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Biomedical subjects

A Rolfs

Publications and source records attributed to A Rolfs.

At least 73 records · Page 4Linked to original sources
PubMed1995

Hypoxia, a novel inducer of acute phase gene expression in a human hepatoma cell line.

Transcriptional regulation of gene expression by hypoxia is an important, but yet only marginally characterized mechanism by which organisms adapt to low oxygen concentrations. The human hepatoma cell line HepG2 is a widely used model for studying hypoxic induction of the hematopoietic growth factor erythropoietin. In an attempt to identify additional genes expressed in HepG2 cells during hypoxia, we differentially screened a cDNA library derived from hypoxic (1% O2) HepG2 cells using probes isolated from either normoxic (21% O2) or hypoxic cells. Two genes were identified, one encoding aldolase, a member of the glycolytic enzymes, and the other encoding alpha 1-antitrypsin which belongs to the family of the acute phase (AP) responsive proteins. Whereas hypoxic induction of glycolytic enzymes is well established, oxygen-dependent regulation of AP genes has not been reported so far. AP proteins are liver-derived plasma proteins whose production during inflammation is either up-regulated (positive AP reactants) or down-regulated (negative AP reactants). In the present study, we demonstrate that on the mRNA level hypoxic stimulation of HepG2 cells led to (i) an induction of the positive AP reactants alpha 1-antitrypsin, alpha 1-antichymotrypsin, complement C3, haptoglobin, and alpha 1-acid glycoprotein; (ii) a down-regulation of the negative AP reactant albumin; (iii) an up-regulation of the negative AP reactant transferrin; and (iv) unchanged levels of the positive AP reactants alpha- and beta-fibrinogen as well as hemopexin. Cycloheximide inhibited hypoxic up-regulation of AP mRNAs demonstrating that de novo protein synthesis is required for hypoxic induction. Nuclear run-on assays indicate that the hypoxic increase in AP mRNAs is mainly due to transcriptional regulation. The hypoxic response was compared to AP stimulation by interleukin 6. The results suggest that the adaptive response to hypoxia overlaps with, but is not identical with, the AP response mediated by interleukin 6.

Acute-Phase Proteins↗
PubMed1995

Regulation, chamber localization, and subtype distribution of angiotensin II receptors in human hearts.

BACKGROUND: To assess the chamber localization, subtype distribution, and regulation of human myocardial angiotensin II receptors in heart failure, we determined the binding of angiotensin II, Sar1Ile8-angiotensin II, and the subtype-specific antagonists Dup 753 (AT1-specific) and PD 123319 (AT2-specific) in atria from patients with normal (left ventricular ejection fraction > 55%) or moderately impaired (left ventricular ejection fraction 30% to 55%) cardiac function and in atria and ventricles from explanted end-stage failing hearts. Sarcolemmal and combined fractions, the latter including internalized receptors, were studied. In addition, AT1 mRNA content was analyzed by polymerase chain reaction after reverse transcription. METHODS AND RESULTS: The number of angiotensin II binding sites (Bmax) in sarcolemmal fractions was significantly reduced in explanted end-stage failing hearts in comparison with control subjects and moderate heart failure (Bmax 3.9 +/- 0.8 versus 11.2 +/- 1.7 and 9.6 +/- 0.8 fmol/mg protein, respectively). A comparable 65% reduction in receptor numbers was found in combined fractions from end-stage failing hearts, indicating that the loss of binding sites was not due to their internalization. The dissociation constants were comparable in sarcolemmal and combined fractions and in nonfailing and failing hearts, ranging from 0.5 +/- 0.2 to 1.2 +/- 0.5 nmol/L. In nonfailing hearts, 69 +/- 4% of binding sites were blocked by the subtype-2-specific inhibitor PD 123319 and were therefore classified as AT2; 33 +/- 5% were blocked by the subtype-1-specific inhibitor DUP 753 and thus classified as subtype 1. In explanted hearts, comparable ratios of 66 +/- 5% AT2 sites and 34 +/- 5% AT1 sites were found. AT1 cDNA amplification signals by polymerase chain reaction were reduced to about one third of the level in control subjects in end-stage failing hearts. CONCLUSIONS: Angiotensin II receptors in human myocardium are present in relatively low numbers, and AT2 is the predominant subtype. A significant loss of angiotensin II receptors occurs in end stage but not in moderate heart failure. The loss of receptors affects both subtypes to a comparable degree. The data suggest that the decrease in receptor density is due to a decrease in steady-state mRNA abundance.

Binding Sites↗
PubMed1995

Dopamine D1, D2 and D3 receptor genes in alcohol dependence.

Hereditary factors play a substantial role in the etiology of alcohol dependence. Alcohol mediates its reinforcing effects by an activation of the mesolimbic dopamine system. These findings suggest that the genes encoding the dopamine receptor (DR) subtypes represent high-ranking candidates for susceptibility genes to addictive disorders. Our present population-based association study investigated whether sequence variants of the dopamine D1, D2 and D3 receptor genes confer susceptibility to alcohol dependence in 278 alcoholics, and clinically more homogeneous subgroups ascertained through positive family history, early age at onset, delirium, withdrawal seizures and antisocial tendencies. No evidence for an allelic association was found for the PCR-based TaqA RFLP fo the DRD2 gene and a Bsp1286I RFLP of the DRD1 gene. Without correction for multiple testing, we found a significantly increased allele frequency of a common DRD3 gene variant expressing a serine at position 9 in the extracellular N-terminal part of the receptor protein in 55 alcohol-dependent individuals with delirium (chi 2 = 4.1, df = 1, p = 0.042). Further studies have to examine whether this amino acid substitution or a nearby mutation confers genetic susceptibility to at least a subgroup of alcohol-dependent individuals with delirium.

Adult↗
PubMed1995

Clinical evaluation of a Mycobacterium tuberculosis PCR assay.

On the basis of previously published PCR primer sequences, we have designed a sensitive system for detecting DNA of the Mycobacterium tuberculosis complex (MTB) in patient sputum samples which employs a fast and simplified sample preparation method appropriate for routine diagnostic testing. In order to evaluate the accuracy of the PCR assay, we performed a prospective study with 103 patients, comparing PCR results with culture results of samples obtained from a parallel culture assay as well as with subsequent culture results. Using two MTB-specific PCR primer systems, we found 48 of 49 tuberculosis (Tb) patients to be PCR positive (PCR sensitivity, 0.98). Sixteen of 54 presumably non-Tb patients showed amplifiable MTB DNA (specificity, 0.7). The study demonstrates that for diagnostic applications of MTB PCR two MTB-specific primer pairs should be used. MTB infection is extremely unlikely in cases of MTB PCR-negative samples: with our method for the exclusion of active Tb, the validity of one PCR assay seems to be equivalent to those of at least three culturing procedures. Positive PCR results do not necessarily reflect active MTB infection. It remains to be shown whether positive PCR results in Tb-negative patients mean false-positivity, an early laboratory finding which predicts a subsequent reactivation of a prior Tb infection, or whether asymptomatic patients may carry PCR-amplifiable MTB DNA without any clinical relevance. It is important to point out that the validity of PCR results in clinical studies depends on the use of contamination controls parallel to all PCR steps and the simplicity of the DNA extraction method as well as on the specificity of the PCR results.

Base Sequence↗
PubMed1995

Amplification of Mycobacterium tuberculosis from peripheral blood.

To determine the value of the PCR assay of lymphomonocytic blood cells for the diagnosis of pulmonary tuberculosis, we compared, in a prospective study, PCR results with results of conventional diagnostic methods of Mycobacterium tuberculosis detection. Clinical investigators who were unaware of the PCR data classified 162 immunocompetent patients, who had been hospitalized because of suspected pulmonary tuberculosis, in accordance with the recommendations of the American Thoracic Society (ATS). By testing blood samples from these patients for mycobacterial DNA with three different PCR systems, we were able to demonstrate that nonquantitative PCR of peripheral blood leukocytes is of little value for the specific diagnosis of pulmonary tuberculosis. However, semiquantitative PCR assays might have some significance since we found an increasing level of mycobacterial DNA copies in blood from patients classified in ATS groups 2 to 5.

Base Sequence↗
PubMed1995

Clinical and immunologic follow-up study of patients with neurocysticercosis after treatment with praziquantel.

In a prospective study, 17 Brazilian patients with parenchymatous neurocysticercosis were monitored for a period of 12 months after treatment with praziquantel. Taenia solium-specific IgG antibodies, interleukin-1 beta (IL-1 beta), tumor necrosis factor alpha, neopterin, and soluble interleukin-2 receptor (sIL-2R) were measured in serum and CSF before starting the therapy, on the last day of treatment, 5 weeks after treatment, and 3, 6, and 12 months after treatment. The most common symptoms and signs found in patients before treatment were headache, neck stiffness, and seizures. Six months after commencement of therapy, 13 of the 17 patients were free of complaints. Clinical normalization was paralleled by a significant decrease (p < 0.05) in the amount of intrathecally produced anti-T solium IgG 1 year after treatment. IL-1 beta was undetectable in serum at all times, whereas in the CSF it was within the normal range during the whole study period. The mean concentration of sIL-2R in the CSF was 65 kU/l (normal, < 50 kU/l) 5 weeks after treatment, whereas in all subsequent investigations sIL-2R was undetectable. The median CSF neopterin level was slightly elevated before treatment (6.8 nmol/l). One year after treatment, it had dropped by 69% (p < 0.001) to a median value of 2.1 nmol/l. The size of planimetrically measured focal cystic brain lesions on CT correlated with the amount of intrathecally synthesized anti-T solium IgG at the end of the study (r = 0.89, p < 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)

Adolescent↗
PubMed1994

Genetic polymorphisms of the angiotensin II type 1 (AT1) receptor gene.

Numerous essential, physiological effects on the cardiovascular system are attributable to angiotensin II (Ang II). Because of this we can assume that genetic changes in the specific receptor of Ang II (Ang II type 1 receptor gene, AT1) play a decisive role in the occurrence of cardiovascular disease associated with blood pressure regulation, vascular tone, cardiac and vascular growth process. To test this hypothesis, we examined the presence of polymorphisms within the coding region of the AT1 gene using polymerase chain reaction (PCR) and subsequent non-radioactive sequencing of samples from a control group with no previous history of cardiovascular complaint in individuals or immediate family. Using the Taq-sequencing procedure we found polymorphic sites, especially in the 5' region of the gene (base pair positions 9, 16, 87, 133, 186), two of which led to an exchange of the amino acid (amino acid 6: Ser<==>Pro, amino acid 45: Gly<==>Arg). Together with the silent polymorphism at base pair position 573, which our group established previously, an additional polymorphism in the 3' region of the gene was discovered. This, however, did not confer any changes in amino acid sequence. In a preliminary study we found no association between the distribution of the C/T573 polymorphic site and cardiovascular disease, such as essential hypertension (n = 20) coronary artery disease (n = 16) hypertrophic cardiomyopathy (n = 12) or dilated cardiomyopathy (n = 21). Further studies will be needed to determine to what extent the polymorphisms described are associated with cardiovascular disease.

Amino Acid Sequence↗
PubMed1994

Guillain-Barré syndrome in pregnancy: reflections on immunopathogenesis.

Here we describe the case of a 29-year-old woman, 36 weeks pregnant, with severe Guillain-Barré syndrome who delivered by Caesarean section because of respiratory insufficiency. The detection of high antibody titers to glycolipids of human peripheral nerve myelin in both the mother and the newborn, as well as differences regarding lymphocyte typing and levels of complement components, give rise to some reflections on the immunopathogenesis of this disorder supporting the hypothesis that cell-mediated immunity is the major mechanism in the development of Guillain-Barré syndrome.

Adult↗
PubMed1994

Guillain-Barré syndrome in pregnancy--an indication for caesarian section?

Guillain-Barré syndrome (GBS) in pregnancy is a rare neurological disorder which confronts the obstetrician with a difficult decision regarding the management of pregnancy and delivery. Two case reports illustrate the problems of obstetrical and neurological patient management. We thereby discuss indications for intervention based on the reciprocal influences of GBS and pregnancy. In acute GBS intervention by induction of labor or caesarian section seems to provoke deterioration of the patient's condition and delayal of recovery.

Adult↗
PubMed1994

Fully-automated, nonradioactive solid-phase sequencing of genomic DNA obtained from PCR.

Nonradioactive sequencing in combination with solid-phase template purification is a powerful method for sequence analysis, especially of PCR-generated fragments. To perform DNA sequencing under optimal conditions, it is necessary to obtain a well-purified and single-stranded (ss) DNA template. The disadvantage of ssDNA sequencing is that additional steps are required to generate ssDNA templates before any sequencing reactions can be carried out and is thus more time-consuming. Here we describe an automated solid-phase system for direct sequencing of ssPCR products that incorporates magnetic beads coated with streptavidin as solid support and a computer-controlled device (PolySeq) with heating, magnetic and mixing functions, all of which are integrated into a robotic workstation (Biomek 1000). This solid-phase method is extremely useful for rapid template purification and strand separation of DNA obtained from PCR as well as for high-quality dideoxyribonucleotide chain termination sequencing with fluorescently labeled primers. The system allows the complete automation of the sequencing procedure starting with PCR amplicons. DNA sequencing results obtained with this system were reproducible and gave an excellent length of readable sequences with low background and an analysis capacity of 30,000-40,000 bp per week.

Base Sequence↗
PubMed1993

Molecular cloning and primary structure of Man9-mannosidase from human kidney.

Man9-mannosidase, a processing enzyme found in the endoplasmic reticulum (ER), catalyses the removal of three distinct mannose residues from peptide-bound Man9-GlcNAc2 oligosaccharides producing a single Man6 isomer [Bause, E., Breuer, W., Schweden, J., Roesser, R. & Geyer, R. (1992) Eur. J. Biochem. 208, 451-457]. We have isolated four Man9-mannosidase-specific clones from a human kidney cDNA library and used these to construct a full-length cDNA of 3250 base pairs. A single open reading frame of 1875 nucleotides encodes a protein of approximately 71 kDa, consistent with data from immunological studies. Analysis of the coding sequence predicts that Man9-mannosidase is a type II transmembrane protein consisting of a short cytoplasmic polypeptide tail, a single transmembrane domain acting as a non-cleavable signal sequence and a large luminal catalytic domain. This domain architecture closely resembles that of other ER and Golgi-located processing enzymes, pointing to common structural motifs involved in membrane insertion and topology. The protein sequence of the Man9-mannosidase contains three potential N-glycosylation sites of which only one site is used. The amino acid sequence of several peptide regions, including a calcium-binding consensus sequence, bears striking similarities to an ER alpha-1,2-mannosidase from yeast, whereas, by contrast, no sequence similarity was detectable with rat liver ER alpha-mannosidase and Golgi alpha-mannosidase II. This finding may indicate that the mammalian alpha-mannosidases, which differ significantly in their substrate specificity, are coded for by evolutionarily unrelated genes, providing an attractive means of regulation and fine-tuning oligosaccharide processing, not only at the enzymic but also at the transcriptional level.

Amino Acid Sequence↗
PubMed1993

Rapid detection of cytomegalovirus structures in cerebrospinal fluid and brain biopsies by polymerase chain reaction.

The joint application of stereotactic brain biopsy and DNA amplification methods, like the polymerase chain reaction (PCR) are diagnostic purposes that allows in approximately 24 h to clarify the viral etiology of central nervous system infections. We demonstrate PCR results from the brain biopsy of an HIV-1 patient with two cerebral lesions. Beside the neuropathological diagnosis of a B-cell non-Hodgkin lymphoma the experimental results are indicative of active transcription of cytomegalovirus (CMV) in cerebral tissue. The markedly higher amount of CMV-specific RNA found in the biopsy specimens and CSF suggest the presence of an active infection with viral replication. CT-guided stereotactic brain biopsies followed by PCR fulfill the demands of a clinician, namely to get the results as fast as possible to initiate adequate treatment.

Adult↗
PubMed1992

Stereotactic brain biopsy in AIDS.

In the hope of finding a treatable condition, the need for rapid diagnosis in HIV-seropositive patients with brain lesions is apparent. In order to evaluate the efficacy of stereotactic brain biopsy in AIDS patients, we retrospectively studied 25 HIV-infected patients undergoing stereotactic biopsy. Brain lesions were identified with gadolinium-enhanced MRI and/or contrast CT. Brain biopsy was performed using the system of Riechert. From 8 up to 15 small tissue samples from one or two targets were obtained in every patient. The biopsy material was examined cytologically, histologically (including electron microscopy), immunohistochemically and, in part, by animal test and polymerase chain reaction (PCR). A definite diagnosis was achieved in 92%. Diagnosis included primary central nervous system lymphoma (PCNSL) (10), toxoplasmosis (10), progressive multifocal leukoencephalopathy (2) and one case of co-existing toxoplasmosis and cytomegalovirus infection. Two biopsies were non-diagnostic. All PCNSLs showed polymorphic B-cell populations of high malignancy; accurate classification according to the Kiel classification was not possible. In 3 lymphomas Epstein-Barr nuclear antigen (EBNA) 2-mRNA could be detected by PCR and confirmed immunohistochemically by EBNA 2 expression. In 6 cases autopsy confirmed the biopsy diagnosis. Conventional histology was not sufficiently decisive for toxoplasmosis and progressive multifocal leukoencephalopathy, so that immunohistochemistry and animal tests became very important for a final diagnosis. With the help of different morphological and molecular biological techniques stereotactic brain biopsy appears to be an effective method in the diagnosis of HIV-associated brain lesions. In view of the marked radio- and chemosensitivity of PCNSLs it is mandatory to establish an early and accurate histological diagnosis for adequate treatment.

Acquired Immunodeficiency Syndrome↗
PubMed1989

[Borrelia burgdorferi-induced pseudolymphoma with pathogen cultivation in an HIV-1 positive patient].

Cutaneous symptoms and skin diseases are common findings in almost all HIV-1-positive patients. In many cases the clinical presentation and course of the skin diseases are atypical, and occasionally the development of the appropriate circulating antibodies is lacking or impaired. In this report we present a patient seen in our multidisciplinary outpatient clinic for HIV patients. This patient had a Borrelia burgdorferi infection with an unusual course. The acute inflammatory phase of the arthropod reaction was maintained over a period of 9 months with development into pseudolymphoma showing unusual cytological characteristics. Immunohistological evaluation revealed an almost complete lack of T-helper and Langerhans cells, but an increased number of activated cytotoxic cells with class II antigen expression. A marked serological response was observed on IgG-ELISA and in the IgG-immunofluorescence test. Borrelia burgdorferi was cultured in vitro from a skin biopsy of the involved area. To our knowledge this is the first reported case of skin borreliosis in an HIV-1-positive patient.

Bacteriological Techniques↗