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Biomedical subjects

M Volkenandt

Publications and source records attributed to M Volkenandt.

105 records · Page 6Linked to original sources
PubMed1991

[Polymerase chain reaction with subsequent direct gene sequence analysis. A possibility for molecular analysis in dermatology].

Molecular analyses are of increasing importance in clinical research as well as for diagnostic evaluations. Gene alterations are involved in the pathogenesis of numerous diseases and are described in an increasing number of conditions. A method is presented which allows for rapid and detailed nucleotide sequence analysis of a segment of a gene. Buccal epithelial cells of a patient are obtained by means of a mouth wash with 10 ml water. DNA molecules are amplified in vitro using the polymerase chain reaction, followed by direct sequence analysis of the product. Using this technique, a sequence analysis of a gene segment is possible within 1-2 days. As an example, the analysis of a segment of the gene for uroporphyrinogen decarboxylase of patients with cutanea tarda is described.

Base Sequence↗
PubMed1991

[Polymerase chain reaction--principle and application in medicine].

The polymerase chain reaction, a new molecular technique, is of increasing importance in many areas of medical research and diagnosis. Within hours DNA sequences can be amplified millionfold with very high specificity, making detection and further analysis possible. Using multiple rounds of exponential amplification, even one copy of a gene of interest can be detected by agarose gel electrophoresis and other methods. Furthermore, minute amounts of DNA even from tissue damaged by embedding in paraffin can be analysed. Polymerase chain reaction methodology has already gained significance in many areas of medical research, e.g. diagnosis of inherited diseases, detection of viral DNA in clinical samples, cancer research and diagnosis, and characterization of gene expression.

DNA, Viral↗
PubMed1990

Identification and characterization of a mutation in the dihydrofolate reductase gene from the methotrexate-resistant Chinese hamster ovary cell line Pro-3 MtxRIII.

A methotrexate-resistant Chinese hamster ovary cell line (Pro-3 MtxRIII), resistant due to a low-level amplified, altered target enzyme, dihydrofolate reductase (DHFR), has been characterized on the molecular level. The cDNA and coding regions of all six DHFR exons were amplified in vitro using Taq polymerase and directly sequenced. Analysis of the Pro-3 MtxRIII DHFR cDNA demonstrated a C----T base transition at nucleotide 67 that results in the substitution of phenylalanine for leucine at residue 22 and the loss of a BsaI site in the Pro-3 MtxRIII cDNA. This mutation results in a decreased binding of methotrexate to the altered enzyme. Molecular modeling of Leu22----Phe supports the concept of the importance of Leu22 in the active site of the enzyme and indicates that replacement with phenylalanine will decrease the binding of methotrexate.

Amino Acid Sequence↗
PubMed1990

Mutational analysis of human NRAS genes in malignant melanoma: rapid methods for oligonucleotide hybridization and manual and automated direct sequencing of products generated by the polymerase chain reaction.

Three methods to detect single base mutations in codon 61 of the human NRAS gene from human melanoma DNA are described and compared: oligonucleotide hybridization analysis and direct manual and automated sequence analysis. Point mutations are detected by oligonucleotide hybridization and direct manual and direct automated sequence analysis of in vitro amplified genomic DNA. Heterozygosity for mutant alleles is reliably detected by oligonucleotide hybridization and by direct manual, but not by direct automated, sequence analysis. Generating single-stranded DNA via "asymmetric polymerase chain reaction (PCR)" and utilizing alpha 35S-dATP as radiolabel for manual sequencing and fluorescent-dye labeled primers for automated sequencing (Applied Biosystems, Inc.), we can obtain sequence information from either strand. The use of several of these methodologies to detect single base changes in the human NRAS gene is illustrated. In addition, the use of these and other related techniques to define the involvement of RAS oncogenes in human melanomas more precisely is reviewed.

Automation↗
PubMed1989

[Pain in tumor patients and indications for psychopharmaceutic agents].

Pain involves both somatic and psychological processes, and often requires in cancer patients a multidisciplinary management approach. Psychotropic medications can be helpful as part of adjuvant pharmacological treatment of pain and can be beneficial especially in cancer patients. Anxiety and depression, which may be associated with cancer, can influence the pain experience and can be managed symptomatically with psychotropic drugs. In addition, some psychotropic drugs have analgetic properties, or may be used to reduce some side effects of narcotics.

Anxiety↗
PubMed1989

[Suicide and cancer].

Epidemiological studies demonstrate that cancer patients are at increased risk of suicide. While affective illness and alcoholism are the most important determinants of suicide in the physically healthy population, vulnerability to suicide in cancer patients is influenced by a number of other factors including psychosocial and psychosomatic effects of advanced illness, pain, organic mental syndromes and preexisting psychopathology. Ways of influencing these risk factors are discussed, together with their use in preventive care and management of suicidal cancer patients.

Humans↗
PubMed1989

Detection of a single base mutation in the human dihydrofolate reductase gene from a methotrexate-resistant cell line using the polymerase chain reaction.

To test the utility of the polymerase chain reaction in identifying single base mutations in a gene known to give rise to an altered enzyme and drug resistance phenotype, a human colon adenocarcinoma cell line resistant to methotrexate, with a known single base mutation (Srimatkandada et al., J. Biol. Chem. 264:3524, 1989) was examined. Poly A+ RNA was used for cDNA synthesis with reverse transcriptase, deoxynucleoside triphosphates, and 5 microM 3' primer that anneals outside the coding region of the human dihydrofolate reductase. The RNA:DNA hybrid was used as a template for the polymerase chain reaction with the addition of a 5' primer and Thermus aquaticus (Taq)I DNA polymerase. These primers flank the coding region of the human dihydrofolate reductase and define a region of 650 bases. The polymerase chain reaction was carried out for 40 cycles resulting in full length transcripts in microgram amounts clearly visible by ethidium bromide staining on agarose gels. DNA was isolated by standard methods, and double-stranded DNA was sequenced by the chain-termination method using TaqI DNA polymerase. A single point mutation was discovered at position 91 (T----C) resulting in a substitution of serine for phenylalanine at codon 31, as determined previously by classical cDNA cloning and sequencing. Sequence analysis indicated that this base transition resulted in the loss of Eco RI and Xmn I sites and the gain of a HinfI site in the cDNA, which were confirmed by restriction digests.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenocarcinoma↗
PubMed1989

Sequence analysis of a human gene responsible for drug resistance: a rapid method for manual and automated direct sequencing of products generated by the polymerase chain reaction.

We developed a system for rapid, manual and automated sequence analysis by utilizing and modifying methods used in conjunction with the polymerase chain reaction (PCR). We are using these techniques to detect single base mutations in the dihydrofolate reductase (DHFR) gene giving rise to methotrexate (MTX) resistance of tumor cells obtained from patients with malignancies. Amplifying in vitro both genomic DNA and transcripts of the human DHFR we are able to reproducibly generate single-stranded templates. Utilizing [alpha-35S]dATP and both the universal and reverse sequencing primers we obtain sequence information from either strand. The methods described have been successfully used for automated sequencing with the Applied Biosystems Model 370A Sequencer using both modified T7 DNA polymerase and Taq I. DNA polymerase for dideoxy-termination sequencing. The use of this methodology to detect a single base change in a human colon carcinoma cell line, HCT-8, is illustrated.

Adenocarcinoma↗
PubMed1983

Pharmacokinetics of isosorbide-5-mononitrate after oral and intravenous administration in patients with liver cirrhosis: first results.

The pharmacokinetics and bioavailability of IS-5-MN after single-dose oral and intravenous application were investigated over 24 h in three patients with liver cirrhosis and compared with normal subjects. The analysis of IS-5-MN in plasma was carried out using a gas-chromatographic method with electron capture detection. A two-compartment open model was taken as a basis for the calculations of the plasma concentration curves and the pharmacokinetic parameters. First results show that plasma concentrations after intravenous administration in the patient group were not higher than in the control group. After oral administration the peak concentrations of IS-5-MN were also no higher, nor did they occur earlier, in patients with liver cirrhosis than in the normal subjects. Our data also show the same complete absolute bioavailability of IS-5-MN in the patient group as in normal subjects, and the same elimination half-life of the plasma concentration response curve. We saw no influence of typical comedication, i.e., cimetidine or spironolactone on pharmacokinetic parameters in the group of liver patients.

Administration, Oral↗
PubMedDate not supplied

Polymerase chain reaction control of antibiotic treatment in dermatoborreliosis.

Assessment of the efficacy of an antibiotic drug used in patients with various manifestations of dermatoborreliosis is crucial. Clinical judgement alone (resolution of the present dermatologic lesion, prevention of later major or minor sequelae) is not sufficient in erythema migrans and acrodermatitis chronica atrophicans. Thus, laboratory tests are desirable to prove the benefit of an antimicrobial agent. It was intended to establish a constant parameter--besides the clinical picture--for assessing the efficacy of antibiotic treatment in patients with dermatoborreliosis in terms of eradication of Borrelia burgdorferi from the site of infection. Polymerase chain reaction (PCR) was therefore performed from pretreatment biopsy specimens from lesional skin of 36 erythema migrans patients (m:f = 15:21, mean age 49 years) and seven acrodermatitis chronica atrophicans patients (m:f = 0:7, mean age 59 years), respectively. After antibiotic therapy with minocycline (100 mg, orally twice daily, 14 days) for erythema migrans, and ceftriaxone (2 g, intravenously once daily, 14 days) for acrodermatitis chronica atrophicans another punch biopsy was obtained and analysed by PCR. In pretreatment specimens, B. burgdorferi-specific DNA was amplified by PCR in 23/36 erythema migrans patients (69%), and in 5/7 acrodermatitis chronica atrophicans patients (71%). After antibiotic therapy, PCR yielded negative results in all of these cases. Clinically, all patients showed complete recovery or at least marked improvement of lesions at this time. PCR appears to be a reliable parameter for the assessment of the efficacy of antibiotic treatment in dermatoborreliosis.

Acrodermatitis↗