Transendothelial migration of human basophils

During allergic reactions, basophils migrate from the blood compartment to inflammatory sites, where they act as effector cells in concert with eosinophils. Because transendothelial migration (TEM) represents an essential step for extravasation of cells, for the first time we have studied basophil TEM using HUVEC. Treatment of HUVEC with IL-1beta significantly enhanced basophil TEM, which was further potentiated by the presence of a CCR3-specific ligand, eotaxin/CCL11. In addition to CCR3 ligands, MCP-1/CCL2 was also active on basophil TEM. Although stromal cell-derived factor-1/CXCL12, a CXCR4 ligand, failed to induce TEM in freshly isolated basophils, it caused strong TEM in 24-h cultured cells. IL-3 enhanced basophil TEM by increasing the chemokinetic response. Spontaneous TEM across activated HUVEC was inhibited by treatment of cells with anti-CD18 mAb, but not with anti-CD29 mAb, and also by treatment of HUVEC with anti-ICAM-1 mAb. Anti-VCAM-1 mAb alone failed to inhibit TEM, but showed an additive inhibitory effect in combination with anti-ICAM-1 mAb. In contrast, eotaxin- and IL-3-mediated TEM was significantly inhibited by anti-CD29 mAb as well as anti-CD18 mAb. These results indicate that beta2 integrins play the primary role in basophil TEM, but beta1 integrins are also involved, especially in TEM of cytokine/chemokine-stimulated basophils. In conclusion, the regulatory profile of basophil TEM is very similar to that reported for eosinophils. Our results thus support the previous argument for a close relationship between basophils and eosinophils and suggest that the in vivo kinetics of these two cell types are similar.     

A plant growth retardant, uniconazole, is a potent inhibitor of ABA catabolism in Arabidopsis

Plant growth retardants (PGRs) reduce the shoot growth of plants by inhibiting gibberellin biosynthesis. In this study, we performed detailed analyses of the inhibitory effects of PGRs on Arabidopsis abscisic acid (ABA) 8'-hydroxylase, a major ABA catabolic enzyme, recently identified as CYP707As. In an in vitro assay with CYP707A3 microsomes expressed in insect cells, uniconazole-P inhibited CYP707A3 activity more effectively than paclobutrazol or tetcyclacis, whereas the other PGRs tested did not inhibit it significantly. Uniconazole-P was found to be a strong competitive inhibitor (K(i)=8.0 nM) of ABA 8'-hydroxylase. Uniconazole-P-treated Arabidopsis plants showed enhanced drought tolerance. In uniconazole-P-treated plants, endogenous ABA levels increased 2-fold as compared with the control, and co-application of GA(4) did not suppress the effects, indicating that the effects were not due to gibberellin deficiency. Thus uniconazole-P effectively inhibits ABA catabolism in Arabidopsis plants. We also discuss the structure-activity relationship of the azole-type compounds on ABA 8'-hydroxylase inhibitory activity.     

Prostaglandin D2 plays an essential role in chronic allergic inflammation of the skin via CRTH2 receptor

PGD(2) plays roles in allergic inflammation via specific receptors, the PGD receptor designated DP and CRTH2 (chemoattractant receptor homologous molecule expressed on Th2 cells). We generated mutant mice carrying a targeted disruption of the CRTH2 gene to investigate the functional roles of CRTH2 in cutaneous inflammatory responses. CRTH2-deficent mice were fertile and grew normally. Ear-swelling responses induced by hapten-specific IgE were less pronounced in mutant mice, giving 35-55% of the responses of normal mice. Similar results were seen in mice treated with a hemopoietic PGD synthase inhibitor, HQL-79, or a CRTH2 antagonist, ramatroban. The reduction in cutaneous responses was associated with decreased infiltration of lymphocytes, eosinophils, and basophils and decreased production of macrophage-derived chemokine and RANTES at inflammatory sites. In models of chronic contact hypersensitivity induced by repeated hapten application, CRTH2 deficiency resulted in a reduction by approximately half of skin responses and low levels (63% of control) of serum IgE production, although in vivo migration of Langerhans cells and dendritic cells to regional lymph nodes was not impaired in CRTH2-deficient mice. In contrast, delayed-type hypersensitivity to SRBC and irritation dermatitis in mutant mice were the same as in wild-type mice. These findings indicate that the PGD(2)-CRTH2 system plays a significant role in chronic allergic skin inflammation. CRTH2 may represent a novel therapeutic target for treatment of human allergic disorders, including atopic dermatitis.     

Developmental changes in the expression pattern of the JNK activator kinase MUK/DLK/ZPK and active JNK in the mouse cerebellum

JNK is one of the key molecules regulating cell differentiation and migration in a variety of cell types, including cerebral cortical neurons. MUK/DLK/ZPK belongs to the MAP kinase-kinase-kinase class of protein kinases for the JNK pathway and is expressed predominantly in neural tissue. We have determined the expression pattern of MUK/DLK/ZPK and active JNK in the cerebellum at different stages of postnatal development. Quantitative analysis by Western blotting has showed that high expression levels of MUK/DLK/ZPK and active JNK are maintained during the postnatal development of the cerebellum, and that these levels decrease in the adult cerebellum. Immunohistochemical staining has revealed, however, that their distribution in the developing cerebellum is considerably different. Although active JNK is highly concentrated in the premigratory zone of the external germinal layer (EGL), high expression of MUK/DLK/ZPK has been observed in the molecular layer and in the premigratory zone. Neither the active JNK nor MUK protein has been detected in the proliferative zone of the EGL. These observations suggest that during the postnatal development of the cerebellum, the MUK-JNK signaling pathway contributes to the regulation of granule cell differentiation and migration; further, the activity of MUK/DLK/ZPK is tightly regulated by posttranslational mechanisms and by its expression level.This work was supported by a Ishizu Shun memorial scholarship and grants from the Ministry of Education, Culture, Sports, Science and Technology of Japan.     

PbGCbeta is essential for Plasmodium ookinete motility to invade midgut cell and for successful completion of parasite life cycle in mosquitoes

When malaria parasites enter to mosquitoes, they fertilize and differentiate to zygotes and ookinetes. The motile ookinetes cross the midgut cells and arrive to the basement membranes where they differentiate into oocysts. The midgut epithelium is thus a barrier for ookinetes to complete their life cycle in the mosquitoes. The ookinetes develop gliding motility to invade midgut cells successfully, but the molecular mechanisms behind are poorly understood. Here, we identified a single molecule with guanylate cyclase domain and N-terminal P-type ATPase like domain in the rodent malaria parasite Plasmodium berghei and named it PbGCbeta. We demonstrated that transgenic parasites in which the PbGCbeta gene was disrupted formed normal ookinetes but failed to produce oocyst. Confocal microscopic analysis showed that the disruptant ookinetes remained on the surface of the microvilli. The disruptant ookinetes showed severe defect in motility, resulting in failure of parasite invasion of the midgut epithelium. When the disruptant ookinetes were cultured in vitro, they transformed into oocysts and sporozoites. These results demonstrate that PbGCbeta is essential for ookinete motility when passing through the midgut cells, but not for further development of the parasites.     

A novel ATP/ADP hydrolysis activity of hyperthermostable group II chaperonin in the presence of cobalt or manganese ion

A novel ATPase activity that was strongly activated in the presence of either cobalt or manganese ion was discovered in the chaperonin from hyperthermophilic Pyrococcus furiosus (Pfu-cpn). Surprisingly, a significant ADPase activity was also detected under the same conditions. A more extensive search revealed similar nucleotide hydrolysis activities in other thermostable chaperonins. Chaperonin activity, i.e., thermal stabilization and refolding of malate dehydrogenase from the guanidine-hydrochloride unfolded state were also detected for Pfu-cpn under the same conditions. We propose that the novel cobalt/manganese-dependent ATP/ADPase activity may be a common trait of various thermostable chaperonins.     

Assignment of 118 novel cDNAs of cynomolgus monkey brain to human chromosomes

In order to isolate genes that may not be represented in current human brain cDNA libraries, we have sequenced about 20,000 sequence tags of cDNA clones derived from cerebellum and parietal lobe of cynomolgus monkeys (Macaca fascicularis). We determined the entire cDNA sequence of approximately 700 clones whose 5'-terminal sequences showed no homology to annotated putative genes or expressed sequence tags in current databases of genetic information. From this, 118 clones with sequences encoding novel open reading frames of more than 100 amino acid residues were selected for further analysis. To localize the genes corresponding to these 118 newly identified cDNA clones on human chromosomes, we performed a homology search using the human genome sequence and fluorescent in situ hybridization. In total, 108 of 118 clones were successfully assigned to specific regions of human chromosomes. This result demonstrates that genes expressed in cynomolgus monkey are highly conserved throughout primate evolution, and that virtually all had human homologs. Furthermore, we will be able to discover novel human genes in the human genome using monkey homologs as probes.     

Prediction of operon-like gene clusters in the Arabidopsis thaliana genome based on co-expression analysis of neighboring genes

Operon-like arrangements of genes occur in eukaryotes ranging from yeasts and filamentous fungi to nematodes, plants, and mammals. In plants, several examples of operon-like gene clusters involved in metabolic pathways have recently been characterized, e.g. the cyclic hydroxamic acid pathways in maize, the avenacin biosynthesis gene clusters in oat, the thalianol pathway in Arabidopsis thaliana, and the diterpenoid momilactone cluster in rice. Such operon-like gene clusters are defined by their co-regulation or neighboring positions within immediate vicinity of chromosomal regions. A comprehensive analysis of the expression of neighboring genes therefore accounts a crucial step to reveal the complete set of operon-like gene clusters within a genome. Genome-wide prediction of operon-like gene clusters should contribute to functional annotation efforts and provide novel insight into evolutionary aspects acquiring certain biological functions as well. We predicted co-expressed gene clusters by comparing the Pearson correlation coefficient of neighboring genes and randomly selected gene pairs, based on a statistical method that takes false discovery rate (FDR) into consideration for 1469 microarray gene expression datasets of A. thaliana. We estimated that A. thaliana contains 100 operon-like gene clusters in total. We predicted 34 statistically significant gene clusters consisting of 3 to 22 genes each, based on a stringent FDR threshold of 0.1. Functional relationships among genes in individual clusters were estimated by sequence similarity and functional annotation of genes. Duplicated gene pairs (determined based on BLAST with a cutoff of E<10(-5)) are included in 27 clusters. Five clusters are associated with metabolism, containing P450 genes restricted to the Brassica family and predicted to be involved in secondary metabolism. Operon-like clusters tend to include genes encoding bio-machinery associated with ribosomes, the ubiquitin/proteasome system, secondary metabolic pathways, lipid and fatty-acid metabolism, and the lipid transfer system.