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.     

Ligand-binding specificities of laminin-binding integrins: a comprehensive survey of laminin-integrin interactions using recombinant alpha3beta1, alpha6beta1, alpha7beta1 and alpha6beta4 integrins.

The interactions of cells with basement membranes are primarily mediated via the engagement of laminins by a group of integrin family proteins, including integrins alpha3beta1, alpha6beta1, alpha7beta1 and alpha6beta4. To explore the ligand-binding specificities of these laminin-binding integrins, we produced these integrins, including two alpha7beta1 splice variants (alpha7X1beta1 and alpha7X2beta1), as soluble recombinant proteins and determined their binding specificities and affinities toward a panel of purified laminin isoforms containing distinct alpha chains. Among the five laminin-binding integrins investigated, alpha3beta1 and alpha6beta4 exhibited a clear specificity for laminin-332 (alpha3beta3gamma2) and laminin-511 (alpha5beta1gamma1)/521 (alpha5beta2gamma1), while integrin alpha6beta1 showed a broad specificity, binding to all laminin isoforms with a preference for laminin-111 (alpha1beta1gamma1), laminin-332 and laminin-511/521. The two alpha7beta1 variants were distinct from alpha3beta1, alpha6beta1 and alpha6beta4 in that they did not bind to laminin-332. alpha7X1beta1 bound to all laminins, except laminin-332, with a preference for laminin-211 (alpha2beta1gamma1)/221 (alpha2beta2gamma1) and laminin-511/521, while alpha7X2beta1 bound preferentially to laminin-111 and laminin-211/221. Laminin-511/521 was the most preferred ligand for all the laminin-binding integrins, except for alpha7X2beta1, whereas laminin-411 was the poorest ligand, capable of binding to alpha6beta1 and alpha7X1beta1 with only modest binding affinities. These comprehensive analyses of the interactions between laminin-binding integrins and a panel of laminins clearly demonstrate that the isoforms of both integrins and laminins differ in their binding specificities and affinities, and provide a molecular basis for better understanding of the adhesive interactions of cells with basement membranes of defined laminin compositions.     

The Vibrio motor proteins, MotX and MotY, are associated with the basal body of Na-driven flagella and required for stator formation

The four motor proteins PomA, PomB, MotX and MotY, which are believed to be stator proteins, are essential for motility by the Na(+)-driven flagella of Vibrio alginolyticus. When we purified the flagellar basal bodies, MotX and MotY were detected in the basal body, which is the supramolecular complex comprised of the rotor and the bushing, but PomA and PomB were not. By antibody labelling, MotX and MotY were detected around the LP ring. These results indicate that MotX and MotY associate with the basal body. The basal body had a new ring structure beneath the LP ring, which was named the T ring. This structure was changed or lost in the basal body from a DeltamotX or DeltamotY strain. The T ring probably comprises MotX and MotY. In the absence of MotX or MotY, we demonstrated that PomA and PomB were not localized to a cell pole. From the above results, we suggest that MotX and MotY of the T ring are involved in the incorporation and/or stabilization of the PomA/PomB complex in the motor.     

Utility of intraperitoneal administration as a route of AAV serotype 5 vector-mediated neonatal gene transfer

BACKGROUND: Gene transfer into a fetus or neonate can be a fundamental approach for treating genetic diseases, particularly disorders that have irreversible manifestations in adulthood. Although the potential utility of this technique has been suggested, the advantages of neonatal gene transfer have not been widely investigated. Here, we tested the usefulness of neonatal gene transfer using adeno-associated virus (AAV) vectors by comparing the administration routes and vector doses. METHODS: To determine the optimal administration route, neonates were subjected to intravenous (i.v.) or intraperitoneal (i.p.) injections of AAV5-based vectors encoding the human coagulation factor IX (hfIX) gene, and the dose response was examined. To determine the distribution of transgene expression, vectors encoding lacZ or luciferase (luc) genes were used and assessed by X-gal staining and in vivo imaging, respectively. After the observation period, the vector distribution across tissues was quantified. RESULTS: The factor IX concentration was higher in i.p.-injected mice than in i.v.-injected mice. All transgenes administered by i.p. injection were more efficiently expressed in neonates than in adults. The expression was confined to the peritoneal tissue. Interestingly, a sex-related difference was observed in transgene expression in adults, whereas this difference was not apparent in neonates. CONCLUSIONS: AAV vector administration to neonates using the i.p. route was clearly advantageous in obtaining robust transgene expression. Vector genomes and transgene expression were observed mainly in the peritoneal tissue. These findings indicate the advantages of neonatal gene therapy and would help in designing strategies for gene therapy using AAV vectors.     

Selective delivery of rifampicin incorporated into poly(DL-lactic-co-glycolic) acid microspheres after phagocytotic uptake by alveolar macrophages, and the killing effect against intracellular Mycobacterium bovis Calmette-Guérin

Macrophages and their phagocytotic abilities play a dominant role for defense against infected organisms. However, Mycobacterium tuberculosis can survive in the phagosomes of macrophages. In this study, the effective delivery of a drug and the killing effect of tubercle bacilli within macrophages were investigated utilizing the phagocytotic uptake of rifampicin (RFP) that had been incorporated into poly(DL-lactic-co-glycolic) acid (PLGA) microspheres. The microspheres were composed of PLGA that had a monomer ratio (lactic acid/glycolic acid) of either 50/50 or 75/25. They had molecular weights from 5000 to 20,000, and diameters of 1.5, 3.5, 6.2 and 8.9 microm. The most significant factor for phagocytotic activity of macrophages was the diameter of the microspheres. By contrast, molecular weight and monomer ratio of PLGA did not influence phagocytosis. The amount of RFP delivered into cells was also investigated. RFP-PLGA microspheres composed of PLGA with a molecular weight of 20,000 and monomer ratio of 75/25 showed the highest amount of delivery (4 microg/1 x 10(6) cells). Fourteen days after infection, the survival rate of treated intracellular bacilli was 1% when compared with untreated cells. There was almost no killing effect of free RFP (4 or 15 microg/ml) on intracellular bacilli. In vivo efficacy of RFP-PLGA was also examined in rats infected with M. tuberculosis Kurono. Intratracheal administration of RFP-PLGA microspheres was shown to be superior to free RFP for killing of intracellular bacilli and preventing granuloma formation in some lobes. These results suggest that phagocytotic activity could be part of a new drug delivery system that selectively targeted macrophages.     

A new cottid species, Icelus sekii (Perciformes: Cottoidei), from Hokkaido, Japan

A new cottid species, Icelus sekii, is described on the basis of six specimens collected from off Rausu and Urakawa, Hokkaido Island, Japan. This species is distinguished from its congeners by the following combination of characters: supraocular and parietal spines absent; nuchal spine obscure; uppermost preopercular spine unbranched; no scales between dorsal scale row and lateral line scale row, and no scales below lateral line scale row; supraocular, parietal, and nuchal cirri present; five dark brown saddles dorsolaterally; anal fin rays 13; pectoral fin rays 15; vertebrae 12 + 24–25 = 36–37. Icelus sekii can be mature at the smallest size among the species of Icelus. As a secondary sexual character, the male holotype has unique ensiform flaps on the distal tips of the first dorsal fin.     

Construction of physical map and mapping of chromosomal virulence genes of the biovar 3 Agrobacterium (Rhizobium vitis) strain K-Ag-1

Most plant pathogenic Agrobacterium strains have been classified into three biovars, "biovar 1 (A. tumefaciens; Rhizobium radiobacter), biovar 2 (A. rhizogenes; R. rhizogenes) and biovar 3 (A. vitis; R. vitis)". The bacteria possess diverse types of genomic organization depending on the biovar. Previous genomic physical maps indicated difference in location of rDNA and chromosomally-coded virulence genes between biovar 1 and 2 genomes. In order to understand biovar 3 genome and its evolution in relation to the biovar 1, 2 and 3 genomes, we constructed physical map of a pathogenic biovar 3 strain K-Ag-1 in this study. Its genome consisted of two circular chromosomes (3.6 and 1.1 Mbp in length), and three plasmids (560, 230 and 70 kbp). Gene mapping based on the physical map showed presence of two rDNA loci in the larger chromosome and at least one rDNA locus in the smaller chromosome. Six chromosomal virulence genes, namely chvA, chvD, chvE, glgP, exoC and ros were found in the larger chromosome and not in the smaller chromosome. The location of rDNA loci is similar with that of biovar 1 genome, whereas the location of chromosomal virulence genes is similar with that of biovar 2 genome despite of the closer 16S-rRNA based phylogenetic relation of biovar 3 with biovar 1 than with biovar 2. Genomic PFGE RFLP analysis revealed that the K-Ag-1 strain, which was isolated on a kiwifruit plant in Japan, has the closest intra-species relation with two strains isolated from grapevine plants in Japan among eight biovar 3 strains examined. This datum suggests that the line of the strain is a major one in biovar 3 in Japan. Evolution of the genome of the strain is discussed based on the data.