Olanzapine promotes fat accumulation in male rats by decreasing physical activity, repartitioning energy and increasing adipose tissue lipogenesis while impairing lipolysis.

Olanzapine and other atypical antipsychotics cause metabolic side effects leading to obesity and diabetes; although these continue to be an important public health concern, their underlying mechanisms remain elusive. Therefore, an animal model of these side effects was developed in male Sprague-Dawley rats. Chronic administration of olanzapine elevated fasting glucose, impaired glucose and insulin tolerance, increased fat mass but, in contrast to female rats, did not increase body weight or food intake. Acute studies were conducted to delineate the mechanisms responsible for these effects. Olanzapine markedly decreased physical activity without a compensatory decline in food intake. It also acutely elevated fasting glucose and worsened oral glucose and insulin tolerance, suggesting that these effects are adiposity independent. Hyperinsulinemic-euglycemic clamp studies measuring (14)C-2-deoxyglucose uptake revealed tissue-specific insulin resistance. Insulin sensitivity was impaired in skeletal muscle, but either unchanged or increased in adipose tissue depots. Consistent with the olanzapine-induced hyperglycemia, there was a tendency for increased (14)C-2-deoxyglucose uptake into fat depots of fed rats and, surprisingly, free fatty acid (FFA) uptake into fat depots was elevated approximately twofold. The increased glucose and FFA uptake into adipose tissue was coupled with increased adipose tissue lipogenesis. Finally, olanzapine lowered fasting plasma FFA, and as it had no effect on isoproterenol-stimulated rises in plasma glucose, it blunted isoproterenol-stimulated in vivo lipolysis in fed rats. Collectively, these results suggest that olanzapine exerts several metabolic effects that together favor increased accumulation of fuel into adipose tissue, thereby increasing adiposity.

Phase I trial on the safety of topical rhVEGF on chronic neuropathic diabetic foot ulcers.

OBJECTIVE: To assess the safety/tolerability and perform a preliminary efficacy evaluation of a multiple-dosing regimen of recombinant human vascular endothelial growth factor (VEGF165 or rhVEGF; telbermin) applied topically to chronic diabetic neuropathic foot ulcers. METHOD: Subjects with type 1 or 2 diabetes mellitus were randomised to receive either topical applied telbermin (72 microg/cm2) (n=29) or placebo (n=26) treatment to the foot ulcer surface in conjunction with standard ulcer care. Subjects received treatment every 48 hours (maximum three doses per week) for up to six weeks. Weekly 35mm photography, quantitative planimetry and physical examinations documented the ulcer appearance, surface area and stage. Safety endpoints included incidence of clinically significant hypotension, adverse events and ulcer infection. Exploratory efficacy endpoints included percentage reduction in total ulcer surface area, incidence of complete ulcer healing and time to complete ulcer healing. RESULTS: Incidence of adverse events was comparable in the two treatment groups. None of the adverse events were attributed to study drug, and no hypotension was observed as a result of telbermin treatment. Occurrence of infected study ulcers appeared to be balanced between the treatment groups. Positive trends suggestive of potential signals of biological activity were observed for incidence of complete ulcer healing (41.4% telbermin versus 26.9% placebo at day 43 [P=0.39]) and time to complete ulcer healing (25th percentile of 32.5 days telbermin versus 43.0 days placebo [log-rank P=0.13]). CONCLUSION: The topical application of telbermin 72 microg/cm2 three times a week for up to six weeks appeared to be well tolerated. Further studies are required to characterise the safety/efficacy of telbermin more completely.

Loss of one p53 allele results in four-fold reduction of p53 mRNA and protein: a basis for p53 haplo-insufficiency.

A haploid genotype may be insufficient to support normal wild-type function. Such haplo-insufficiency has recently been documented for numerous tumour suppressor genes. p53 is a crucial tumour suppressor governing DNA repair, cell cycle arrest and apoptosis via its role as a stress-responsive transcription factor. p53 haplo-insufficiency has been observed in vivo with human familial cancer in Li-Fraumeni Syndrome (LFS) and in mouse p53-knockout models of LFS. The increased tumorigenesis associated with loss of one p53 allele has been attributed to reduced p53-dependent stress responses. However, the underlying biochemical basis for such attenuated responses in p53+/- cells remains unclear. Here we have determined basal p53 messenger RNA (mRNA) and protein levels, and compared the p53 stress response in p53+/+, p53+/- and p53-/- isogenic clones derived from HCT116 cells. Basal expression of p53 in p53+/- cells was 25% relative to p53+/+ cells, and this differential was maintained following oncogenic stress. This deficiency was manifested at both p53 mRNA and protein levels and resulted in attenuated p53 stress responses, in particular for p21waf1 upregulation and survivin downregulation, and reduced G1 arrest and apoptosis. These observations identify a molecular basis for wild-type p53 haplo-insufficiency, which may explain the attenuated tumour-suppressive phenotype observed in cells with a single wild-type p53 allele and in humans with LFS.

Guanine nucleotide binding regulatory proteins and adenylate cyclase in livers of streptozotocin- and BB/Wor-diabetic rats. Immunodetection of Gs and Gi with antisera prepared against synthetic peptides.

Adenylate cyclase in liver plasma membranes from streptozotocin-diabetic (STZ) or BB/Wor spontaneously diabetic rats showed increased responsiveness to GTP, glucagon, fluoroaluminate, and cholera toxin. Basal or forskolin-stimulated activity was unchanged in STZ rats, but increased in BB/Wor rats. No change in the alpha-subunit of Gi (alpha i) was observed in STZ or BB/Wor rats using pertussis toxin-stimulated [32P]ADP-ribosylation. Immunodetection using antibodies against the COOH-terminal decapeptides of alpha T and alpha i-3 showed no change in alpha i in STZ rats and a slight decrease in BB/Wor rats. Angiotensin II inhibition of hepatic adenylate cyclase was not altered in either diabetic rat. In both models of diabetes, Gs alpha-subunits were increased as measured by cholera toxin-stimulated [32P]-ADP-ribosylation of 43-47.5-kD peptides, reconstitution with membranes from S49 cyc- cells or immunoreactivity using antibodies against the COOH-terminal decapeptide of alpha s. These data indicate that STZ-diabetes increases hepatic Gs but does not change Gi or adenylate cyclase catalytic activity. In contrast, BB/Wor rats show increased hepatic Gs and adenylate cyclase. These changes could explain the increase in hepatic cAMP and related dysfunctions observed in diabetes.

Simultaneous loss and reappearance of alpha 1-adrenergic responses and [3H]prazosin binding sites in rat liver after irreversible blockade by phenoxybenzamine.

The relative influences of the in vivo administration of phenoxybenzamine on in vitro binding to alpha 1-adrenergic receptors and alpha 1-receptor-mediated responses were studied. Phenoxybenzamine treatment reduced maximal specific binding of the alpha 1-selective antagonist [3H]prazosin to liver cell membranes. This response was rapid (less than 90 min) and half-maximal following a phenoxybenzamine dose of approx. 10 mg/kg. A similar decrease in the ability of phenylephrine to stimulate glucose release and 45Ca2+ efflux from liver slices was also noted after phenoxybenzamine treatment. During the recovery period following administration of 30 mg/kg phenoxybenzamine, [3H]prazosin specific binding and phenylephrine-stimulated glucose release and 45Ca2+ efflux returned to their respective control levels with t 1/2 values of 42, 49 and 38 h, respectively. At all times studied during the recovery period, alpha 1-binding and both of the alpha 1-responses were similar fractions of their respective control values. These observations indicate that a close relationship exists between the density of [3H]prazosin binding sites and the ability of rat liver to respond to alpha 1-stimulation. We suggest that the binding sites identified in studies using the antagonist [3H]prazosin and those through which the agonist phenylephrine stimulates glucose release and 45Ca2+ efflux are either identical or in equilibrium with each other.

An endogenous Ca2+-sensitive proteinase converts the hepatic alpha 1-adrenergic receptor to guanine nucleotide-insensitive forms.

An iodoazido[125I]prazosin analogue was employed to photoaffinity label alpha 1-adrenergic receptors in rat liver plasma membranes. Labeled proteins were separated by gradient polyacrylamide gel electrophoresis in sodium dodecyl sulfate, and (-)-epinephrine displacement of [3H]prazosin binding was concurrently measured in the presence or absence of guanosine 5'-O-(gamma-thiotriphosphate) (GTP[gamma S]). Inclusion of EGTA and/or proteinase inhibitors during membrane preparation and incubation increased the effect of GTP[gamma S] on alpha 1-adrenergic agonist binding and this could be correlated with increased concentrations of a 78 kDa photoaffinity labeled protein. In contrast, omission of EGTA or addition of exogenous Ca2+ diminished or abolished the effect of GTP[gamma S] on binding and caused loss of the 78 kDa form and the appearance of lower molecular weight labeled proteins. Age-dependent differences in GTP[gamma S] effects on alpha 1-adrenergic agonist binding were abolished when membranes were prepared and incubated in the presence of EGTA and proteinase inhibitors. However, the 78 kDa photoaffinity labeled protein observed in adult rats (over 225 g body weight) was not apparent in membranes from younger rats (50-75 g), even when the membranes were prepared and incubated in the presence of EGTA and proteinase inhibitors. Instead, a 68 kDa species was the major labeled protein. These data suggest that GTP effects on alpha 1-adrenergic agonist binding in rat liver membranes require the presence of either a 68 or 78 kDa alpha 1-adrenergic binding protein. Failure to inhibit proteolysis in the membranes leads to the generation of lower-molecular-weight binding proteins and the loss of GTP effects on alpha 1-adrenergic agonist binding, although [3H]prazosin binding characteristics are not changed. It is suggested that either the proteolyzed forms of the alpha 1-adrenergic receptor are unable to couple to a putative guanine nucleotide-binding regulatory protein, or that such a protein is concurrently proteolyzed and is thus unable to couple to the receptor.

Erythropoietin modulation of intracellular calcium: a role for tyrosine phosphorylation.

We have reported that erythropoietin induces a dose-dependent increase in cytosolic calcium ([Cai]) in single human peripheral blood BFU-E derived erythroblasts which is specific for stage of differentiation and that this increase is modulated by erythropoietin through an ion channel permeable to Ca2+. Here, the role of protein phosphorylation in the increase in intracellular free calcium [Cai] stimulated by erythropoietin was studied with digital video imaging. Preincubation of day 10 erythroblasts with a broad inhibitor of serine/threonine and tyrosine kinases, staurosporine (100 nM), blocked the increase in [Cai] over 20 min following erythropoietin stimulation. However, erythropoietin-induced calcium influx was unaffected by preincubation of cells with specific inhibitions of protein kinase C (calphostin C) or the cAMP- or cGMP-dependent kinases (KT 5720, HA 1004), and [Cai] did not increase following stimulation with phorbol 12-myristate 13-acetate (PMA) or dibutyryl cAMP. These results suggest that neither protein kinase C nor protein kinase A mediate the erythropoietin-induced [Cai] increase. In contrast, preincubation with genistein, a tyrosine kinase inhibitor, blocked the erythropoietin induced increase in [Cai]. To further study calcium entry in erythroblasts, we determined mastoparan, a peptide from wasp venom, induced a dose-dependent rise in [Cai] in erythroblasts which required external calcium. Stimulation of erythroid precursors with 10 microM mastoparan resulted in an increase in [Cai] from 52 +/- 3 nM to 214 +/- 36 nM which peaked at 20 min. The mastoparan-induced [Cai] increase was also dependent on tyrosine phosphorylation since it was blocked by preincubation with genistein. These results demonstrate that both erythropoietin and mastoparan stimulate calcium entry by a mechanism which has a genistein sensitive step and suggest that tyrosine kinase activation is required for the rise in [Cai] to occur.

Alpha-subunits of Ns are released from the plasma membrane following cholera toxin activation.

Cholera toxin (CT) and islet-activating protein (IAP, a Bordetella pertussis toxin) were employed to test the hypothesis that GTP-binding regulatory proteins are released from plasma membranes to a greater extent when 'activated' than when 'inactivated'. CT, which activates Ns (the stimulatory GTP-binding regulatory protein of the adenylate cyclase system), catalyzed the incorporation of radioactivity from [32P]NAD into 45 and 47.5 kDa peptides associated with rat liver plasma membranes. Following ADP-ribosylation and centrifugation at 100000 X g for 1 h, approx. 30-35% of these CT-labelled peptides were no longer associated with the plasma membranes, but were recovered from the supernatant fraction. IAP, which inactivates Ni (the inhibitory GTP-binding regulatory protein of the adenylate cyclase system) catalyzed the incorporation of radioactivity from [32P]NAD into a 41 kDa peptide associated with the membranes. However, in contrast to the CT-labelled peptides, typically less than 5% of the IAP-labelled peptide was found in the 100000 X g supernatant fraction, but rather was almost exclusively associated with the membrane pellet. The data indicate that the alpha-subunits of Ns are released from the plasma membrane following activation, and support the hypothesis that the beta gamma-subunits act to anchor the alpha-subunits to the plasma membrane.

Formation of the high-affinity agonist state of the alpha 1-adrenergic receptor at cold temperatures does not require a G-protein.

Two methods were employed to uncouple hepatic alpha 1-adrenergic receptors from their associated G-protein (termed Gp) in order to determine whether locking of the alpha 1-receptor in a high-affinity agonist state at cold temperatures (2 degrees C) represents formation of a ternary complex. Uncoupling is defined as the inability to observe the GppNHp-sensitive, high-affinity agonist state of the receptor in [3H]prazosin competition binding studies performed at 25 degrees C. The first method for achieving uncoupling involved brief alkalinization and resulted in greater than 95% loss of several G-proteins. The second method involved proteolytic cleavage of either part or all of the alpha 1-receptor coupling domain from the binding domain. Following either treatment, receptors were converted to the high-affinity agonist state at 2 degrees C. Thus, while formation of the high-affinity state of the receptor at higher temperatures may require Gp, formation of this state at 2 degrees C does not require Gp or even the entire alpha 1-adrenergic receptor.

Okadaic acid stimulates ouabain-sensitive 86Rb(+)-uptake and phosphorylation of the Na+/K(+)-ATPase alpha-subunit in rat hepatocytes.

Ca(2+)-mobilizing and cAMP-dependent hormones rapidly increase sodium, potassium-dependent adenosine triphosphatase (Na+/K(+)-ATPase)-mediated transport in rat hepatocytes. To explore the possible role of protein phosphatases in these responses we used a protein phosphatase inhibitor, okadaic acid. Okadaic acid stimulation of ouabain-sensitive 86Rb(+)-uptake was maximal between two and three minutes and displayed an EC50 of 41 +/- 1 nM. Inhibition of Na+/H+ exchange with an amiloride analog abolished the response to insulin, but had no effect on okadaic acid-mediated stimulation of Na+/K(+)-ATPase transport. In hepatocytes metabolically-radiolabeled with 32Pi, okadaic acid stimulated the incorporation of radioactivity into several 95 kDa peptides, one of which reacted with anti-LEAVE peptide antisera, that recognizes Na+/K(+)-ATPase alpha-subunits. In other experiments Na+/K(+)-ATPase was immunoprecipitated from detergent-solubilized membrane fractions of metabolically-radiolabeled cells with an antisera to purified rat kidney Na+/K(+)-ATPase. A 95 kDa phosphoprotein was immunoprecipitated using anti-Na+/K(+)-ATPase antisera, but not by preimmune serum. Okadaic acid stimulated incorporation of radioactivity into this band by 220 +/- 28%. These findings provide support for the hypothesis that rapid stimulation of hepatic Na+/K(+)-ATPase by hormones may be related to protein kinase/phosphatase-mediated changes in the phosphorylation state of the Na+/K(+)-ATPase alpha-subunit.

Evidence for a decrease in the efficiency of beta-receptor coupling to adenylate cyclase in liver membranes from sucrose-fed rats.

Sucrose feeding has been shown previously to alter the plasma concentration of several factors which may regulate beta-adrenergic receptors, including corticosteroids and insulin as well as altered sympathetic nervous system (SNS) tone. For this reason we initiated a study of the effects of sucrose feeding on the beta-adrenergic receptor-adenylate cyclase system in rat liver plasma membranes. Beta-Adrenergic responsiveness was monitored by measuring isoproterenol stimulation of adenylate cyclase activity, while beta-adrenergic receptor characteristics were evaluated by analyzing [125I]iodocyanopindolol [( 125I]CYP) binding. Rats fed rat chow ad lib. supplemented by drinking water containing 10% sucrose solution exhibited a 50-75% reduction in hepatic isoproterenol-sensitive adenylate cyclase activity. This effect of sucrose was also observed in adrenalectomized (ADX) and 6-hydroxydopamine-pretreated animals, ruling out a causal role for corticosteroids or the sympathetic nervous system respectively. No effect was observed on basal, glucagon-, fluoride- or GTP-stimulated adenylate cyclase. A small but significant decrease in [125I]CYP specific binding capacity was observed in liver membranes prepared from sucrose-fed ADX rats, whereas no change in [125I]CYP binding capacity was observed in in sucrose-fed normal rats. These observations suggest that beta-receptor to adenylate cyclase coupling efficiency is decreased by the sucrose diet. The activities of two membrane-associated phospholipid methyltransferases and the content of endogenous S-adenosylmethionine in liver were reduced by sucrose feeding, implying a defect in the methylation pathway for phosphatidylcholine synthesis. The possible relationship between this latter finding and the observed decrease in beta-adrenergic receptor to adenylate cyclase coupling efficiency is discussed.

Altered expression of skeletal muscle proteins during sepsis.

Sepsis induces a net catabolic state in gastrocnemius by increasing protein degradation and decreasing protein synthesis. To determine whether or not sepsis induces a preferential effect on the expression of individual proteins, proteins from gastrocnemius muscle of control and septic rats were separated by two-dimensional isoelectric focusing/sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Laser densitometry of proteins stained with silver provided evidence that the relative abundance of thirty-five proteins was significantly (p < .05) and reproducibly increased during sepsis compared to control. No individual protein underwent significant down-regulation in their relative abundance during sepsis. Twenty-three of the 35 proteins identified in two-dimensional gels of the gastrocnemius were also present in the plasma of septic rats. The remaining 12 proteins, therefore, were taken to represent skeletal muscle proteins. One of the 12 proteins was identified by immunoblot analysis to be carbonic anhydrase III. Another of the proteins was identified as triosephosphate isomerase based upon microsequencing of the N terminus.

Lack of correlation between [3H]ouabain binding and Na-K ATPase inhibition in rat aorta.

The binding of [3H]ouabain to intact strips of rat aorta was compared with the ability of ouabain to inhibit the uptake of 86Rb by the same preparation. When a cold temperature wash was used to process tissues after binding of [3H]ouabain, a class of relatively high affinity binding sites was found (KD = 1.2 X 10(-7) M). Binding was saturable and sensitive to both ATP depletion and elevated potassium. Elevation of cytoplasmic Ca2+ levels by phenylephrine or c-AMP levels by theophylline and terbutaline had no influence on [3H]ouabain binding. Ouabain inhibition of 86Rb uptake progressed to 60% of the total 86Rb uptake at 2 X 10(-3) from a threshold of about 10(-5) M. Half-maximal inhibition by ouabain occurred at a concentration of 10(-4) M. The disparity between [3H]ouabain binding and inhibition of 86Rb uptake indicates that the high affinity binding site in the rat does not contribute to inhibition of Na-K ATPase function.

Mouth versus deep airways absorption of nicotine in cigarette smokers.

Nicotine from the alkaline smoke of cigars is absorbed through the buccal mucosa, but such absorption from the more acidic smoke of American cigarettes has not been reported. Forty-one male and 52 female smokers were studied under normal ventilation and smoking conditions, and under high ventilation and controlled smoking conditions that restricted intake to the mouth only, with no inhalation. The major finding is that there is virtually no intake of nicotine through the buccal mucosa while smoking American cigarettes. Confirming prior reports, plasma nicotine and expired CO levels showed no correlation with the analytical yields of nicotine and CO of the cigarettes smoked. Fifteen nonsmokers (7 male, 8 female) participated in this study as controls. Data from these subjects provided additional information regarding absorption of nicotine and carbon monoxide during passive smoking. Within the highly ventilated environment, there was no significant change of CO and nicotine levels of nonsmokers. However, within the normally ventilated environment, there was minimal increase in both substances, statistically significant only for nicotine. These results suggest that nicotine may be a better indicator of exposure to second-hand smoke than carbon monoxide.

Role of guanine nucleotide regulatory proteins and inositol phosphates in the hormone induced mobilization of hepatocyte calcium.

Treatment of isolated hepatocytes with F- produced a concentration-dependent activation of phosphorylase, efflux of Ca2+, rise in [Ca2+]i, increase in Ins 1,4,5-P3 levels, decrease in PI-4,5-P2 levels, and increase in DAG levels. The levels of intracellular cAMP were decreased by NaF. The effects of NaF were potentiated by AlCl3. This potentiation was abolished by the Al3+ chelator deferoxamine. These results illustrate that AlF4- can mimic the effects of Ca2+-mobilizing hormones in hepatocytes and suggest that the coupling of the receptors for these hormones to the hydrolysis of PI-4,5-P2 is through a guanine nucleotide-binding regulatory protein. This is because AlF4- is known to modulate the activity of other guanine nucleotide regulatory proteins (Gi, Gs, and transducin). Calcium-sensitive inositide release in a purified rat liver plasma membrane preparation was increased by calcium-mobilizing hormones in the presence of guanine nucleotides. Vasopressin-stimulated inositide release was evident in the presence of GTP or GTP gamma S. The guanine nucleotide and hormonal stimulation was evident on both inositide production and PI 4,5-P2 degradation. Treatment of plasma membranes with cholera toxin or islet activating protein or prior injection of animals with islet activating protein did not affect stimulation of inositide release by GTP gamma S or GTP gamma S plus vasopressin. The results suggest that calcium-mobilizing hormones stimulate polyphosphoinositide breakdown in rat liver plasma membranes through a novel guanine nucleotide binding protein. The GTPase activity of rat liver plasma membranes was stimulated 20% by 10(-8) M vasopressin. The vasopressin-stimulated GTPase activity was not inhibited in plasma membranes that had been ADP-ribosylated with either cholera toxin or pertussis toxin. When membranes that had been solubilized after preincubation with [3H]vasopressin were subjected to sucrose gradient centrifugation, most of the protein-bound [3H]vasopressin migrated as a single band, also, there was a GTPase activity that migrated with the bound [3H]vasopressin. This peak of bound [3H]vasopressin was decreased 90% when the sucrose gradient centrifugation was run in the presence of 10 M GTP gamma S. Direct evidence that a GTP-binding protein was present in the [3H]vasopressin peak was obtained by the immuno-detection of a 35 kDa beta subunit of a GTP-binding protein and a 40 kDa alpha subunit. These results support the conclusion that liver plasma membranes contain a GTP-binding protein that can complex with the vasopressin receptor.(ABSTRACT TRUNCATED AT 400 WORDS)

The acoustic characteristics of the cry of an infant with unilateral vocal fold paralysis.

We have previously shown the acoustic similarity among well, full-term infants of the same post-conceptional and post-partum ages (Int. J. Pediat. Otorhinolaryngol., 10 (1985) 1-8). The objective of that research was to provide a template against which the cries of any given infant may be compared. This paper shows a more detailed analysis of both the normal infants' cries and the cry of an infant with a unilateral vocal fold paralysis. It is evident from this analysis that this infant's cry is not normal as it displays a spectrum not found in the cries of normal infants. What this research shows is that a technique is available with which it is possible to make a screening identification of those infants who merit further investigation. It is an objective of such research to determine if the cry of a given infant is abnormal in specifiable, and therefore diagnostic, ways. In this instance, we can conclude that the cry of this infant differs from that of one with a normal airway.

Crosstalk between site-specific modifications on p53 and histone H3.

Previously, we have observed a link between p53 expression and histone H3 post-translational modifications. Here, we ask if specific post-translational modifications of p53 impact upon histone H3 modifications in a selective manner. We have also screened for internal co-operative effects within the repertoire of p53 modifications. Exogenous p53 constructs were expressed in HCT116 p53-/- cells. Four mutant p53 constructs were used, with single 'phosphorylation' mutations at serines 15 and 37 (S15A, S15D, S37A and S37D) and compared with exogenously expressed wild-type p53. The results showed that the replacement of serine 15 with either alanine (S15A) or aspartic acid (S15D) induced phosphorylation at S33P, S37P and S46P. In contrast, phosphorylation mutants p53(S37A) and p53(S37D) were not phosphorylated on S33. S46 phosphorylation appeared specifically enhanced by p53(S37D) relative to p53(S37A). Distal induction of S392 phosphorylation was observed for each of the p53 N-terminal phosphorylation mutants. Analysis of endogenous histone H3 (from the transfected cells) revealed loss of di-methylated K9 following expression of wild type and mutant p53 constructs. Expression of p53 (S15A), (S15D) and (S37A) selectively induced acetylation at K9 and K14. In contrast, wt p53 and p53(S37D) had no effect upon K9 or K14 acetylation. K18 acetylation status was unaffected throughout.

Role of leucine in the regulation of mTOR by amino acids: revelations from structure-activity studies.

In this study an overview is presented of the mTOR signaling pathway and its regulation by amino acids, particularly L-leucine. Our laboratory is studying amino acid regulation of mTOR in adipocytes. Potential roles for mTOR in adipocytes that were previously posited include hypertrophic growth, leptin secretion, protein synthesis and adipose tissue morphogenesis. A current area of interest in the field is how amino acids regulate mTOR and which amino acids are regulatory. Revelations concerning mechanism and recognition are emerging from different laboratories that examined the structural requirements for stimulation and inhibition of the mTOR signaling pathway by leucine and amino acid analogs. In adipocytes and some other cell types, leucine appears to be the main regulatory amino acid. However, this is not uniformly the case. In those cells where mTOR is regulated by several amino acids, there is evidence that the mechanism of mTOR activation may be different from cells where mainly leucine is regulatory. Furthermore, in tissues where leucine regulates mTOR, the possible existence of different tissue-specific leucine recognition sites may be indicated.

Spontaneous cigarette brand switching: consequences for nicotine and carbon monoxide exposure.

A group of smokers who had participated in smoking-related studies three to six years earlier were re-studied to assess changes in their smoking practices. Individuals who smoked the same brands of cigarettes showed no change in plasma cotinine (reflecting exposure to nicotine) or expired carbon monoxide (CO) concentration. Those who switched to cigarettes of lower nicotine yield (average decrease 38 per cent) showed reduced plasma cotinine concentrations, due primarily to smoking fewer cigarettes per day. The intake of nicotine per cigarette was not different. Subjects who smoked cigarettes of higher yield (102 per cent increase) had higher cotinine and CO levels, due to greater intake per cigarette.