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Biomedical subjects

J N Hawthorne

Publications and source records attributed to J N Hawthorne.

At least 19 recordsLinked to original sources
PubMed1991

The latent membrane protein-1 in Epstein-Barr virus-transformed lymphoblastoid cells is found with ubiquitin-protein conjugates and heat-shock protein 70 in lysosomes oriented around the microtubule organizing centre.

Immunofluorescence studies on Epstein-Barr virus (EBV)-transformed lymphoblastoid cells have previously shown that the latent membrane transforming protein (LMP-1) is found in patch-like inclusions which also immunostain for vimentin. We now show that EBV transformation causes a major reorganization of intermediate filaments, microtubules, mitochondria, and lysosomal elements, which generally become oriented around the microtubule organizing centre. Immunogold electron microscopy shows that LMP-1 is primarily concentrated in secondary lysosomes together with ubiquitin-protein conjugates and heat-shock protein 70. Intermediate filament inclusion formation with the above characteristics may be a general response triggered by other membrane glycoproteins; as seen, for example, in major human neurodegenerative diseases such as diffuse Lewy body disease.

Antigens, Viral↗
PubMed1991

Purification, properties, and phosphorylation by protein kinase C of two phosphoinositidase C isozymes from rat brain.

Two forms of phosphoinositidase C have been purified from the soluble fraction of rat brain. The purification scheme included gel filtration followed by chromatography on cellulose phosphate, phenyl-Sepharose, and Mono Q. Gradient sodium dodecyl sulphate-polyacrylamide gel electrophoresis gave apparent molecular masses of 151 kDa and 147 kDa. Western blotting with monoclonal antibodies showed that the isozymes corresponded to PLC-beta-1 and PLC-gamma of bovine brain. With both enzymes phosphatidylinositol 4,5-bisphosphate was a better substrate than phosphatidylinositol at neutral pH and low calcium ion concentrations. Both enzymes produced a proportion of inositol 1:2-cyclic phosphates from each substrate, particularly at acid pH. Some GTPase activity was seen in the early stages of purification, but was separated from PLC-beta-1 and PLC-gamma on Mono Q. Purified rat brain protein kinase C phosphorylated PLC-gamma but not PLC-beta-1. Incubation with the kinase increased the activity of both enzymes however, possibly by phosphorylation of another protein in the preparations.

Animals↗
PubMed1989

Second messengers involved in the muscarinic control of the heart: the role of the phosphoinositide response.

Two heart muscarinic responses are compared, the reduction of cyclic AMP concentration and the hydrolysis of phosphoinositides. It is suggested that the former is more important physiologically, since the latter is a slow response requiring much higher agonist concentrations. Inositol trisphosphate released from phosphoinositides is unlikely to produce a positive inotropic effect by releasing calcium from the sarcoplasmic reticulum. However, long term muscarinic effects may involve activation of protein kinase C by diacylglycerol released from phosphoinositides.

Animals↗
PubMed1988

The muscarinic receptor of rat pituitary GH3 cells is coupled with adenylate cyclase inhibition, but not with phosphoinositide turnover.

The effects of muscarinic stimulation on cyclic AMP accumulation and on basal and thyrotropin-releasing hormone (TRH)-induced phosphoinositide turnover have been studied in rat pituitary GH3 tumour cells. Carbachol produced a dose-dependent atropine-sensitive inhibition of basal cyclic AMP accumulation. It had no effect on basal or TRH-stimulated production of inositol phosphates. The majority of the muscarinic receptors had a low affinity (Ki 0.22 microM) for pirenzepine and can be classified as M2 type, inhibiting adenylate cyclase but having no links with phosphoinositide metabolism.

1-Methyl-3-isobutylxanthine↗
PubMed1988

Polyphosphoinositide hydrolysis in response to light stimulation of rat and chick retina and retinal rod outer segments.

Phosphoinositides of chick and rat retina were labelled with [3H]inositol. Exposure of retinal preparations to light for 30 s caused loss of labelled phosphatidylinositol 4,5-bisphosphate and to a smaller extent of the other phosphoinositides. Similar light-induced changes were seen when rod outer segment preparations were used and, when these were illuminated in calcium-free media, phosphatidylinositol 4,5-bisphosphate was the only lipid affected. No inositol 1,4,5-trisphosphate was seen after either 30 s or 5 s of illumination of retina or 30 s illumination of rod outer segments. It is concluded that this compound plays no direct part in vertebrate photoreceptor light transduction, though phosphoinositide metabolism might relate to adaptation mechanisms.

Animals↗
PubMed1988

Phospholipid turnover during cell-cycle traverse in synchronous Chinese-hamster ovary cells. Mitogenesis without phosphoinositide breakdown.

The turnover of phospholipids was investigated in quiescent serum-starved Chinese-hamster ovary (CHO-K1) cells stimulated to progress through the cell cycle by the addition of dialysed bovine serum. A variety of radiolabelling techniques were employed to study the rapid effects of serum on phospholipids and later events during G1 and S phases of the cell cycle. Pulse-labelling studies using [32P]Pi revealed that there was a stimulation of the synthesis rate of all phospholipids investigated during the initial few hours after serum addition. The greatest stimulation (20-fold) was observed in phosphatidylcholine, and the smallest in the polyphosphoinositides (PPIs). Mock stimulation with serum-free medium caused a similar increase in PPI turnover, but little or no effect on turnover of other phospholipids. This effect could be accounted for by a stimulation of the turnover of cellular ATP pools increasing [32P]ATP specific radioactivity. Late G1 and S phases were associated with a decrease in the rate of synthesis of all phospholipids. Phosphatidic acid was the only phospholipid whose labelling fell below that in mock-stimulated cells during the period of the cell cycle. Stimulation of serum-starved cells that had been prelabelled with myo-[2-3H]inositol caused no change in the amounts of inositol trisphosphate, but both serum-stimulated and mock-stimulated cells exhibited similar small decreases in both inositol bisphosphate and inositol monophosphate, of approx. 30% after 30 s. When cells were serum-stimulated in the presence of 10 mM-Li+, there was no increase in the size of the total inositol phosphate pool. We conclude that mitogenic stimulation and cell-cycle traverse cause profound and complex effects on phospholipid turnover in CHO-K1 cells, but there is no evidence for a role of inositol lipid turnover in the proliferative response to serum in this cell line.

Adenosine Triphosphate↗
PubMed1988

Reduced Na+ + K+-ATPase activity in peripheral nerve of streptozotocin-diabetic rats: a role for protein kinase C?

Six weeks after induction of diabetes, the rate of ouabain-sensitive 86Rb+ accumulation, a parameter which reflects Na+ + K+-ATPase pumping activity, was significantly reduced in endoneurial preparations of sciatic nerve from untreated diabetic rats compared with that in control rats (Trial, 1, 0.19 +/- 0.09 versus 0.48 +/- 0.13 pmol/min per mg wet weight of tissue, p less than 0.001; Trial 2, 0.27 +/- 0.16 versus 0.47 +/- 0.18, p less than 0.01). This decrease in ouabain-sensitive 86Rb+ uptake was not observed in nerves from diabetic rats maintained on sorbinil (an aldose reductase inhibitor) or myo-inositol diets. Protein kinase C activity was demonstrated in the soluble fraction of a sciatic nerve homogenate by assaying for lipid-activated, Ca+-dependent phosphorylation of calf thymus histone. No significant difference in the time course of kinase C activity was observed between cytosol fractions of nerve homogenates from control and diabetic rats (control, 6.22 +/- 0.97 pmol 32P incorporated/mg cytosol protein in 50 min; diabetic, 5.32 +/- 0.71). Three low molecular weight neural proteins (each with Mr less than 29,000) were identified as substrates for protein kinase C.

Animals↗
PubMed1987

Elevated phosphatidylinositol kinase activity in Rous sarcoma virus-transformed cells. Lack of evidence for enzyme translocation.

Phosphatidylinositol kinase (E.C. 2.7.1.67) activity of rat fibroblasts transformed by Rous sarcoma virus (RSV) was measured and compared with immunoprecipitated protein tyrosine kinase activity associated with pp60v-src. Both enzyme activities were elevated in the particulate fractions from wild-type RSV-transformed cells and cells transformed by a temperature-sensitive mutant of RSV when grown at the permissive temperature. The presence of the non-ionic detergent Nonidet P-40 in the phosphatidylinositol kinase assays stimulated the soluble and particulate forms of the enzyme to different degrees but did not affect the relative differences between transformed and untransformed cells. Our results indicate that phosphatidylinositol kinase activity is a good correlate of RSV transformation and suggest a functional relationship between pp60v-src and phosphatidylinositol kinase.

1-Phosphatidylinositol 4-Kinase↗
PubMed1987

Reduced phosphoinositide concentrations in anterior temporal cortex of Alzheimer-diseased brains.

Samples of brain anterior temporal cortex from 17 patients with Alzheimer's disease and 18 age-matched controls have been analysed for myo-inositol and the three phosphoinositides. There was significantly less phosphatidylinositol in the Alzheimer samples (1.36 mumol/g wet weight) than in the controls (2.28 mumol/g). The concentrations of phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate were also lower in Alzheimer cortex, but differences from the control group were not statistically significant. Free myo-inositol concentrations were 5.11 mumol/g (Alzheimer) and 4.44 mumol/g (control) and again the difference was not significant. The lack of phosphoinositides in Alzheimer temporal cortex may impair receptor function.

Aged↗
PubMed1986

Changes in sorbitol, myo-inositol and lipid inositol in dorsal root and sympathetic ganglia from streptozotocin-diabetic rats.

Sorbitol, fructose, myo-inositol and lipid inositol concentrations were measured in excised dorsal root and sympathetic ganglia from rats with streptozotocin-induced diabetes, both in the acute stage (1 and 2 weeks after the induction of diabetes) and chronically (after 2 months of diabetes). In comparison with age-matched controls, myo-inositol concentrations were decreased by 26-32% after 1 and 2 weeks but had returned to normal levels at 2 months. Lipid inositol concentrations were normal both in the acutely and chronically diabetic animals. Sorbitol was not detectable in ganglia from diabetic or control animals except for a small quantity (0.05 mumol/g wet weight) in dorsal root ganglia at the 2-month stage. Fructose was present in dorsal root ganglia (1.71-3.53 mumol/g wet weight) at all stages and in sympathetic ganglia (2.18 mumol/g wet weight) at the 8-week stage. The differences in these results from those obtained in peripheral nerve trunks are possibly related to the lack of a blood-nerve barrier in sensory and autonomic ganglia.

Animals↗
PubMed1986

Muscarinic release of inositol trisphosphate without mobilization of calcium in bovine adrenal chromaffin cells.

Chromaffin cells of bovine adrenal medulla release catecholamines in response to activation of nicotinic ACh receptors which open voltage-sensitive calcium channels. Catecholamine secretion by exocytosis requires an increase in cytosolic free calcium. The cells also possess muscarinic ACh receptors but muscarinic agents do not provoke catecholamine release. Quin-2 studies show that they do not increase cytosolic free Ca2+ concentration, but unlike the nicotinic agents, they cause phosphoinositide hydrolysis. Muscarinic stimulation leads to rapid loss of labelled phosphatidylinositol 4-phosphate and of phosphatidylinositol 4,5-bisphosphate. At the same time there is release of inositol trisphosphate, inositol bisphosphate and inositol phosphate. In a number of other cells inositol trisphosphate may act as a second messenger releasing Ca2+ from storage sites in the endoplasmic reticulum but this is not its function in bovine chromaffin cells.

Adrenal Medulla↗
PubMed1985

Stimulation of specific GTPase activity by vasopressin in isolated membranes from cultured rat hepatocytes.

Membranes were isolated by isotonic homogenization and differential centrifugation from rat hepatocytes cultured overnight. The specific GTPase activity of the membranes was 1-1.3 pmol gamma-labelled GTP hydrolysed/mg protein per min in the presence of 1.2 mM Na+, 2 mM EGTA, 1 mM ATP and 0.2 mM 5-adenylyl imidodiphosphate. Under these conditions there was a stimulation of specific GTPase activity of no more than 20% by 11-115 nM vasopressin. No effect of vasopressin was seen in the presence of 1.7 microM free Ca2+ or 100 mM Na+. The findings indicate that vasopressin is able to influence GTPase activity as well as accelerate phosphoinositide breakdown in rat hepatocytes.

Adenosine Triphosphate↗
PubMed1979

Enzymes of myo-inositol and inositol lipid metabolism in rats with streptozotocin-induced diabetes.

Diabetes, with only mild ketosis, was induced in male rats by a single injection of streptozotocin. After 12 weeks the specific activities of enzymes concerned with the metabolism of inositol and of inositol lipids were measured in various tissues. Inositol 1-phosphate synthase (EC 5.5.1.4) was most active in testis and the activity was significantly less in diabetic rats than in controls on a similar diet. Inositol oxygenase (EC 1.13.99.1), which converts myo-inositol into glucuronic acid, was also less active in kidney from diabetic animals. CDP-diacylglycerol-inositol phosphatidyltransferase (EC 2.7.8.11) and phosphatidylinositol 4-phosphate kinase (EC 2.7.1.68) showed decreased specific activities in brain and sciatic nerve of diabetic rats. By contrast the diabetic state did not affect the specific activities of phosphatidylinositol kinase (EC 2.7.1.67) or phosphatidylinositol 4,5-bisphosphate phosphatase (EC 3.1.3.36) in these tissues. The results are discussed in relation to diabetic neuropathy.

Animals↗