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J C Lawrence

Publications and source records attributed to J C Lawrence.

At least 91 records · Page 5Linked to original sources
PubMed1994

Haematology reports of routine blood films in patients with burns.

A retrospective pilot study of 99 peripheral blood films from 27 patients with burns is reported. Abnormalities of the granular leucocyte series were more common in the more extensive burns and usually preceded bacteriological evidence of wound pathogens or a clinical decision to take a blood culture. The evidence suggests that a prospective study is needed to determine the possible clinical value of reporting such granulocyte abnormalities. Abnormalities of the myelo-monocytic and lymphocyte cell lines were sufficiently frequent to permit fundamental research of possible relevance not only to patients with burns but in other host responses such as in sepsis, malaria or AIDS.

Adolescent↗
PubMed1994

Glucose transport and phosphorylation in single cardiac myocytes: rate-limiting steps in glucose metabolism.

Microanalytic methods were used to investigate the regulation of glucose metabolism by insulin in single myocytes isolated from adult rat ventricles. Cultured myocytes were incubated with or without insulin and, with either glucose or 2-deoxyglucose (2-DG), rinsed, and freeze-dried. Individual cells were weighed and levels of 2-DG-6-phosphate (2-DG-6-P) or glucose and glucose 6-phosphate (G-6-P) were determined after enzymatic amplification. In cells incubated with 2-DG, insulin increased the level of 2-DG-6-P by as much as 30-fold, indicative of dramatic activation of glucose transport. In cells incubated with glucose, insulin increased the levels of G-6-P by approximately threefold. Increasing extracellular glucose without insulin also increased G-6-P; however, intracellular glucose concentrations were not increased, indicating that glucose transport is rate limiting in nonstimulated myocytes. In contrast, intracellular glucose concentrations were increased by over an order of magnitude by insulin, reaching 60% of the extracellular glucose concentration. Measurements of glucose and G-6-P in the same insulin-treated cells revealed that accumulation of G-6-P reached a plateau when extracellular glucose was increased > 2 mM. At this point the estimated intracellular glucose concentration was 300 microM, or approximately 10 times the Michaelis constant of hexokinase for glucose. These results indicate that in the presence of insulin and physiological concentrations of glucose, hexokinase is saturated with glucose. Consequently, the rate-limiting step for insulin-stimulated glucose utilization is glucose phosphorylation rather than glucose transport.

Animals↗
PubMed1993

Mitogen-activated protein kinase activation is not sufficient for stimulation of glucose transport or glycogen synthase in 3T3-L1 adipocytes.

The role of mitogen-activated protein (MAP) kinase in the regulation of glucose metabolism has been investigated by comparing the effects of insulin and epidermal growth factor (EGF) on MAP kinase activation, glucose transport, and glycogen synthase in 3T3-L1 adipocytes. Insulin or EGF treatment for 5 min increased p42mapk and p44mapk activity to the same extent as determined by myelin basic protein kinase activity measurements and phosphotyrosine immunoblotting. The profiles of myelin basic protein kinase activity following MonoQ chromatography of extracts obtained from cells incubated with insulin or EGF were almost identical. Insulin increased glucose transport and GLUT4 translocation to the cell surface by 15- and 7-fold, respectively. EGF had no significant effect on these processes. Insulin increased the glycogen synthase ratio (-Glc-6-P/+Glc-6-P) by 7.5- and 3.5-fold in the presence and absence of glucose, respectively. EGF increased the ratios by only 2- and 1.3-fold, respectively. EGF did not appear to inhibit downstream of MAP kinase, because when adipocytes were incubated with insulin plus EGF, the stimulation of glucose transport and glycogen synthase was similar to that observed with insulin alone. These findings indicate that activation of the MAP kinase isoforms p42mapk and p44mapk is not sufficient for the activation of glucose transport and glycogen synthase in 3T3-L1 adipocytes.

3T3 Cells↗
PubMed1993

Increasing cAMP attenuates activation of mitogen-activated protein kinase.

Activation of the mitogen-activated protein kinase (MAP kinase) isoforms ERK1 and ERK2 was investigated in rat adipocytes. Kinase activities were measured by using myelin basic protein as substrate after the isoforms were resolved by Mono Q chromatography or by immunoprecipitation with specific antibodies. Insulin increased the activity of both isoforms by 3- to 4-fold. The beta-adrenergic agonist isoproterenol was without effect in the absence of insulin but markedly reduced the increases in ERK1 and ERK2 activities produced by the hormone. MAP kinase activation was also attenuated by forskolin and glucagon, which increase intracellular cAMP, and by dibutyryl-cAMP, 8-bromo-cAMP, and 8-(4-chlorophenylthio)-cAMP. Thus, increasing cAMP is associated with decreased activation of MAP kinase by insulin. Forskolin also inhibited activation of MAP kinase by several agents (epidermal growth factor, phorbol 12-myristate 13-acetate, and okadaic acid) that act independently of insulin receptors. Moreover, forskolin did not inhibit insulin-stimulated tyrosine phosphorylation of the insulin receptor substrate IRS-1. Therefore, the inhibitory effect on MAP kinase did not result from compromised functioning of the insulin receptor. The inhibitory effect was not confined to adipocytes, as forskolin and dibutyryl-cAMP inhibited the increase in MAP kinase activity by phorbol 12-myristate 13-acetate in wild-type CHO cells. In contrast, these agents did not inhibit MAP kinase activity in mutant CHO cells (line 10248) that express a cAMP-dependent protein kinase resistant to activation by cAMP. Our results suggest that activation of cAMP-dependent protein kinase represents a general counter-regulatory mechanism for opposing MAP kinase activation.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1993

Glucose transport in L6 myoblasts overexpressing GLUT1 and GLUT4.

The roles of the glucose transporter isoforms, GLUT1 and GLUT4, in mediating insulin-stimulated glucose transport were investigated by stably overexpressing the transporters in L6 myoblasts. Levels of GLUT1 and GLUT4 in myoblasts from the cell lines having the highest content of these transporters were approximately 16- and 30-fold higher, respectively, than levels in nontransfected cells. The basal rate of 2-deoxy[3H]glucose uptake was severalfold higher in cells overexpressing GLUT1 than in the parent L6 myoblasts or in control cell lines that were generated by transfecting cells with expression vectors lacking transporter insert. The basal rate was not elevated in any of the lines expressing GLUT4. The net increase in 2-deoxy[3H]glucose uptake produced by insulin was larger in both the GLUT1 and GLUT4 cells than in the control cells. Insulin increased uptake in GLUT4 cells by as much as 6-fold; whereas, the fold increase over basal uptake produced by insulin in GLUT1 cells was comparable to that (2-fold) observed in the control myocytes. Thus, both GLUT1 and GLUT4 can mediate insulin-stimulated glucose transport in L6 myoblasts, although GLUT4 is needed to observe large percentage increases comparable to those observed in skeletal muscle fibers in vivo. In contrast to insulin, the protein phosphatase inhibitors, okadaic acid and calyculin A, inhibited glucose transport in cells expressing either GLUT1 or GLUT4. Calyculin A, which produced a half-maximum effect at 10 nM, was approximately 100 times more potent than okadaic acid in decreasing both basal and insulin-stimulated 2-deoxyglucose uptake. Inhibition of uptake by calyculin A was associated with a decrease in the cell surface concentration of both GLUT1 and GLUT4. These results indicate that increased protein phosphorylation can lead to inhibition of transport mediated by both GLUT1 and GLUT4.

Animals↗
PubMed1993

GLUT4 phosphorylation and inhibition of glucose transport by dibutyryl cAMP.

To investigate the mechanism responsible for the inhibition of glucose transport by dibutyryl cAMP (Bt2cAMP), two different transporter isoforms (GLUT1 and GLUT4) and several GLUT1/4 chimeric transporters were expressed in Chinese hamster ovary (CHO) cells by using a Sindbis virus expression system. Bt2cAMP inhibited GLUT4-mediated 2-deoxy[3H]glucose (2DOG) uptake by 50% but was without effect on GLUT1-mediated uptake. When the subcellular distribution of GLUT4 was assessed by quantitative immunocytochemistry, neither the overall concentration of GLUT4 nor the regional distribution of GLUT-4 within the plasma membrane was found to be altered by Bt2cAMP. Thus, inhibition of 2DOG uptake by Bt2cAMP appears to be due to a decrease in transporter activity rather than a decrease in the number of transporters exposed at the plasma membrane. By using chimeric transporters, a region of GLUT4 necessary for the inhibitory effect of Bt2cAMP was localized to the last 29 amino acids in the COOH terminus. This intracellular region contains the site (Ser488) phosphorylated in vitro by cAMP-dependent protein kinase (cAdPK). Changing Ser488 to an Ala abolished phosphorylation of GLUT4; however, the inhibitory effect of Bt2cAMP on glucose transport was not diminished by this mutation. Therefore, phosphorylation of GLUT4 was not required for the inhibition. The effects of other nucleotides on GLUT4 transport activity were assessed to investigate the role of cAdPK. Uptake of 2DOG by GLUT4 was inhibited by 8-bromo-AMP, but not by 8-bromo-cAMP, suggesting that the inhibitory effect did not involve activation of cAdPK. Results consistent with this interpretation were obtained with CHO cells (line 10248), which express a cAdPK that is resistant to activation by cAMP. No difference in the concentrations of Bt2cAMP required to inhibit GLUT4-mediated transport was observed in normal CHO cells and 10248 cells. The results presented suggest that the inhibitory effects of Bt2cAMP could be mediated by direct binding of a nucleotide to GLUT4 at a site involving the intracellular COOH terminus of the transporter.

Animals↗
PubMed1992

GLUT4 facilitates insulin stimulation and cAMP-mediated inhibition of glucose transport.

The glucose transporter isoform GLUT4 is found only in cells that exhibit insulin-sensitive glucose transport. To investigate the function of this transporter, L6 myoblasts were stably transfected with GLUT4 cDNA. GLUT4 underwent insulin-dependent movement to the cell surface in myoblasts overexpressing the transporter. One cell line (243-6) expressed sufficient levels of the GLUT4 protein to study insulin-dependent glucose transport. Unlike wild-type L6 cells, 243-6 myoblasts exhibited two features that are characteristic of differentiated muscle fibers and adipocytes in vivo: a large insulin-stimulated component of glucose transport and inhibition of this stimulated component by cAMP. Relative to normal L6 cells, 243-6 cells responded to insulin or insulin-like growth factor 1 with a 5-fold larger increase in 2-deoxy[3H]glucose uptake. N6,O2'-Dibutyryladenosine 3',5'-cyclic monophosphate (Bt2cAMP) did not inhibit transport in normal L6 myoblasts, which express only GLUT1, but inhibited IGF-1/insulin-stimulated transport by 50% in 243-6 cells. The effect of cAMP was investigated further by using Chinese hamster ovary cells transiently expressing GLUT1 and GLUT4. Bt2cAMP inhibited glucose transport only in Chinese hamster ovary cells expressing GLUT4. These results indicate that cAMP-mediated inhibition of glucose transport is dependent on expression of the GLUT4 isozyme.

Animals↗
PubMed1992

Bilateral metastatic endophthalmitis as a complication of major burns.

Metastatic infection of the eye is a rare complication of burns. The following report describes a patient with endophthalmitis occurring as a complication of major burns. The diagnostic difficulties that arose are discussed and the recommended treatment outlined.

Accidents, Occupational↗
PubMed1991

Wound infection under occlusive dressings.

It is often supposed that occlusive dressings potentiate wound infection. However, even though heavy colonization by skin and wound flora is often seen under certain types of occlusion, clinical infection is not a frequent occurrence. Commensal wound flora consists of a variety of Gram-positive and Gram-negative organisms and fungi which do not appear to be detrimental to healing. Certain aspects of wound healing may in fact be promoted by bacterial colonization, although clinical infection can lead to wound breakdown and systemic infection. Wounds compromised by devitalized tissue, drains or sutures are more susceptible than clean wounds to clinical infection. Occlusive dressings may help prevent infection by presenting a barrier to potential pathogens, and hydrocolloid occlusive dressings have been shown to prevent dissemination of methicillin-resistant Staphylococcus aureus. The rate of clinical infection as deduced from published trials of dressings is lower under occlusion than when non-occlusive dressings are used, and this is likely to be a result of normal activity of the host defences under occlusive dressings.

Bacteria↗
PubMed1991

The use of GORE-TEX bags for hand burns.

Clinical and laboratory studies were made to compare the water vapour permeability, bacteriological properties and clinical performance of polythene and polytetrafluoroethylene fabric (GORE-TEX) bags in the treatment of hand burns. Polythene bags are virtually impermeable to saline, whereas GORE-TEX bags containing silver sulphadiazine cream show a water vapour permeability of 0.53 ml/cm2/day, resulting in a 30 per cent weight reduction of added water after 48 h. Clinically, hand maceration and accumulation of exudate are significantly reduced in hands treated in GORE-TEX bags. The mean daily volume of accumulated exudate for GORE-TEX bags was 37 ml compared to 83 ml for polythene (P less than 0.01). When adjusted for the percentage area of the hand surface burned, this reduction remained significant (P less than 0.005). A tendency for less pain and better hand movement was noted with GORE-TEX bags. There were no significant differences in rate of healing or bacterial colonization of the burned hand between the two type of bags. GORE-TEX bags prevent skin maceration and accumulation of exudate, allowing ease of burn assessment and improved hand function. They are also durable and non-slip, thus increasing patient independence.

Adult↗
PubMed1991

Treating burns.

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Burns↗
PubMed1990

Stimulation of glucose transport and glucose transporter phosphorylation by okadaic acid in rat adipocytes.

Okadaic acid, an inhibitor of Type I and IIa protein phosphatases, was recently found to stimulate 2-deoxyglucose uptake in rat adipocytes (Haystead, T. A. J., Sim, A. T. R., Carling, D., Honnor, R. C., Tsukitani, Y., Cohen, P., and Hardie, D. G. (1989) Nature 337, 78-81). In the present experiments the effect of okadaic acid on the phosphorylation and subcellular distribution of the insulin-regulatable glucose transporter (IRGT) was investigated. At maximally effective concentrations, insulin and okadaic acid increased the amount of IRGT in the plasma membrane by 10- and 4-fold, respectively. Thus, the stimulation of glucose transport by okadaic acid was apparently due to an increase in the surface concentration of the IRGT. However, despite its stimulatory actions, okadaic acid partially inhibited the ability of insulin to enhance glucose transport and translocation of the transporter. When cells were incubated with okadaic acid alone or in combination with insulin, phosphorylation of the IRGT in the plasma membrane was increased by approximately 3-fold relative to the intracellular pool of transporters in control cells. Phosphorylation of the IRGT was confined to the presumed cytoplasmic domain at the COOH terminus of the protein. Glucose transporters were dephosphorylated in vitro by Type I or Type IIa protein phosphatases, indicating that inhibition of one or both of these phosphatases could account for the increased phosphorylation produced by okadaic acid. The observation that okadaic acid stimulated translocation of the IRGT implicated a serine/threonine phosphorylation event in triggering movement of the intracellular IRGT-containing vesicles (GTV) to the cell surface. Immunoadsorption of GTV from 32P-labeled adipocytes revealed that the IRGT was the major phosphoprotein in these vesicles. The phosphorylation of at least three other GTV proteins was increased by okadaic acid, and these species would appear to be candidates for regulators of GTV movement to the plasma membrane. It is unlikely that phosphorylation of the IRGT is the signal for translocation because insulin did not increase phosphorylation of the protein. Rather, the inhibitory effect of okadaic acid on insulin-stimulated translocation is consistent with the hypothesis that phosphorylation of the IRGT promotes its internalization.

Adipose Tissue↗
PubMed1990

Phosphorylation of the glucose transporter in rat adipocytes. Identification of the intracellular domain at the carboxyl terminus as a target for phosphorylation in intact-cells and in vitro.

Phosphorylation of the insulin-regulatable glucose transporter (IRGT) is increased by incubating rat adipocytes with isoproterenol or by incubating microsomal membranes with cAMP-dependent protein kinase. To attempt to locate the sites of phosphorylation, the IRGT (apparent Mr = 46,000) was immunoprecipitated from 32P-labeled adipocytes and cleaved with CNBr or trypsin. Essentially all of the 32P could be recovered in a single CNBr fragment, denoted CB-T (Mr = 8,000), which bound a polyclonal antibody (R820) against a peptide having the sequence of the last 12 amino acids in the COOH terminus of the IRGT. 32P-Labeling of the IRGT was also confined to CB-T when membranes were incubated with [gamma-32P]ATP and cAMP-dependent protein kinase. Isoproterenol increased phosphorylation of CB-T, but insulin was without effect. To resolve phosphorylation sites further, IRGT from 32P-labeled cells was subjected to exhaustive proteolysis with trypsin. Samples were applied to a C-18 column, and 32P-labeled fragments were resolved into three peak fractions by elution with an increasing gradient of acetonitrile. [32P]Phosphoserine was the only phosphoamino acid detected in any of the peaks. Peak III contained approximately 80% of the 32P and was increased by isoproterenol. Almost all of the 32P introduced by cAMP-dependent protein kinase in vitro eluted in Peak III. In all cases, the 32P-labeled species in Peak III were quantitatively immunoprecipitated by R820. Digesting the peptide(s) in Peak III with V8 protease generated a single peak of 32P which eluted at lower acetonitrile than Peak III and contained 32P-labeled species that did not interact with R820. Automated Edman degradation indicated that the serine residue in Peak III phosphorylated by cAMP-dependent protein kinase was the 3rd or 4th residue from the NH2 terminus of the peptide. These findings indicate that phosphorylation of the IRGT is restricted to the presumed intracellular domain at the COOH terminus and that Ser488 is a major site phosphorylated both by cAMP-dependent protein kinase in vitro and in response to isoproterenol in vivo.

Adipose Tissue↗
PubMed1990

Phosphorylase kinase isozymes and phosphorylase in denervated skeletal muscles.

The effects of motor denervation on levels of phosphorylase kinase isozymes and phosphorylase were investigated in rat epitrochlearis, hemidiaphragm, and soleus muscles. Amounts of the proteins were measured after quantitative immunoprecipitation and found to be decreased by as much as 70% 2 weeks after denervation. Unexpectedly, denervation had little, if any, effect on the relative proportions of the two phosphorylase kinase isozymes. Phosphorylase and phosphorylase kinase were decreased by essentially the same extent after denervation, and the effects of denervation were comparable in all three muscles. The decreases in these enzymes explain, at least in part, the marked alterations in glycogen metabolism that occur after motor denervation.

Animals↗
PubMed1989

Insulin stimulates dephosphorylation of phosphorylase in rat epitrochlearis muscles.

We have investigated the effects of insulin on the phosphorylation of glycogen phosphorylase in skeletal muscle. Rat epitrochlearis muscles were incubated in vitro with 32Pi to label cellular phosphoproteins, before being treated with hormones. Phosphorylase, phosphorylase kinase, and glycogen synthase were immunoprecipitated under conditions that prevented changes in their phosphorylation states. Based on measurements of the activity ratio (-AMP/+AMP) and the 32P content of phosphorylase, 4-8% of the phosphorylase in untreated muscles appeared to be phosphorylated. Epinephrine promoted increases of approximately 4-fold in the 32P content and activity ratio. Neither these effects nor the epinephrine-stimulated increases in phosphorylation of glycogen synthase and phosphorylase kinase were attenuated by insulin. However, insulin at physiological concentrations rapidly decreased the 32P content of phosphorylase in muscles incubated without epinephrine. Results from peptide mapping experiments indicate that phosphorylase was phosphorylated at a single site in both control and insulin on phosphorylase represented a decrease in 32P of approximately 50%. By comparison, the 32P content of glycogen synthase and the beta subunit of phosphorylase kinase were decreased by only 20 and 16%, respectively; the 32P content of the kinase alpha subunit was not affected by insulin. The results provide direct evidence that insulin decreases the amount of phosphate in phosphorylase and phosphorylase kinase. These findings have important implications with respect to both the regulation of glycogen metabolism in skeletal muscle and the mechanism of insulin action.

Animals↗