Exacerbation of experimental allergic asthma by augmented Th2 responses in WSX-1-deficient mice

WSX-1 (IL-27R) is a class I cytokine receptor with homology to gp130 and IL-12 receptors and is typically expressed on CD4+ T lymphocytes. Although previous reports have clarified that IL-27/WSX-1 signaling plays critical roles in both Th1 differentiation and attenuation of cell activation and proinflammatory cytokine production during some bacterial or protozoan infections, little is known about the importance of WSX-1 in cytokine-mediated diseases of allergic origin. To this aim, we took advantage of WSX-1-deficient (WSX-1(-/-)) mice and induced experimental asthma, in which Th2 cytokines are central modulators of the pathology. OVA-challenged WSX-1(-/-) mice showed marked enhancement of airway responsiveness with goblet cell hyperplasia, pulmonary eosinophil infiltration, and increased serum IgE levels compared with wild-type mice. Production of Th2 cytokines, which are largely responsible for the pathogenesis of asthma, was augmented in the lung or in the culture supernatants of peribronchial lymph node CD4+ T cells from WSX-1(-/-) mice compared with those from wild-type mice. Surprisingly, IFN-gamma production was also enhanced in WSX-1(-/-) mice, albeit at a low concentration. The cytokine overproduction, thus, seems independent from the Th1-promoting property of WSX-1. These results demonstrated that IL-27/WSX-1 also plays an important role in the down-regulation of airway hyper-reactivity and lung inflammation during the development of allergic asthma through its suppressive effect on cytokine production.     

Processing and stability of type IIc sodium-dependent phosphate cotransporter mutations in patients with hereditary hypophosphatemic rickets with hypercalciuria

Mutations in the apically located Na(+)-dependent phosphate (NaPi) cotransporter, SLC34A3 (NaPi-IIc), are a cause of hereditary hypophosphatemic rickets with hypercalciuria (HHRH). We have characterized the impact of several HHRH mutations on the processing and stability of human NaPi-IIc. Mutations S138F, G196R, R468W, R564C, and c.228delC in human NaPi-IIc significantly decreased the levels of NaPi cotransport activities in Xenopus oocytes. In S138F and R564C mutant proteins, this reduction is a result of a decrease in the V(max) for P(i), but not the K(m). G196R, R468W, and c.228delC mutants were not localized to oocyte membranes. In opossum kidney (OK) cells, cell surface labeling, microscopic confocal imaging, and pulse-chase experiments showed that G196R and R468W mutations resulted in an absence of cell surface expression owing to endoplasmic reticulum (ER) retention. G196R and R468W mutants could be partially stabilized by low temperature. In blue native-polyacrylamide gel electrophoresis analysis, G196R and R468W mutants were either denatured or present in an aggregation complex. In contrast, S138F and R564C mutants were trafficked to the cell surface, but more rapidly degraded than WT protein. The c.228delC mutant did not affect endogenous NaPi uptake in OK cells. Thus, G196R and R468W mutations cause ER retention, while S138F and R564C mutations stimulate degradation of human NaPi-IIc in renal epithelial cells. Together, these data suggest that the NaPi-IIc mutants in HHRH show defective processing and stability.     

Arabidopsis lonely guy (LOG) multiple mutants reveal a central role of the LOG-dependent pathway in cytokinin activation

Cytokinins are phytohormones that play key roles in the maintenance of stem cell activity in plants. Although alternative single-step and two-step activation pathways for cytokinin have been proposed, the significance of the single-step pathway which is catalyzed by LONELY GUY (LOG), is not fully understood. We analyzed the metabolic flow of cytokinin activation in Arabidopsis log multiple mutants using stable isotope-labeled tracers and characterized the mutants' morphological and developmental phenotypes. In tracer experiments, cytokinin activation was inhibited most pronouncedly by log7, while the other log mutations had cumulative effects. Although sextuple or lower-order mutants did not show drastic phenotypes in vegetative growth, the log1log2log3log4log5log7log8 septuple T-DNA insertion mutant in which the LOG-dependent pathway is impaired, displayed severe retardation of shoot and root growth with defects in the maintenance of the apical meristems. Detailed observation of the mutants showed that LOG7 was required for the maintenance of shoot apical meristem size. LOG7 was also suggested to play a role for normal primary root growth together with LOG3 and LOG4. These results suggest a dominant role of the single-step activation pathway mediated by LOGs for cytokinin production, and overlapping but differentiated functions of the members of the LOG gene family in growth and development.     

Kendrin is a novel substrate for separase involved in the licensing of centriole duplication

The centrosome, consisting of a pair of centrioles surrounded by pericentriolar material, directs the formation of bipolar spindles during mitosis. Aberrant centrosome number can promote chromosome instability, which is implicated in tumorigenesis. Thus, centrosome duplication needs to be tightly regulated to occur only once per cell cycle. Separase, a cysteine protease that triggers sister chromatid separation, is involved in centriole disengagement, which licenses centrosomes for the next round of duplication. However, at least two questions remain unsolved: what is the substrate relevant to the disengagement, and how does separase, activated at anaphase onset, act on the disengagement that occurs during late mitosis. Here, we show that kendrin, also named pericentrin, is cleaved by activated separase at a consensus site in vivo and in vitro, and this leads to the delayed release of kendrin from the centrosome later in mitosis. Furthermore, we demonstrate that expression of a noncleavable kendrin mutant suppresses centriole disengagement and subsequent centriole duplication. Based on these results, we propose that kendrin is a novel and crucial substrate for separase at the centrosome, protecting the engaged centrioles from premature disengagement and thereby blocking reduplication until the cell passes through mitosis.     

Nonsynonymous single-nucleotide polymorphisms of the human apoptosis-related endonuclease--DNA fragmentation factor beta polypeptide, endonuclease G, and Flap endonuclease-1--genes show a low degree of genetic heterogeneity

DNA fragmentation factor beta (DFFB) polypeptide, endonuclease G (EndoG), and Flap endonuclease-1 (FEN-1) are responsible for DNA fragmentation, a hallmark of apoptosis. Although the human homologs of these genes show three, four, and six nonsynonymous single-nucleotide polymorphisms (SNPs), respectively, data on their genotype distributions in populations worldwide are limited. In this context, the objectives of this study were to elucidate the genetic heterogeneity of all these SNPs in wide-ranging populations, and thereby to clarify the genetic background of these apoptosis-related endonucleases in human populations. We investigated the genotype distribution of their SNPs in 13 different populations of healthy Asians, Africans, and Caucasians using novel genotyping methods. Among the 13 SNPs in the 3 genes, only 3 were found to be polymorphic: R196K and K277R in the DFFB gene, and S12L in the EndoG gene. All 6 SNPs in the FEN-1 gene were entirely monoallelic. Although it remains unclear whether each SNP would exert any effect on endonuclease functions, these genes appear to exhibit low degree of genetic heterogeneity with regard to nonsynonymous SNPs. These findings allow us to conclude that human apoptosis-related endonucleases, similarly to other human DNase genes, revealed previously, are well conserved at the protein level during the course of human evolution.     

Suppression of α‐amylase genes improves quality of rice grain ripened under high temperature

High temperature impairs rice (Oryza sativa) grain filling by inhibiting the deposition of storage materials such as starch, resulting in mature grains with a chalky appearance, currently a major problem for rice farming in Asian countries. Such deterioration of grain quality is accompanied by the altered expression of starch metabolism‐related genes. Here we report the involvement of a starch‐hydrolyzing enzyme, α‐amylase, in high temperature‐triggered grain chalkiness. In developing seeds, high temperature induced the expression of α‐amylase genes, namely Amy1A, Amy1C, Amy3A, Amy3D and Amy3E, as well as α‐amylase activity, while it decreased an α‐amylase‐repressing plant hormone, ABA, suggesting starch to be degraded by α‐amylase in developing grains under elevated temperature. Furthermore, RNAi‐mediated suppression of α‐amylase genes in ripening seeds resulted in fewer chalky grains under high‐temperature conditions. As the extent of the decrease in chalky grains was highly correlated to decreases in the expression of Amy1A, Amy1C, Amy3A and Amy3B, these genes would be involved in the chalkiness through degradation of starch accumulating in the developing grains. The results show that activation of α‐amylase by high temperature is a crucial trigger for grain chalkiness and that its suppression is a potential strategy for ameliorating grain damage from global warming.