Differential role of programmed death-ligand 1 [corrected] and programmed death-ligand 2 [corrected] in regulating the susceptibility and chronic progression of experimental autoimmune encephalomyelitis

Programmed death-1 (PD-1) is a negative costimulatory molecule, and blocking the interaction of PD-1 with its ligands, PD-L1 (B7-H1) and PD-L2 (B7-DC), enhances autoimmune disease in several animal models. We have studied the role of PD-1 ligands in disease susceptibility and chronic progression in experimental autoimmune encephalomyelitis (EAE). In BALB/c mice immunized with myelin oligodendrocyte glycoprotein (MOG) peptide 35-55, PD-L1 but not PD-L2 blockade significantly increased EAE incidence. In B10.S mice immunized with myelin proteolipid protein (PLP) peptide 139-151, both PD-L1 and PD-L2 blockade markedly enhanced EAE severity. In prediabetic NOD mice immunized with PLP48-70, PD-L2 blockade worsened EAE but did not induce diabetes, whereas PD-L1 blockade precipitated diabetes but did not worsen EAE, suggesting different regulatory roles of these two ligands in EAE and diabetes. B6 mice immunized with MOG35-55 developed chronic persistent EAE, and PD-L2 blockade in the chronic phase exacerbated EAE, whereas PD-L1 blockade did not. In contrast, SJL/J mice immunized with PLP139-151 developed chronic relapsing-remitting EAE, and only PD-L1 blockade during remission precipitated EAE relapse. The strain-specific effects of PD-1 ligand blockade did not correlate with the expression of PD-L1 and PD-L2 on dendritic cells and macrophages in lymphoid tissue, or on inflammatory cells in the CNS. However, EAE enhancement is correlated with less prominent Th2 cytokine induction after specific PD-1 ligand blockade. In conclusion, PD-L1 and PD-L2 differentially regulate the susceptibility and chronic progression of EAE in a strain-specific manner.     

Effects of colonization of a bacterial endophyte,Azospirillum sp. B510, on disease resistance in tomato

A plant growth-promoting bacteria, Azospirillum sp. B510, isolated from rice, can enhance growth and yield and induce disease resistance against various types of diseases in rice. Because little is known about the interaction between other plant species and this strain, we have investigated the effect of its colonization on disease resistance in tomato plants. Treatment with this strain by soil-drenching method established endophytic colonization in root tissues in tomato plant. The endophytic colonization with this strain-induced disease resistance in tomato plant against bacterial leaf spot caused by Pseudomonas syringae pv. tomato and gray mold caused by Botrytis cinerea. In Azospirillum-treated plants, neither the accumulation of SA nor the expression of defense-related genes was observed. These indicate that endophytic colonization with Azospirillum sp. B510 is able to activate the innate immune system also in tomato, which does not seem to be systemic acquired resistance.     

Isolation of putative glycoprotein gene from early somatic embryo of carrot and its possible involvement in somatic embryo development

Somatic embryogenesis is a unique process in plant cells. For example, embryogenic cells (EC) of carrot (Daucus carota) maintained in a medium containing 2,4-dichlorophenoxyacetic acid (2,4-D) regenerate whole plants via somatic embryogenesis after the depletion of 2,4-D. Although some genes such as C-ABI3 and C-LEC1 have been found to be involved in somatic embryogenesis, the critical molecular and cellular mechanisms for somatic embryogenesis are unknown. To characterize the early mechanism in the induction of somatic embryogenesis, we isolated genes expressed during the early stage of somatic embryogenesis after 2,4-D depletion. Subtractive hybridization screening and subsequent RNA gel blot analysis suggested a candidate gene, Carrot Early Somatic Embryogenesis 1 (C-ESE1). C-ESE1 encodes a protein that has agglutinin and S-locus-glycoprotein domains and its expression is highly specific to primordial cells of somatic embryo. Transgenic carrot cells with reduced expression of C-ESE1 had wide intercellular space and decreased polysaccharides on the cell surface and showed delayed development in somatic embryogenesis. The importance of cell-to-cell attachment in somatic embryogenesis is discussed.     

Vacuolar processing enzymes are essential for proper processing of seed storage proteins in Arabidopsis thaliana

The proprotein precursors of storage proteins are post-translationally processed to produce their respective mature forms within the protein storage vacuoles of maturing seeds. To investigate the processing mechanism in vivo, we isolated Arabidopsis mutants that accumulate detectable amounts of the precursors of the storage proteins, 12 S globulins and 2 S albumins, in their seeds. All six mutants isolated have a defect in the beta VPE gene. VPE (vacuolar processing enzyme) is a cysteine proteinase with substrate specificity toward an asparagine residue. We further generated various mutants lacking different VPE isoforms: alpha VPE, beta VPE, and/or gamma VPE. More than 90% of VPE activity is abolished in the beta vpe-3 seeds, and no VPE activity is detected in the alpha vpe-1/beta vpe-3/gamma vpe-1 seeds. The triple mutant seeds accumulate no properly processed mature storage proteins. Instead, large amounts of storage protein precursors are found in the seeds of this mutant. In contrast to beta vpe-3 seeds, which accumulate both precursors and mature storage proteins, the other single (alpha vpe-1 and gamma vpe-1) and double (alpha vpe-1/gamma vpe-1) mutants accumulate no precursors in their seeds at all. Therefore, the vegetative VPEs, alpha VPE and gamma VPE, are not necessary for precursor processing in the presence of beta VPE, but partly compensates for the deficiency in beta VPE in beta vpe-3 seeds. In the absence of functional VPEs, a proportion of pro2S albumin molecules are alternatively cleaved by aspartic proteinase. This cleavage by aspartic proteinase is promoted by the initial processing of pro2S albumins by VPE. Our overall results suggest that seed-type beta VPE is most essential for the processing of storage proteins, and that the vegetative-type VPEs and aspartic proteinase complement beta VPE activity in this processing.     

Rhythmic expression of circadian clock genes in human leukocytes and beard hair follicle cells

Evaluating individual circadian rhythm traits is crucial for understanding the human biological clock system. The present study reports characterization of physiological and molecular parameters in 13 healthy male subjects under a constant routine condition, where interfering factors were kept to minimum. We measured hormonal secretion levels and examined temporal expression profiles of circadian clock genes in peripheral leukocytes and beard hair follicle cells. All 13 subjects had prominent daily rhythms in melatonin and cortisol secretion. Significant circadian rhythmicity was found for PER1 in 9 subjects, PER2 in 3 subjects, PER3 in all 13 subjects, and BMAL1 in 8 subjects in leukocytes. Additionally, significant circadian rhythmicity was found for PER1 in 5 of 8 subjects tested, PER2 in 2 subjects, PER3 in 6 subjects, and BMAL1 in 3 subjects in beard hair follicle cells. The phase of PER1 and PER3 rhythms in leukocytes correlated significantly with that of physiological rhythms. Our results demonstrate that leukocytes and beard hair follicle cells possess an endogenous circadian clock and suggest that PER1 and PER3 expression would be appropriate biomarkers and hair follicle cells could be a useful tissue source for the evaluation of biological clock traits in individuals.     

Effects of the combined substitutions of amino acid residues on thermal properties of cold-adapted monomeric isocitrate dehydrogenases from psychrophilic bacteria

In the two cold-adapted monomeric isocitrate dehydrogenases from psychrophilic bacteria, Colwellia maris and Colwellia psychrerythraea (CmIDH and CpIDH, respectively), the combined substitutions of amino acid residues between the Leu693, Leu724 and Phe735 residues of CmIDH and the corresponding Phe693, Gln724 and Leu735 residues of CpIDH were introduced by site-directed mutagenesis. A double mutant of CmIDH substituted its Leu724 and Phe735 residues by the corresponding ones of CpIDH, CmL724Q/F735L, and the triple mutant of CpIDH, CpF693L/Q724L/L735F, showed the most decrease and increase of activity, respectively, of each wild-type and its all mutated enzymes. In the case of CmIDH, the substitutions of these three amino acid residues resulted in the decrease of catalytic activity and thermostability for activity, but the combined substitutions of amino acid residues did not necessarily exert additive effects on these properties. On the other hand, similar substitutions in CpIDH had quite opposite effects to CmIDH, and the effects of the combined substitutions were additive. All multiple mutants of CmIDH and CpIDH showed lower and higher catalytic efficiency (k _cat/K _m) values than the respective wild-type enzymes. Single and multiple mutations of the substituted amino acid residues in the CmIDH and CpIDH led to the increase and decrease of sensitivity to tryptic digestion, indicating that the stability of protein structure was decreased and increased by the mutations, respectively.     

The Tom40 assembly process probed using the attachment of different intramitochondrial sorting signals

The TOM40 complex is a protein translocator in the mitochondrial outer membrane and consists of several different subunits. Among them, Tom40 is a central subunit that constitutes a protein-conducting channel by forming a β-barrel structure. To probe the nature of the assembly process of Tom40 in the outer membrane, we attached various mitochondrial presequences to Tom40 that possess sorting information for the intermembrane space (IMS), inner membrane, and matrix and would compete with the inherent Tom40 assembly process. We analyzed the mitochondrial import of those fusion proteins in vitro. Tom40 crossed the outer membrane and/or inner membrane even in the presence of various sorting signals. N-terminal anchorage of the attached presequence to the inner membrane did not prevent Tom40 from associating with the TOB/SAM complex, although it impaired its efficient release from the TOB complex in vitro but not in vivo. The IMS or matrix-targeting presequence attached to Tom40 was effective in substituting for the requirement for small Tim proteins in the IMS for the translocation of Tom40 across the outer membrane. These results provide insight into the mechanism responsible for the precise delivery of β-barrel proteins to the outer mitochondrial membrane.     

Identification of QTLs for resistance to powdery mildew and SSR markers diagnostic for powdery mildew resistance genes in melon (Cucumis melo L.)

Powdery mildew caused by Podosphaera xanthii is an important foliar disease in melon. To find molecular markers for marker-assisted selection, we constructed a genetic linkage map of melon based on a population of 93 recombinant inbred lines derived from crosses between highly resistant AR 5 and susceptible ‘Earl’s Favourite (Harukei 3)’. The map spans 877 cM and consists of 167 markers, comprising 157 simple sequence repeats (SSRs), 7 sequence characterized amplified region/cleavage amplified polymorphic sequence markers and 3 phenotypic markers segregating into 20 linkage groups. Among them, 37 SSRs and 6 other markers were common to previous maps. Quantitative trait locus (QTL) analysis identified two loci for resistance to powdery mildew. The effects of these QTLs varied depending on strain and plant stage. The percentage of phenotypic variance explained for resistance to the pxA strain was similar between QTLs (R ² = 22–28%). For resistance to pxB strain, the QTL on linkage group (LG) XII was responsible for much more of the variance (41–46%) than that on LG IIA (12–13%). The QTL on LG IIA was located between two SSR markers. Using an independent population, we demonstrated the effectiveness of these markers. This is the first report of universal and effective markers linked to a gene for powdery mildew resistance in melon.     

Adipose‐derived mesenchymal stem cells and regenerative medicine

Adipose tissue‐derived mesenchymal stem cells (ADSCs) are multipotent and can differentiate into various cell types, including osteocytes, adipocytes, neural cells, vascular endothelial cells, cardiomyocytes, pancreatic β‐cells, and hepatocytes. Compared with the extraction of other stem cells such as bone marrow‐derived mesenchymal stem cells (BMSCs), that of ADSCs requires minimally invasive techniques. In the field of regenerative medicine, the use of autologous cells is preferable to embryonic stem cells or induced pluripotent stem cells. Therefore, ADSCs are a useful resource for drug screening and regenerative medicine. Here we present the methods and mechanisms underlying the induction of multilineage cells from ADSCs.     

A dual role of FGF10 in proliferation and coordinated migration of epithelial leading edge cells during mouse eyelid development

The development of the eyelid requires coordinated cellular processes of proliferation, cell shape changes, migration and cell death. Mutant mice deficient in the fibroblast growth factor 10 (Fgf10) gene exhibit open-eyelids at birth. To elucidate the roles of FGF10 during eyelid formation, we examined the expression pattern of Fgf10 during eyelid formation and the phenotype of Fgf10-null eyelids in detail. Fgf10 is expressed by mesenchymal cells just beneath the protruding epidermal cells of the nascent eyelid. However, Fgf10-null epithelial cells running though the eyelid groove do not exhibit typical cuboid shape or sufficient proliferation. Furthermore, peridermal clumps are not maintained on the eyelid leading edge, and epithelial extension does not occur. At the cellular level, the accumulation of actin fibers is not observed in the mutant epithelial leading edge. The expression of activin/inhibin betaB (ActbetaB/Inhbb) and transforming growth factor alpha (Tgfa), previously reported to be crucial for eyelid development, is down-regulated in the mutant leading edge, while the onset of sonic hedgehog (Shh) expression is delayed on the mutant eyelid margin. Explant cultures of mouse eyelid primordia shows that the open-eyelid phenotype of the mutant is reduced by exogenous FGF10 protein, and that the expression of ActbetaB and Tgfa is ectopically induced in the thickened eyelid epithelium by the FGF10 protein. These results indicate a dual role of FGF10 in mouse eyelid development, for both proliferation and coordinated migration of eyelid epithelial cells by reorganization of the cytoskeleton, through the regulation of activin, TGFalpha and SHH signaling.