Phylogenetic relationship within the Erythrobasidium clade: molecular phylogenies, secondary structure, and intron positions inferred from partial sequences of ribosomal RNA and elongation factor-1alpha genes

Phylogenetic relationships within the Erythrobasidium clade as a lineage of the urediniomycetous yeasts were examined using partial regions of 18S rDNA, 5.8S rDNA, 26S rDNA, internal transcribed spacers (ITSs), and elongation factor (EF)-1alpha. Combined data analysis of all segments successfully yielded a reliable phylogeny and confirmed the cohesion of species characterized by Q-10(H2) as a major ubiquinone. Differences in secondary structure predicted for a variable region in 26S rDNA corresponded to major divergences in the phylogenetic tree based on the primary sequence. The common presence of a shortened helix in this region was considered to be evidence of monophyly for species with Q-10(H2), Sakaguchia dacryoides, Rhodotorula lactosa, and Rhodotorula lamellibrachiae, although it was not as strongly supported by the combined data tree. The information on intron positions in the EF-1alpha gene had potential usefulness in the phylogenetic inference between closely related species.     

Crucial role of the small GTPase ARF6 in hepatic cord formation during liver development

The mammalian small GTPase ADP-ribosylation factor 6 (ARF6) plays important roles in a wide variety of cellular events, including endocytosis, actin cytoskeletal reorganization, and phosphoinositide metabolism. However, physiological functions for ARF6 have not previously been examined. Here, we described the consequence of ARF6 ablation in mice, which manifests most obviously in the context of liver development. Livers from ARF6-/- embryos are smaller and exhibit hypocellularity, due to the onset of midgestational liver cell apoptosis. Preceding the apoptosis, however, defective hepatic cord formation is observed; the liver cells migrate abnormally upon exiting the primordial hepatic epithelial sheet and clump rather than becoming dispersed. Consistent with this observation, the ability of hepatocyte growth factor/scatter factor (HGF) to induce hepatic cord-like structures from ARF6-/- fetal hepatocytes cultured in vitro in collagen gel matrix is impaired. Finally, we show that endogenous ARF6 in wild-type fetal hepatocytes is activated in response to HGF stimulation. These results provide evidence that ARF6 is an essential component in the signaling pathway coupling HGF signaling to hepatic cord formation.     

The allocation of attention on a perpendicular visual display during reaching movements in depth

We investigated how visual attentional resources are allocated during reaching movements. Particularly, this study examined whether or not the direction of the reaching movement affected visual attention resource allocation. Participants held a stylus pen to reach their hand toward a target stimulus on a graphics tablet as quickly and accurately as possible. The direction of the hand movement was either from near to far space or the reverse. They observed visual stimuli and a cursor, which represented the hand position, on a perpendicularly positioned display, instead of directly seeing their hand movements. Regardless of the movement direction, the participants tended with quickly responding to the target stimuli located far from the start position as compared with those located near to the start position. These results led us to conclude that attentional resources were preferentially allocated in the areas far from the start position of reaching movements. These findings may provide important information for basic research on attention, but also contribute to a decrease of human errors in manipulation tasks performed with visual feedback on visual display terminals.     

Discovery of 4,5-diphenyl-1,2,4-triazole derivatives as a novel class of selective antagonists for the human V(1A) receptor

In the search for a novel class of selective antagonists for the human V(1A) receptor, high-throughput screening (HTS) of the Yamanouchi chemical library using CHO cells expressing the cloned human V(1A) (hV(1A)) receptor led to the discovery of 5-(4-biphenyl)-4-(2-methoxyphenyl)-3-methyl-1,2,4-triazole (3) which possessed the novel 4,5-diphenyl-1,2,4-triazole structure. Subsequent structure-activity relationships studies on a series of the 4,5-diphenyl-1,2,4-triazole derivatives related to 3 revealed that the 4,5-diphenyl-1,2,4-triazole structure played an essential role in exerting high affinity for the hV(1A) receptor and that introduction of a basic amine moiety to the methoxy part of the 4-phenyl ring was effective in the improvement of both affinity for the hV(1A) receptor and selectivity versus the hV(2) receptor. Compound 3 and the 2-(morphorino)ethoxy derivative (11b) were shown to be antagonists for the hV(1A) receptor, from their effects on AVP-induced [Ca(2+)](i) response in CHO cells expressing the hV(1A) receptor.     

Nicorandil as a novel therapy for advanced diabetic nephropathy in the eNOS-deficient mouse

Nicorandil is an orally available drug that can act as a nitric oxide donor, an antioxidant, and an ATP-dependent K channel activator. We hypothesized that it may have a beneficial role in treating diabetic nephropathy. We administered nicorandil to a model of advanced diabetic nephropathy (the streptozotocin-induced diabetes in mice lacking endothelial nitric oxide synthase, eNOSKO); controls included diabetic eNOS KO mice without nicorandil and nondiabetic eNOS KO mice treated with either nicorandil or vehicle. Mice were treated for 8 wk. Histology, blood pressure, and renal function were determined. Additional studies involved examining the effects of nicorandil on cultured human podocytes. Here, we found that nicorandil did not affect blood glucose levels, blood pressure, or systemic endothelial function, but significantly reduced proteinuria and glomerular injury (mesangiolysis and glomerulosclerosis). Nicorandil protected against podocyte loss and podocyte oxidative stress. Studies in cultured podocytes showed that nicorandil likely protects against glucose-mediated oxidant stress via the ATP-dependent K channel as opposed to its NO-stimulating effects. In conclusion, nicorandil may be beneficial in diabetic nephropathy by preserving podocyte function. We recommend clinical trials to determine whether nicorandil may benefit diabetic nephropathy or other conditions associated with podocyte dysfunction.     

Utilization of a multiple antigenic peptide as a calibration standard in the BAN50 single antibody sandwich ELISA for Aβ oligomers

Soluble amyloid-β (Aβ) oligomers are thought to be a cause of neurodegeneration and memory loss in Alzheimer disease (AD). We recently reported a newly developed enzyme linked immunosorbent assay (ELISA) for high molecular weight (HMW) Aβ oligomers in which the same Aβ monoclonal antibody, BAN50, was used for both capture and detection in a single antibody sandwich ELISA (SAS-ELISA) system. Our previous data suggest that this assay will be useful for the early diagnosis of AD, but its practical application to large-scale or longitudinal studies has been limited because of lack of a reliable calibration standard. In order to develop such a standard, we have now constructed a novel peptide using the multiple antigenic peptide (MAP) technique, where multiple epitopes of BAN50 were linked, via a spacer, to a branching lysine core. We show that the standard curve constructed from a 16-mer MAP covered the physiological range of signals obtained in the BAN50 SAS-ELISA from samples of human CSF, serum, and plasma. Furthermore, this 16-mer MAP is available in large quantities and is stable against freeze-thawing. We estimate that the signal per 1 pM of this standard corresponds to 1.54-5.0 pM of HMW Aβ oligomers. This MAP approach could also be used to provide an effective calibration standard for other SAS-ELISAs.     

The ciliary protein Nek8/Nphp9 acts downstream of Inv/Nphp2 during pronephros morphogenesis and left-right establishment in zebrafish

Nephronophthisis (NPHP) is an autosomal recessive cystic kidney disease. Among 12 reported Nphp gene products, Inv/Nphp2, Nphp3 and Nek8/Nphp9 are localized to the proximal segment in the primary cilium. However, the functional relationships are unknown. This study focused on phenotype analysis of nek8 knockdown embryos and the genetic relationship between nek8 and inv in zebrafish. Knockdown of nek8 produced both pronephric cysts and abnormal cardiac looping. Simultaneous knockdown of nek8 and inv synergistically increased the incidence of these defects. Interestingly, nek8 mRNA rescued inv morphant phenotypes, although inv mRNA could not rescue nek8 morphant phenotypes. These results suggest that Nek8 acts downstream of Inv function.     

GlmS and NagB regulate amino sugar metabolism in opposing directions and affect Streptococcus mutans virulence

Streptococcus mutans is a cariogenic pathogen that produces an extracellular polysaccharide (glucan) from dietary sugars, which allows it to establish a reproductive niche and secrete acids that degrade tooth enamel. While two enzymes (GlmS and NagB) are known to be key factors affecting the entrance of amino sugars into glycolysis and cell wall synthesis in several other bacteria, their roles in S. mutans remain unclear. Therefore, we investigated the roles of GlmS and NagB in S. mutans sugar metabolism and determined whether they have an effect on virulence. NagB expression increased in the presence of GlcNAc while GlmS expression decreased, suggesting that the regulation of these enzymes, which functionally oppose one another, is dependent on the concentration of environmental GlcNAc. A glmS-inactivated mutant could not grow in the absence of GlcNAc, while nagB-inactivated mutant growth was decreased in the presence of GlcNAc. Also, nagB inactivation was found to decrease the expression of virulence factors, including cell-surface protein antigen and glucosyltransferase, and to decrease biofilm formation and saliva-induced S. mutans aggregation, while glmS inactivation had the opposite effects on virulence factor expression and bacterial aggregation. Our results suggest that GlmS and NagB function in sugar metabolism in opposing directions, increasing and decreasing S. mutans virulence, respectively.     

Synthesis and biological evaluation of (4H-1,2,4-triazol-4-yl)isoquinoline derivatives as selective glycine transporter 1 inhibitors

To identify novel glycine transporter 1(GlyT1) inhibitors with greater selectivity relative to GlyT2 and improved aqueous solubility, we synthesized a series of 4H-1,2,4-triazole derivatives with heteroaromatic rings at the 4-position and investigated their structure-activity relationships. Replacement of the 2-fluorophenyl group of lead compound 5 with various aromatic groups led to the identification of 5-(3-biphenyl-4-yl-5-ethyl-4H-1,2,4-triazol-4-yl)isoquinoline (15) with 38-fold selectivity between GlyT1 and GlyT2. 15 also showed improved aqueous solubility and in vivo efficacy on (+)-HA966-induced hyperlocomotion in mice over the lead compound.     

Identification,characterization and interaction of <Emphasis Type="Italic">HAP</Emphasis> family genes in rice

A HAP complex, which consists of three subunits, namely HAP2 (also called NF-YA or CBF-B), HAP3 (NF-YB/CBF-A) and HAP5 (NF-YC/CBF-C), binds to CCAAT sequences in a promoter to control the expression of target genes. We identified 10 HAP2 genes, 11 HAP3 genes and 7 HAP5 genes in the rice genome. All the three HAP family genes encode a protein with a conserved domain in each family and various non-conserved regions in both length and amino acid sequence. These genes showed various expression patterns depending on genes, and various combinations of overlapped expression of the HAP2, HAP3 and HAP5 genes were observed. Furthermore, protein interaction analyses showed interaction of OsHAP3A, a ubiquitously expressed HAP3 subunit of rice, with specific members of HAP5. These results indicate that the formation of specific complex with various HAP subunits combinations can be achieved by both tissue specific expression of three subunit genes and specific interaction of three subunit proteins. This may suggest that the HAP complexes may control various aspects of rice growth and development through tissue specific expression and complex formation of three subunit members. Nucleotide sequence data reported are available in the DDBJ/EMBL/GenBank databases under the accession numbers AB288027 to AB288048 and BR000373 to BR000375.