 receptor gene. We conclude that activating mutations of the AT1 receptor are not a major cause of the development of adrenal adenomas, if at all. In addition, polymorphic subtypes of AT1 receptor do not seem to play a major role in the pathogenesis of these tumors, even though a tendency towards a higher frequency of the polymorphic base substitution at position 573 (T573-->C) in cortisol-producing tumors needs to be further evaluated.

Adenoma↗

PubMed1997

Use of manganese in RT-PCR eliminates PCR artifacts resulting from DNase I digestion.

The precise quantification of rare mRNA copies from intronless genes by reverse transcription polymerase chain reaction (RT-PCR) requires the complete removal of genomic DNA because discrimination of cDNA and DNA amplification products by differing sizes of PCR products is not possible. Elimination of DNA is achieved by treating the RNA sample with RNase-free DNase I before RT-PCR. The lack of a PCR product from DNase-treated RNA samples before RT is usually accepted as a proof of efficient DNA destruction. However, this may vary depending on the metal cofactor used in the DNase I cleavage. Treating DNA-contaminated RNA samples with DNase I and magnesium as a cofactor creates a negative PCR control after digestion without further RT. Paradoxically, after additional RT-PCR, the original intron-containing DNA fragment size may be produced again. In the presence of manganese as cofactor, RT-created DNA fragments do not appear. This is because in the presence of manganese, DNase I cleaves both DNA strands at approximately the same site, yielding DNA fragments that are blunt-ended or that have protruding termini of only one or two nucleotides in length. However, overlapping fragments with the potential to recombine are created by DNase digestion with magnesium as cofactor. Because one cannot differentiate between a PCR signal produced by RNA and one produced by recombined DNA after DNase I digestion and RT, all such DNase I assays should be performed with manganese instead of magnesium.

Artifacts↗

PubMed1996

Dopamine D2 receptor gene (DRD2) haplotypes in Caucasians.

The human dopamine D2 receptor gene (DRD2) is considered a candidate gene for neuro-psychiatric diseases. We typed three new DNA sequence variants in DRD2 intron 4, intron 6 and exon 8, in combination with the known TaqI A restriction fragment length polymorphism (RFLP) and exon 7 311Ser/Cys in 106 unrelated psychiatrically healthy Caucasians. Based on the genotypic data we delineated 10 distinct DRD2 haplotypes and their genetic relationship. Our data provide evidence that the Taq A1 allele and the 311Cys variant are components of different groups of haplotypes though both variants have been speculated to be associated with alcoholism or schizophrenia in recent studies. Therefore we conclude that the prior knowledge of the frequencies and genetic relationships of DRD2 haplotypes will lead to the selection of more suitable intragenic markers for future association studies.

Gene Frequency↗

PubMed1996

Species-specific detection of Legionella using polymerase chain reaction and reverse dot-blotting.

Legionella pneumophila and some other Legionella species are capable of causing Legionnaire's disease, a potentially fatal pneumonia. The identification of legionellae by standard laboratory techniques such as culture is difficult and time consuming. In the present work, the DNA sequence of the 23S-5S spacer region was determined for 43 Legionella isolates, and the sequence information was used to develop a species-specific detection system using PCR and reverse dot-blotting which employs just one PCR amplicon to perform genus- and species-specific detection. L. pneumophila serogroups 1-16 as well as 21 non-pneumophila isolates could be identified and differentiated at the species level using this system.

Base Sequence↗

PubMed1996

Nucleotide sequence, chromosomal assignment and mRNA expression of mouse hypoxia-inducible factor-1 alpha.

The heterodimeric hypoxia-inducible transcription factor HIF-1 is involved in the oxygen-regulated transcription of several genes including erythropoietin. Cloning and sequencing of the alpha-subunit of mouse HIF-1 cDNA revealed a 90% overall homology to human HIF-l alpha but lack of any similarity in the 5' untranslated region and translational start site. Mouse HIF-1 alpha is encoded by an evolutionary conserved single-copy gene located on chromosome 12. We found a widespread constitutive expression of mouse HIf-1 alpha mRNA which was particularly high in lung and kidney. Despite a strong erythropoietin induction, HIF-1 alpha mRNA concentrations were not upregulated in hypoxic mouse tissues.

Amino Acid Sequence↗

PubMed1996

Low frequency of p53 gene mutation and protein expression in mucinous colorectal carcinomas.

Immunohistochemical data indicate that the frequency of p53 protein overexpression is consistently lower in the mucinous than in the non-mucinous carcinomas of the breast, ovary, pancreas and colon. This peculiar immunohistochemical behavior of the mucinous phenotype could be due to the effect of large amounts of mucus on the staining or to an actual mutation frequency difference between mucinous and non-mucinous carcinomas. This question was investigated on a group of mucinous colorectal carcinomas. DNA was extracted from paraffin sections of 16 human mucinous colorectal carcinomas and the mutation frequency was determined by sequencing of p53 exons amplified in PCR. The expression of p53 protein was determined with the avidin-biotin complex-peroxidase staining procedure and CM-1 antiserum. Twenty-five percent of the tumors, exhibited p53 protein overexpression and in 31% a mutation was detected. Concordance between the two techniques was found in 69% of tumors. Overexpression without mutation was observed in 12% and mutation without overexpression in 19%. G:C --> A:T transitions represented the most frequent lesion (80%), as previously observed in non-mucinous colorectal carcinomas. These data indicate that the mutation pattern in the p53 gene is similar in mucinous and non-mucinous colorectal carcinomas. The low frequency of p53 overexpression in the mucinous phenotype is not due to a mucus effect on the staining but is related to the low mutation frequency of p53 gene. These results lead to the hypothesis that in contrast to the nonmucinous tumors the development of the majority of colonic carcinomas with the mucinous phenotype may be independent from p53 mutations.

Adenocarcinoma, Mucinous↗

PubMed1996

Reduced atrial angiotensin receptor type 1 mRNA content in end-stage human heart failure: assessment by a novel quantitative PCR-ELISA technique.

The number of atrial angiotensin II binding sites is reduced in end-stage human heart failure. The goals of our study were the development of a quantitative polymerase chain reaction for angiotensin II receptor type 1 mRNA to determine the angiotensin receptor type 1 (AT1) mRNA content in the atria of patients with end-stage heart failure. We established a quantitative PCR based on coamplification of AT1 wild-type and an internal standard in the same PCR, followed by liquid-phase hybridization of PCR products in microtiter plates and quantitation by ELISA. Glyceraldehyde phosphate dehydrogenase mRNA in the same samples was used to relate the AT1 mRNA content to a stably expressed reference gene. Atrial samples from 11 patients with end-stage heart failure obtained at cardiac transplantation were compared with atrial samples from 11 patients with normal cardiac function undergoing routine cardiac surgery. A PCR/ELISA system with a variance of about 6% after reverse transcription and a linear measuring range was established. In the samples from 11 patients with end-stage heart failure a 58% decrease in AT1 mRNA content was found in comparison with 11 controls (heart failure: 185,680 +/- 196,912 AT1 mRNA copies/microgram RNA, controls: 440,555 +/- 268,456, P < 0.02). When AT1 mRNA content was related to glyceraldehyde phosphate dehydrogenase mRNA, a 65% decrease was detected (AT1/glyceraldehyde phosphate dehydrogenase: heart failure: 4.84 +/- 5.18; controls: 13.74 +/- 7.77; P < 0.005). Standardization of PCR resulting in a low coefficient of variance, high reproducibility, and large sample capacity is possible using optimal internal standardization and the liquid-phase hybridization/ELISA system for detection. The optimized PCR procedure indicated downregulation of atrial AT1 in end-stage human heart failure, suggesting a reduced capacity of the atria to respond to angiotensin II stimulation in end-stage heart failure.

Angiotensin-Converting Enzyme Inhibitors↗

PubMed1996

Controversies in the treatment of ductal carcinoma in situ.

In summary, certain subgroups of DCIS appear not to require radiation. Corroboration of these results from retrospective reviews and prospective trials is necessary to confirm the safety and efficacy of individualized treatment strategies. Even though the current standard of treatment is (1) lumpectomy with radiation therapy, (2) mastectomy, or (3) mastectomy with reconstruction, it is possible in the future to say that patients with low-grade DCIS (the exact criteria to be defined) may be eligible for breast conservation without radiation, and all patients with high-grade DCIS or perhaps low-grade DCIS with necrosis would be treated best by lumpectomy plus radiation. It is possible that a small subgroup of patients may be best treated by mastectomy, or perhaps, as the results of B-24 become available, by radiation therapy plus tamoxifen. The use of tumor markers such as c-erbB-2, cathepsin D, and NM 23 may help us to better define these subgroups, but much study is necessary before a definite treatment strategy is reached.

Biomarkers, Tumor↗

PubMed1996

Chlamydia pneumoniae as a cause of community-acquired pneumonia in hospitalized patients in Berlin.

The incidence of community-acquired pneumonia due to Chlamydia pneumoniae was evaluated in a 1-year prospective study of 236 hospitalized patients with 237 manifestations of pneumonia. The microbiological diagnosis was based on results of cultures of blood and sputum or bronchoalveolar lavage fluid and results of complement fixation tests and indirect immunofluorescence of acute- and convalescent-phase sera for C. pneumoniae, Mycoplasma pneumoniae, Legionella species, Coxiella burnetii, and respiratory viruses. Diagnosis of acute C. pneumoniae infection was made on the basis of the results of microimmunofluorescence of paired serum samples. A microbiological diagnosis was obtained in 160 cases (67.5%). C. pneumoniae was the causative agent in 27 patients (11.4%). The following organisms were the other etiologic agents of pneumonia: Streptococcus pneumoniae, 30 cases (12.7%) (bacteremia occurred in 53.3%); Mycoplasma pneumoniae, 22 (9.3%); respiratory viruses, 22 (9.3%); and Enterobacteriaceae, 18 (7.6%). The prevalence of C. pneumoniae antibody in our study population was 47.5%. As has been increasingly reported in recent years and confirmed by this study, C. pneumoniae appears to be a common etiologic agent of community-acquired pneumonia and upper respiratory tract infection in Germany.

Adolescent↗

PubMed1996

No association of the structural dopamine D2 receptor (DRD2) variant 311Cys with alcoholism.

The human dopamine D2 receptor (DRD2) has been implied in the vulnerability for alcoholism and/or the modification of its severity. This is supported through animal experimental and pharmacological data. We analyzed the DRD2 311Ser/Cys polymorphism in 312 German alcoholics and 131 ethnically matched controls to investigate the association of genetic DRD2 variants with alcoholism or clinical characteristics of homogeneous subgroups of alcoholics. We observed no association between the 311Cys variant and alcoholism, and none of the clinical characteristics evaluated was significantly associated with 311Cys. The allele frequencies of the 311Cys variant were 0.026 and 0.031 in the alcoholics and controls, respectively. These are the highest reported 311Cys frequencies in Caucasians. The DRD2 TaqI A1/A2 restriction fragment length polymorphism was analyzed simultaneously in our samples. In most cases, the 311Cys allele is associated with the TaqI A2-allele. Data do not suggest a clinical relevance of the 311Cys variant in alcoholism. However, the relevance of this variant in other diseases or the existence of other DRD2 variants with altered receptor function or expression cannot be excluded.

Adult↗

PubMed1996

Lack of allelic association of dopamine D1 and D2 (TaqIA) receptor gene polymorphisms with reduced dopaminergic sensitivity to alcoholism.

Our study tested the hypothesis of whether the sensitivity of central dopamine receptors corresponds to the genotypic constitution of DNA-polymorphisms of the dopamine D1 and D2 receptor (DRD1, DRD2) genes and is associated with poor treatment outcome. Therefore, 97 alcohol-dependent patients were assessed according to their sensitivity of central dopamine receptors (apomorphine-induced secretion of growth hormone), clinical outcome during a 6-month observation period, and genotypic constitution of the TaqIA restriction fragment length polymorphism (RFLP) at the DRD2 locus and of the Bsp1286I RFLP at the DRD1 locus. On the 1st day of detoxification, dopamine receptor hyposensitivity was found in treatment nonresponders, but not in responders. Apomorphine-induced growth hormone release did not differ significantly in alcoholics with different genotypes of the DRD1 and DRD2 RFLPs. Neither did we find a significant allelic association with treatment response. Thus, we did not find evidence for a genetic determination of dopamine receptor hyposensitivity in alcoholics with poor treatment outcome.

Adult↗

PubMed1996

Two new missense mutations in a non-Jewish Caucasian family with type 3 Gaucher disease.

Gaucher disease is an autosomal recessive, lysosomal storage disease caused by a deficiency of the enzyme glucocerebrosidase. The prevalence of this disorder differs significantly among various populations and is highest in some Jewish populations. More than 35 disease-producing mutations of the gene have been reported. The change of the amino acid leucine at position 444 to proline (444Leu-->Pro) is the most common mutation in non-Jewish populations, and a second mutation at amino acid 370 (370Asn-->Ser) is the most common one in Jewish populations. However, most of the mutations have been reported in very few cases or even in only one pedigree. We performed a direct DNA sequencing analysis of the complete functional glucocerebrosidase gene in a 22-year-old neuronopathic non-Jewish patient with Gaucher disease type 3 with myoclonic epilepsy and slight mental disturbances and in most members of his family. After selective amplification of the complete coding region of the patient's functional glucocerebrosidase gene, we identified two hitherto unreported mutations in exon 9 (genomic nucleotide 5224G-->C; 417Val-->Leu) and in exon 11 (genomic nucleotide 6668C-->T; 510Thr-->Ile). Each parent showed one of these mutations. Once we sequenced the complete active gene, we could rule out the existence of further mutations. Data that show that in about 15 to 25% of non-Jewish patients with Gaucher disease no mutations within the glucocerebrosidase gene are found may be due to inappropriate screening methods, mostly used for the genotyping. Until we have precise information about the frequency and distribution of single-point mutations in patients with Gaucher disease, it is necessary to analyze the complete glucocerebrosidase gene.

Adult↗

PubMed1996

Allele-specific PCR for simultaneous amplification of both alleles of a deletion polymorphism in intron 6 of the human dopamine 2 receptor gene (DRD2).

The human dopamine 2 receptor gene (DRD2) is an important candidate gene for drug addiction and alcoholism. So far, no mutations within the coding region of DRD2 have been found to be associated with addiction disorders. To identify sequence polymorphisms for further haplotype analyses and to analyze the importance of possible intron sequence variations of the human DRD2 gene (>260kb) in greater cohorts and in a routine manner we established an optimized methodological procedure for polymerase chain reaction (PCR) amplification and direct non-radioactive sequencing followed by a bidirectional allele-specific PCR protocol; the latter one allows the simultaneous amplification of several alleles in one reaction tube. Overall, the sequences of the DRD2 introns 3-7 are highly conserved. Nevertheless, in each of the analyzed intron sequences we found substitution variants as well as a one base-pair deletion polymorphism in intron 6. The allele-specific PCR allowed the reliable testing of 95 healthy control individuals and 270 alcoholics for analyzing a possible genetic association of this newly characterized polymorphic DRD2 marker with alcoholism in an ethnically and clinically homogenous group of patients. However, the observed allele frequencies for the 1bp deletion polymorphism were 15.9% for the alcoholics and 15.3% for the controls suggesting no association of the deletion to alcoholism.

Alleles↗

PubMed1996

Absence of human T-lymphotrophic virus type I in patients with systemic lupus erythematosus.

The role of the human T-cell lymphotropic/leukaemia virus type I (HTLV-I) in the pathogenesis of autoimmune diseases of unknown cause, such as systemic lupus erythematosus (SLE), multiple sclerosis (MS) or Sjögren's syndrome (SS) has been discussed extensively. We have investigated whether SLE is in any way associated with exogenous HTLV-I. Using enzyme immunoassay (EIA), we found no seroreactivity against HTLV-I antigens in any of 24 SLE patients under investigation. Using a radioimmunoprecipitation assay (RIPA), there was also no expression of retroviral tax-protein demonstrable in 24 individuals with SLE. DNA preparations of peripheral blood cells, as well as isolated CD4- and CD8-positive cells, were examined for HTLV-I sequences (pol-, env-, gag-, LTR- and tax-region) using polymerase chain reaction (PCR). We were unable to demonstrate any specific HTLV-I PCR products in SLE specimens. Our data suggest that exogenous HTLV-I is not involved in the pathogenesis of SLE. Systemic lupus erythematosus (SLE) is a multisystem disease of unknown cause characterized by B-cell hyperactivity with hypergammaglobulinaemia and the formation of pathogenic autoantibodies. Patients may also show altered suppressor/helper T-cell ratios. Abnormalities in T-cell function include T-cell lymphopenia, expression of activation antigens and alteration of responses to mitogens and lymphokines. Human retroviruses are known to cause immune aberrations, such as diminished T-cell function and polyclonal B-cell stimulation, which are observed in patients with leukaemias, lymphomas and the acquired immunodeficiency syndrome (AIDS). Human T-cell lymphotropic/leukaemia (HTLV-I) is aetiologically linked with adult virus type I T-cell leukaemia (ATL) and tropical spastic paraparesis. A common feature of the HTLV family is an LTR encoded protein (tax protein, p40tax) which triggers viral protein production in the early stages of a retroviral infection. Detection of p40tax may indicate transcriptional activity of a provirus. Several investigators have examined the possible involvement of HTLV-I in SLE and have produced conflicting results, especially so far as seroreactivity against HTLV-I antigens is concerned. The discovery of HTLV-I like particles in murine lupus might also indicate an important role of exogenous viruses in SLE. Olsen and colleagues describe a high seropositivity to anti-HTLV-I- and anti-HTLV-III antibodies and evidence of viral replication in patients with SLE. Lin et al. also demonstrated anti-HTLV-I antibodies in SLE patients. However, other authors failed to detect anti-HTLV-I antibodies, proviral structures or viral transcripts in SLE. We investigated whether or not there are any indications of the presence of the exogenous retrovirus, HTLV-I, in patients with SLE, at immunological and genetic levels. We found neither immunological nor molecular biological evidence for the existence of HTLV-I in SLE patients. There were some weak suggestions of the presence of possibly endogenous retroviral sequences.

Antibodies, Viral↗