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Biomedical subjects

D Werner

Publications and source records attributed to D Werner.

At least 127 records · Page 7Linked to original sources
PubMed1985

Purification of a phospholipase B inhibitor from Saccharomyces cerevisiae.

The phospholipase B activity of plasma membrane vesicles from Saccharomyces cerevisiae is inhibited by the 100 000 X g supernatant of mechanically disrupted yeast cells. A 1850-fold purification of the inhibitor activity was achieved by gel filtration, ion exchange chromatography with DEAE-cellulose and hydrophobic interaction chromatography with Octyl-Sepharose. SDS-polyacrylamide gel electrophoresis of the purified inhibitor revealed two main bands with an apparent Mr of 60 000 and 26 500. The phospholipase B activity was strongly reduced but not completely blocked by this preparation, while the lysophospholipase and transacylase reactions, which are catalyzed by the same membrane-bound enzymes (Witt, W. et al. (1984) Biochim. Biophys. Acta 795, 108-116), were not affected.

Cell Membrane↗
PubMed1985

Screening of isolated DNA for sequences released from anchorage sites in nuclear matrix.

Isolated chromosomal DNA is associated with polypeptides that are not released from DNA by several methods designed to purify DNA, e.g. treatment with sodium dodecyl sulphate. DNA fragments associated with these very tight DNA/protein complexes show high affinity to nitrocellulose filters in the presence of salt concentrations of 500 mM or greater. Consequently, a fraction of AluI-fragmented native DNA comprising the complexes and 0.2 to 0.3 micron of vicinal DNA can be isolated by one filtration step. This fraction of DNA shows characteristics of residual DNA sequences retained in nuclei after extraction with nucleases and high salt (nuclear matrix). The DNA fragments retained on filters are highly enriched in replicative DNA; and their degree of hybridization with poly(A)+ RNA points to enrichment in actively transcribed sequences. The results support previous work indicating that the very tight DNA/polypeptide complexes co-isolating with DNA under conditions that release other peptide materials from DNA may be anchorage sites of DNA in the nuclear matrix. Moreover, the method described here allows isolation of replicating and actively transcribed DNA sequences directly from isolated total genomic DNA by skipping artefact-prone isolations of the nuclear matrix.

Animals↗
PubMed1985

Acute hemolytic anemia caused by severe hypophosphatemia in diabetic ketoacidosis.

Hypophosphatemia in diabetic ketoacidosis is well recognized, but is believed to be usually of moderate severity. We describe 2 patients in whom acute hemolytic anemia secondary to severe (0.19-0.35 mmol/l) hypophosphatemia has developed 1-2 days following treatment for diabetic ketoacidosis. Our experience indicates that severe hypophosphatemia requiring phosphate supplementation does occur in diabetic patients, and calls for increased awareness for the clinical and laboratory manifestations of this complication of diabetic ketoacidosis.

Acidosis↗
PubMed1984

DNA polymerases in isolated 'nuclear matrix' of Ehrlich ascites cells.

Isolated nuclei from Ehrlich ascites tumor cells continue a replicative-like in vitro DNA synthesis. Polymerase alpha is the major dNTP polymerizing enzyme in nuclei. Following complete achromatinization dNTP polymerizing activities are still associated with the residual structure termed 'nuclear matrix'. In contrast to DNA synthesis in native nuclei, 'nuclear matrix' DNA synthesis is mainly due to polymerase beta-like activity.

Animals↗
PubMed1984

Hodgkin's disease in children. A ten-year experience in South Africa.

Twenty-seven children with Hodgkin's disease were treated with MVOPP (mustine, Velban [vinblastine], Oncovin [vincristine], procarbazine, prednisone) combination chemotherapy. All 11 children with Stage I or Stage II disease achieved complete remission and no relapses have occurred between 34 and 179 months of continuous follow-up. Of the 16 children with Stage III or Stage IV disease, two with partial remission and three nonresponding patients died with a median survival of 18 months. The remaining 11 (68%) achieved complete remission. Of these, 6 (55%) have relapsed with a median remission duration of 36 months; five have been retreated with MVOPP, and one with total nodal irradiation, and all achieved second complete remission. Three of the children have died of Hodgkin's disease, with a median survival of 55 months; one child died of acute myeloblastic leukemia while in remission; and one child remains disease-free off all therapy at 165 months; the sixth child treated with total nodal irradiation is disease-free at 166 months. The initial complete remission has been sustained in all 11 children with Stage I or Stage II Hodgkin's disease, suggesting that combination chemotherapy is an alternative to radiotherapy as the initial form of treatment in this group of individuals.

Adolescent↗
PubMed1984

Highly sensitive sites for guanine-O6 ethylation in rat brain DNA exposed to N-ethyl-N-nitrosourea in vivo.

Brain chromosomal DNA isolated from fetal BDIX-rats 1 h after i.v. administration of the ethylating N-nitroso carcinogen N-ethyl-N-nitrosourea (75 micrograms/g body weight), statistically contained one molecule of O6-ethyl-2'-deoxyguanosine (O6-EtdGuo) per 81 micron of DNA, as determined in enzymatic DNA hydrolysates by competitive radio-immunoassay using a high-affinity anti-(O6-EtdGuo) monoclonal antibody (ER-6). After fragmentation of the DNA by the restriction enzyme AluI (average fragment length, Lav = 0.28 micron = 970 bp; length range, Lr = 1.87-0.02 micron = 6540 - 60 bp), a small (approximately 2%) fraction of DNA enriched in specific polypeptides tightly associated with DNA was separated from the bulk DNA by a glass fiber binding technique. As analyzed by immune electron microscopy, approximately 1% of the DNA molecules in this fraction contained clusters of 2-10 (O6-EtdGuo)-antibody binding sites (ABS). On the cluster-bearing fragments (Lav, 0.85 micron +/- 0.50 micron S.D.; corresponding to 2970 +/- 1760 bp) the average ABS-ABS interspace distance was 110 nm (= 390 bp; range approximately 9-600 nm), indicating a highly non-random distribution of O6-EtdGuo in target cell DNA.

Alkylating Agents↗
PubMed1984

Isolation of poly(A)+ RNA by paper affinity chromatography.

Poly(A)+ RNA was isolated from in vitro short-term-labeled total cytoplasmic RNA of Ehrlich ascites tumor cells by oligo(dT) cellulose chromatography. This poly(A)+ RNA fraction was compared with a poly(A)+ RNA fraction isolated by a new procedure which involves specific binding of poly(A)+ RNA to messenger affinity paper (mAP) and its release in hot (70 degrees C) water. In typical experiments 10-11 micrograms (2.3%) of poly(A)+ RNA can be retained from 500 micrograms of total cytoplasmic RNA per cm2 of mAP in a quick one-step procedure. The poly(A)+ RNA preparations isolated by the two methods proved to be almost identical with respect to their fraction in total cytoplasmic RNA, specific radioactivities, sucrose gradient profiles, and translation assays. Since the isolation of poly(A)+ RNA by mAP is much less time consuming than that by oligo(dT) column chromatography and since the poly(A)+ RNA can be recovered from mAP in small volumes, which avoids further loss during precipitations, it can be advantageously used for preparative isolation of poly(A)+ RNA.

Animals↗
PubMed1984

Antibodies to the most tightly bound proteins in eukaryotic DNA. Formation of immuno-complexes with 'nuclear matrix' components.

Chromosomal DNA is associated with polypeptides covalently bound to internal DNA ends. Since these polypeptides can only be released from chromosomal DNA by enzymes or other agents hydrolysing phosphodiester bonds they were termed 'the most tightly bound' (MTB) polypeptides in DNA. Antibodies developed against the MTB polypeptides are shown to form immunocomplexes with major 'nuclear matrix' polypeptides as well as with polypeptides which are still associated with 'nuclear matrix' DNA isolated by means of SDS/proteinase K and phenol. Immuno-complex formation is revealed by immunoblotting and by indirect immunofluorescence. Thus, since MTB polypeptides, major 'nuclear matrix' polypeptides and 'nuclear matrix' DNA-associated polypeptides share common antigenic sites they can be considered to be identical or at least closely related. This suggests that a fraction of distinct 'nuclear matrix' polypeptides is either transiently or permanently linked to DNA by covalent bonds. Consistently, isolated eukaryotic 'bulk' DNA is inevitably associated with residual 'nuclear matrix' polypeptides.

Animals↗
PubMed1984

Effects of dietary sucrose on factors influencing cholesterol gall stone formation.

Twelve subjects with radiolucent gall stones and bile supersaturated with cholesterol were studied after six weeks each on diets which contained 112 g and 16 g respectively of refined (fibre depleted) sucrose but which allowed free access to other foods. Energy intake was 24.5% higher on the high sugar diet and body weight ended 1.4 kg higher than on the low sugar diet. Biliary secretion rates of cholesterol, phospholipid and bile acid, measured by a perfusion technique, were similar on the two diets, as were the bile acid pool sizes measured by isotope dilution. Cholesterol saturation index of fasting 'gall bladder' bile was 1.30 +/- 0.11 and 1.37 +/- 0.14 on high and low sugar respectively (NS). Plasma triglycerides were 36% higher and plasma high density lipoprotein cholesterol concentrations were 9% lower on the high sugar diet. These findings indicate that over a six week period refined sugar in normally consumed amounts does not adversely affect the lipid composition of bile.

Adult↗
PubMed1983

Phosphodiester bonds between polypeptides and chromosomal DNA.

Polypeptides co-purifying with DNA in alkali are covalently bound to DNA. DNA purified by treatment with alkali, sodium dodecyl sulphate and phenol absorbed 125I under conditions designed to radioiodinate exclusively tyrosine and histidine in peptides. A significant amount of the absorbed 125I remained associated with DNA during treatment with phenol as well as during precipitation with ethanol from neutral and alkaline solutions. However, after prolonged digestion with proteinase K, most of the radiolabelled material could be removed from 125I-treated DNA. Further treatment with a second protease (Pronase) released no larger fraction of the 125I label. The residual radiolabelled material could be precipitated together with DNA by ethanol and it remained associated with DNA also in the presence of alkali (95 degrees C), acid (37 degrees C) and hydroxylamine (37 degrees C). In contrast, radiolabelled peptides were released from DNA by treatment with hot piperidine (10% at 95 degrees C) and by agents that hydrolyse peptides and modify DNA, e.g. strong acid (95 degrees C) and formic acid/diphenylamine. The radiolabelled peptides, once released from DNA by these chemical methods, could be further cleaved by Pronase. This shows that the residual DNA/peptide complex isolated after prolonged protease digestion is protease-resistant unless it is cleaved or otherwise modified by harsh chemical treatment. The linking groups between deoxynucleotides and the radiolabelled residual peptides could be isolated by digestion of DNA in the DNA/peptide complex. Radiolabelled peptides could be released from this linking group material by phosphodiesterases, indicating the involvement of phosphodiesters in the linking groups.

Acids↗
PubMed1983

Fragmentation of 'nuclear matrix' on a mica target.

Chromatin depleted nuclei ('nuclear matrix') of Ehrlich ascites cells were characterized and fragmented by glycerol shot technique (particle fragmentation). The preparations reveal that 'nuclear matrix' is entirely composed of granules and fibres. Prominent size classes of granules are 10 to 20 nm and 25 to 40 nm, respectively. Most of the granules remain attached to fibres during the fragmentation process. The diameter of the fibres corresponds with double-stranded DNA visualized under identical conditions. The RNP-like nature of the particles is shown by their proteinase K/RNase sensitivity. Since the 'nuclear matrix' architecture becomes instable in high salt buffer after pretreatment with RNase which changes the RNP-particle-like material it must be inferred that the RNP/DNA interaction is a prerequisite for the high salt stability of the 'nuclear matrix' complex.

Animals↗
PubMed1982

A comparison of diazepam and phenoperidine in premedication for upper gastrointestinal endoscopy: a randomized double blind controlled study.

A variety of agents are used as premedication for upper gastrointestinal endoscopy (U.G.E.). To our knowledge, no double blind studies have been performed to compare their value. In this study phenoperidine (2 mg i.v.) was compared with diazepam (t mg i.v.) in 200 consecutive patients undergoing elective U.G.E. The study was randomized and double blind in regard to both endoscopists and patients. All patients were given atropine (0.4 mg i.v.) and a throat spray with 2% amethocaine. Patients who needed supplemental medication were given diazepam and excluded from final analysis. A graded questionnaire was recorded by endoscopists and patients after U.G.E., and a further anonymous questionnaire was returned by patients four days later. Statistical analysis revealed that phenoperidine was superior at facilitating intubation and providing more relaxation as judged by the endoscopist. Patient questionnaires, four days after U.G.E., indicated less distress during intubation and examination with phenoperidine. Nausea, vomiting, amnesia and phlebitis were uncommon after either phenoperidine or diazepam.

Clinical Trials as Topic↗