Prostaglandin E2-EP4 signaling initiates skin immune responses by promoting migration and maturation of Langerhans cells

Antigen-specific immune responses in the skin are initiated by antigen uptake into Langerhans cells and the subsequent migration of these cells to draining lymph nodes. Although prostaglandin E2 (PGE2) is produced substantially in skin exposed to antigen, its role remains unclear. Here we show that although Langerhans cells express all four PGE receptor subtypes, their migration to regional lymph nodes was decreased only in EP4-deficient (Ptger4-/-) mice and in wild-type mice treated with an EP4 antagonist. An EP4 agonist promoted the migration of Langerhans cells, increased their expression of costimulatory molecules and enhanced their ability to stimulate T cells in the mixed lymphocyte reaction in vitro. Contact hypersensitivity to antigen was impaired in Ptger4-/- mice and in wild-type mice treated with the EP4 antagonist during sensitization. PGE2-EP4 signaling thus facilitates initiation of skin immune responses by promoting the migration and maturation of Langerhans cells.     

Gene expression and sensitivity in response to copper stress in rice leaves

Gene expression in response to Cu stress in rice leaves was quantified using DNA microarray (Agilent 22K Rice Oligo Microarray) and real-time PCR technology. Rice plants were grown in hydroponic solutions containing 0.3 (control), 10, 45, or 130 microM of CuCl(2), and Cu accumulation and photosynthesis inhibition were observed in leaves within 1 d of the start of treatment. Microarray analysis flagged 305 Cu-responsive genes, and their expression profile showed that a large proportion of general and defence stress response genes are up-regulated under excess Cu conditions, whereas photosynthesis and transport-related genes are down-regulated. The Cu sensitivity of each Cu-responsive gene was estimated by the median effective concentration value (EC50) and the range of fold-changes (F) under the highest (130 microM) Cu conditions (|log(2)F|(130)). Our results indicate that defence-related genes involved in phytoalexin and lignin biosynthesis were the most sensitive to Cu, and that plant management of abiotic and pathogen stresses has overlapping components, possibly including signal transduction.     

Single-chain Fv fragments derived from an anti-11-deoxycortisol antibody. Affinity,specificity, and idiotype analysis

Single-chain Fv fragments (scFvs) against a corticosteroid, 11-deoxycortisol (11-DC), have been generated as a template antibody fragment from which a comprehensive mutated antibody library containing various anti-steroid antibodies could be constructed. The cDNAs encoding variable heavy (V(H)) and light (V(L)) domains of a mouse anti-11-DC antibody (CET-M8), were amplified by RT-PCR, combined via a common linker to construct the sequence of 5'-V(H)-(Gly(4)Ser)(3)-V(L)-3', and cloned into a phagemid vector, pEXmide 5. The phage clones exhibiting binding activity to 11-DC were isolated after single panning against a hapten-immobilizing immunotube. The scFv gene in one of these clones was reamplified to introduce the ochre codons, and then expressed in the bacterial periplasm as the soluble antibody fragment. Two different scFvs (#6 and #12) were cloned, whose binding characteristics were examined by a radioimmunoassay using a tritium-labeled 11-DC. Both of them showed high affinity (K(a)=1.3x10(10)M(-1)) and practical specificity (cross-reactivity: cortisol, <0.2%; cortisone, <0.3%) to 11-DC, and furthermore, strong reactivity with an anti-idiotype antibody which recognizes the paratope of CET-M8. These results suggest that the present scFvs retain the three-dimensional structure of the paratope of the original monoclonal antibody.     

Evidence for linkage of human primary systemic carnitine deficiency with D5S436: a novel gene locus on chromosome 5q.

Primary systemic carnitine deficiency (SCD) is a rare hereditary disorder transmitted by an autosomal recessive mode of inheritance. The disorder includes cardiomyopathy, muscle weakness, hypoketotic coma with hypoglycemia, and hyperammonemia. In this study, we conducted a linkage analysis of a Japanese SCD family with a proband-a 9-year-old girl-and 26 members. The serum and urinary carnitine levels were determined for all members. The entire genome was searched for linkage to the gene locus for SCD, by use of a total of approximately 300 polymorphic markers located approximately 15-20 cM apart. In the family, there were two significantly different phenotypes, in terms of serum free-carnitine levels: low serum free-carnitine level (29.5+/-5.0 microM; n=14) and normal serum free-carnitine level (46.8+/-6.2 microM; n=12). There was no correlation of urinary free-carnitine levels with the low serum-level phenotype (putative heterozygote), but in normal phenotypes (wild type) urinary levels decreased as the serum levels decreased; renal resorption of free carnitine appeared to be complete in wild-type individuals, when the serum free-carnitine level was <36 microM. Linkage analysis using an autosomal dominant mode of inheritance of heterozygosity revealed a tight linkage between the disease allele and D5S436 on chromosome 5q, with a two-point LOD score of 4.98 and a multipoint LOD score of 5.52. The haplotype analysis revealed that the responsible genetic locus lies between D5S658 and D5S434, which we named the "SCD" locus. This region was syntenic with the jvs locus, which is responsible for murine SCD. Phylogenic conversion of the SCD locus strongly suggests involvement of a single gene, in human SCD.     

An Integrated High-density Linkage Map of Soybean with RFLP, SSR, STS, and AFLP Markers Using A Single F2 Population

Soybean [Glycine max (L.) Merrill] is the most important leguminouscrop in the world due to its high contents of high-quality proteinand oil for human and animal consumption as well as for industrialuses. An accurate and saturated genetic linkage map of soybeanis an essential tool for studies on modern soybean genomics.In order to update the linkage map of a F₂ population derivedfrom a cross between Misuzudaizu and Moshidou Gong 503 and tomake it more informative and useful to the soybean genome researchcommunity, a total of 318 AFLP, 121 SSR, 108 RFLP, and 126 STSmarkers were newly developed and integrated into the frameworkof the previously described linkage map. The updated geneticmap is composed of 509 RFLP, 318 SSR, 318 AFLP, 97 AFLP-derivedSTS, 29 BAC-end or EST-derived STS, 1 RAPD, and five morphologicalmarkers, covering a map distance of 3080 cM (Kosambi function)in 20 linkage groups (LGs). To our knowledge, this is presentlythe densest linkage map developed from a single F₂ populationin soybean. The average intermarker distance was reduced to2.41 from 5.78 cM in the earlier version of the linkage map.Most SSR and RFLP markers were relatively evenly distributedamong different LGs in contrast to the moderately clusteredAFLP markers. The number of gaps of more than 25 cM was reducedto 6 from 19 in the earlier version of the linkage map. Thecoverage of the linkage map was extended since 17 markers weremapped beyond the distal ends of the previous linkage map. Inparticular, 17 markers were tagged in a 5.7 cM interval betweenCE47M5a and Satt100 on LG C2, where several important QTLs wereclustered. This newly updated soybean linkage map will enableto streamline positional cloning of agronomically importanttrait locus genes, and promote the development of physical maps,genome sequencing, and other genomic research activities.     

Involvement of Rho/Rho kinase pathway in regulation of apoptosis in rat hepatic stellate cells

Hepatic stellate cells (HSCs) play a central role in the development of hepatic fibrosis. Recent evidence has revealed that HSCs also play a role in its resolution, where HSC apoptosis was determined. Moreover, induction of HSC apoptosis caused a reduction of experimental hepatic fibrosis in rats. Thus knowing the mechanism of HSC apoptosis might be important to clarify the pathophysiology and establish the therapeutic strategy for hepatic fibrosis. In HSCs, Rho and Rho kinase are known to regulate contraction, migration, and proliferation with modulation of cell morphology. Controversy exists as to the participation of Rho and Rho kinase on cell survival, and little is known regarding this matter in HSCs. In this study, we directed our focus on the role of the Rho pathway in the regulation of HSC survival. C3, an inhibitor of Rho, increased histone-associated DNA fragmentation and caspase 3 activity with enhanced condensation of nuclear chromatin in rat cultured HSCs. Moreover, Y-27632, an inhibitor of Rho kinase, had the same effects, suggesting that inhibition of the Rho/Rho kinase pathway causes HSC apoptosis. On the other hand, lysophosphatidic acid, which stimulates the Rho/Rho kinase pathway, decreased histone-associated DNA fragmentation in HSCs. Inhibition of the Rho/Rho kinase pathway did not affect p53, Bcl-2, or Bax levels in HSCs. Thus we concluded that the Rho/Rho kinase pathway may play a role in the regulation of HSC survival.     

Design,synthesis and biological evaluation of novel 7-azaspiro[3.5]nonane derivatives as GPR119 agonists

The design and synthesis of a novel class of 7-azaspiro[3.5]nonane GPR119 agonists are described. In this series, optimization of the right piperidine N-capping group (R²) and the left aryl group (R³) led to the identification of compound 54g as a potent GPR119 agonist. Compound 54g showed a desirable PK profile in Sprague-Dawley (SD) rats and a favorable glucose lowering effect in diabetic rats.