Hydrogen peroxide triggers the proteolytic cleavage and the inactivation of calcineurin

Increases in the levels of reactive oxygen species (ROS) are correlated with a decrease in calcineurin (CN) activity under oxidative or neuropathological conditions. However, the molecular mechanism underlying this ROS-mediated CN inactivation remains unclear. Here, we describe a mechanism for the inactivation of CN by hydrogen peroxide. The treatment of mouse primary cortical neuron cells with Abeta(1-42) peptide and hydrogen peroxide triggered the proteolytic cleavage of CN and decreased its enzymatic activity. In addition, hydrogen peroxide was found to cleave CN in different types of cells. Calcium influx was not involved in CN inactivation during hydrogen peroxide-mediated cleavage, but CN cleavage was partially blocked by chloroquine, indicating that an unidentified lysosomal protease is probably involved in its hydrogen peroxide-mediated cleavage. Treatment with hydrogen peroxide triggered CN cleavage at a specific sequence within its catalytic domain, and the cleaved form of CN had no enzymatic ability to dephosphorylate nuclear factor in activated T cells. Thus, our findings suggest a molecular mechanism by which hydrogen peroxide inactivates CN by proteolysis in ROS-related diseases.     

Beneficial effects of beta-sitosterol on glucose and lipid metabolism in L6 myotube cells are mediated by AMP-activated protein kinase

AMP-activated protein kinase (AMPK) is an energy-sensing enzyme that has been implicated as a key factor for controlling intracellular lipids and glucose metabolism. β-Sitosterol, a plant sterol known to prevent cardiovascular disease was identified from Schizonepeta tenuifolia to an AMPK activator. In L6 myotube cells, β-sitosterol significantly increased phosphorylation of the AMPKα subunit and acetyl-CoA carboxylase (ACC) with stimulating glucose uptake. In contrast, β-sitosterol treatment reduced intracellular levels of triglycerides and cholesterol in L6 cells. These effects were all reversed by pretreatment with AMPK inhibitor Compound C or LKB1 destabilizer radicicol. Similarly, β-sitosterol-induced phosphorylation of AMPK and ACC was not increased in HeLa cells lacking LKB1. These results together suggest that β-sitosterol-mediated enhancement of glucose uptake and reduction of triglycerides and cholesterol in L6 cells is predominantly accomplished by LKB1-mediated AMPK activation. Our findings further reveal a molecular mechanism underlying the beneficial effects of β-sitosterol on glucose and lipid metabolism.     

Replication of an African-American GWAS on blood pressure and hypertension in the Korean population

Hypertension is a complex disease that is caused by the interaction of multiple genetic and environmental risk factors, affecting 30% adult in industrialized countries. The identification of genetic factors that impact one’s predisposition to hypertension and its progression is an ongoing challenge. A genome-wide association study of African-Americans, who have one of the highest rates of hypertension in the world, was reported. We replicated the GWAS results in 8842 unrelated Koreans. Fifteen of the 30 reported SNPs were analyzed for their association with blood pressure and hypertension. Linear regression was used to analyze blood pressure as a quantitative trait in 7551 subjects, and a case-control study was performed using 1968 hypertensive cases and 4452 normotensive controls by logistic regression analysis. The quantitative trait study demonstrated a moderate association of 2 SNPs, rs9301196 (p=4.9×10^?3 for diastolic blood pressure) and rs2823756 (p=0.04 for systolic blood pressure), which were also associated with hypertension (p=0.042 and p=6.3×10^?3, respectively). Further, 3 SNPs were associated with hypertension (p=0.042 for rs7902529, p=0.027 for rs10135446, and p=0.01 for rs4613079) but not with blood pressure. Based on the moderate association signals and the low proportion of positive signals, this cross validation between African-Americans and Asians suggests that association studies of blood pressure traits require a larger number of subjects and a more refined design.     

A Cdo-Bnip-2-Cdc42 signaling pathway regulates p38alpha/beta MAPK activity and myogenic differentiation

The p38alpha/beta mitogen-activated protein kinase (MAPK) pathway promotes skeletal myogenesis, but the mechanisms by which it is activated during this process are unclear. During myoblast differentiation, the promyogenic cell surface receptor Cdo binds to the p38alpha/beta pathway scaffold protein JLP and, via JLP, p38alpha/beta itself. We report that Cdo also interacts with Bnip-2, a protein that binds the small guanosine triphosphatase (GTPase) Cdc42 and a negative regulator of Cdc42, Cdc42 GTPase-activating protein (GAP). Moreover, Bnip-2 and JLP are brought together through mutual interaction with Cdo. Gain- and loss-of-function experiments with myoblasts indicate that the Cdo-Bnip-2 interaction stimulates Cdc42 activity, which in turn promotes p38alpha/beta activity and cell differentiation. These results reveal a previously unknown linkage between a cell surface receptor and downstream modulation of Cdc42 activity. Furthermore, interaction with multiple scaffold-type proteins is a distinctive mode of cell surface receptor signaling and provides one mechanism for specificity of p38alpha/beta activation during cell differentiation.     

CO2 enhances effects of hypoxia on mortality,development, and gene expression in cowpea bruchid, Callosobruchus maculatus

Modified atmosphere based on lack of O₂ offers a safe, residue-free alternative to chemical fumigants for pest control in stored grains. In this study, we intended to determine whether elevated CO₂ (at a biologically achievable level) has an enhanced suppressive effect over low O₂ atmosphere alone on the cowpea bruchid (Callosobruchus maculatus), a storage pest of cowpea and other legumes. Experiments were performed under two modified atmospheric conditions, (1) 2% O₂ + 18% CO₂ + 80% N₂ and (2) 2% O₂ + 98% N₂. Both hypoxic environments significantly affected the development and survival of all insect developmental stages. Eggs were most vulnerable to hypoxia, particularly at the early stage (4–6 h old), surviving only up to a maximum of 2 days in both treatments. These were followed by adults, pupae and larvae, in order of decreasing susceptibility. The 3rd and 4th instar larvae were most resilient to hypoxia and could survive up to 20 days of low O₂. The presence of 18% CO₂ significantly increased the mortality of adults, the later stage of eggs, as well as 1st and 4th instar larvae caused by hypoxia. However, the surviving insects exhibited faster development, evidenced by their earlier emergence from cowpea seeds compared to those without CO₂. One interesting observation was the frequent, premature opening of the emergence windows in the 4th instar larvae when CO₂ was involved. This phenomenon was not observed at all in insects stressed by low O₂ alone. Differential expression profiling of metabolic genes and proteolytic activity of midgut digestive enzymes suggested that the rate of metabolic activity could contribute in part to the difference in insect development and survival under hypoxia in the presence and absence of CO₂.     

Osthenol,a prenylated coumarin,as a monoamine oxidase A inhibitor with high selectivity

Osthenol (6), a prenylated coumarin isolated from the dried roots of Angelica pubescens, potently and selectively inhibited recombinant human monoamine oxidase-A (hMAO-A) with an IC₅₀ value of 0.74?µM and showed a high selectivity index (SI?>?81.1) for hMAO-A versus hMAO-B. Compound 6 was a reversible competitive hMAO-A inhibitor (K_i?=?0.26?µM) with a potency greater than toloxatone (IC₅₀?=?0.93?µM), a marketed drug. Isopsoralen (3) and bakuchicin (1), furanocoumarin derivatives isolated from Psoralea corylifolia L., showed slightly higher IC₅₀ values (0.88 and 1.78?µM, respectively) for hMAO-A than 6, but had low SI values (3.1 for both). Other coumarins tested did not effectively inhibit hMAO-A or hMAO-B. A structural comparison suggested that the 8-(3,3-dimethylallyl) group of 6 increased its inhibitory activity against hMAO-A compared with the 6-methoxy group of scopoletin (4). Molecular docking simulations revealed that the binding affinity of 6 for hMAO-A (?8.5?kcal/mol) was greater than that for hMAO-B (?5.6?kcal/mol) and that of 4 for hMAO-A (?7.3?kcal/mol). Docking simulations also implied that 6 interacted with hMAO-A at Phe208 and with hMAO-B at Ile199 by carbon hydrogen bondings. Our findings suggest that osthenol, derived from natural products, is a selective and potent reversible inhibitor of MAO-A, and can be regarded a potential lead compound for the design of novel reversible MAO-A inhibitors.     

Association analysis of v-AKT murine thymoma viral oncogene homolog 1 (AKT1) polymorphisms and type 2 diabetes mellitus in the Korean population

V-AKT murine thymoma viral oncogene homolog 1 (AKT1) is an important downstream target of the insulin-signaling pathway and may be an important regulator of pancreatic beta cell growth. This study investigated the association of theAKT1 gene with susceptibility to type 2 diabetes mellitus and its related traits. By sequencing theAKT1 gene in 24 unrelated individuals, we iden-tified 32 genetic variations including 30 single nucleotide polymorphisms and 2 deletions. For the association analysis, we selected seven single nucleotide polymorphisms (rs10138227, ?726G>A; rs3730358, +12574C>T; rs2494737, +12656T>A; rs2498796, +15761T>C; rs2498799, +19087 A>G; rs2494732, +19789G>A; rs3803304, +19835G>C) based on minor allele frequency (>0.05) and linkage disequilibrium status. The study included 483 type 2 diabetes patients (206 men and 277 women with mean age 64±2.8 years and mean age at onset 56 ± 8.1 years) and 1,138 non-diabetic control subjects (516 men and 622 women with mean age 64 ±2.9 years). Two single nucleotide polymorphisms (rs2498796, +15761T>C and rs2494732, +19789G>A) were found to be associated with risk of type 2 diabetes mellitus, and showed an increased risk of type 2 diabetes mellitus in a recessive model (OR=1.343, 95% CI 1.021–1.765,p=0.035 and OR=1.534, 95% CI 1.058–2.225,p=0.024, respectively). These SNPs were also associated with diabetes-related traits such as levels of fasting blood glucose and hemoglobin A1c. In addition, type 2 diabetes mellitus patients who also have dyslipidemia or high blood pressure showed significant association with single nucleotide polymorphisms in AKT1 when compared with healthy controls. These results indicate that genetic variation in AKT1 influences the development of type 2 diabetes mellitus in the Korean population.