Genome-Wide Association Studies Using Single Nucleotide Polymorphism Markers Developed by Re-Sequencing of the Genomes of Cultivated Tomato

With the aim of understanding relationship between genetic and phenotypic variations in cultivated tomato, single nucleotide polymorphism (SNP) markers covering the whole genome of cultivated tomato were developed and genome-wide association studies (GWAS) were performed. The whole genomes of six tomato lines were sequenced with the ABI-5500xl SOLiD sequencer. Sequence reads covering ∼13.7× of the genome for each line were obtained, and mapped onto tomato reference genomes (SL2.40) to detect ∼1.5 million SNP candidates. Of the identified SNPs, 1.5% were considered to confer gene functions. In the subsequent Illumina GoldenGate assay for 1536 SNPs, 1293 SNPs were successfully genotyped, and 1248 showed polymorphisms among 663 tomato accessions. The whole-genome linkage disequilibrium (LD) analysis detected highly biased LD decays between euchromatic (58 kb) and heterochromatic regions (13.8 Mb). Subsequent GWAS identified SNPs that were significantly associated with agronomical traits, with SNP loci located near genes that were previously reported as candidates for these traits. This study demonstrates that attractive loci can be identified by performing GWAS with a large number of SNPs obtained from re-sequencing analysis.     

Development of Capsicum EST–SSR markers for species identification and in silico mapping onto the tomato genome sequence

Capsicum spp. are widely cultivated for use as vegetables and spices. The Kihara Institute for Biological Research, Yokohama City University, Japan, has stocks of approximately 800 lines of Capsicum spp. collected from various regions of Central and South America, the regions of origin for Capsicum spp. In this study, 5,751 primer pairs for simple sequence repeat markers, based on 118,060 publicly available sequences of expressed sequence tags of Capsicum annuum, were designed and subjected to a similarity search against the genomic sequence of tomato, a model Solanaceae species. Nucleotide sequences spanning 2,245 C. annuum markers were successfully mapped onto the tomato genome, and 96 of these, which spanned the entire tomato genome, were selected for further analysis. In genotyping analysis, 60 out of the 77 markers that produced specific DNA amplicons showed polymorphism among the Capsicum lines examined. On the basis of the resulting data, the 192 tested lines were grouped into five main clusters. The additional sequencing analysis of the plastid genes, matK and rbcL, divided the resources into three groups. As a result, 19 marker loci exhibited genotypes specific to species and cluster, suggesting that the DNA markers are useful for species identification. Information on the DNA markers will contribute to Capsicum genetics, genomics, and breeding.     

Fission yeast TOR signaling is essential for the down-regulation of a hyperactivated stress-response MAP kinase under salt stress

TOR (target of rapamycin) signaling regulates cell growth and division in response to environmental stimuli such as the availability of nutrients and various forms of stress. The vegetative growth of fission yeast cells, unlike other eukaryotic cells, is not inhibited by treatment with rapamycin. We found that certain mutations including pmc1Δ (Ca²⁺-ATPase), cps9-193 (small GTPase, Ryh1) and cps1-12 (1,3-β-d-glucan synthase, Bgs1) confer a rapamycin-sensitive phenotype to cells under salt stress with potassium chloride (>0.5 M). Cytometric analysis revealed that the mutant cells were unable to enter the mitotic cell cycle when treated with the drug under salt stress. Gene cloning and overexpression experiments revealed that the sensitivity to rapamycin was suppressed by the ectopic expression of tyrosine phosphatases, Pyp1 and Pyp2, which are negative regulators of Spc1/Sty1 mitogen-activated protein kinase (MAPK). The level of tyrosine phosphorylation on Spc1 was higher and sustained substantially longer in these mutants than in the wild type under salt stress. The hyperphosphorylation was significantly suppressed by overexpression of pyp1 ⁺ with concomitant resumption of the mutant cells’ growth. In fission yeast, TOR signaling has been thought to stimulate the stress-response pathway, because mutations of TORC2 components such as Tor1, Sin1 and Ste20 result in similar sensitive phenotypes to environmental stress. The present study, however, strongly suggests that TOR signaling is required for the down-regulation of a hyperactivated Spc1 for reentry into the mitotic cell cycle. This finding may shed light on our understanding of a new stress-responsive mechanism in TOR signaling in higher organisms.     

Receptor-like cytoplasmic kinases are pivotal components in pattern recognition receptor-mediated signaling in plant immunity

Innate immunity is generally initiated with recognition of conserved pathogen-associated molecular patterns (PAMPs). PAMPs are perceived by pattern recognition receptors (PRRs), leading to activation of a series of immune responses, including the expression of defense genes, ROS production and activation of MAP kinase. Recent progress has indicated that receptor-like cytoplasmic kinases (RLCKs) are directly activated by ligand- activated PRRs and initiate pattern -triggered immunity (PTI) in both Arabidopsis and rice. To suppress PTI, pathogens inhibit the RLCKs by many types of effectors, including AvrAC, AvrPphB and Xoo1488. In this review, we summarize recent advances in RLCK-mediated PTI in plants.     

Mutation of GDP-Mannose-4,6-Dehydratase in Colorectal Cancer Metastasis

Fucosylation is a crucial oligosaccharide modification in cancer. The known function of fucosylation in cancer is to mediate metastasis through selectin ligand-dependent processes. Previously, we found complete loss of fucosylation in the colon cancer cell line HCT116 due to a mutation in the GDP-fucose synthetic enzyme, GDP-mannose-4,6-dehydratase (GMDS). Loss of fucosylation led to escape of cancer cells from tumor immune surveillance followed by tumor progression and metastasis, suggesting a novel function of fucosylation in tumor progression pathway. In the present study, we investigated the frequency of GMDS mutation in a number of clinical colorectal cancer tissue samples: 81 samples of primary colorectal cancer tissue and 39 samples of metastatic lesion including liver and lymph node. Four types of deletion mutation in GMDS were identified in original cancer tissues as well as metastatic lesions. The frequency of GMDS mutation was slightly higher in metastatic lesions (12.8%, 5/39 samples) than in original cancer tissues (8.6%, 7/81 samples). No mutation of the GMDS gene was observed in normal colon tissues surrounding cancer tissues, suggesting that the mutation is somatic rather than in the germline. Immunohistochemical analysis revealed complete loss of fucosylation in three cases of cancer tissue. All three cases had GMDS mutation. In one of three cases, loss of fucosylation was observed in only metastatic lesion, but not its original colon cancer tissue. These data demonstrate involvement of GMDS mutation in the progression of colorectal cancer.     

Development and characterization of ten novel microsatellite loci for the emu (Dromaius novaehollandiae) and genetic diversity of Japanese farm populations

Molecular Biology Reports - The emu (Dromaius novaehollandiae) is a useful poultry animal farmed for fat, meat, and eggs. Genetic structure and relationships among farmed emu populations in Japan...     

Initial Contact of Glioblastoma Cells with Existing Normal Brain Endothelial Cells Strengthen the Barrier Function via Fibroblast Growth Factor 2 Secretion: A New In Vitro Blood–Brain Barrier Model

Glioblastoma multiforme (GBM) cells invade along the existing normal capillaries in brain. Normal capillary endothelial cells function as the blood–brain barrier (BBB) that limits permeability of chemicals into the brain. To investigate whether GBM cells modulate the BBB function of normal endothelial cells, we developed a new in vitro BBB model with primary cultures of rat brain endothelial cells (RBECs), pericytes, and astrocytes. Cells were plated on a membrane with 8 μm pores, either as a monolayer or as a BBB model with triple layer culture. The BBB model consisted of RBEC on the luminal side as a bottom, and pericytes and astrocytes on the abluminal side as a top of the chamber. Human GBM cell line, LN-18 cells, or lung cancer cell line, NCI-H1299 cells, placed on either the RBEC monolayer or the BBB model increased the transendothelial electrical resistance (TEER) values against the model, which peaked within 72 h after the tumor cell application. The TEER value gradually returned to baseline with LN-18 cells, whereas the value quickly dropped to the baseline in 24 h with NCI-H1299 cells. NCI-H1299 cells invaded into the RBEC layer through the membrane, but LN-18 cells did not. Fibroblast growth factor 2 (FGF-2) strengthens the endothelial cell BBB function by increased occludin and ZO-1 expression. In our model, LN-18 and NCI-H1299 cells secreted FGF-2, and a neutralization antibody to FGF-2 inhibited LN-18 cells enhanced BBB function. These results suggest that FGF-2 would be a novel therapeutic target for GBM in the perivascular invasive front.     

Macrophage lipoprotein lipase modulates the development of atherosclerosis but not adiposity

The role of macrophage lipoprotein lipase (LpL) in the development of atherosclerosis and adiposity was examined in macrophage LpL knockout (MLpLKO) mice. MLpLKO mice were generated using cre-loxP gene targeting. Loss of LpL in macrophages did not alter plasma LpL activity or lipoprotein levels. Incubation of apolipoprotein E (ApoE)-deficient β-VLDL with peritoneal macrophages from ApoE knockout mice lacking macrophage LpL (MLpLKO/ApoEKO) led to less cholesteryl ester formation than that found with ApoEKO macrophages. MLpLKO/ApoEKO macrophages had reduced intracellular triglyceride levels, with decreased CD36 and carnitine palmitoyltransferase-1 mRNA levels compared with ApoEKO macrophages, when incubated with VLDL. Although both MLpLKO/ApoEKO and ApoEKO mice developed comparable hypercholesterolemia in response to feeding with a Western-type diet for 12 weeks, atherosclerosis was less in MLpLKO/ApoEKO mice. Epididymal fat mass and gene expression levels associated with inflammation did not differ between the two groups. In conclusion, macrophage LpL plays an important role in the development of atherosclerosis but not adiposity.     

Structure–activity relationships of 6-(aminomethylphenoxy)-benzoxaborole derivatives as anti-inflammatory agent

A series of novel 6-(aminomethylphenoxy)benzoxaborole analogs was synthesized for the investigation of the structure–activity relationship of the inhibition of TNF-alpha, IL-1beta, and IL-6, from lipopolysaccharide stimulated peripheral blood mononuclear cells. Compounds 9d and 9e showed potent activity against all three cytokines with IC₅₀ values between 33 and 83 nM. Chloro substituted analog 9e (AN3485) is considered to be a promising lead for novel anti-inflammatory agent with a favorable pharmacokinetic profile.