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P Watt

Publications and source records attributed to P Watt.

At least 37 records · Page 2Linked to original sources
PubMed1994

The C-terminal domain of Saccharomyces cerevisiae DNA topoisomerase II.

A set of carboxy-terminal deletion mutants of Saccharomyces cerevisiae DNA topoisomerase II were constructed for studying the functions of the carboxyl domain in vitro and in vivo. The wild-type yeast enzyme is a homodimer with 1,429 amino acid residues in each of the two polypeptides; truncation of the C terminus to Ile-1220 has little effect on the function of the enzyme in vitro or in vivo, whereas truncations extending beyond Gln-1138 yield completely inactive proteins. Several mutant enzymes with C termini in between these two residues were found to be catalytically active but unable to complement a top2-4 temperature-sensitive mutation. Immunomicroscopy results suggest that the removal of a nuclear localization signal in the C-terminal domain is likely to contribute to the physiological dysfunction of these proteins; the ability of these mutant proteins to relax supercoiled DNA in vivo shows, however, that at least some of the mutant proteins are present in the nuclei in a catalytically active form. In contrast to the ability of the catalytically active mutant proteins to relax supercoiled intracellular DNA, all mutants that do not complement the temperature-dependent lethality and high frequency of chromosomal nondisjunction of top2-4 were found to lack decatenation activity in vivo. The plausible roles of the DNA topoisomerase II C-terminal domain, in addition to providing a signal for nuclear localization, are discussed in the light of these results.

Adenosine Triphosphatases↗
PubMed1994

DETERMINATION OF PROTEIN SYNTHESIS IN RAINBOW TROUT, ONCORHYNCHUS MYKISS, USING A STABLE ISOTOPE

It has been suggested (Houlihan, 1991) that the consumption of 1 g of protein in a variety of species of fish stimulates the synthesis of, approximately, an equal amount of protein. Although synthesis of protein may account for as much as 40 % of the whole-animal oxygen consumption (Lyndon et al. 1992), only about 30 % of the synthesized proteins are retained as growth (Houlihan et al. 1988; Carter et al. 1993a,b). Thus, one focus of attention is the potential advantage gained by fish in allocating a considerable proportion of assimilated energy to protein turnover in contrast to relatively low-cost, low-turnover protein growth (Houlihan et al. 1993). Rates of protein synthesis in several species of fish have been measured using radioactively labelled amino acids, frequently given as a flooding dose (reviewed by Fauconneau, 1985; Houlihan, 1991). These measurements cannot be made for longer than a few hours because of the decline in specific radioactivity in the amino acid free pool. However, as protein synthesis rates vary during the course of a day as a result of the post-prandial stimulation, and since radiolabelled amino acid methodology is invasive, short-term and terminal, it has been difficult to be certain of the relationship between protein growth measured in the long term and protein synthesis rates measured in the short term. This paper addresses these problems by developing a method using 15N in orally administered protein to measure protein synthesis rates in fish over relatively long periods, the aim being to use procedures that are as non-invasive and repeatable as possible. The use of stable isotopes to measure protein metabolism is well established in terrestrial mammals (see Rennie et al. 1991; Wolfe, 1992), but to our knowledge the only published data for aquatic ectotherms are on the blue mussel (Mytilus edulis L.) (Hawkins, 1985). In the present study, rates of protein synthesis of individual rainbow trout [Oncorhynchus mykiss (Walbaum)] were calculated from the enrichment of excreted ammonia with 15N over the 48 h following the feeding of a single meal (dose) containing protein uniformly labelled with 15N by use of an end-point stochastic model (Waterlow et al. 1978; Wolfe, 1992). Application of this type of modelling would appear to be ideal for measuring ammonotelic fish nitrogen metabolism since, unlike the situation in mammals, the catabolic flux of amino acids through urea is very small. Further, ammonia is excreted directly into the surrounding water via the gills and is not stored for any length of time, in contrast to the situation in mammals, so the rate of tracer appearance is easily measurable.

Journal Article↗
PubMed1993

Distinct negative regulation of the human embryonic globin genes zeta and epsilon.

The human embryonic globin genes zeta and epsilon are expressed when erythropoiesis is initiated at about the third week of development but are subsequently repressed as expression of the fetal globins, alpha and gamma, begins. We have examined the promoter region of the human zeta-globin and epsilon-globin genes in order to identify regulatory sequences that may be involved in this process. Stable transfection of the human erythroid cell line K562 with either a truncated form of the zeta-globin gene, containing 112 base pairs (bp) of 5'-flanking sequences, or a larger zeta-globin construct, containing several hundred bp of 5'-flanking sequence, revealed that the zeta-globin gene is subject to negative regulation by its 5'-flanking region. We have defined the sequences responsible for this negative regulation to a 22 bp region immediately upstream of the proximal promoter sequence of the zeta-globin gene. A 22 bp oligonucleotide including this negative element was found to inhibit both the zeta-globin and HSV TK promoters. We have also analyzed the promoter of the human epsilon-globin gene, since it is coordinately expressed with zeta-globin. We show that it is likewise subject to negative regulation, though in this case from a distal silencer element. Gel retardation and methylation interference assays have provided evidence of a factor which binds specifically to the epsilon-globin silencer. However, no obvious sequence homology exists between the zeta and epsilon negative elements, and at least some of the factors that recognize these elements are distinct. We postulate that the negative transcriptional control elements in the human embryonic globin gene promoters contribute to the observed reduction in zeta- and epsilon-globin gene expression that occurs during development.

Base Sequence↗
PubMed1992

Regulation of human embryonic globin genes zeta 2 and epsilon in stably transformed mouse erythroleukemia cells.

Previous work has suggested that the promoter regions of the human embryonic zeta 2 and epsilon globin genes contain negative regulatory regions that could play a role in the repression of these genes in postembryonic erythroblasts. We have examined this possibility by studying the expression of these genes in mouse erythroleukemia cells, an adult erythroid cell line that might be expected to contain repressor molecules that would bind to the putative negative regulatory regions. When attached to appropriate upstream regulatory elements (alpha HS-40 and beta HS1,2) both the zeta and epsilon genes were expressed in these cells at a low level, but no increase in expression was observed when similar constructs lacking the proposed negative regulatory sequences were introduced into these cells. These results cast doubt on the possibility that these sequences play a major role in the developmental repression of the embryonic globin genes, unless they function only in a normal chromosomal organization.

Adenine Phosphoribosyltransferase↗
PubMed1990

The molecular mechanism by which insulin stimulates glycogen synthesis in mammalian skeletal muscle.

The ability of insulin to promote the phosphorylation of some proteins and the dephosphorylation of others is paradoxical. An insulin-stimulated protein kinase is shown to activate the type-1 protein phosphatase that controls glycogen metabolism, by phosphorylating its regulatory subunit at a specific serine. Furthermore, the phosphorylation of this residue is stimulated by insulin in vivo. Increased and decreased phosphorylation of proteins by insulin can therefore be explained through the same basic underlying mechanism.

Amino Acid Sequence↗
PubMed1990

Further studies on the role of glycogenin in glycogen biosynthesis.

About 90% of the glycogenin in skeletal muscle extracts prepared from fed, 24-h starved or alloxan-diabetic rabbits sedimented at 140,000 x g with the glycogen/sarcovesicular fraction, from which it was released by glycogenolysis, but not by 1% SDS. Glycogenin in the glycogen/sarcovesicular fraction is therefore bound covalently to glycogen, and not associated (covalently or non-covalently) with the sarcoplasmic reticulum. The same proportion of glycogen synthase was also recovered in the glycogen/sarcovesicle fraction, was solubilised by glycogenolysis, and copurified with glycogenin to yield a heterodimer composed of a 1:1 complex between these proteins. Glycogen synthase and glycogenin are therefore present in equimolar amounts in skeletal muscle and there is an average of one glycogen synthase catalytic subunit associated with each molecule of glycogen in vivo. Glycogenin and glycogen synthase released into the muscle cytosol by degradation of glycogen did not form a complex initially, and only 50% reassociation took place after storage for several hours or overnight dialysis. This suggests that the muscle cytosol may contain a factor(s) which regulates glycogen biogenesis by modulating the association of glycogenin and glycogen synthase. Only glycogen synthase that was complexed to glycogenin was capable of elongating the primer formed by incubation of glycogenin with Mn2+ and micromolar concentrations of UDP-glucose, demonstrating the critical importance of this complex for glycogen biogenesis.

Animals↗
PubMed1990

A factor binding GATAAG confers tissue specificity on the promoter of the human zeta-globin gene.

We describe the characterisation of cis-acting sequences which control the tissue specific expression of the human zeta globin gene. An extensive search for enhancer sequences in the vicinity of this gene proved negative. Instead our data demonstrate that the minimal promoter of the zeta gene is itself tissue specific. Sequences close to and possibly including the -100 CACCC and -70 CCAAT boxes display some erythroid specificity. However the principal tissue specific element is a GATAA sequence at -120 directly adjacent to the minimal promoter. Specific deletion of GATAA reduces zeta promoter activity 5 fold in erythroid but not non-erythroid cells. We also demonstrate that an erythroid specific factor binds to this GATAA sequence. Furthermore this factor forms a complex with the transcription factor CP1 which we show interacts with the zeta CCAAT box. We present evidence that the zeta GATAA binding factor is equivalent to GF1 recently purified and cloned by Tsai et al [1]. The erythroid specific GATAA sequence has been found in the promoters and enhancers of a number of erythroid specific genes. Similarly we show here that the zeta globin gene relies on a GATAA sequence in its promoter to specify its expression in erythroid cells.

Animals↗
PubMed1988

Hypercholesterolemia in patients undergoing coronary bypass surgery: are they aware, under treatment, and under control?

Hypercholesterolemia is an important determinant of progressive atherosclerosis in both vein grafts and native vessels after coronary artery bypass surgery. We demonstrated that nearly half of all patients admitted for coronary bypass have elevated cholesterol levels before surgery. Of these, half are unaware of the elevations in cholesterol level, and few are undergoing any treatment. The results suggest that lipids should be measured before coronary bypass surgery to optimize postoperative risk reduction education.

Adult↗
PubMed1986

Effect of stretch combined with electrical stimulation on the type of sarcomeres produced at the ends of muscle fibers.

Stretching a muscle results in a rapid addition of sarcomeres at the ends of the muscle fibers. The effect of a pattern of electrical stimulation resembling that of a slow motoneuron on the newly formed muscle tissue in a stretched, fast-contracting muscle was investigated. We found that after a period as short as 4 days, the type of sarcomeres which were added on to the ends of the existing myofibrils differed from those in the middle regions of the experimental muscles: there was a much higher proportion of type I and type IIA sarcomeres in the stretch-stimulated ends. This study showed that reprogramming of the synthesis of fiber type-specific contractile proteins can be achieved and detected within a very short time by using electrical stimulation combined with stretch.

Animals↗
PubMed1984

Acute lower respiratory tract infections in children admitted to Port Moresby General Hospital.

The clinical pattern of acute lower respiratory tract infection (ALRTI) in children admitted to Port Moresby General Hospital (PMGH) was studied. Most patients (60%) were less than twelve months of age. Common symptoms were cough, fever and shortness of breath. Common signs were crepitations, chest recession, elevated temperature and tachypnoea. Concurrent illness was common, with evidence of malnutrition in 62% patients. Most patients were anaemic (haemoglobin less than 10g per dl). Blood cultures isolated pathogens in 13% of patients in which it was done, the most common isolate being Haemophilus influenzae. Chest radiograph showed most patients had multisegmental changes, with the lower lobes commonly involved. Of the 129 patients, discharges accounted for 106 (82%), while 15 (12%) absconded and eight (6%) died. Of those 121 discharged or absconding, 15 (12%) were readmitted within three months of departure. Sixty-six (51%) patients stayed in hospital for four days or less. Of the eight patients who died, six (75%) were malnourished, six (75%) were less than eighteen months of age, seven (87.5%) were sick for one week or less before admission, five (62.5%) had received antibiotics before admission and chest radiograph showed more lung zones affected than in those not dying. Of the eight patients who died, six had white cell counts (WCC) performed and none of these was more than 30,000.

Anti-Bacterial Agents↗
PubMed1982

Comparison of the effects of potassium and pH on the calibre of cerebral veins and arteries.

The vasomotor response of individual pial veins and arteries on the convexity of the cerebral cortex to perivascular microinjection of mock cerebrospinal fluid (CSF) containing various concentrations of potassium (K+) and of various pH (achieved by altering the bicarbonate, HCO-3 concentration) have been examined in cats anaesthetised with alpha-chloralose. Microapplication of CSF containing 0 mM HCO-3 (pH 4.80) effected significant increases in calibre of pial veins and arteries of 9.3 +/- 2.4% and 38.2 +/- 4% respectively (mean calibre change +/- SE), whereas CSF containing 22 mM HCO-3 (pH 7.45) which constricted pial arteries significantly (-18.5 +/- 2.9%) minimally altered venous calibre (-4.3 +/- 2.2%). Microapplication of CSF containing 0 mM potassium resulted in a significant reduction in pial arterial calibre (-11.4 +/- 2.8%) but failed to alter pial venous calibre (-0.3 +/- 0.6%). Perivascular microapplication of CSF containing moderately elevated potassium concentrations (10 mM) which effected marked, significant increases in pial arterial calibre (49.3 +/- 3.9%) did not significantly alter the calibre of the pial veins (mean response -1.6 +/- 2.4%). The perivascular administration of CSF containing a high concentration of potassium (40 mM) resulted in the significant constriction of both pial veins (-13.5 +/- 0.9%) and pial arteries (-47.2 +/-6.3%). The magnitude of the response was significantly smaller in the pial veins. The relative insensitivity to K+ and pH of the pial veins as compared to pial arteries suggests that alteration in the chemical composition of of the perivascular fluid are of lesser importance in the control of cerebrovascular capacitance than for the regulation of cerebrovascular resistance.

Animals↗