A novel yeast cell-based screen identifies flavone as a tankyrase inhibitor

The telomere-associated protein tankyrase 1 is a poly(ADP-ribose) polymerase and is considered to be a promising target for cancer therapy, especially for BRCA-associated cancers. However, an efficient assay system for inhibitor screening has not been established, mainly due to the difficulty of efficient preparation of the enzyme and its substrate. Here, we report a cell-based assay system for detecting inhibitory activity against tankyrase 1. We found that overexpression of the human tankyrase 1 gene causes a growth defect in the fission yeast Schizosaccharomyces pombe. Chemicals that restore the growth defect phenotype can be identified as potential tankyrase 1 inhibitors. We performed a high-throughput screen using this system, and identified flavone as a compound that restores the growth of yeast cells overexpressing tankyrase 1. Indeed, flavone inhibited poly(ADP-ribosyl)ation of proteins caused by overexpression of tankyrase 1 in yeast cells. This system allows rapid identification of inhibitory activity against tankyrase 1 and is amenable to high-throughput screening using robotics.     

Genetic analysis of abdominal fat distribution in SM/J and A/J mice

Each abdominal fat depot, such as mesenteric or epididymal, differently contributes to the development of insulin resistance. The aim of this study was to identify the genetic regions that contribute to fat accumulation in epididymal/mesenteric fat and to examine whether or not the genetic regions that affect glucose metabolism and body fat distribution are coincident. We previously mapped a major quantitative trait locus (QTL) (T2dm2sa) for impaired glucose tolerance on chromosome 2 and revealed that SM.A-T2dm2sa congenic mice showed not only glucose tolerance but also fat accumulation. In the present study, to identify the loci/genes that control the accumulation of abdominal fat, we perfomed QTL analyses of epididymal/mesenteric fat weight by using (A/J×SM.A-T2dm2sa)F2 mice in which the effect of T2dm2sa was excluded. As a result, two highly significant QTLs for mesenteric fat, as well as three significant QTLs for epididymal/mesenteric fat, were mapped on the different chromosomal regions. This suggests that the fat accumulations in individual fat depots are controlled by distinct genomic regions. Our comparison of these QTLs for abdominal fat distribution with those for glucose metabolism revealed that the major genetic factors affecting body fat distribution do not coincide with genetic factors affecting glucose metabolism in (A/J×SM.A-T2dm2sa)F2.     

Closely Linked Polymorphic Markers for Determining the Autosomal Dominant Allele (Cy) in Rat Polycystic Kidney Disease

The Han:SPRD strain is an SD-background strainknown to be a model of polycystic kidney disease (PKD)expressed through an autosomal dominant gene (Cy).However, different genotypes of this strain cannot be identified in the neonatal period. First, toestablish an accurate method of determining thegenotypes (Cy/Cy, Cy/+, +/+) which cause differentdisease progressions, we used polymorphic markers on rat chromosome 5. PCR products of tissue DNAtemplated with D5Rat9 showed distinct patterns onelectrophoresis indicating three genotypes. Second, todetermine whether the same locus plays a major role inexpressing PKD, we performed linkage analyses in a [BN X(BN X Han:SPRD)F₁] backcross. Cy/Cy and Cy/+also caused PKD in a BN background. In this backcross,we discovered that D5Rat11 is located closer to the Cy locus than D5Mgh10, which is regardedas one of the closest loci. We conclude that D5Rat9 andD5Rat11 are useful markers for determining the presenceof the Cy allele, which is regarded as the gene responsible for PKD.     

Roles of two ATPase-motif-containing domains in cyanobacterial circadian clock protein KaiC

Cyanobacterial clock protein KaiC has a hexagonal, pot-shaped structure composed of six identical dumbbell-shaped subunits. Each subunit has duplicated domains, and each domain has a set of ATPase motifs. The two spherical regions of the dumbbell are likely to correspond to two domains. We examined the role of the two sets of ATPase motifs by analyzing the in vitro activity of ATPgammaS binding, AMPPNP-induced hexamerization, thermostability, and phosphorylation of KaiC and by in vivo rhythm assays both in wild type KaiC (KaiCWT) and KaiCs carrying mutations in either Walker motif A or deduced catalytic Glu residues. We demonstrated that 1) the KaiC subunit had two types of ATP-binding sites, a high affinity site in N-terminal ATPase motifs and a low affinity site in C-terminal ATPase motifs, 2) the N-terminal motifs were responsible for hexamerization, and 3) the C-terminal motifs were responsible for both stabilization and phosphorylation of the KaiC hexamer. We proposed the following reaction mechanism. ATP preferentially binds to the N-terminal high affinity site, inducing the hexamerization of KaiC. Additional ATP then binds to the C-terminal low affinity site, stabilizing and phosphorylating the hexamer. We discussed the effect of these KaiC mutations on circadian bioluminescence rhythm in cells of cyanobacteria.     

The carbohydrate-binding domain of Lafora disease protein targets Lafora polyglucosan bodies

Lafora's disease (LD) is an autosomal recessive and fatal form of epilepsy with onset in late childhood or adolescence. One of the characteristic features of LD pathology is the presence of periodic acid-Schiff (PAS) positive Lafora inclusion bodies. Lafora bodies are present primarily in neurons, but they have also been found in other organs. Histochemical and biochemical studies have indicated that Lafora bodies are composed mainly of polysaccharides. The LD gene, EPM2A, encodes a 331 amino acid long protein named laforin that contains an N-terminal carbohydrate-binding domain (CBD) and a C-terminal dual-specificity phosphatase domain (DSPD). Here we demonstrate that the CBD of laforin targets the protein to Lafora inclusion bodies and this property could be evolutionarily conserved. We also tested in vitro the effects of five LD missense mutations on laforin's affinity to Lafora body. While the missense mutant W32G failed to bind to purified Lafora body, four other mutants (S25P, E28L, F88L, and R108C) did not show any effect on the binding affinity. Based on these observations we propose the existence of a laforin-mediated glycogen metabolic pathway regulating the disposal of pathogenic polyglucosan inclusions. This is the first report demonstrating a direct association between the LD gene product and the disease-defining storage product, the Lafora bodies.     

Calcium restriction allows cAMP activation of the B-Raf/ERK pathway, switching cells to a cAMP-dependent growth-stimulated phenotype

cAMP can be either mitogenic or anti-mitogenic, depending on the cell type. We demonstrated previously that cAMP inhibited the proliferation of normal renal epithelial cells and stimulated the proliferation of cells derived from the cysts of polycystic kidney disease (PKD) patients. The protein products of the genes causing PKD, polycystin-1 and polycystin-2, are thought to regulate intracellular calcium levels, suggesting that abnormal polycystin function may affect calcium signaling and thus cause a switch to the cAMP growth-stimulated phenotype. To test this hypothesis, we disrupted intracellular calcium mobilization by treating immortalized mouse M-1 collecting duct cells and primary cultures of human kidney epithelial cells with calcium channel blockers and by lowering extracellular calcium with EGTA. Calcium restriction for 3-5 h converted both cell types from a normal cAMP growth-inhibited phenotype to an abnormal cAMP growth-stimulated phenotype, characteristic of PKD. In M-1 cells, we showed that calcium restriction was associated with an elevation in B-Raf protein levels and cAMP-stimulated, Ras-dependent activation of B-Raf and ERK. Moreover, the activity of Akt, a negative regulator of B-Raf, was decreased by calcium restriction. Inhibition of Akt or phosphatidylinositol 3-kinase also allowed cAMP-dependent activation of B-Raf and ERK in normal calcium. These results suggest that calcium restriction causes an inhibition of the phosphatidylinositol 3-kinase/Akt pathway, which relieves the inhibition of B-Raf to allow the cAMP growth-stimulated phenotypic switch. Finally, M-1 cells stably overexpressing an inducible polycystin-1 C-terminal cytosolic tail construct were shown to exhibit a cAMP growth-stimulated phenotype involving B-Raf and ERK activation, which was reversed by the calcium ionophore A23187. We conclude that disruption of calcium mobilization in cells that are normally growth-inhibited by cAMP can derepress the B-Raf/ERK pathway, thus converting these cells to a phenotype that is growth-stimulated by cAMP.     

The diversity of vacA and cagA genes of Helicobacter pylori in East Asia

It has been reported that Helicobacter pylori infection with the type I strain, which expresses the VacA and CagA antigens, is associated with duodenal ulcer. We examined the diversity of vacA and cagA genes in 143 isolates obtained from patients with duodenal ulcer or chronic gastritis in East Asia (two different areas of Japan, Fukui and Okinawa, and also in Hangzhou, China) by polymerase chain reaction (PCR) and sequence analysis. Diversities of cagA and vacA genes were detected in East Asia. The prevalence of cagA-positive H. pylori was significantly different between Fukui and Okinawa (P=0.0032). The prevalence of Western type CagA was significantly higher in Okinawa than in Fukui (P<0.0001). However, there was no significant association between the genotype of cagA and clinical outcome. In Japan, the predominant vacA genotype was s1c/m1b. In contrast, in Hangzhou, the predominant vacA genotype was s1c/m2, and they were all East Asian CagA-positive. These findings suggest that a distinct distribution of the vacA and cagA genotypes is present in East Asia, regardless of clinical outcome.     

Detection of a new cerebral malaria susceptibility locus,using CBA mice

Human cerebral malaria (CM) during acute Plasmodium falciparum infection is a serious neurological complication that leads to coma and death. P. berghei ANKA infection of CBA mice is a useful experimental model of CM. To identify host susceptibility loci, we performed chromosomal mapping in crossbred populations of both CM-susceptible CBA and CM-resistant DBA/2 mice. One significant region for a CM-susceptible locus in CBA mice was mapped to H2 region on Chromosome 17, tentatively designated cmsc. cmsc was mapped to a different chromosomal region from that previously reported in the C57BL/6 mouse model of CM. It is possible that different loci contribute to CM in CBA and C57BL/6 mouse strains. Comparison of the function of CM susceptibility loci between CBA and C57BL/6 mice could have important implications for the study of the complex pathogenesis of CM in humans.