PKB-mediated PHF20 phosphorylation on Ser291 is required for p53 function in DNA damage

PHD finger protein 20 (PHF20) is a transcription factor, which was originally identified in glioma patients. PHF20 appears to be a novel antigen in glioma, and has also termed glioma-expressed antigen 2. PHF20 is thought to contribute to the development of cancers, including glioblastoma, lung cancer, colon cancer and ovarian cancer. However, little is known about the function of PHF20 in various cancers. Here we report that PHF20 contains two consensus sites for protein kinase B (PKB) phosphorylation (RxRxxS/T). PKB can directly phosphorylate PHF20 on Ser291 in vitro and in vivo. It has been shown that PKB participates in the tumor suppressor p53 regulated gene expression program and has a direct effect on p21 regulation after DNA damage. UV-induced DNA damage results in accumulation of p53 and PKB activation. Interestingly, PKB-mediated PHF20 phosphorylation led to an inhibition of p53 induction following UV treatment, leading to the reduction of p21 transcriptional activity. Using anti PHF20 and anti pPKB (S473) antibodies, these events were mapped in various human cancer tissues. Taken together, these data suggest that PHF20 is a novel substrate for PKB and its phosphorylation by PKB plays an important role in tumorigenesis via regulating of p53 mediated signaling. © 2012 Elsevier Inc.

Immunochemical characterization of a polysaccharide antigen of Bacteroides fragilis with an IgM monoclonal antibody

An IgM mouse monoclonal antibody (McAb) Bf4 was produced to a surface polysaccharide of Bacteroides fragilis NCTC 9343. Immunoblotting showed that McAb Bf4 reacted strongly with a high molecular mass structure which was sensitive to oxidation with periodate but resisted protease treatment. An inhibition enzyme-linked immunosorbent assay (ELISA) indicated that McAb Bf4 did not cross react with the sixteen Bacteroides species and strains tested. Cells of B. fragilis NCTC 9343 recovered from the various interfaces of a Percoll discontinuous density gradient were tested in the inhibition ELISA. Bacteria from the 0-20%, 20-40% and 40-60% interfaces inhibited the ELISA; however, cells from the 60-80% interface did not. Electron microscopy with immunogold labelling showed that McAb Bf4 did not react with the extracellular fibrous network on bacteria recovered from the 0-20% interface, or the extracellular electron dense layer on cells from the 60-80% interface; however, it was associated with a surface structure on cells from the 20-40% interface. Growth in vivo did not enrich for bacteria with this structure.

Immunotoxicity of aflatoxin B1: Impairment of the cell-mediated response to vaccine antigen and modulation of cytokine expression

Aflatoxin B1 (AFB1), a mycotoxin produced by Aspergillus flavus or A. parasiticus, is a frequent contaminant of food and feed. This toxin is hepatotoxic and immunotoxic. The present study analyzed in pigs the influence of AFB1 on humoral and cellular responses, and investigated whether the immunomodulation observed is produced through interference with cytokine expression. For 28 days, pigs were fed a control diet or a diet contaminated with 385, 867 or 1807 mu g pure AFB1/kg feed. At days 4 and 15, pigs were vaccinated with ovalbumin. AFB1 exposure, confirmed by an observed dose-response in blood aflatoxin-albumin adduct, had no major effect on humoral immunity as measured by plasma concentrations of total IgA, IgG and IgM and of anti-ovalbumin IgG. Toxin exposure did not impair the mitogenic response of lymphocytes but delayed and decreased their specific proliferation in response to the vaccine antigen, suggesting impaired lymphocyte activation in pigs exposed to AFB1. The expression level of pro-inflammatory (TNF-alpha, IL-1 beta, IL-6, IFN-gamma) and regulatory (IL-10) cytokines was assessed by real-time PCR in spleen. A significant up-regulation of all 5 cytokines was observed in spleen from pigs exposed to the highest dose of AFB1. In pigs exposed to the medium dose, IL-6 expression was increased and a trend towards increased IFN-gamma and IL-10 was observed. In addition we demonstrate that IL-6 impaired in vitro the antigenic- but not the mitogenic-induced proliferation of lymphocytes from control pigs vaccinated with ovalbumin. These results indicate that AFB1 dietary exposure decreases cell-mediated immunity while inducing an inflammatory response. These impairments in the immune response could participate in failure of vaccination protocols and increased susceptibility to infections described in pigs exposed to AFB1. (C) 2008 Elsevier Inc. All rights reserved.

Glutathione peroxidase 1 genotype is associated with an increased risk of coronary artery disease

Objective Oxidative stress is implicated in the pathogenesis of many human diseases including atherosclerosis. Human glutathione peroxidase 1 (hgpx1) participates in limiting cellular damage caused by oxidation. A characteristic polyalanine sequence polymorphism in exon 1 of hgpx1 produces three alleles with five, six or seven alanine (ALA) repeats in this sequence. The objective of this study was to determine whether hgpx1 genotype is associated with an altered risk of coronary artery disease (CAD).

Methods The frequency of the ALA6 allele was determined in 207 men with angiographic evidence of significant CAD compared to a control group (n = 146), by analysing the lengths of polymerase chain reaction fragments containing the ALA repeat polymorphism. Additional information was collected on severity of CAD, presence or absence of a prior acute myocardial infarction (AMI), smoking status, body mass index (BMI) and other clinical data.

Results There was a significant association between individuals with at least one ALA6 allele and an increased risk of CAD after adjustment for age, BMI and smoking status (odds ratio, 2.07, 95% confidence interval, 1.08-3.99, P = 0.029). However, there was no association between hgpx1 genotype and a previous history of AMI or hgpx1 genotype and severity of CAD.

Conclusion We conclude that individuals possessing one or two ALA6 alleles appear to be at a modest increased risk of CAD. This observation merits further investigation in other patient populations.

Activation of the ACE2/Angiotensin-(1-7)/Mas Receptor Axis Enhances the Reparative Function of Dysfunctional Diabetic Endothelial Progenitors

We tested the hypothesis that activation of the protective arm of the renin angiotensin system, the angiotensin-converting enzyme 2 (ACE2)/angiotensin-(1-7) [Ang-(1-7)]/Mas receptor axis, corrects the vasoreparative dysfunction typically seen in the CD34(+) cells isolated from diabetic individuals. Peripheral blood CD34(+) cells from patients with diabetes were compared with those of nondiabetic controls. Ang-(1-7) restored impaired migration and nitric oxide bioavailability/cGMP in response to stromal cell-derived factor and resulted in a decrease in NADPH oxidase activity. The survival and proliferation of CD34(+) cells from diabetic individuals were enhanced by Ang-(1-7) in a Mas/phosphatidylinositol 3-kinase (PI3K)/Akt-dependent manner. ACE2 expression was lower, and ACE2 activators xanthenone and diminazine aceturate were less effective in inducing the migration in cells from patients with diabetes compared with controls. Ang-(1-7) overexpression by lentiviral gene modification restored both the in vitro vasoreparative functions of diabetic cells and the in vivo homing efficiency to areas of ischemia. A cohort of patients who remained free of microvascular complications despite having a history of longstanding inadequate glycemic control had higher expression of ACE2/Mas mRNA than patients with diabetes with microvascular complications matched for age, sex, and glycemic control. Thus, ACE2/Ang-(1-7)\Mas pathway activation corrects existing diabetes-induced CD34(+) cell dysfunction and also confers protection from development of this dysfunction.

Misfolding of galactose 1-phosphate uridylyltransferase can result in type I galactosemia

Type I galactosemia is a genetic disorder that is caused by the impairment of galactose-1-phosphate uridylyltransferase (GALT; EC 2.7.7.12). Although a large number of mutations have been detected through genetic screening of the human GALT (hGALT) locus, for many it is not known how they cause their effects. The majority of these mutations are missense, with predicted substitutions scattered throughout the enzyme structure and thus causing impairment by other means rather than direct alterations to the active site. To clarify the fundamental, molecular basis of hGALT impairment we studied five disease-associated variants p.D28Y, p.L74P, p.F171S, p.F194L and p.R333G using both a yeast model and purified, recombinant proteins. In a yeast expression system there was a correlation between lysate activity and the ability to rescue growth in the presence of galactose, except for p.R333G. Kinetic analysis of the purified proteins quantified each variant's level of enzymatic impairment and demonstrated that this was largely due to altered substrate binding. Increased surface hydrophobicity, altered thermal stability and changes in proteolytic sensitivity were also detected. Our results demonstrate that hGALT requires a level of flexibility to function optimally and that altered folding is the underlying reason of impairment in all the variants tested here. This indicates that misfolding is a common, molecular basis of hGALT deficiency and suggests the potential of pharmacological chaperones and proteostasis regulators as novel therapeutic approaches for type I galactosemia.

A strategy for sensitivity and specificity enhancements in prostate specific antigen-[alpha] 1-antichymotrypsin detection based on surface plasmon resonance

A biochip based on surface plasmon resonance was fabricated to detect prostate specific antigen-a1-antichymotrypsin (PSA-ACT complex) in both HBS buffer and human serum. To reduce non-specific binding and steric hindrance effect, the chemical surface of the sensor chips was constructed by using various oligo(ethylene glycol) mixtures of different molar ratios of HS(CH2)11(OCH2CH2)6OCH2COOH and HS(CH2)11(OCH2CH2)3OH. The self-assembled monolayers were biotinylated to facilitate the immobilization of streptavidin. Using the chip surfaces, PSA-ACT complex in HBS buffer and human serum was detected at 20.7 and 47.5 ng/ml by primary immunoresponse, respectively. However, the limit of detection could be simply enhanced by a sandwich strategy to improve the sensitivity and specificity of the immunoassay. An intact PSA polyclonal antibody was used as an amplifying agent in the strategy. As a result, PSA-ACT complex concentrations as low as 10.2 and 18.1 ng/ml were found in the HBS buffer and human serum sample, respectively. The result indicates that this approach could satisfy our goal without modifying the secondary interactant.

The Arabidopsis microtubule-associated protein AtMAP65-1: Molecular analysis of its microtubule bundling activity

The 65-kD microtubule-associated protein (MAP65) family is a family of plant microtubule-bundling proteins. Functional analysis is complicated by the heterogeneity within this family: there are nine MAP65 genes in Arabidopsis thaliana, AtMAP65-1 to AtMAP65-9. To begin the functional dissection of the Arabidopsis MAP65 proteins, we have concentrated on a single isoform, AtMAP65-1, and examined its effect on the dynamics of mammalian microtubules. We show that recombinant AtMAP65-1 does not promote polymerization and does not stabilize microtubules against cold-induced microtubule depolymerization. However, we show that it does induce microtubule bundling in vitro and that this protein forms 25-nm cross-bridges between microtubules. We further demonstrate that the microtubule binding region resides in the C-terminal half of the protein and that Ala409 and Ala420 are essential for the interaction with microtubules. Ala420 is a conserved amino acid in the AtMAP65 family and is mutated to Val in the cytokinesis-defective mutant pleiade-4 of the AtMAP65-3/PLEIADE gene. We show that AtMAP65-1 can form dimers and that a region in the N terminus is responsible for this activity. Neither the microtubule binding region nor the dimerization region alone could induce microtubule bundling, strongly suggesting that dimerization is necessary to produce the microtubule cross-bridges. In vivo, AtMAP65-1 is ubiquitously expressed both during the cell cycle and in all plant organs and tissues with the exception of anthers and petals. Moreover, using an antiserum raised to AtMAP65-1, we show that AtMAP65-1 binds microtubules at specific stages of the cell cycle.

Clinical and technical factors associated with skin intrinsic fluorescence in subjects with type 1 diabetes from the Diabetes Control and Complications Trial/Epidemiology of Diabetes Interventions and Complications Study

The Diabetes Control and Complications Trial (DCCT)/Epidemiology of Diabetes Interventions and Complications(EDIC) studies have established multiyear mean hemoglobin A1c (HbA1c) as predictive of microvascular complications in persons with type 1 diabetes. However, multiyear mean HbA1c is not always available in the clinical setting. Skin advanced glycation end products (AGEs) are thought to partially reflect effects of hyperglycemia over time, and measurement of skin AGEs might be a surrogate for multiyear mean HbA1c. As certain AGEs fluoresce and skin fluorescence has been demonstrated to correlate with the concentration of skin AGEs, noninvasive measurement by skin intrinsic fluorescence(SIF) facilitates the exploration of the association of mean HbA1c and other clinical/technical factors with SIF using the detailed phenotypic database available in DCCT/EDIC.

High concentrations of AGE-LDL and oxidized LDL in circulating immune complexes are associated with progression of retinopathy in type 1 diabetes

To determine whether immunocomplexes (ICs) containing advanced glycation end product (AGE)-LDL (AGE-LDL) and oxidized LDL (oxLDL) contribute to the development of retinopathy over a 16-year period in subjects with type 1 diabetes.

Increased serum pigment epithelium-derived factor is associated with microvascular complications, vascular stiffness and inflammation in Type 1 diabetes

To determine in Type 1 diabetes patients if levels of pigment epithelium-derived factor (PEDF), an anti-angiogenic, anti-inflammatory and antioxidant factor, are increased in individuals with complications and positively related to vascular and renal dysfunction, body mass index, glycated haemoglobin, lipids, inflammation and oxidative stress.

Immune complexes containing modified lipoproteins are related to the progression of internal carotid intima-media thickness in patients with type 1 diabetes

Modified lipoproteins induce autoimmune responses including the synthesis of autoantibodies with pro-inflammatory characteristics. Circulating modified lipoprotein autoantibodies combine with circulating antigens and form immune complexes (IC). We now report the results of a study investigating the role of circulating IC containing modified lipoproteins in the progression of carotid intima-media thickness (IMT) in patients enrolled in the Epidemiology of Diabetes Interventions and Complications (EDIC) Trial, a follow-up study of the Diabetes Control and Complications Trial (DCCT). This cohort includes 1229 patients with type 1 diabetes in whom B-mode ultrasonography of internal and common carotid arteries was performed in 1994-1996 and in 1998-2000. Conventional CHD risk factors, antibodies against modified forms of LDL and modified lipoprotein IC were determined in 1050 of these patients from blood collected in 1996-1998. Cholesterol and apolipoprotein B content of IC (surrogate markers of modified ApoB-rich lipoproteins) were significantly higher in patients who showed progression of the internal carotid IMT than in those showing no progression, regression or mild progression. Multivariate linear and logistic regression modeling using conventional and non-conventional risk factors showed that the cholesterol content of IC was a significant positive predictor of internal carotid IMT progression. In conclusion these data demonstrate that increased levels of modified ApoB-rich IC are associated with increased progression of internal carotid IMT in the DCCT/EDIC cohort of type 1 diabetes.

Pigmented paravenous chorioretinal atrophy is associated with a mutation within the crumbs homolog 1 (CRB1) gene

Pigmented paravenous chorioretinal atrophy (PPCRA) is an unusual retinal degeneration characterized by accumulation of pigmentation along retinal veins. The purpose of this study was to describe the phenotype of a family with PPCRA, determine the mode of inheritance, and identify the causal mutation.

A cell surface antigen associated with meiosis in male Staurdoderus scalaris (Orthopteran)

Immunofluorescence has identified seven monoclonal antibodies reactive with the surface of meiotic cells and absent in premeiotic cells. Analysis by immunogold electron microscopy indicated that these antigens were present on the external surface of the cells and were coincident with the presence of synaptonemal complexes in the nucleus. On immunoblots a common glycosylated protein of 205 kDa was recognized, in addition to smaller subunits, suggesting the presence of a protein complex comprised of smaller peptides.

Caveolin-1 single nucleotide polymorphism in antineutrophil cytoplasmic antibody associated vasculitis

Immunosuppression is cornerstone treatment of antineutrophil cytoplasmic antibody associated vasculitis (AAV) but is later complicated by infection, cancer, cardiovascular and chronic kidney disease. Caveolin-1 is an essential structural protein for small cell membrane invaginations known as caveolae. Its functional role has been associated with these complications. For the first time, caveolin-1 (CAV1) gene variation is studied in AAV.