Introduction and verification of a novel method for measuring wood fiber length using a single cross section in Acacia mangium

A novel method for calculating the wood fiber length using a single cross section was devised and verified in Acacia mangium. This method is based on the ratio of cell tips to total cell number in a cross section related to the wood fiber. The fiber length was calculated using the single cross-section method and was compared with the measurements obtained using the conventional maceration method and the serial cross-section method. There was no significant difference among the three methods.     

A role of LIM kinase 1/cofilin pathway in regulating endocytic trafficking of EGF receptor in human breast cancer cells

We have previously shown that overexpression of LIM kinase1 (LIMK1) resulted in a marked retardation of the internalization of the receptor-mediated endocytic tracer, Texas red-labeled epidermal growth factor (EGF) in low-invasive human breast cancer cell MCF-7. We thereby postulate that LIMK1 signaling plays an important role in the regulation of ligand-induced endocytosis of EGF receptor (EGFR) in tumor cells by reorganizing and influencing actin-filament dynamics. In the present study, we further assessed the effect of wild-type LIMK1, a kinase-deficient dominant negative mutant of LIMK1 (DN-LIMK1) and an active, unphosphorylatable cofilin mutant (S3A cofilin) on internalization of EGF-EGFR in MDA-MB-231, a highly invasive human breast cancer cell line. We demonstrate here that a marked delay in the receptor-mediated internalization of Texas red-labeled EGF was observed in the wild-type LIMK1 transfectants, and that most of the internalized EGF staining were accumulated within transferrin receptor-positive early endosomes even after 30 min internalization. In contrast, the expression of dominant-negative LIMK1 mutant rescued the efficient endocytosis of Texas red-EGF, and large amounts of Texas red-EGF staining already reached LIMPII-positive late endosomes/lysosomal vacuoles after 15 min internalization. We further analyzed the effect of S3A cofilin mutant on EGFR trafficking, and found an efficient delivery of Texas red-EGF into late endosomes/lysosomes at 15–30 min after internalization. Taken together, our novel findings presented in this paper implicate that LIMK1 signaling indeed plays a pivotal role in the regulation of EGFR trafficking through the endocytic pathway in invasive tumor cells.     

Phosphorylation of tau at Ser214 mediates its interaction with 14-3-3 protein: implications for the mechanism of tau aggregation

The microtubule associated protein tau is a major component of neurofibrillary tangles in Alzheimer disease brain, however the neuropathological processes behind the formation of neurofibrillary tangles are still unclear. Previously, 14-3-3 proteins were reported to bind with tau. 14-3-3 Proteins usually bind their targets through specific serine/threonine –phosphorylated motifs. Therefore, the interaction of tau with 14-3-3 mediated by phosphorylation was investigated. In this study, we show that the phosphorylation of tau by either protein kinase A (PKA) or protein kinase B (PKB) enhances the binding of tau with 14-3-3 in vitro . The affinity between tau and 14-3-3 is increased 12- to 14-fold by phosphorylation as determined by real time surface plasmon resonance studies. Mutational analyses revealed that Ser214 is critical for the phosphorylation-mediated interaction of tau with 14-3-3. Finally, in vitro aggregation assays demonstrated that phosphorylation by PKA/PKB inhibits the formation of aggregates/filaments of tau induced by 14-3-3. As the phosphorylation at Ser214 is up-regulated in fetal brain, tau's interaction with 14-3-3 may have a significant role in the organization of the microtubule cytoskeleton in development. Also as the phosphorylation at Ser214 is up-regulated in Alzheimer's disease brain, tau's interaction with 14-3-3 might be involved in the pathology of this disease.     

Infrared laser‐mediated local gene induction in medaka,zebrafish and Arabidopsis thaliana

Heat shock promoters are powerful tools for the precise control of exogenous gene induction in living organisms. In addition to the temporal control of gene expression, the analysis of gene function can also require spatial restriction. Recently, we reported a new method for in vivo, single‐cell gene induction using an infrared laser‐evoked gene operator (IR‐LEGO) system in living nematodes (Caenorhabditis elegans). It was demonstrated that infrared (IR) irradiation could induce gene expression in single cells without incurring cellular damage. Here, we report the application of IR‐LEGO to the small fish, medaka (Japanese killifish; Oryzias latipes) and zebrafish (Danio rerio), and a higher plant (Arabidopsis thaliana). Using easily observable reporter genes, we successfully induced gene expression in various tissues in these living organisms. IR‐LEGO has the potential to be a useful tool in extensive research fields for cell/tissue marking or targeted gene expression in local tissues of small fish and plants.     

Synthesis, SAR studies, and evaluation of 1,4-benzoxazepine derivatives as selective 5-HT1A receptor agonists with neuroprotective effect: Discovery of Piclozotan

A new series of 1,4-benzoxazepine derivatives was designed, synthesized, and evaluated for binding to 5-HT1A receptor and cerebral anti-ischemic effect. A lot of compounds exhibited nanomolar affinity for 5-HT1A receptor with good selectivity over both dopamine D2 and alpha1-adrenergic receptors. Among these compounds, 3-chloro-4-[4-[4-(2-pyridinyl)-1,2,3,6-tetrahydropyridin-1-yl]butyl]-1, 4-benzoxazepin-5(4H)-one (50: SUN N4057 (Piclozotan) as 2HCl salt) showed remarkable neuroprotective activity in a transient middle cerebral artery occlusion (t-MCAO) model.     

QTL mapping of clubroot resistance in radish (Raphanus sativus L.)

A QTL analysis for clubroot resistance (CR) of radish was performed using an F₂ population derived from a crossing of a CR Japanese radish and a clubroot-susceptible (CS) Chinese radish. F₃ plants obtained by selfing of F₂ plants were used for the CR tests. The potted seedlings were inoculated and the symptom was evaluated 6 weeks thereafter. The mean disease indexes of the F₃ plants were used for the phenotype of the F₂. The results of two CR tests were analyzed for the presence of QTL. A linkage map was constructed using AFLP and SSR markers; it spanned 554 cM and contained 18 linkage groups. A CR locus was observed in the top region of linkage group 1 in two tests. Therefore, the present results suggest that a large part of radish CR is controlled by a single gene or closely linked genes in this radish population, although minor effects of other genomic areas cannot be ruled out. The CR locus was named Crs1. Markers linked to Crs1 showed sequence homology to the genomic region of the top of chromosome 3 of Arabidopsis, as in the case of Crr3, a CR locus in Brassica rapa. These markers should be useful for breeding CR cultivars of radish. As Japanese radishes are known to be highly resistant or immune to clubroot, these markers may also be useful in the introgression of this CR gene to Brassica crops.     

Deletion of CnLIG4 DNA ligase gene in the fungal pathogen Cryptococcus neoformans elevates homologous recombination efficiency

In eukaryotes from yeasts to human, DNA double-strand breaks are repaired by nonhomologous end-joining (NHEJ) or homologous integration (HI). In the human pathogenic yeast Cryptococcus neoformans, gene manipulation by HI does not occur frequently because ectopic integration by NHEJ is predominant, and it has been necessary to screen 30–100 transformants per experiment to obtain transformants with the desired genotypes. To overcome this problem, we constructed a strain in which one of the NHEJ-related genes, CnLIG4, was deleted. CnLIG4 encodes a homologue of the human DNA ligase IV involved in the last step of DNA repair by NHEJ. Gene targeting in the URA5 locus of a URA5-lacking strain TAD1 with URA5 gene fragments having 1-kb flanking sequences achieved 80% HI efficiency, which is higher than that of the wild-type control (50%). Growth phenotypes and virulence were not attenuated by deletion of the CnLIG4 gene. Such results suggest that the CnLIG4 knockout strain created in this study provides an additional alternative for the molecular genetics study of C. neoformans.