Solid-Phase synthesis of a library constructed of aromatic phosphate, long alkyl chains and tryptophane components, and identification of potent dipeptide telomerase inhibitors

Telomerase inhibitors are expected as a new candidate of therapeutic agents for cancer. Recently, we have found novel inhibitors based on the bisindole skeleton. In this study, solid-phase synthesis was applied to construct a library of inhibitors having aromatic phosphate, long alkyl chain and tryptophane components, from which a D,D-ditryptophane derivative has been identified as a new potent telomerase inhibitor with IC(50) values of 0.3 microM. A hypothetical binding model for the new inhibitors has been proposed based on the structure-activity relationship.     

Acidic extracellular pH-activated outwardly rectifying chloride current in mammalian cardiac myocytes

Extracellular acidic pH was found to induce an outwardly rectifying Cl- current (I(Cl,acid)) in mouse ventricular cells, with a half-maximal activation at pH 5.9. The current showed the permeability sequence for anions to be SCN- > Br- > I- > Cl- > F- > aspartate, while it exhibited a time-dependent activation at large positive potentials. Similar currents were also observed in mouse atrial cells and in atrial and ventricular cells from guinea pig. Some Cl- channel blockers (DIDS, niflumic acid, and glibenclamide) inhibited ICl,acid, whereas tamoxifen had little effect on it. Unlike volume-regulated Cl- current (ICl,vol) and CFTR Cl- current (ICl,CFTR), ICl,acid was independent of the presence of intracellular ATP. Activation of ICl,acid appeared to be also independent of intracellular Ca2+ and G protein. ICl,acid and ICl,vol could develop in an additive fashion in acidic hypotonic solutions. Isoprenaline-induced ICl,CFTR was inhibited by acidification in a pH-dependent manner in guinea pig ventricular cells. Our results support the view that ICl,acid and ICl,vol stem from two distinct populations of anion channels and that the ICl,acid channels are present in cardiac cells. ICl,acid may play a role in the control of action potential duration or cell volume under pathological conditions, such as ischemia-related cardiac acidosis.     

Diurnal expression of Dnmt3b mRNA in mouse liver is regulated by feeding and hepatic clockwork

DNA methyltransferase 3B (DNMT3B) is critically involved in de novo DNA methylation and genomic stability, while the regulatory mechanism in liver is largely unknown. We previously reported that diurnal variation occurs in the mRNA expression of Dnmt3b in adult mouse liver. The aim of this study was to determine the mechanism underlying the diurnal expression pattern. The highest level and the lowest level of Dnmt3b mRNA expression were confirmed to occur at dawn and in the afternoon, respectively, and the expression pattern of Dnmt3b closely coincided with that of Bmal1. Since the diurnal pattern of Dnmt3b mRNA expression developed at weaning and scheduled feeding to separate the feeding cycle from the light/dark cycle led to a phase-shift in the expression, it could be assumed that feeding plays a critical role as an entrainment signal. In liver-specific Bmal1 knockout (L-Bmal1 KO) mice, L-Bmal1 deficiency resulted in significantly higher levels of Dnmt3b at all measured time points, and the time when the expression was the lowest in wild-type mice was shifted to earlier. Investigation of global DNA methylation revealed a temporal decrease of 5-methyl-cytosine percentage in the genome of wild-type mice in late afternoon. By contrast, no such decrease in 5-methyl-cytosine percentage was detected in L-Bmal1 KO mice, suggesting that altered Dnmt3b expression affects the DNA methylation state. Taken together, the results suggest that the feeding and hepatic clockwork generated by the clock genes, including Bmal1, regulate the diurnal variation in Dnmt3b mRNA expression and the consequent dynamic changes in global DNA methylation.     

Construction of an SSR and RAD-Marker Based Molecular Linkage Map of Vigna vexillata (L.) A. Rich

Vigna vexillata (L.) A. Rich. (tuber cowpea) is an underutilized crop for consuming its tuber and mature seeds. Wild form of V. vexillata is a pan-tropical perennial herbaceous plant which has been used by local people as a food. Wild V. vexillata has also been considered as useful gene(s) source for V. unguiculata (cowpea), since it was reported to have various resistance gene(s) for insects and diseases of cowpea. To exploit the potential of V. vexillata, an SSR-based linkage map of V. vexillata was developed. A total of 874 SSR markers successfully amplified single DNA fragment in V. vexillata among 1,336 SSR markers developed from Vigna angularis (azuki bean), V. unguiculata and Phaseolus vulgaris (common bean). An F₂ population of 300 plants derived from a cross between salt resistant (V1) and susceptible (V5) accessions was used for mapping. A genetic linkage map was constructed using 82 polymorphic SSR markers loci, which could be assigned to 11 linkage groups spanning 511.5 cM in length with a mean distance of 7.2 cM between adjacent markers. To develop higher density molecular linkage map and to confirm SSR markers position in a linkage map, RAD markers were developed and a combined SSR and RAD markers linkage map of V. vexillata was constructed. A total of 559 (84 SSR and 475 RAD) markers loci could be assigned to 11 linkage groups spanning 973.9 cM in length with a mean distance of 1.8 cM between adjacent markers. Linkage and genetic position of all SSR markers in an SSR linkage map were confirmed. When an SSR genetic linkage map of V. vexillata was compared with those of V. radiata and V. unguiculata, it was suggested that the structure of V. vexillata chromosome was considerably differentiated. This map is the first SSR and RAD marker-based V. vexillata linkage map which can be used for the mapping of useful traits.     

A Novel Satellite RNA of Cucumber Mosaic Virus Induces Unique Line-pattern Mosaic Symptoms in Tobacco

The CMV(YW) isolate of cucumber mosaic virus (CMV) induced unique line‐pattern mosaic symptoms in systemically infected leaves of tobacco (Nicotiana tabacum cv. Ky57). By northern hybridization analysis using cDNA to CMV(Y) satellite RNA as a probe, it was confirmed that CMV(YW) contained a satellite RNA. which was designated sat‐YW RNA; this was 388 nucleotides in length and did not have either a conserved domain that induces necrosis in tomato or chlorosis in tobacco. CMV(YW) free of sat‐YW RNA. which was isolated by the single lesion isolation method using Chenopodium amaranticolor, did not induce the unique line‐pattern mosaic symptom. Furthermore, the sat‐YW RNA‐mediated line‐pattern mosaic symptom was also induced by in vitro transcribed infectious sat‐YW RNA in tobaccos infected with either CMV(YW) or CMV(Y) genomic RNA. These results clearly demonstrated that sat‐YW RNA induces the unique line‐pattern mosaic symptom on CMV‐infected tobaccos.     

Mapping the virus and host genes involved in the resistance response in cucumber mosaic virus-Infected Arabidopsis thaliana

A yellow strain of cucumber mosaic virus (CMV) [CMV(Y)] induces a resistance response characterized by inhibition of virus systemic movement with development of necrotic local lesions in the virus-inoculated leaves of Arabidopsis thaliana ecotype C24. In this report, the avirulence determinant in the virus genome was defined and the resistance gene (RCY1) of C24 was genetically mapped. The response of C24 to CMV containing the chimeric RNA3 between CMV(Y) and a virulent strain of CMV indicated that the coat protein gene of CMV(Y) determined the localization of the virus in the inoculated leaves of C24. The RCY1 locus was mapped between two CAPS markers, DFR and T43968, which were located in the region containing genetically defined disease resistance genes and their homologues. These results indicate that the resistance response to CMV(Y) in C24 is determined by the combination of the coat protein gene and RCY1 on chromosome 5.     

Development of hyperfimbriated strains of Vibrio cholerae O1

The Vibrio cholerae O1 and O139 fimbrillin genes (fimA or mshA) were amplified by polymerase chain reaction and cloned into an Escherichia coli pCR vector. These clones were sequenced. The fimA sequences were found to be identical between V cholerae O1 and O139. One of the plasmids was digested with EcoR I and inserted into the EcoR I site of pGEX-3X. The plasmid pVPP thus obtained was transferred into strains of wild-type V cholerae O1 Bgd17 (classical in biotype) and its fimbriated strain by electroporation. The recombinant plasmid pVPP overexpressed mature fimbriae following induction of the tac promoter with isopropyl-beta-D-thiogalactopyranoside. The cloned gene product was purified to homogeneity by sucrose-linear gradient centrifugation (7.8 mg of fimbriae/L-culture). All the properties of the recombinant fimbriae (e.g., subunit structure, hydrophobicity, hemagglutinating activity sensitive to D-mannose and D-glucose and immunogenicity) were identical to those of the wild-type fimbriae. This overexpression system will be extremely useful for rapid, inexpensive preparation of large amounts of fimbriae for vaccine design and development.     

Association of IL-10 receptor 2 (IL10RB) SNP with systemic sclerosis

Interleukin-10 (IL-10) signaling has been suggested to play a role in systemic sclerosis (SSc). IL10RB codes for IL-10 receptor 2 (IL-10R2), a component shared in receptor complexes for IL-10, IL-22, IL-26 and interferon (IFN)-λ. In this study, we examined association of IL10RB polymorphism with susceptibility to SSc. Genotype A/A at rs2834167 (47K/K) was significantly increased in diffuse cutaneous SSc (dcSSc) (41.3% in dcSSc, 20.9% in controls, P = 0.0018, odds ratio = 2.67). A SNP in the 5′ flanking region of IL10RB, rs999788, also showed association with dcSSc; however, this association was shown to be secondarily caused by linkage disequilibrium with rs2834167. Significant association was not observed in limited cutaneous SSc (lcSSc). Presence of anti-topoisomerase I antibody was also associated with rs2834167A/A genotype (P = 0.0019). Serum IL-10 level was significantly associated with the number of rs2834167A allele (P = 0.007). These findings suggested that signaling through IL-10R2 may play a causative role in dcSSc.     

4-Amino-2-cyanopyrimidines: novel scaffold for nonpeptidic cathepsin S inhibitors

We describe here a novel 4-amino-2-cyanopyrimidine scaffold for nonpeptidomimetic cathepsin S selective inhibitors. Some of the synthesized compounds have sub-nanomolar potency and high selectivity toward cathepsin S along with promising pharmacokinetic and physicochemical properties. The key structural features of the inhibitors consist of a combination of a spiro[2.5]oct-6-ylmethylamine P2 group at the 4-position, a small or polar P3 group at the 5-position and/or a polar group at the 6-position of the pyrimidine.