Functional characterization of a B-type cell cycle switch 52 in rice (OsCCS52B)

Plant growth and development depend on a precise coordination between cell division and cell expansion. In this study, a rice cell cycle switch 52 B (OsCCS52B) was functionally characterized using two approaches: overexpression of the gene product in fission yeast and characterization of an insertion mutant line 1B-10423. In wild-type plants, OsCCS52B is highly expressed in generative organs such as flowers and kernels. Overexpression of OsCCS52B induces cell elongation and slower cell proliferation in fission yeast. Characterization of the mutant line 1B-10423 revealed that the mutant exhibits semi-dwarf and smaller kernel phenotypes. In addition, microscopic analysis of mutant kernels showed that the reduced kernel size was due to a reduced cell size. However, the nuclear size and ploidy level were unaffected. These results suggest that OsCCS52B may be involved in cell expansion regulation in rice endosperm.     

Bacteroides thetaiotaomicron VPI-5482 glycoside hydrolase family 66 homolog catalyzes dextranolytic and cyclization reactions

Bacteroides?thetaiotaomicron VPI-5482 harbors a gene encoding a putative cycloisomaltooligosaccharide glucanotransferase (BT3087) belonging to glycoside hydrolase family?66. The goal of the present study was to characterize the catalytic properties of this enzyme. Therefore, we expressed BT3087 (recombinant endo-dextranase from Bacteroides?thetaiotaomicron VPI-5482) in Escherichia?coli and determined that recombinant endo-dextranase from Bacteroides?thetaiotaomicron VPI-5482 preferentially synthesized isomaltotetraose and isomaltooligosaccharides (degree of polymerization >?4) from dextran. The enzyme also generated large cyclic isomaltooligosaccharides early in the reaction. We conclude that members of the glycoside hydrolase?66 family may be classified into three types: (a) endo-dextranases, (b) dextranases possessing weak cycloisomaltooligosaccharide glucanotransferase activity, and (c) cycloisomaltooligosaccharide glucanotransferases.     

Ginkgo biloba extract (EGb 761) prevents the ischemic brain injury-induced decrease in parvalbumin expression

Ginkgo biloba extract (EGb 761) exerts a neuroprotective effect against ischemic brain injury through an anti-apoptotic mechanism. Parvalbumin is a calcium buffering protein that plays an important role in modulating intracellular calcium concentration and regulating apoptotic cell death. The aim of this study was to investigate whether EGb 761 affects parvalbumin expression in cerebral ischemic injury. Adult male Sprague-Dawley rats were treated with vehicle or EGb 761 (100 mg/kg) prior to middle cerebral artery occlusion (MCAO) and cerebral cortex tissues were collected 24 h after MCAO. A proteomic approach revealed a reduction in parvalbumin expression in the vehicle-treated animals, whereas EGb 761 pretreatment attenuates the ischemic injury-induced decrease in parvalbumin expression. RT-PCR and Western blot analyses clearly confirmed the fact that EGb 761 prevents the injury-induced decrease in parvalbumin. Moreover, the results of immunohistochemical staining showed that the number of parvalbumin-positive cells was lower in vehicle-treated animals than in sham-operated animals, and EGb 761 averted this decrease. Thus, these results suggest that the maintenance of parvalbumin expression is associated with the neuroprotective function of EGb 761 against neuronal damage induced by ischemia.     

Inhibitory effects of epigallocatechin gallate and its glucoside on the human intestinal maltase inhibition

Human intestinal maltase (HMA) is an ??-glucosidase responsible for the hydrolysis of ??-1,4-linkages from the non-reducing end of malto-oligosaccharides. HMA has become an important target in the treatment of type-2 diabetes. In this study, epigallocatechin gallate (EGCG) and EGCG glucoside (EGCG-G1) were identified as inhibitors of HMA by an in vitro assay with IC₅₀ of 20 ± 1.0 and 31.5 ± 1.0 ??M, respectively. A Lineweaver-Burk plot confirmed that EGCG and EGCG-G1 were competitive inhibitors of maltose substrate against HMA and inhibition kinetic constants (K _i ) calculated from a Dixon plot were 5.93 ± 0.26 and 7.88 ± 0.57 ??M, respectively. Both EGCG and EGCG-G1 bound to the active site of HMA with numerous hydrophobic and hydrogen bond interactions.     

Biological and genetic properties of SA14-14-2, a live-attenuated Japanese encephalitis vaccine that is currently available for humans

Japanese encephalitis virus (JEV), a mosquito-borne flavivirus, is a major cause of acute encephalitis, a disease of significance for global public health. In the absence of antiviral therapy to treat JEV infection, vaccination is the most effective method of preventing the disease. In JE-endemic areas, the most widely used vaccine to date is SA(14)-14-2, a live-attenuated virus derived from its virulent parent SA(14). In this study, we describe the biological properties of SA(14)-14-2, both in vitro and in vivo, and report the genetic characteristics of its genomic RNA. In BHK-21 (hamster kidney) cells, SA(14)-14-2 displayed a slight delay in plaque formation and growth kinetics when compared to a virulent JEV strain, CNU/LP2, with no decrease in maximum virus production. The delay in viral growth was also observed in two other cell lines, SH-SY5Y (human neuroblastoma) and C6/36 (mosquito larva), which are potentially relevant to JEV pathogenesis and transmission. In 3-week-old ICR mice, SA(14)-14-2 did not cause any symptoms or death after either intracerebral or peripheral inoculation with a maximum dose of up to 1.5×10(3) plaque-forming units (PFU) per mouse. The SA(14)-14-2 genome consisted of 10977 nucleotides, one nucleotide longer than all the previously reported genomes of SA(14)-14-2, SA(14) and two other SA(14)-derived attenuated viruses. This difference was due to an insertion of one G nucleotide at position 10701 in the 3 noncoding region. Also, we noted a significant number of nucleotide and/or amino acid substitutions throughout the genome of SA(14)-14-2, except for the prM protein-coding region, that differed from SA(14) and/or the other two attenuated viruses. Our results, together with others', provide a foundation not only for the study of JEV virulence but also for the development of new and improved vaccines for JEV.     

Phosphorylation of Arabidopsis response regulator 7 (ARR7) at the putative phospho-accepting site is required for ARR7 to act as a negative regulator of cytokinin signaling

Cytokinins are plant hormones that regulate diverse aspects of plant growth and development. Arabidopsis cytokinin signal transduction utilizes a multi-step two-component signaling (TCS) system by histidyl–aspartidyl phosphorelays. We here show that phosphorylation of ARR7, an A-type response regulator that acts as a negative regulator of cytokinin signaling, is required for its function in plants. Phosphorylation of ARR7 is inhibited in vitro by mutation in a putative phospho-accepting Asp residue into an Asn residue (ARR7^D85N). While ectopic expression of ARR7 decreases root-growth inhibition, callus formation, and cytokinin-inducible gene expression, overexpression of ARR7 ^D85N at the similar level does not generate these phenotypes. ARR7^D85N is localized to the nucleus and the half-life of this mutant protein is similar to that of ARR7 in Arabidopsis mesophyll protoplasts. These results suggest that the phosphorylation of ARR7 is necessary for ARR7-mediated cytokinin response.     

Characterization of a novel type of adherens junction in meningiomas and the derived cell line HBL-52

Adhering junctions are generally grouped into desmosomes and adherens junctions based on their ultrastructural appearance and molecular composition. The armadillo-protein plakoglobin is common to both types of junctions, which are otherwise composed of mutually exclusive proteins. This view is based on observations in epithelial tissues but cannot easily be transferred to other cell types and tissues, as has become apparent during the last decade with the identification of new junctional proteins and the investigation of further non-epithelial junctions. Using a broad array of well-characterized specific antibodies against key junctional proteins in immunoblot reactions, high-resolution double-label laser scanning confocal microscopy, and immunoelectron microscopy, we describe a new type of adherens junction in human meningiomas and the human meningioma cell line HBL-52. This novel junction has a unique composition of proteins not found in any other tissue; it contains the desmosomal armadillo-protein plakophilin 2 together with the classic proteins of “epithelial” adherens junctions, i.e., E-cadherin (in some instances replaced by N-cadherin), alpha-catenin, beta-catenin, plakoglobin, and p120^ctn. Ultrastructurally, it is formed between two or three neighboring cells. For pragmatic reasons, we suggest the name “meningeal junction” for this new structure. All authors declare the absence of conflicts of interest.     

Variation of ginkgolides and bilobalide contents in leaves and cell cultures of<Emphasis Type="Italic">Ginkgo biloba</Emphasis> L.

Ginkgolides (GK) and bilobalide are valuable compounds that belong to the lactone terpene. The contents of these metabolites were determined by HPLC from female and male tree ofGinkgo biloba L. The productivity ofG. biloba cells was also compared with the corresponding individual trees. High variations in the ginkgolides and bilobalide were observed from different individuals, plant parts, and cultured cells. The ginkgolides and bilobalide contents were different depending on the plant parts. Callus was obtained from various plant tissues, and NAA was better at callogenesis than 2,4-D in both the female and male trees. The plants and their corresponding cells showed considerable variation in their ginkgolides and bilobalide concentrations. The ginkgolides and bilobalide contents were not correlated with the production between dominant trees and their corresponding cells. Light irradiation enhanced the production of GK-A and GK-B, however, the concentration of bilobalide decreased under dark conditions.     

Effects of oleamide on choline acetyltransferase and cognitive activities

We screened 50 Korean traditional natural plants to measure the activation effect on choline acetyltransferase and attenuation of scopolamine-induced amnesia. The methanolic extracts from Zizyphus jujuba among the tested 50 plants, showed the highest activatory effect (34.1%) on choline acetyltransferase in vitro. By sequential fractionation of Zizyphus jujuba, the active component was finally identified as cis-9-octadecenoamide (oleamide). After isolation, oleamide showed a 65% activation effect. Administration of oleamide (0.32%) to mice significantly reversed the scopolamine-induced memory and/or cognitive impairment in the passive avoidance test and Y-maze test. Injection of scopolamine to mice impaired performance on the passive avoidance test (31% decrease in step-through latency), and on the Y-maze test (16% decrease in alternation behavior). In contrast, mice treated with oleamide before scopolamine injection were protected from these changes (12-25% decrease in step-through latency; 1-10% decrease in alternation behavior). These results suggest that oleamide should be a useful chemo-preventive agent against Alzheimer's disease.     

Random homologous pairing and incomplete sister chromatid alignment are common in angiosperm interphase nuclei

The chromosome arrangement in interphase nuclei is of growing interest, e.g., the spatial vicinity of homologous sequences is decisive for efficient repair of DNA damage by homologous recombination, and close alignment of sister chromatids is considered as a prerequisite for their bipolar orientation and subsequent segregation during nuclear division. To study the degree of homologous pairing and of sister chromatid alignment in plants, we applied fluorescent in situ hybridisation with specific bacterial artificial chromosome inserts to interphase nuclei. Previously we found in Arabidopsis thaliana and in A. lyrata positional homologous pairing at random, and, except for centromere regions, sister chromatids were frequently not aligned. To test whether these features are typical for higher plants or depend on genome size, chromosome organisation and/or phylogenetic affiliation, we investigated distinct individual loci in other species. The positional pairing of these loci was mainly random. The highest frequency of sister alignment (in >93% of homologues) was found for centromeres, some rDNA and a few other high copy loci. Apparently, somatic homologous pairing is not a typical feature of angiosperms, and sister chromatid aligment is not obligatory along chromosome arms. Thus, the high frequency of chromatid exchanges at homologous positions after mutagen treatment needs another explanation than regular somatic pairing of homologues (possibly an active search of damaged sites for homology). For sister chromatid exchanges a continuous sister chromatid alignment is not required. For correct segregation, permanent alignment of sister centromeres is sufficient.