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Biomedical subjects

J E Watson

Publications and source records attributed to J E Watson.

At least 19 recordsLinked to original sources
PubMed1992

Direct evidence for protein kinase C involvement in insulin-stimulated hexose uptake.

Insulin has been reported to translocate protein kinase C (PKC) in rat adipocytes, and activation of PKC by phorbol esters is known to increase hexose uptake in these cells (1.2). To test the hypothesis that PKC may participate in insulin-stimulated hexose uptake, adipocytes were partially depleted of protein kinase C by overnight phorbol ester treatment, thereby impairing insulin effects on hexose uptake. Purified PKC was then introduced into these PKC-depleted adipocytes by electropermeabilization, and this fully restored insulin-stimulated hexose uptake. These findings provide direct evidence that PKC is required for insulin-stimulated hexose uptake.

Adipose Tissue↗
PubMed1992

Characteristics associated with compliance to cholesterol lowering eating patterns.

The achievement of high levels of adherence is the most important objective of nutrition intervention programs. In order to determine characteristics which were most highly related to adherence to a cholesterol lowering eating pattern, a group of 264 men were sampled. Participants had been enrolled for six years in a multi-risk reduction program for cardiovascular disease which included dietary intervention for blood cholesterol. They were asked to respond to 35 statements, each of which was designed to reflect one of seven characteristics: perception of threat of disease, cost-benefit of therapy, quality of care, social support, external environmental media, and internal as well as external health locus of control. There were seven possible responses to each statement, from strongly agree to strongly disagree. Using a reduced rank regression analysis the numerical answers to the items were treated as a set of predictors for the criterion measure, the food record rating (FRR) score which reflected compliance to a cholesterol lowering eating pattern. Overall the seven characteristics accounted for almost half of the variance in the FRR score (multiple R = 0.48). The most highly related characteristics were cost-benefit, quality of care and external environmental media. These results are highly consistent with those obtained in another population, and indicate the importance of minimizing the cost and increasing the benefits of cholesterol lowering programs by providing high quality treatment programs which emphasize tailoring the regimen to the individual. These results also support the importance of public information efforts such as the National Cholesterol Education Program.

Cholesterol, Dietary↗
PubMed1992

Inactivation of the remaining allele of the WT1 gene in a Wilms' tumour from a WAGR patient.

A candidate gene (WT1) has recently been described for the 11p13 tumour-suppressor gene involved in the development of Wilms' tumour. This gene encodes a zinc finger protein which can bind to a specific DNA sequence. We have found a 226 base deletion in the mRNA from a unilateral Wilms' tumour, which would cause a frameshift that completely deletes the zinc finger domain. The tumour developed in a patient suffering from the WAGR syndrome, who had a constitutional 11p13 deletion, and so the 226 base deletion represents the inactivation of the remaining WT1 allele in the tumour. This provides further direct evidence that loss of function of WT1 is an essential step in the development of Wilms' tumour.

Abnormalities, Multiple↗
PubMed1992

Incidence of positive aspiration in the Gow-Gates mandibular block.

Evidence strongly suggests that rigorous precautions should be taken to avoid accidental intravascular injection of local anesthetic solutions. In this study, 3,000 patients each received a Gow-Gates mandibular block, resulting in a positive aspiration frequency of 1.6%. In the conventional inferior alveolar block technique, a range of positive aspirations from 3.6% to 22% has been noted. The Gow-Gates block has other important advantages over the conventional block. By observing the landmarks and the depth of penetration, it is possible to reinsert the needle with a reasonable assurance of a negative aspiration at the second attempt. The conventional technique suffers the major disadvantage that needle insertion techniques which produce the highest rates of clinically successful pain control run the greatest risk of vascular penetration. Operators skilled in the Gow-Gates technique should be able to achieve a positive aspiration rate of less than 2%. The technique is unique among oral nerve block procedures in that the preferred injection site for maximum efficacy is also the site that is least likely to result in vascular penetration.

Accidents↗
PubMed1991

Linear order of new and established DNA markers around the fragile site at Xq27.3.

We have used recombinant clones derived from microdissection of the fragile X region to characterize breakpoints around the fragile site at Xq27.3. So far, no microdissection markers derived from Xq28 material have been found, thus allowing a rapid screening for clones surrounding the fragile site by their presence in a somatic cell hybrid containing Xq27.2-Xqter. A total of 43 new DNA markers from Xq27 have been sublocalized within this chromosome band. Of these new DNA markers, 5 lie in an interval defined as containing the fragile X region. The saturation of Xq27 with DNA markers by microdissection demonstrates the power of this technique and provides the resources for generating a complete physical map of the region.

Blotting, Southern↗
PubMed1991

Concentration-dependent effects of protein kinase C-activating and -nonactivating phorbol esters on myocardial contractility, coronary resistance, energy metabolism, prostacyclin synthesis, and ultrastructure in isolated rat hearts. Effects of amiloride.

An extensive investigation of the cardiac actions of phorbol esters and the potential role of the Na(+)-H+ exchanger in those actions was carried out using isolated rat hearts. Sixty minutes of perfusion with 10(-9) M phorbol 12-myristate 13-acetate (PMA) or 10(-8) M phorbol 12,13-dibutyrate (PDBu) produced marked cardiac dysfunction associated with depressed contractility, coronary constriction, and elevated resting tension, the latter being particularly evident with PMA. These effects were also associated with disturbances in tissue levels of energy metabolites manifested primarily by a reduction in ATP and an elevation in lactate. Furthermore, both phorbols produced a sustained stimulation of the release of 6-ketoprostaglandin F1 alpha (6-keto PGF1 alpha), the hydrolysis product of prostacyclin (prostaglandin I2). Amiloride, an inhibitor of the Na(+)-H+ exchanger, significantly attenuated the loss in contractility and elevation in coronary pressure as well as the stimulated release of 6-keto PGF1 alpha but was without effect on elevations in resting tension or on changes in energy metabolites. Increasing concentrations of PMA or PDBu 10-fold resulted in a much more rapid and severe (greater than 80% loss in contractile function after 30 minutes) effect that was nonetheless qualitatively identical to that seen with the lower concentrations of phorbol. However, the effects were not prevented by amiloride. Surprisingly, 4 alpha-phorbol 12,13-didecanoate (alpha-PDD, 10(-6) M), which does not activate protein kinase C, was found to be a potent inhibitor of cardiac function (greater than 80% loss in contractility and 50% increase in resting tension) after 30 minutes of perfusion, although these effects were not associated with changes in levels of energy metabolites or with elevations in coronary pressure. Similarly, none of the actions of this compound were attenuated by amiloride. In contrast to the sustained effects of other phorbols on 6-keto PGF1 alpha release, the effect of alpha-PDD was transient (less than 10 minutes). In all hearts studied, the marked depression in contractile function caused by all phorbol esters occurred in the absence of any ultrastructural changes. 4 alpha-Phorbol (10(-6) M), which does not activate protein kinase C, was without effect on any parameter studied. Our results demonstrate very complex effects of phorbol esters on numerous parameters of cardiac function, including an amiloride-sensitive component that occurs at low concentrations. The latter observation suggests the involvement of Na(+)-H+ exchange activation, possibly occurring as a consequence of protein kinase C stimulation, in mediation of the effects of phorbol esters at low concentrations.(ABSTRACT TRUNCATED AT 400 WORDS)

Amiloride↗
PubMed1990

Protein kinase C activation patterns are determined by methodological variations. Studies of insulin action in BC3H-1 myocytes and rat adipose tissue.

In BC3H-1 myocytes, insulin has been reported to (a) increase diacyglycerol (DAG) production and provoke increases in protein kinase C enzyme activity of crude or DEAE-Sephacel-purified cytosol and membrane fractions in BC3H-1 myocytes (Cooper et al. (1987) J. Biol. Chem. 262, 3633-3739), but (b) decrease cytosolic, and transiently increase membrane, immunoreactive protein kinase C (Acevedo-Duncan et al. (1989) FEBS Lett. 244, 174-176). Presently, we used a Mono-Q column to purify protein kinase C and found that, similar to immunoblot findings, enzyme activity decreased in the cytosol, and increased in the membrane during insulin treatment. Similar differences in protein kinase C activation patterns were observed in rat adipose tissue: insulin stimulated cytosolic protein kinase C enzyme activity as measured after DEAE-Sephacel chromatography, but decreased cytosolic enzyme activity when measured after Mono-Q chromatography or by immunoblotting. We presently evaluated the possibility that insulin-induced increases in endogenous DAG may influence protein kinase C during assay in vitro. Crude cytosol from BC3H-1 myocytes contained 25-35% of total and [3H]glycerol-labelled DAG and insulin increased this DAG. Considerable amounts of [3H]glycerol-labelled DAG were present in insulin-stimulated protein kinase C-containing column fractions following DEAE-Sephacel chromatography of cytosol fractions, whereas lesser amounts were recovered after Mono-Q column chromatography. This difference in recovery of DAG and activation of the enzyme by this endogenous DAG may explain why we were able to discern insulin-induced (presumably translocation 'provoked') decreases in cytosolic protein kinase C in the present Mono-Q column preparations of both BC3H-1 myocytes and rat adipose tissue.

Adipose Tissue↗
PubMed1990

Programme evaluation in nursing education: the state of the art.

Programme evaluation is a complex but integral component of a nursing education programme. It is an ongoing process of collecting and describing data which provides the basis for decision making. Specifically, evaluative data can be used to prepare for accreditation visits; account for budgetary expenditures; answer requests for information; develop faculty and staff; and examine the planned and actual effects of the programme within the community and make changes accordingly. There are several programme evaluation models available to guide the evaluation process, but no single model is best and nurse educators must consider a variety of variables. Ideally, the selection of a model should be based on the purpose of the evaluation, programme needs, material and spatial resources, and personnel time, as well as the needs and desires of key interest groups. Several considerations are also required before the model is implemented. These include determining specific priorities for evaluation since all aspects of a programme (conceptual framework, philosophy, programme goals, student characteristics, graduates' performance, faculty and administrative expertise, as well as adequacy of resources) usually cannot be evaluated simultaneously; how the evaluation should proceed; time frames for specific evaluation projects; and personnel responsible for the evaluative activities.

Curriculum↗
PubMed1990

Mechanisms for cardiac depression induced by phorbol myristate acetate in working rat hearts.

1. The effects of the phorbol ester, phorbol myristate acetate (PMA) were examined on function and energy metabolism in the isolated working heart of the rat. 2. At a concentration of 10(-9) M PMA produced a rapid loss in cardiac function in terms of aortic flow rate (AFR) and coronary flow rates (CFR) whereas a similar concentration of 4 alpha-phorbol 12,13-didecanoate was ineffective. At a concentration of 10(-10) M, the PMA-induced depression was more gradual but nevertheless very pronounced with an almost total loss in AFR after 30 min perfusion. The reduction in CFR was more moderate than that observed with respect to AFR. 3. The protein kinase C (PKC) inhibitor (+/-)-1-O-hexadecyl-2-O-acylglycerol significantly attenuated the loss in AFR and CFR following addition of PMA. 4. Two inhibitors of Na+/H+ exchange, amiloride and quinacrine, totally prevented the reduction in AFR. Although the PMA-induced depression in CFR was also attenuated by both amiloride and quinacrine, these effects were not significant, probably reflecting the less pronounced effect of PMA on this parameter. 5. Nifedipine, a dihydropyridine calcium channel blocker reduced PMA toxicity to a similar degree as Na+/N+ exchange inhibition whereas the calcium channel agonist Bay K 8644 was without effect. 6. Tissue content of energy metabolites including high energy phosphates, total adenine nucleotides or lactate were not significantly affected by PMA perfusion. 7. We conclude that PKC activation is necessary for phorbol ester-induced cardiac dysfunction. The consequence of PKC stimulation includes (1) Na+/H+ exchange activation and a subsequent elevation in intracellular calcium [Ca2+]i via Na+/Ca2+ exchange and (2) PKC-dependent phosphorylation of the calcium channel, both of which would produce toxicity by elevation of [Ca21]i. Pharmacological manipulation of any of these steps prevents PMA toxicity by virtue of a reduction in the accumulation of [Ca21]i. PMA effects or their prevention are unrelated to any changes in energy metabolism.

Amiloride↗
PubMed1990

Sulfonylurea-stimulated glucose transport association with diacylglycerollike activation of protein kinase C in BC3H1 myocytes.

The extrapancreatic effects of sulfonylurea drugs include increased glucose uptake by certain peripheral tissues. To study this effect, we used BC3H1 myocytes, which are reported to respond to these drugs. Within 30 min, tolbutamide and glyburide increased [3H]-2-deoxyglucose uptake in a dose-dependent manner. The inactive analogue carboxytolbutamide had no effect on glucose transport. Because increases in glucose transport may be mediated by activation of the diacylglycerol-protein kinase C signaling system, we examined the effects of these drugs on lipid metabolism and protein kinase C activity. Unlike insulin, tolbutamide and glyburide failed to increase [3H]glycerol labeling of diacylglycerol or labeling of phospholipids by 32P. After 30 min of treatment with tolbutamide or glyburide, however, membrane-associated and cytosolic protein kinase C activity were each increased. When cells were treated with 12-O-tetradecanoylphorbol-13-acetate (TPA) for 48 h to deplete certain isoforms of protein kinase C, glyburide, tolbutamide, and acute TPA treatment failed to increase glucose uptake, suggesting that TPA and sulfonylureas operate through activation of a common pathway. The effect of glyburide was additive to TPA in stimulating glucose uptake at low but not high TPA concentrations. As with insulin and TPA, extracellular Ca2+ was not essential for sulfonylurea-stimulated glucose uptake. Staurosporine, a protein kinase C inhibitor, blocked glyburide-, tolbutamide-, and insulin-stimulated glucose uptake. In intact cells, glyburide stimulated the phosphorylation of both 80,000-Mr and 40,000-Mr proteins, which are markers for protein kinase C activation. Addition of sulfonylureas directly to the protein kinase C assay system in vitro provoked dioleinlike effects, in that sensitivity of the enzyme to Ca2+ was increased.(ABSTRACT TRUNCATED AT 250 WORDS)

Alkaloids↗
PubMed1990

Microdissection of the fragile X region.

We have microdissected and cloned the region around the fragile site at Xq27.3 on the human X chromosome. All of the clones tested map to the Xq27-Xq28 region, and detailed mapping on a panel of somatic cell hybrids indicates that the microdissected library contains sequences derived from both sides of the fragile X mutation. Some of these clones give signals in rodent DNA. This library demonstrates the power of microdissection for the identification of potential coding sequences near a disease locus and provides a promising resource for the identification of the fragile X mutation.

Animals↗
PubMed1989

Glucose-induced synthesis of diacylglycerol de novo is associated with translocation (activation) of protein kinase C in rat adipocytes.

Addition of glucose (5-20 mM) to rat adipocytes provoked dose-related increases in diacylglycerol, without increasing production of [3H]inositol phosphates. Cytosolic protein kinase C enzyme activity and immunoreactivity decreased within 1-5 min of 5 mM glucose addition, and further over 20 min. Membrane protein kinase C increased stoichiometrically during the first 5 min and then decreased. Higher concentrations (10 and 20 mM) of glucose provoked greater and more rapid decreases of cytosolic and membrane protein kinase C. Our findings suggest that glucose stimulates diacylglycerol production by providing substrate for phosphatidic acid synthesis de novo, and this is associated with translocative activation of protein kinase C.

Adipose Tissue↗
PubMed1989

Retention of specific protein kinase C isozymes following chronic phorbol ester treatment in BC3H-1 myocytes.

Since insulin effects on glucose transport persist in phorbol ester "desensitized" or "down-regulated" BC3H-1 myocytes, we reexamined the evidence for protein kinase C (PKC) depletion. After 24 hrs of 5 microM 12-0-tetradecanoyl phorbol-13-acetate (TPA) treatment, PKC-directed histone phosphorylation and acute TPA effects on glucose transport were lost, but PKC-dependent vinculin phosphorylation was still evident. Hydroxylapatite (HAP) chromatography revealed loss of a type III, but not a type II, PKC-dependent vinculin phosphorylation. Immunoblots of cytosolic preparations of PKC-"depleted" myocytes confirmed the retention of PKC. Our findings indicate that TPA "down-regulated" BC3H-1 myocytes contain immunoreactive and functionally active PKC. The latter may explain the continued effectiveness of both insulin and diacylglycerol (DiC8) for stimulating glucose transport in "down-regulated" cells.

Animals↗