Importin/exportin-mediated nucleocytoplasmic shuttling of cucumber mosaic virus 2b protein is required for 2b’s efficient suppression of RNA silencing

The 2b protein (2b) of cucumber mosaic virus (CMV), an RNA-silencing suppressor (RSS), is a major pathogenicity determinant of CMV. 2b is localized in the nucleus and cytoplasm, and its nuclear import is determined by two nuclear localization signals (NLSs); a carrier protein (importin [IMPα]) is predicted to be involved in 2b’s nuclear transport. Cytoplasmic 2bs play a role in suppression of RNA silencing by binding to small RNAs and AGO proteins. A putative nuclear export signal (NES) motif was also found in 2b, but has not been proved to function. Here, we identified a leucine-rich motif in 2b’s C-terminal half as an NES. We then showed that NES-deficient 2b accumulated abundantly in the nucleus and lost its RSS activity, suggesting that 2b exported from the nucleus can play a role as an RSS. Although two serine residues (S40 and S42) were previously found to be phosphorylated, we also found that an additional phosphorylation site (S28) alone can affect 2b’s nuclear localization and RSS activity. Alanine substitution at S28 impaired the IMPα-mediated nuclear/nucleolar localization of 2b, and RSS activity was even stronger compared to wild-type 2b. In a subcellular fractionation assay, phosphorylated 2bs were detected in the nucleus, and comparison of the accumulation levels of nuclear phospho-2b between wild-type 2b and the NES mutant showed a greatly reduced level of the phosphorylated NES mutant in the nucleus, suggesting that 2bs are dephosphorylated in the nucleus and may be translocated to the cytoplasm in a nonphosphorylated form. These results suggest that 2b manipulates its nucleocytoplasmic transport as if it tracks down its targets, small RNAs and AGOs, in the RNA silencing pathway. We infer that 2b’s efficient RSS activity is maintained by a balance of phosphorylation and dephosphorylation, which are coupled to importin/exportin-mediated shuttling between the nucleus and cytoplasm.     

Cloning of bovine LYST gene and identification of a missense mutation associated with Chediak-Higashi syndrome of cattle

An inheritable bleeding disorder with light coat color caused by an autosomal recessive gene has been reported in a population of Japanese black cattle. The disease has been diagnosed as Chediak-Higashi Syndrome (CHS) of cattle which correspond to a human inheritable disorder caused by mutation in LYST gene. To characterize the molecular lesion causing CHS in cattle, cDNAs encoding bovine LYST were isolated from a bovine brain cDNA library. The nucleotide and deduced amino acid sequences of bovine LYST had 89.6 and 90.2% identity with those of the human LYST gene, respectively. In order to identify the mutation within the LYST gene causing CHS in cattle, cDNA fragments of the LYST gene were amplified from an affected animal by RT-PCR and their nucleotide sequences were completely determined. Notably, a nucleotide substitution of A to G transition, resulting in an amino acid substitution of histidine to arginine (H2015R) was identified in the affected animal. The presence of the substitution was completely corresponding with the occurrence of the CHS phenotype among 105 members of pedigrees of the Japanese black cattle and no cattle of other populations had this substitution. These findings strongly suggested that H2015R is the causative mutation in CHS of Japanese black cattle. Received: 25 May 1999 / Accepted: 26 July 1999     

Larger Right Posterior Parietal Volume in Action Video Game Experts: A Behavioral and Voxel-Based Morphometry (VBM) Study

Recent studies suggest that action video game players exhibit superior performance in visuospatial cognitive tasks compared with non-game players. However, the neural basis underlying this visuospatial cognitive performance advantage remains largely unknown. The present human behavioral and imaging study compared gray matter volume in action video game experts and non-experts using structural magnetic resonance imaging and voxel-based morphometry analysis. The results revealed significantly larger gray matter volume in the right posterior parietal cortex in experts compared with non-experts. Furthermore, the larger gray matter volume in the right posterior parietal cortex significantly correlated with individual performance in a visual working memory task in experts. These results suggest that differences in brain structure may be linked to extensive video game play, leading to superior visuospatial cognitive performance in action video game experts.     

N1, N14-diferuloylspermine as an antioxidative phytochemical contained in leaves of Cardamine fauriei

Most Brassicaceae vegetables are ideal dietary sources of antioxidants beneficial for human health. Cardamine fauriei (Ezo-wasabi in Japanese) is a wild, edible Brassicaceae herb native to Hokkaido, Japan. To clarify the main antioxidative phytochemical, an 80% methanol extraction from the leaves was fractionated with Diaion® HP-20, Sephadex® LH-20, and Sep-Pak® C18 cartridges, and the fraction with strong antioxidant activity depending on DPPH method was purified by HPLC. Based on the analyses using HRESIMS and MS/MS, the compound might be N¹, N¹⁴-diferuloylspermine. This rare phenol compound was chemically synthesized, whose data on HPLC, MS and ¹H NMR were compared with those of naturally derived compound from C. fauriei. All results indicated they were the same compound. The radical-scavenging properties of diferuloylspermine were evaluated by ORAC and ESR spin trapping methods, with the diferuloylspermine showing high scavenging activities of the ROO^·, O₂^·?, and HO^· radicals as was those of conventional antioxidants.     

Overexpression of chitinase 3-like 1/YKL-40 in lung-specific IL-18-transgenic mice, smokers and COPD

We analyzed the lung mRNA expression profiles of a murine model of COPD developed using a lung-specific IL-18-transgenic mouse. In this transgenic mouse, the expression of 608 genes was found to vary more than 2-fold in comparison with control WT mice, and was clustered into 4 groups. The expression of 140 genes was constitutively increased at all ages, 215 genes increased gradually with aging, 171 genes decreased gradually with aging, and 82 genes decreased temporarily at 9 weeks of age. Interestingly, the levels of mRNA for the chitinase-related genes chitinase 3-like 1 (Chi3l1), Chi3l3, and acidic mammalian chitinase (AMCase) were significantly higher in the lungs of transgenic mice than in control mice. The level of Chi3l1 protein increased significantly with aging in the lungs and sera of IL-18 transgenic, but not WT mice. Previous studies have suggested Chi3l3 and AMCase are IL-13-driven chitinase-like proteins. However, IL-13 gene deletion did not reduce the level of Chi3l1 protein in the lungs of IL-18 transgenic mice. Based on our murine model gene expression data, we analyzed the protein level of YKL-40, the human homolog of Chi3l1, in sera of smokers and COPD patients. Sixteen COPD patients had undergone high resolution computed tomography (HRCT) examination. Emphysema was assessed by using a density mask with a cutoff of -950 Hounsfield units to calculate the low-attenuation area percentage (LAA%). We observed significantly higher serum levels in samples from 28 smokers and 45 COPD patients compared to 30 non-smokers. In COPD patients, there was a significant negative correlation between serum level of YKL-40 and %FEV(1). Moreover, there was a significant positive correlation between the serum levels of YKL-40 and LAA% in COPD patients. Thus our results suggest that chitinase-related genes may play an important role in establishing pulmonary inflammation and emphysematous changes in smokers and COPD patients.     

Autophagy as a Neuroprotective Mechanism Against 3-Nitropropionic Acid-Induced Murine Astrocyte Cell Death

Huntington’s disease (HD) is a genetic neurodegenerative disorder that is characterized by severe striatal atrophy with extensive neuronal loss and gliosis. Although the molecular mechanism is not well understood, experimental studies use the irreversible mitochondrial inhibitor 3-nitropropionic acid (3-NP) to mimic the neuropathological features of HD. In this study, the role of autophagy as a neuroprotective mechanism against 3-NP-induced astrocyte cytotoxicity was evaluated. Autophagy is a catabolic process that is essential for the turnover of cytosolic proteins and organelles and is involved in the modulation of cell death and survival. We showed that 3-NP-induced apoptosis, which was accompanied by Bax and Beclin-1 upregulation, was dependent on acidic vesicular organelle (AVO) formation after a continuous exposure to 3-NP for 12 h. The upregulation of Bax and Beclin-1 as well as AVO formation were normalized 24 h after 3-NP exposure.     

Enhanced Nox1 expression and oxidative stress resistance in c-kit-positive hematopoietic stem/progenitor cells

Although stem cells are generally thought to be resistant to oxidative stress, the fact and in detail molecular mechanism are still to be clearly identified. We herein tried to understand the overall characterization of redox regulatory signaling in hematopoietic stem cells. We purified c-kit-positive hematopoietic stem/progenitor cells from the bone marrow of healthy mice, and then evaluated their redox regulatory property. Compared to the c-kit-negative matured mononuclear cells, c-kit-positive stem/progenitor cells showed lower basic levels of intracellular reactive oxygen species, faster clearance of the accumulated intracellular reactive oxygen species, and higher resistant to oxidative stress. An overall view on the gene expression profile associated with redox regulation showed to be widely differed between cell types. We confirmed that the c-kit-positive stem/progenitor cells expressed significantly higher of Nox1 and catalase, but less of lactoperoxidase than these matured mononuclear cells. Our data suggests that stem cells keep specific redox regulatory property for defensing against oxidative stress.     

Systematic Regional Variations in Purkinje Cell Spiking Patterns

In contrast to the uniform anatomy of the cerebellar cortex, molecular and physiological studies indicate that significant differences exist between cortical regions, suggesting that the spiking activity of Purkinje cells (PCs) in different regions could also show distinct characteristics. To investigate this possibility we obtained extracellular recordings from PCs in different zebrin bands in crus IIa and vermis lobules VIII and IX in anesthetized rats in order to compare PC firing characteristics between zebrin positive (Z+) and negative (Z−) bands. In addition, we analyzed recordings from PCs in the A2 and C1 zones of several lobules in the posterior lobe, which largely contain Z+ and Z− PCs, respectively. In both datasets significant differences in simple spike (SS) activity were observed between cortical regions. Specifically, Z− and C1 PCs had higher SS firing rates than Z+ and A2 PCs, respectively. The irregularity of SS firing (as assessed by measures of interspike interval distribution) was greater in Z+ bands in both absolute and relative terms. The results regarding systematic variations in complex spike (CS) activity were less consistent, suggesting that while real differences can exist, they may be sensitive to other factors than the cortical location of the PC. However, differences in the interactions between SSs and CSs, including the post-CS pause in SSs and post-pause modulation of SSs, were also consistently observed between bands. Similar, though less strong trends were observed in the zonal recordings. These systematic variations in spontaneous firing characteristics of PCs between zebrin bands in vivo, raises the possibility that fundamental differences in information encoding exist between cerebellar cortical regions.