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K A Webster

Publications and source records attributed to K A Webster.

68 records · Page 4Linked to original sources
PubMed1988

Adenovirus E1A products suppress myogenic differentiation and inhibit transcription from muscle-specific promoters.

The primary function of the adenovirus E1A-region genes is to activate other adenoviral genes during a permissive viral infection by modifying the host cell transcriptional apparatus. Host cell immortalization, or transformation by the whole adenoviral early region, presumably results as a consequence of these modifications. Both transcriptional activation and transcriptional repression of non-adenoviral genes by the E1A proteins have been reported. It is currently not clear which, if either, of these activities contributes to host cell transformation and immortalization. Although there may be a physiological impact of some E1A-stimulated host cell genes, in many cases the functional significance is unclear. No common target sequences have been recognized in stimulated cellular genes and it has recently been proposed that in many cases, particularly involving newly transfected genes, available 'TATA-box' sequences may be the opportunistic beneficiaries of E1A assistance as a secondary consequence of E1A primary functions within the host cell nucleus. E1A-mediated transcriptional repression appears to be a more specific process insofar as common core elements are shared by the E1A-suppressed SV40, polyoma B, IgG heavy-chain and insulin enhancers. In the present communication we report that the complete myogenic programme of L8 and C2 myoblasts can be blocked by the introduction of constitutively expressing E1A genes, and show that the transcriptional induction of muscle-specific genes is inhibited. In particular, the promoter-inducing activities of well-defined elements that are required for the muscle-specific expression of the two sarcomeric alpha-actins, and which normally bind cellular trans-acting factors, become targets for E1A suppression. The results support the hypothesis that the suppression of differentiation by E1A products is effected by an E1A-mediated block in the transcriptional activation of cellular genes by specific developmentally regulated cis-acting promoter elements.

Adenovirus Early Proteins↗
PubMed1988

Use of counter immunoelectrophoresis in detection of antibodies to tickborne fever.

A micromethod using the counter immunoelectrophoresis technique for the detection of antibodies to Cytoecetes phagocytophila, the causative agent of tickborne fever (TBF) is described. Antibodies were first detected nine to 11 days after experimental infection of lambs with TBF and persisted for six to 10 weeks after infection. The test was also applied to approximately 440 field samples of ovine sera collected from tick infested farms in Scotland and the north of England, 16 per cent of which proved positive and showed a marked seasonal variation.

Animals↗
PubMed1987

Regulation of glycolytic enzyme RNA transcriptional rates by oxygen availability in skeletal muscle cells.

Cytoplasmic beta-actin and five glycolytic enzyme cDNAs were isolated from a rat skeletal muscle cDNA library and together with a genomic clone of rat cytochrome c were used as probes to quantitate the respective RNA transcription rates in isolated nuclei run off transcription assays from stationary cells cultured under normal or 2% oxygen. The transcription rates of lactate dehydrogenase, pyruvate kinase, triosephosphate isomerase and aldolase increased by 2-5 fold during the 72 hr exposure to 2% oxygen. There was a small increase in actin RNA transcription while both cytochrome c and glyceraldehyde-3-phosphate dehydrogenase RNA transcription rates decreased. Since previous studies demonstrated an increase in steady state glyceraldehyde-3-phosphate dehydrogenase RNA during low O2 exposure it is concluded that the level of this RNA is regulated post transcriptionally whereas the other four glycolytic enzyme RNAs are regulated at least partially at the level of transcription by oxygen availability. The relative transcriptional rates of the RNAs in this study are related to their cellular RNA and protein concentrations.

Actins↗
PubMed1987

Sequence analysis of cDNAs for the human and bovine ATP synthase beta subunit: mitochondrial DNA genes sustain seventeen times more mutations.

We have cloned and sequenced human and bovine cDNAs for the beta subunit of the ATP synthase (ATP-syn beta), a nuclear DNA (nDNA) encoded oxidative phosphorylation (OXPHOS) gene. The two cDNAs were found to share 99% amino acid homology and 94% nucleotide homology. The evolutionary rate of ATPsyn beta was then compared with that of two mitochondrial DNA (mtDNA) ATP synthase genes (ATPase 6 and 8), seven other mtDNA OXPHOS genes, and a number of nuclear genes. The synonymous substitution rate for ATPsyn beta proved to be 1.9 x 10(-9) substitutions per site per year (substitutions x site-1 x year-1) (SSY). This is less than 1/2 that of the average nDNA gene, 1/12 the rate of ATPase 6 and 8, and 1/17 the rate of the average mtDNA gene. The synonymous and replacement substitution rates were used to calculate a new parameter, the "selective constraint ratio". This revealed that even the most variable mtDNA protein was more constrained than the average nDNA protein. Thus, the high substitution mutation rate and strong selective constraints of mammalian mtDNA proteins suggest that mtDNA mutations may result in a disproportionately large number of human hereditary diseases of OXPHOS.

Amino Acid Sequence↗
PubMed1982

Assignment of an oligomycin-resistance locus to human chromosome 10.

An oligomycin-resistant variant of human fibrosarcoma HT1080 was isolated and characterized as nuclear and codominant. The mutant was stable, was not cross-resistant to respiratory inhibitors, and it contained a mitochondrial ATPase which was less sensitive to oligomycin. Hybrids formed between the human mutant and a mouse cell line expressed the resistance phenotype. By a detailed karyotypic analysis of these hybrids using trypsin-Giemsa banding it was found that resistance to oligomycin correlated with the retention of two human chromosomes 10. The hybrid lines contained only mouse mitochondrial DNA as shown by analyses of mitochondrially synthesized proteins and mitochondrial DNA. The study assigns an ATPase oligomycin-resistance locus to human chromosome 10 and suggests that mouse and human subunits can combine in a functional enzyme complex.

Adenosine Triphosphatases↗
PubMed1980

Hydrophobic interaction between the monomer of mitochondrial malate dehydrogenase and phospholipid membranes.

Porcine mitochondrial malate dehydrogenase (EC 1.1.1.37) dissociates into subunits on dilution. The enzyme monomer caused large increases in the surface pressure of monolayers of 1:1 phosphatidylserine/phosphatidylcholine at air/water and oil/water interfaces. The monomer increased the permeability of phospholipid vesicles to 22Na+. Both effects were significantly greater than the corresponding effects of ribonuclease A, cytochrome c and the dimeric form of malate dehydrogenase. Changes in the circular-dichroism spectra of the enzyme indicated that conformational changes may be associated with dimer formation or when monomer interacts with lysophosphatidyl-choline. Similar interactions to those described may occur in situ when mitochondrial malate dehydrogenase is transported to the mitochondrial matrix from its site of synthesis on cytosolic ribosomes.

Animals↗
PubMed1979

Interaction of mitochondrial malate dehydrogenase monomer with phospholipid vesicles.

The association between bovine and porcine mitochondrial malate dehydrogenase (EC 1.1.1.37) and phospholipid vesicles was investigated. At concentrations at which malate dehydrogenase exists as a dimer, entrapment within the aqueous compartment but not binding of the 14C-labelled enzyme was observed. The dissociated enzyme was labile to moderate heat and to p-chloromercuribenzoate, but in both cases inactivation was decreased by incubation with suspensions of charged phospholipid vesicles. This suggested an interaction between enzyme subunits and phospholipid, and this was confirmed by direct binding measurements and by studies that followed changes in the fluorescein-labelled enzyme. The circular-dichroism spectra of the enzyme indicated a high alpha-helix content, and suggested that a small conformational change occurred when the enzyme dissociated. Fluorescence data also suggested less-rigid molecules after dissociation. A possible mechanism, based on the flexibility of enzyme monomer and its interaction with phospholipids, by which mitochondrial matrix enzymes are specifically localized in cells, is discussed.

Animals↗
PubMed1979

Binding of mitochondrial malate dehydrogenase to mitoplasts.

The binding of 14C-labelled bovine and porcine malate dehydrogenase (EC 1.1.1.37) to rat liver mitochondria and mitoplasts was examined. The bovine enzyme was found to associate nonspecifically with isolated mitochondria and sonicated mitoplasts. Scatchard plot analysis suggested a specific binding to mitoplasts of the order of 5 pmol malate dehydrogenase per milligram of mitoplast protein. Porcine malate dehydrogenase dimer but not monomer exhibited a similar binding. The results are discussed in relation to the mechanism of uptake of the enzyme by mitochondria after synthesis on cytosolic ribosomes.

Animals↗
PubMed1978

Ion movements during energy-linked mitochondrial structural changes.

The structure of isolated rat liver mitochondria has been observed in the electron microscope following incubation of the mitochondria in vitro under a variety of conditions. The results show that ultrastructural changes are only associated with the energization and deenergization of isolated mitochondria if the composition of the incubation medium permits ion movements in or out of the matrix. The mechanism of energy coupling does not appear to depend on these major mitochondrial structural changes. The addition of low levels of valinomycin greatly increases the rate at which the matrix compartment swells and shrinks on energization and deenergization even at low K+ concentrations.

Adenosine Diphosphate↗
PubMedDate not supplied

Missing data in quality of life research in Eastern Cooperative Oncology Group (ECOG) clinical trials: problems and solutions.

Incorporation of quality of life (QOL) investigation into Eastern Cooperative Oncology Group (ECOG) multi-centre clinical trials has led to innovative strategies for protocol design and high quality data collection. A scientific advisory committee reviews protocol design components, measurement selection, timing of assessments and compliance issues. Extensive educational programmes provide information about the scientific and clinical relevance of QOL protocols, as well as practical strategies for data collection and management. Compliance with QOL data collection standards is prospectively monitored and evaluated. Preliminary results from eight ECOG-run protocols found overall compliance to be approximately 85 per cent (94 per cent at baseline and 73 per cent during treatment). Selected patient and institutional factors were evaluated for their association with compliance.

Adult↗
PubMedDate not supplied

Modulation of cytokine-induced cardiac myocyte apoptosis by nitric oxide, Bak, and Bcl-x.

-Cytokine-induced NO production depresses myocardial contractility and has been shown to be cytotoxic to cardiac myocytes. However, the mechanisms of cytokine-induced cardiac myocyte cell death are unclear. To analyze these mechanisms in detail, we treated neonatal cardiac myocytes in serum-free culture with a combination of the macrophage-derived cytokines interleukin-1beta, tumor necrosis factor-alpha, and interferon-gamma. These cytokines caused a time-dependent induction of cardiac myocyte apoptosis, but not necrosis, beginning 72 hours after treatment, as determined by nuclear morphology, DNA internucleosomal cleavage, and cleavage of poly(ADP-ribose) polymerase, reflecting caspase activation. Apoptosis was preceded by a >50-fold induction of inducible NO synthase mRNA and the release of large amounts (5 to 8 nmol/ microgram protein) of NO metabolites (NOx) into the medium. Cell death was completely blocked by an NO synthase inhibitor and attenuated by antioxidants (N-acetylcysteine and DTT) and the caspase inhibitor ZVAD-fmk. Cytokines also mediated an NO-dependent, sustained increase in myocyte expression of the Bcl-2 homologs Bak and Bcl-x(L). The NO donor S-nitrosoglutathione also induced apoptosis and cell levels of Bak, but not of Bcl-x(L). All effects of cytokines, including poly(ADP-ribose) polymerase cleavage, could be attributed to interleukin-1beta; interferon-gamma and tumor necrosis factor-alpha had no independent effects on apoptosis or on NOx production. We conclude that cytokine toxicity to neonatal cardiac myocytes results from the induction of NO and subsequent activation of apoptosis, at least in part through the generation of oxygen free radicals. The rate and extent of this apoptosis is modulated by alterations in the cellular balance of Bak and Bcl-x(L), which respond differentially to cytokine-induced and exogenous NO and by the availability of oxidant species.

Animals↗