Real-Time Visualization of Neuronal Activity during Perception

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Discovery and structure–activity relationship of thienopyridine derivatives as bone anabolic agents

A cell-based assay was performed for the discovery of novel bone anabolic agents. Alkaline phosphatase (ALPase) activity of ST2 cells was utilized as an indicator of osteoblastic differentiation, and thienopyridine derivative 1 was identified as a hit compound. 3-Aminothieno[2,3-b]pyridine-2-carboxamide was confirmed to be a necessary core structure for the enhancement of ALPase activity, and then optimization of the C4-substituent on the thienopyridine ring was carried out. Introduction of cyclic amino groups to the C4-position of the thienopyridine ring improved the activity. Especially, N-phenyl-homopiperazine derivatives were found to be strong enhancers of ALPase among this new series. Furthermore, 3-amino-4-(4-phenyl-1,4-diazepan-1-yl)thieno[2,3-b]pyridine-2-carboxamide (15k) was orally administered to ovariectomized (OVX) rats over 6 weeks for evaluating the effects on areal bone mineral density (aBMD), and statistically significant improvements in aBMD were observed from the dosage of 10 mg/kg/day.     

Purification and Characterization of a Thermostable Carboxypeptidase (Carboxypeptidase Taq) from Thermus aquaticus YT-1

Brassinosteroid (BR) and auxin co-regulate plant growth in a process termed cross-talking. Based on the assumption that their signal transductions are partially shared, inhibitory chemicals for both signal transductions were screened from a commercially available library. A chemical designated as NJ15 (ethyl 2-[5-(3,5-dichlorophenyl)-1,2,3,4-tetrazole-2-yl]acetate) diminished the growth promotion of both adzuki bean epicotyls and Arabidopsis seedlings, by the application of either BR or auxin. To understand its target site(s), bioassays with a high dependence on the signal transduction of either BR (BR-signaling) or auxin (AX-signaling) were performed. NJ15 inhibited the photomorphogenesis of Arabidopsis seedlings grown in the dark, which mainly depends on BR-signaling, while NJ15 also inhibited their gravitropic responses mainly depending on AX-signaling. On the study for the structure–activity relationships of NJ15 analogs, they showed strong correlations on the inhibitory profiles between BR- and AX-signalings. These correlations imply that NJ15 targets the downstream pathway after the integration of BR- and AX-signals.     

Functional and Structural Analysis of a β-Glucosidase Involved in β-1,2-Glucan Metabolism in Listeria innocua

Despite the presence of β-1,2-glucan in nature, few β-1,2-glucan degrading enzymes have been reported to date. Recently, the Lin1839 protein from Listeria innocua was identified as a 1,2-β-oligoglucan phosphorylase. Since the adjacent lin1840 gene in the gene cluster encodes a putative glycoside hydrolase family 3 β-glucosidase, we hypothesized that Lin1840 is also involved in β-1,2-glucan dissimilation. Here we report the functional and structural analysis of Lin1840. A recombinant Lin1840 protein (Lin1840r) showed the highest hydrolytic activity toward sophorose (Glc-β-1,2-Glc) among β-1,2-glucooligosaccharides, suggesting that Lin1840 is a β-glucosidase involved in sophorose degradation. The enzyme also rapidly hydrolyzed laminaribiose (β-1,3), but not cellobiose (β-1,4) or gentiobiose (β-1,6) among β-linked gluco-disaccharides. We determined the crystal structures of Lin1840r in complexes with sophorose and laminaribiose as productive binding forms. In these structures, Arg572 forms many hydrogen bonds with sophorose and laminaribiose at subsite +1, which seems to be a key factor for substrate selectivity. The opposite side of subsite +1 from Arg572 is connected to a large empty space appearing to be subsite +2 for the binding of sophorotriose (Glc-β-1,2-Glc-β-1,2-Glc) in spite of the higher K_m value for sophorotriose than that for sophorose. The conformations of sophorose and laminaribiose are almost the same on the Arg572 side but differ on the subsite +2 side that provides no interaction with a substrate. Therefore, Lin1840r is unable to distinguish between sophorose and laminaribiose as substrates. These results provide the first mechanistic insights into β-1,2-glucooligosaccharide recognition by β-glucosidase.     

A soluble form of human nectin-2 impairs exocrine secretion of pancreas and formation of zymogen granules in transgenic mice

Transgenic mouse lines expressing a soluble form of human nectin-2 (hNectin-2Ig Tg) exhibited distinctive elevation of amylase and lipase levels in the sera. In this study, we aimed to clarify the histopathology and to propose the transgenic mouse lines as new animal model for characteristic pancreatic exocrine defects. The significant increase of amylase and lipase levels in sera of the transgenic lines approximately peaked at 8 weeks old and thereafter, plateaued or gradually decreased. The histopathology in transgenic acinar cells was characterized by intracytoplasmic accumulation of abnormal proteins with decrease of normal zymogen granules. The hNectin-2Ig expression was observed in the cytoplasm of pancreatic acinar cells, which was consistent with zymogen granules. However, signals of hNectin-2Ig were very weak in the transgenic acinar cells with the abnormal cytoplasmic accumulaion. The PCNA-positive cells increased in the transgenic pancreas, which suggested the affected acinar cells were regenerated. Acinar cells of hNectin-2Ig Tg had markedly small number of zymogen granules with remarkable dilation of the endoplasmic reticulum (ER) lumen containing abundant abnormal proteins. In conclusion, hNectin-2Ig Tg is proposed as a new animal model for characteristic pancreatic exocrine defects, which are due to the ER stress induced by expression of mutated cell adhesion molecule that is a soluble form of human nectin-2.     

Carbon accumulation and vertical accretion in a restored versus historic salt marsh in southern Puget Sound,Washington, United States

Few comparisons exist between vertical accretion (VA) and carbon accumulation rates (CARs) in restored versus historic (i.e. reference) marshes. Here, we compare these processes in a formerly diked, sparsely vegetated, restored salt marsh (Six Gill Slough, SG), whose surface is subsided relative to the tidal frame, to an adjacent, relatively pristine, historic salt marsh (Animal Slough, AS). Six sediment cores were collected at both AS and SG approximately 6 years after restoration. Cores were analyzed for bulk density (BD), % loss of ignition, % organic carbon, and ²¹⁰Pb. We found that sharp changes in BD in surface layers of SG cores were highly reliable markers for the onset of restoration. The mean VA since restoration at SG (0.79 [SD = 0.29] cm/year) was approximately twice that of AS (0.41 [SD = 0.16] cm/year). In comparison, the VA at AS over 50 years was 0.30 (SD = 0.09) cm/year. VA consisted almost entirely of inorganic sediment at SG whereas at AS it was approximately 55%. Mean CARs at SG were somewhat greater than at AS, but the difference was not significant due to high variability (SG: 81–210 g C m^?2 year^?1; AS: 115–168 g C m^?2 year^?1). The mean CAR at AS over the past 50 years was 118 (SD = 23) g C m^?2 year^?1. This study demonstrates that a sparsely vegetated, restored salt marsh can quickly begin to accumulate carbon and that historic and restored marshes can have similar CARs despite highly divergent formation processes.     

Characterization of ScORK28, a transmembrane functional protein receptor kinase predominantly expressed in ovaries from the wild potato species Solanum chacoense

Solanum chacoense ovule receptor kinase 28 (ScORK28) was found among 30 receptor kinases from an ovule cDNA library enriched for weakly expressed mRNAs. This LRR-RLK displayed high level of tissue specificity at the RNA and protein levels and was predominantly expressed in female reproductive tissues. Protein expression analyses in planta revealed that ScORK28 was N-glycosylated and ScORK28::GFP fusion analyses showed that it was localized at the plasma membrane. Bacterial expression of ScORK28 catalytic domain followed by kinase activity assays revealed that ScORK28 is an active Mg2+-dependent protein kinase and that the juxtamembrane domain is necessary for kinase activity.     

alpha-Synuclein stimulates differentiation of osteosarcoma cells: relevance to down-regulation of proteasome activity

Because a limited study previously showed that alpha-synuclein (alpha-syn), the major pathogenic protein for Parkinson disease, was expressed in differentiating brain tumors as well as various peripheral cancers, the main objective of the present study was to determine whether alpha-syn might be involved in the regulation of tumor differentiation. For this purpose, alpha-syn and its non-amyloidogenic homologue beta-syn were stably transfected to human osteosarcoma MG63 cell line. Compared with beta-syn-overexpressing and vector-transfected cells, alpha-syn-overexpressing cells exhibited distinct features of differentiated osteoblastic phenotype, as shown by up-regulation of alkaline phosphatase and osteocalcin as well as inductive matrix mineralization. Further studies revealed that proteasome activity was significantly decreased in alpha-syn-overexpressing cells compared with other cell types, consistent with the fact that proteasome inhibitors stimulate differentiation of various osteoblastic cells. In alpha-syn-overexpressing cells, protein kinase C (PKC) activity was significantly decreased, and reactivation of PKC by phorbol ester significantly restored the proteasome activity and abrogated cellular differentiation. Moreover, activity of lysosome was up-regulated in alpha-syn-overexpressing cells, and treatment of these cells with autophagy-lysosomal inhibitors resulted in a decrease of proteasome activity associated with up-regulation of alpha-syn expression, leading to enhance cellular differentiation. Taken together, these results suggest that the stimulatory effect of alpha-syn on tumor differentiation may be attributed to down-regulation of proteasome, which is further modulated by alterations of various factors, such as protein kinase C signaling pathway and a autophagy-lysosomal degradation system. Thus, the mechanism of alpha-syn regulation of tumor differentiation and neuropathological effects of alpha-syn may considerably overlap with each other.     

Prevalence and species richness of trematode parasites only partially recovers after the 2011 Tohoku,Japan, earthquake tsunami

Trematode parasites have complex life cycles and use a variety of host species across different trophic levels. Thus, they can be used as indicators of disturbance and recovery of coastal ecosystems. Estuaries on the Pacific coast of northeastern Japan were heavily affected by the 2011 Tohoku earthquake tsunami. To evaluate the effect of the tsunami on the trematode community, we examined trematodes in the mud snail, Batillaria attramentaria, at five study sites (three sites severely exposed to the tsunami and two sites sheltered from the tsunami) in Sendai Bay for 2 years prior to and 8 years after the tsunami. While the trematode prevalence decreased at all study sites, the species richness decreased only at the sites exposed to the tsunami. Although parasitism increased over the study period post-tsunami, the community had not fully recovered 8 years after the event. Trematode community structure has changed every year since the tsunami and has not stabilised. This could be explained by the alteration of first and second intermediate host communities. Our study suggests that it will take more time for the recovery of the trematode community and the associated coastal ecosystem in the Tohoku region.