A Screen for Extracellular Signal-Regulated Kinase-Primed Glycogen Synthase Kinase 3 Substrates Identifies the p53 Inhibitor iASPP.

UNLABELLED: The Kaposi's sarcoma-associated herpesvirus (KSHV) LANA protein is essential for the replication and maintenance of virus genomes in latently KSHV-infected cells. LANA also drives dysregulated cell growth through a multiplicity of mechanisms that include altering the activity of the cellular kinases extracellular signal-regulated kinase (ERK) and glycogen synthase kinase 3 (GSK-3). To investigate the potential impact of these changes in enzyme activity, we used protein microarrays to identify cell proteins that were phosphorylated by the combination of ERK and GSK-3. The assays identified 58 potential ERK-primed GSK-3 substrates, of which 23 had evidence for in vivo phosphorylation in mass spectrometry databases. Two of these, SMAD4 and iASPP, were selected for further analysis and were confirmed as ERK-primed GSK-3 substrates. Cotransfection experiments revealed that iASPP, but not SMAD4, was targeted for degradation in the presence of GSK-3. iASPP interferes with apoptosis induced by p53 family members. To determine the importance of iASPP to KSHV-infected-cell growth, primary effusion lymphoma (PEL) cells were treated with an iASPP inhibitor in the presence or absence of the MDM2 inhibitor Nutlin-3. Drug inhibition of iASPP activity induced apoptosis in BC3 and BCBL1 PEL cells but did not induce poly(ADP-ribose) polymerase (PARP) cleavage in virus-negative BJAB cells. The effect of iASPP inhibition was additive with that of Nutlin-3. Interfering with iASPP function is therefore another mechanism that can sensitize KSHV-positive PEL cells to cell death. IMPORTANCE: KSHV is associated with several malignancies, including primary effusion lymphoma (PEL). The KSHV-encoded LANA protein is multifunctional and promotes both cell growth and resistance to cell death. LANA is known to activate ERK and limit the activity of another kinase, GSK-3. To discover ways in which LANA manipulation of these two kinases might impact PEL cell survival, we screened a human protein microarray for ERK-primed GSK-3 substrates. One of the proteins identified, iASPP, showed reduced levels in the presence of GSK-3. Further, blocking iASPP activity increased cell death, particularly in p53 wild-type BC3 PEL cells.

Antioxidant capacity of potato chips and snapshot trends in acrylamide content in potato chips and cereals on the Canadian market.

The concentration of acrylamide was measured in selected varieties of five brands of potato chips and breakfast cereals over a 5-year period. Most of the products were purchased in one locality in Canada. Samples were analysed by an isotope dilution ((13)C(3)) acrylamide method. They were extracted with water, partitioned with dichloromethane, filtered through a 5 kDa centrifuge filter, cleaned-up on HLB Oasis polymeric and Accucat mixed mode anion and cation exchange SPE columns, and analysed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The acrylamide concentration in potato chips varied from 106 to 4630 ng g(-1), while values in cereals varied from 50 to 347 ng g(-1). Wide variations were observed between brands, within brands over time, and between lots of the same brand. A subset of potato chip samples was analysed for in vitro antioxidant activity. No relationship was found between antioxidative capacity of potato chips and their acrylamide content.

Vegetable oils high in phytosterols make erythrocytes less deformable and shorten the life span of stroke-prone spontaneously hypertensive rats.

Previous studies have shown that canola oil (CA), compared with soybean oil (SO), shortens the life span of stroke-prone spontaneously hypertensive (SHRSP) rats, a widely used model for hemorrhagic stroke. SHRSP rats are highly sensitive to dietary cholesterol manipulations because a deficiency of membrane cholesterol makes their cell membranes weak and fragile. Phytosterols, abundant in CA but not in SO, can inhibit the absorption of cholesterol and also replace a part of cholesterol in cell membranes. This study was performed to determine whether the high concentration of phytosterols in CA might account for its life-shortening effect on SHRSP rats. Male, 35-d-old SHRSP rats (n = 28/group) were fed semipurified diets containing CA, SO, CA fortified with phytosterols (canola oil + phytosterols, CA + P), SO fortified with phytosterols (soybean oil + phytosterols, SO + P), corn oil (CO), olive oil (OO) or a fat blend that mimicked the fat composition of a representative Canadian diet (Canadian fat mimic, CFM; 10 g/100 g diet). These fats provided 97, 36, 207, 201, 114, 27 and 27 mg phytosterols/100 g diet, respectively. Ten rats from each group were killed after 30-32 d for blood and tissue analyses. The remaining rats (18/group) were used for determination of life span. The life span of SHRSP rats fed the high phytosterol oils (CA, CA + P, SO + P and CO) was significantly (P<0.05) shorter than that of CFM- and SO-fed rats. At 30-32 d, the groups fed the high phytosterol oils had greater levels of phytosterols and significantly (P<0.05) higher ratios of phytosterols/cholesterol in plasma, RBC, liver and kidney, and a significantly (P<0.05) lower RBC membrane deformabilty index than the groups fed oils low in phytosterols (SO, OO and CFM). The mean survival times were correlated with RBC deformability index (r(2) = 0.91, P = 0.0033) and cholesterol concentration (r(2) = 0.94, P = 0.0016), and inversely correlated with RBC phytosterol concentration (r(2) = 0.58, P = 0.0798) and phytosterols/cholesterol (r(2) = 0.65, P = 0.0579), except in the OO group. This study suggests that the high concentration of phytosterols in CA and the addition of phytosterols to other fats make the cell membrane more rigid, which might be a factor contributing to the shortened life span of SHRSP rats.

Vitamin C, Mood and Cognitive Functioning in the Elderly.

The present study examined the effects of anti-oxidant vitamin supplementation on mood and cognitive functioning in 205 volunteers (110 females, 95 males; age range: 60-80 years). In this randomised, double-blind, placebo-controlled study, the volunteers received either anti-oxidant supplementation (daily dosage 12mg/d ß-carotene, 400 mg/d α-tocopherol and 500mg/d ascorbic acid) or placebo. The volunteers were followed up for 12 months. Vitamin levels were assessed from plasma samples. The primary outcome measures were subjective mood, self-reported cognitive failures, and measures of intelligence. These were measured at 4, 8 and 12 months. There were very few significant differences between the placebo and vitamin groups. Analysis of the effects of changes in vitamin levels on mood and cognition revealed significant effects of changes in vitamin C but not the other anti-oxidants. Increases in vitamin C were associated at 12 months with more positive mood, greater improvements in global assessments of intellectual functioning and a reduction in everyday errors of memory, attention and action. These effects were greatest for those volunteers who had a more negative mood and lower levels of cognitive function at baseline. Overall, results support earlier findings based on examination of dietary intake.

The ability of the rat to metabolize myristoyl-methionine: an acylamino acid with potentially useful antibacterial properties.

Two experiments with Sprague Dawley rats tested their ability to hydrolyse myristoyl-methionine (M-M) into myristic acid and L-methionine (M). In the first experiment, lasting for 3 days. male rats were orally administered [9,10-3H]myristoyl-L-[35S]methionine. The recovery of radioactivity was approximately 90% for both isotopes; 19% of the administered 3H was recovered in the urine and 16% in the faeces, while the recovered 35S activity was 13 and 12%, respectively. The balance of the radioactivity was found among the tissues, organs and blood. In the second experiment, male and female rats received soybean-based diets which were supplemented with either 0.305% M-M or 0.2% M (both diets contained equal amounts of M) for periods up to 4 weeks. The growth rate of the rats receiving the 0.305% M-M diets was slightly slower than that for the rats on the 0.2% M diet, but the difference was not statistically significant (P > 0.05). The M-M rats had a transitory decrease in feed consumption, suggesting that palatability may have contributed to the growth difference and that a somewhat greater amount of M-M was necessary for the rat to attain the same growth rate as that produced by 0.2% M. When the amount of dietary M-M was increased to 3.05% M-M, a greater reduction in feed consumption and body weight gain was observed. This latter diet was an initial attempt to study the potential toxicity of M-M. None of the haematological, clinical chemistry or organ weight data suggested that M-M was overtly toxic per se, but longer-term feeding studies are needed to evaluate the potential toxicity of M-M more fully.

P32/TAP, a cellular protein that interacts with EBNA-1 of Epstein-Barr virus.

The Epstein-Barr virus (EBV) EBNA-1 protein has a central role in the maintenance of a latent EBV infection and is the only virus-encoded protein expressed in all EBV-associated tumors. EBNA-1 is required for replication of the episomal form of the latent viral genome and transactivates the latency C and LMP-1 promoters. The mechanisms by which EBNA-1 performs these functions are not known. Here we describe the cloning, expression, and characterization of a cellular protein, P32/TAP, which strongly interacts with EBNA-1. We show that P32/TAP is expressed at high levels in Raji cells and is synthesized as a proprotein of 282 amino acids (aa) that is posttranslationally processed by a two-step cleavage process to yield a mature protein of 209 aa. It has been previously reported that P32/TAP is expressed on the cell surface. Our transient expression assays detected full-length P32/TAP (1-282 aa) in the cytoplasm while mature P32/TAP protein localized to the nucleus. Three lines of evidence support P32/TAP interaction with EBNA-1. First, in the yeast two-hybrid system we mapped two interactive N-terminal regions of EBNA-1, aa 40-60 and aa 325-376, each of which contains arginine-glycine repeats. These regions interact with the C-terminal half of P32/TAP. Second, the full-length cytoplasmic P32/TAP protein can translocate nuclear EBNA-1 into the cytoplasm. Third, P32/TAP co-immunoprecipitated with EBNA-1. We have confirmed that a Gal4 fusion protein containing the C-terminal region of P32/TAP (aa 244-282) transactivates expression from a reporter containing upstream Gal4-binding sites. Deletion of the P32/TAP interactive regions of EBNA-1 severely diminished EBNA-1 transactivation of FRTKCAT in transient expression assays. Our data suggest that interaction with P32/TAP may contribute to EBNA-1-mediated transactivation.