Application of Digital Image Analysis System for Fine Evaluation of Varietal Differences and the Role of Ethylene in Visible Petal Senescence of Morning Glory

We detected differences in both onset and progression of visible petal senescence among morning glory cultivars by application of a digital image analysis system. The system is based on semiautomated time-lapse measurement of corolla areas. The system could also be applied to evaluate the effects of ethylene and its inhibitor on visible petal senescence. Both onset and progression of visible petal senescence were accelerated by ethylene treatment in all six cultivars tested. Treatment with aminooxyacetic acid (AOA), an ethylene biosynthesis inhibitor, prolonged time to onset of visible petal senescence in three of the six tested cultivars. In contrast, AOA treatment had no effect on duration of visible petal senescence in any tested cultivars. These data suggested differences among morning glory cultivars in the role of endogenous ethylene in controlling onset of visible petal senescence. In addition, we propose a new application of image analysis to fine quantification of time-lapse changes in the shape of plant organs.     

A model for triplet mutation formation based on error-prone translesional DNA synthesis opposite UV photolesions

A triplet mutation is defined as multiple base substitutions or frameshifts within a three-nucleotide sequence which includes a dipyrimidine sequence. Triplet mutations have recently been identified as a new type of UV-specific mutation, although the mechanism of their formation is unknown. A total of 163 triplet mutations were identified through an extensive search of previously published data on UV-induced mutations, including mutations from skin, skin cancer, and cultured mammalian cells. Seven common patterns of sequence changes were found: Type I, NTC-->TTT; Type IIa, NCC-->PyTT or PyCT (Py, pyrimidine); Type IIb, TCC-->PuTT or PuCT (Pu, purine); Type III, NCC-->NAT or NTA; Type IV, NTT-->AAT; Type Va, NCT-->NTX; and Type Vb, PuCT-->XTT (N and X, independent anonymous bases). Furthermore, it is suggested that the type of UV lesion responsible for each of these triplet mutation classes are (a) pyrimidine(6-4)pyrimidone photoproducts for Types I, IIb, III, IV and Vb, (b) cyclobutane pyrimidine dimers for Type Va, and (c) Dewar valence isomers for Types IIa and IIb. These estimations are based primarily on results from previous studies using photolyases specific for each type of UV lesion. A model is proposed to explain the formation of each type of triplet mutation, based on error-prone translesional DNA synthesis opposite UV-specific photolesions. The model is largely consistent with the 'A-rule', and predicts error-prone insertions not only opposite photolesions but also opposite the undamaged template base one-nucleotide downstream from the lesions.     

Comparison of sequence reads obtained from three next-generation sequencing platforms

Next-generation sequencing technologies enable the rapid cost-effective production of sequence data. To evaluate the performance of these sequencing technologies, investigation of the quality of sequence reads obtained from these methods is important. In this study, we analyzed the quality of sequence reads and SNP detection performance using three commercially available next-generation sequencers, i.e., Roche Genome Sequencer FLX System (FLX), Illumina Genome Analyzer (GA), and Applied Biosystems SOLiD system (SOLiD). A common genomic DNA sample obtained from Escherichia coli strain DH1 was applied to these sequencers. The obtained sequence reads were aligned to the complete genome sequence of E. coli DH1, to evaluate the accuracy and sequence bias of these sequence methods. We found that the fraction of "junk" data, which could not be aligned to the reference genome, was largest in the data set of SOLiD, in which about half of reads could not be aligned. Among data sets after alignment to the reference, sequence accuracy was poorest in GA data sets, suggesting relatively low fidelity of the elongation reaction in the GA method. Furthermore, by aligning the sequence reads to the E. coli strain W3110, we screened sequence differences between two E. coli strains using data sets of three different next-generation platforms. The results revealed that the detected sequence differences were similar among these three methods, while the sequence coverage required for the detection was significantly small in the FLX data set. These results provided valuable information on the quality of short sequence reads and the performance of SNP detection in three next-generation sequencing platforms.     

Interleukin-19 Acts as a Negative Autocrine Regulator of Activated Microglia

Activated microglia can exert either neurotoxic or neuroprotective effects, and they play pivotal roles in the pathogenesis and progression of various neurological diseases. In this study, we used cDNA microarrays to show that interleukin-19 (IL-19), an IL-10 family cytokine, is markedly upregulated in activated microglia. Furthermore, we found that microglia are the only cells in the nervous system that express the IL-19 receptor, a heterodimer of the IL-20Rα and IL-20Rβ subunits. IL-19 deficiency increased the production of such pro-inflammatory cytokines as IL-6 and tumor necrosis factor-α in activated microglia, and IL-19 treatment suppressed this effect. Moreover, in a mouse model of Alzheimer’s disease, we observed upregulation of IL-19 in affected areas in association with disease progression. Our findings demonstrate that IL-19 is an anti-inflammatory cytokine, produced by activated microglia, that acts negatively on microglia in an autocrine manner. Thus, microglia may self-limit their inflammatory response by producing the negative regulator IL-19.     

The Effect of Hydrophilic Ionic Liquids 1-Ethyl-3-Methylimidazolium Lactate and Choline Lactate on Lipid Vesicle Fusion

Ionic liquids (ILs) are room-temperature molten salts that have applications in both physical sciences and more recently in the purification of proteins and lipids, gene transfection and sample preparation for electron microscopy (EM) studies. Transfection of genes into cells requires membrane fusion between the cell membrane and the transfection reagent, thus, ILs may be induce a membrane fusion event. To clarify the behavior of ILs with cell membranes the effect of ILs on model membranes, i.e., liposomes, were investigated. We used two standard ILs, 1-ethyl-3-methylimidazolium lactate ([EMI][Lac]) and choline lactate ([Ch][Lac]), and focused on whether these ILs can induce lipid vesicle fusion. Fluorescence resonance energy transfer and dynamic light scattering were employed to determine whether the ILs induced vesicle fusion. Vesicle solutions at low IL concentrations showed negligible fusion when compared with the controls in the absence of ILs. At concentrations of 30% (v/v), both types of ILs induced vesicle fusion up to 1.3 and 1.6 times the fluorescence intensity of the control in the presence of [Ch][Lac] and [EMI][Lac], respectively. This is the first demonstration that [EMI][Lac] and [Ch][Lac] induce vesicle fusion at high IL concentrations and this observation should have a significant influence on basic biophysical studies. Conversely, the ability to avoid vesicle fusion at low IL concentrations is clearly advantageous for EM studies of lipid samples and cells. This new information describing IL-lipid membrane interactions should impact EM observations examining cell morphology.     

Blockade of B7-H1 suppresses the development of chronic intestinal inflammation

A newly identified costimulatory molecule, programmed death-1 (PD-1), provides a negative signal that is essential for immune homeostasis. However, it has been suggested that its ligands, B7-H1 (PD-L1) and B7-dendritic cells (B7-DC; PD-L2), could also costimulate T cell proliferation and cytokine secretion. Here we demonstrate the involvement of PD-1/B7-H1 and B7-DC interaction in the development of colitis. We first examined the expression profiles of PD-1 and its ligands in both human inflammatory bowel disease and a murine chronic colitis model induced by adoptive transfer of CD4(+)CD45RB(high) T cells to SCID mice. Second, we assessed the therapeutic potential of neutralizing anti-B7-H1 and/or B7-DC mAbs using this colitis model. We found significantly increased expression of PD-1 on T cells and of B7-H1 on T, B, and macrophage/DCs in inflamed colon from both inflammatory bowel disease patients and colitic mice. Unexpectedly, the administration of anti-B7-H1, but not anti-B7-DC, mAb after transfer of CD4(+)CD45RB(high) T cells suppressed wasting disease with colitis, abrogated leukocyte infiltration, and reduced the production of IFN-gamma, IL-2, and TNF-alpha, but not IL-4 or IL-10, by lamina propria CD4(+) T cells. These data suggest that the interaction of PD-1/B7-H1, but not PD-1/B7-DC, might be involved in intestinal mucosal inflammation and also show a possible role of interaction between B7-H1 and an as yet unidentified receptor for B7-H1 in inducing T cell activation.     

Effects of soccer matches on neutrophil and lymphocyte functions in female university soccer players

In this study, changes in physical fatigue and biological functions of Japanese female soccer players were investigated by determining changes in neutrophil and lymphocyte functions. Study subjects included 18 female soccer players. Body composition, serum myogenic enzymes, neutrophil function, including reactive oxygen species (ROS) production capability, phagocytic activity (PA) and serum opsonic activity, as well as lymphocyte subpopulation were measured before and after a soccer match. Levels of myogenic enzymes (AST, ALT, CK and LDH) and immunoglobulins (IgG and IgA) and complements (C3) increased significantly after the match. In addition, leukocyte, neutrophils and lymphocyte counts increased whereas total PA decreased significantly. The number of T and Th1 cells (subsets of T helper cells) decreased whereas Th2 increased significantly. In addition, the number of B cells increased and NK cells decreased significantly after the match. The match was found to result in degenerative changes in and damage to athlete muscle tissues together with damage‐ and change‐mediated stress. These data also suggest a post‐match accelerated inflammatory reaction and potential immunosuppression as indicated by reductions in neutrophil PA and lymphocyte functions. Copyright © 2012 John Wiley & Sons, Ltd.     

Full Conversion of the Hemagglutinin-Neuraminidase Specificity of the Parainfluenza Virus 5 Fusion Protein by Replacement of 21 Amino Acids in Its Head Region with Those of the Simian Virus 41 Fusion Protein

For most parainfluenza viruses, a virus type-specific interaction between the hemagglutinin-neuraminidase (HN) and fusion (F) proteins is a prerequisite for mediating virus-cell fusion and cell-cell fusion. The molecular basis of this functional interaction is still obscure partly because it is unknown which region of the F protein is responsible for the physical interaction with the HN protein. Our previous cell-cell fusion assay using the chimeric F proteins of parainfluenza virus 5 (PIV5) and simian virus 41 (SV41) indicated that replacement of two domains in the head region of the PIV5 F protein with the SV41 F counterparts bestowed on the PIV5 F protein the ability to induce cell-cell fusion on coexpression with the SV41 HN protein while retaining its ability to induce fusion with the PIV5 HN protein. In the study presented here, we furthered the chimeric analysis of the F proteins of PIV5 and SV41, finding that the PIV5 F protein could be converted to an SV41 HN-specific chimeric F protein by replacing five domains in the head region with the SV41 F counterparts. The five SV41 F-protein-derived domains of this chimera were then divided into 16 segments; 9 out of 16 proved to be not involved in determining its specificity for the SV41 HN protein. Finally, mutational analyses of a chimeric F protein, which harbored seven SV41 F-protein-derived segments, revealed that replacement of at most 21 amino acids of the PIV5 F protein with the SV41 F-protein counterparts was enough to convert its HN protein specificity.