Mitochondrial haplogroup N9a confers resistance against type 2 diabetes in Asians

Because mitochondria play pivotal roles in both insulin secretion from the pancreatic beta cells and insulin resistance of skeletal muscles, we performed a large-scale association study to identify mitochondrial haplogroups that may confer resistance against or susceptibility to type 2 diabetes mellitus (T2DM). The study population comprised 2,906 unrelated Japanese individuals, including 1,289 patients with T2DM and 1,617 controls, and 1,365 unrelated Korean individuals, including 732 patients with T2DM and 633 controls. The genotypes for 25 polymorphisms in the coding region of the mitochondrial genome were determined, and the haplotypes were classified into 10 major haplogroups (i.e., F, B, A, N9a, M7a, M7b, G, D4a, D4b, and D5). Multivariate logistic-regression analysis with adjustment for age and sex revealed that the mitochondrial haplogroup N9a was significantly associated with resistance against T2DM (P=.0002) with an odds ratio of 0.55 (95% confidence interval 0.40-0.75). Even in the modern environment, which is often characterized by satiety and physical inactivity, this haplogroup might confer resistance against T2DM.     

Modulation of drug-stimulated ATPase activity of human MDR1/P-glycoprotein by cholesterol

MDR1 (multidrug resistance 1)/P-glycoprotein is an ATP-driven transporter which excretes a wide variety of structurally unrelated hydrophobic compounds from cells. It is suggested that drugs bind to MDR1 directly from the lipid bilayer and that cholesterol in the bilayer also interacts with MDR1. However, the effects of cholesterol on drug-MDR1 interactions are still unclear. To examine these effects, human MDR1 was expressed in insect cells and purified. The purified MDR1 protein was reconstituted in proteoliposomes containing various concentrations of cholesterol and enzymatic parameters of drug-stimulated ATPase were compared. Cholesterol directly binds to purified MDR1 in a detergent soluble form and the effects of cholesterol on drug-stimulated ATPase activity differ from one drug to another. The effects of cholesterol on K(m) values of drug-stimulated ATPase activity were strongly correlated with the molecular mass of that drug. Cholesterol increases the binding affinity of small drugs (molecular mass <500 Da), but does not affect that of drugs with a molecular mass of between 800 and 900 Da, and suppresses that of valinomycin (molecular mass >1000 Da). V(max) values for rhodamine B and paclitaxel are also increased by cholesterol, suggesting that cholesterol affects turnover as well as drug binding. Paclitaxel-stimulated ATPase activity of MDR1 is enhanced in the presence of stigmasterol, sitosterol and campesterol, as well as cholesterol, but not ergosterol. These results suggest that the drug-binding site of MDR1 may best fit drugs with a molecular mass of between 800 and 900 Da, and that cholesterol may support the recognition of smaller drugs by adjusting the drug-binding site and play an important role in the function of MDR1.     

Dietary flavonoid apigenin is a potential inducer of intracellular oxidative stress: the role in the interruptive apoptotic signal

Apigenin is a representative dietary flavone (2-phenyl-4H-1-benzopyran-4-one) inhibiting cancer cell growth both in cell culture systems and in vivo. The prooxidant potential of apigenin was confirmed by the observations using flowcytometric and immunoblotting techniques that the intracellular accumulations of reactive oxygen species (ROS) and protein carbonyls were detected in the cells treated with apigenin in a dose-dependent manner. Conversely, chrysin (5,7-dihydroxyflavone) did not show any prooxidant effect. A structure-activity relationship data thus indicated that a 4'-monohydroxyl group, which can be oxidized to semiquinone radical but not up to quinone-like metabolite, is essential for prooxidant effect. When HL-60 cells were treated with not only a heme synthesis inhibitor succinyl acetone (SA) but also myeloperoxidase (MPO) inhibitors, the ROS level enhanced by apigenin was significantly reduced. The gathered data suggested that peroxidase-catalyzed production of apigenin B-ring phenoxyl radicals might be responsible for the prooxidant effect. This is supported by the observation that MPO is able to catalyze production of apigenin phenoxyl radicals, detected by an electron spin resonance-spin trapping technique. We also reveal that both SA and alpha-tocopherol enhance cellular susceptibility to apoptosis-inducing stimuli by apigenin. In conclusion, the prooxidant effect of apigenin is likely to oxidize a variety of thiols through the formation of phenoxyl radicals and thus seems to play a significant role in the abortive apoptotic pathway switching to necrotic cell death.     

Reduced Contamination by the Fusarium Mycotoxin Zearalenone in Maize Kernels through Genetic Modification with a Detoxification Gene

Maize is subject to ear rot caused by toxigenic Aspergillus and Fusarium species, resulting in contamination with aflatoxins, fumonisins, trichothecenes, and zearalenone (ZEN). The trichothecene group and ZEN mycotoxins are produced by the cereal pathogen Fusarium graminearum. A transgenic detoxification system for the elimination of ZEN was previously developed using an egfp::zhd101 gene (gfzhd101), encoding an enhanced green fluorescent protein fused to a ZEN-degrading enzyme. In this study, we produced a transgenic maize line expressing an intact copy of gfzhd101 and examined the feasibility of transgene-mediated detoxification in the kernels. ZEN-degrading activity has been detected in transgenic kernels during seed maturation (for a period of 6 weeks after pollination). The level of detoxification activity was unaltered after an additional storage period of 16 weeks at 6°C. When the seeds were artificially contaminated by immersion in a ZEN solution for 48 h at 28°C, the total amount of the mycotoxin in the transgenic seeds was uniformly reduced to less than 1/10 of that in the wild type. The ZEN in the transgenic maize kernels was also efficiently decontaminated under conditions of lower water activity (a_w) and temperature; e.g., 16.9 μg of ZEN was removed per gram of seed within 48 h at an a_w of 0.90 at 20°C. F. graminearum infection assays demonstrated an absence of ZEN in the transgenic maize seeds, while the mycotoxin accumulated in wild-type kernels under the same conditions. Transgene-mediated detoxification may offer simple solutions to the problems of mycotoxin contamination in maize.     

Enrichment of longevity phenotype in mtDNA haplogroups D4b2b, D4a, and D5 in the Japanese population

We report new results from the re-analysis of 672 complete mitochondrial (mtDNA) genomes of unrelated Japanese individuals stratified into seven equal sized groups by the phenotypes: diabetic patients, diabetic patients with severe angiopathy, healthy non-obese young males, obese young males, patients with Alzheimer’s disease, patients with Parkinson’s disease and centenarians. Each phenotype had 96 samples over 27 known haplogroups: A, B4a, B4b, B4c, B*, B5, D*, F1, F2, M*, M7a, M7b, M8, M9, D4a, D4b1, D4b2, D4d, D4e, D4g, D4h, D5, G, Z, M*, N9a, and N9b. A t-test comparing the fraction of samples in a haplogroup to healthy young males showed a significant enrichment of haplogroups D4a, D5, and D4b2 in centenarians. The D4b2 enrichment was limited to a subgroup of 40 of 61 samples which had the synonymous mutation 9296C > T. We identified this cluster as a distinct haplogroup and labeled it as D4b2b. Using an exhaustive procedure, we constructed the complete list of “mutation patterns” for centenarians and showed that the most significant patterns were in D4a, D5, and D4b2b. We argue that if a selection for longevity appeared only once, it was probably an autosomal event which could be dated to after the appearance of the D mega-group but before the coalescent time of D4a, D5, and D4b2b. Using a simple procedure, we estimated that this event occurred 24.4 ± 0.9 kYBP. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. Gabriela Alexe and Noriyuki Fuku are joint first authors.     

Angiotensin II type 1 receptor signaling contributes to platelet-leukocyte-endothelial cell interactions in the cerebral microvasculature

Angiotensin II type 1 (AT(1)) receptor signaling has been implicated in cerebral microvascular alterations associated with ischemia, diabetes mellitus, hypercholesterolemia, and atherosclerosis. Platelets, which express AT(1) receptors, also appear to contribute to the thrombogenic and inflammatory responses that are elicited by these pathological conditions. This study assesses the role of AT(1) receptor activation on platelet-leukocyte-endothelial cell interactions elicited in cerebral microvasculature by ischemia and reperfusion. Intravital microscopy was used to monitor the adhesion of platelets and leukocytes that were labeled with different fluorochromes, whereas dihydrorhodamine-123 was used to quantify oxygen radical production in cerebral surface of mice that were either treated with the AT(1) receptor agonist Val-angiotensin II (ANG II) or subjected to bilateral common carotid artery occlusion (BCCAO) followed by reperfusion. ANG II elicited a dose- and time- dependent increase in platelet-leukocyte-endothelial cell interactions in cerebral venules that included rolling platelets, adherent platelets on the leukocytes and the endothelial cells, rolling leukocytes, and adherent leukocytes. All of these interactions were attenuated by treatment with either P-selectin or P-selectin glycoprotein ligand 1 (PSGL-1) antibody. The AT(1) receptor antagonist candesartan and losartan as well as diphenyleneiodonium, an inhibitor of flavoproteins including NAD(P)H oxidase, significantly reduced the platelet-leukocyte-endothelial cell interactions elicited by either ANG II administration or BCCAO/reperfusion. The increased oxygen radical generation elicited by BCCAO/reperfusion was also attenuated by candesartan. These findings are consistent with an AT(1) receptor signaling mechanism, which involves oxygen radical production and ultimately results in P-selectin- and PSGL-1-mediated platelet-leukocyte-endothelial cell interactions in the cerebral microcirculation.     

Mycophenolic acid as a latent agonist of PPARgamma

Mycophenolic acid (MPA), known as an inhibitor of inosine monophosphate dehydrogenase (IMPDH), was found to inhibit the differentiation of 3T3-L1 pre-adipocytes into mature adipocytes. Although the effect of MPA was attributed to inhibition of IMPDH, we uncovered a hidden biological property of MPA as an agonist of peroxisome proliferator activated receptor gamma (PPARgamma).     

Establishment and characterization of CAG/EGFP transgenic rabbit line

Cell marking is a very important procedure for identifying donor cells after cell and/or organ transplantation in vivo. Transgenic animals expressing marker proteins such as enhanced green fluorescent protein (EGFP) in their tissues are a powerful tool for research in fields of tissue engineering and regenerative medicine. The purpose of this study was to establish transgenic rabbit lines that ubiquitously express EGFP under the control of the cytomegalovirus immediate early enhancer/beta-actin promoter (CAG) to provide a fluorescent transgenic animal as a bioresource. We microinjected the EGFP expression vector into 945 rabbit eggs and 4 independent transgenic candidate pups were obtained. Two of them died before sexual maturation and one was infertile. One transgenic male candidate founder rabbit was obtained and could be bred by artificial insemination. The rabbit transmitted the transgene in a Mendelian manner. Using fluorescence in situ hybridization analysis, we detected the transgene at 7q11 on chromosome 7 as a large centromeric region in two F1 offspring (one female and one male). Eventually, one transgenic line was established. Ubiquitous EGFP florescence was confirmed in all examined organs. There were no gender-related differences in fluorescence. The established CAG/EGFP transgenic rabbit will be an important bioresource and a useful tool for various studies in tissue engineering and regenerative medicine.     

Antitumor studies. Part 1: design, synthesis, antitumor activity, and AutoDock study of 2-deoxo-2-phenyl-5-deazaflavins and 2-deoxo-2-phenylflavin-5-oxides as a new class of antitumor agents

Novel 2-deoxo-2-phenyl-5-deazaflavins and 2-deoxo-2-phenylflavin-5-oxides were prepared as a new class of antitumor agents and showed significant antitumor activities against NCI-H 460, HCT 116, A 431, CCRF-HSB-2, andKB cell lines. In vivo investigation, 2-deoxo-10-methyl-2-phenyl-5-deazaflavin exhibited the effective antitumor activity against A 431 human adenocarcinoma cells transplanted subcutaneously into nude mouse. Furthermore, AutoDock study has been done by binding of the flavin analogs into PTK pp60(c-src), where a good correlation between their IC(50) and AutoDock binding free energy was exhibited. In particular, 2-deoxo-2-phenylflavin-5-oxides exhibited the highest potential binding affinity within the binding pocket of PTK.