An objective classification system of air mass types for Szeged, Hungary, with special attention to plant pollen levels

This paper discusses the characteristic air mass types over the Carpathian Basin in relation to plant pollen levels over annual pollination periods. Based on the European Centre for Medium-Range Weather Forecasts dataset, daily sea-level pressure fields analysed at 00 UTC were prepared for each air mass type (cluster) in order to relate sea-level pressure patterns to pollen levels in Szeged, Hungary. The database comprises daily values of 12 meteorological parameters and daily pollen concentrations of 24 species for their pollination periods from 1997 to 2001. Characteristic air mass types were objectively defined via factor analysis and cluster analysis. According to the results, nine air mass types (clusters) were detected for pollination periods of the year corresponding to pollen levels that appear with higher concentration when irradiance is moderate while wind speed is moderate or high. This is the case when an anticyclone prevails in the region west of the Carpathian Basin and when Hungary is under the influence of zonal currents (wind speed is high). The sea level pressure systems associated with low pollen concentrations are mostly similar to those connected to higher pollen concentrations, and arise when wind speed is low or moderate. Low pollen levels occur when an anticyclone prevails in the region west of the Carpathian Basin, as well as when an anticyclone covers the region with Hungary at its centre. Hence, anticyclonic or anticyclonic ridge weather situations seem to be relevant in classifying pollen levels.     

IL-17A promotes the growth of airway epithelial cells through ERK-dependent signaling pathway

The effects of IL-17A on mucin production and growth of airway epithelial cells were examined. Histological and immunohistochemical analyses revealed that IL-17A increased the mucin production and number of tracheal epithelial cells in air-liquid interface cultures. The biological property of IL-17A to stimulate the mucin production by tracheal epithelial cells was determined using an ELISA. The mitogenic effect of IL-17A on tracheal epithelial cells was confirmed with Calcein-AM assay. The growth-stimulatory effect of IL-17A was dose-dependent and mediated via the ERK MAP kinase pathway. Inhibitors of MEK abrogated the mitogenic effect of IL-17A, whereas an inhibitor of p38 or JNK displayed no significant inhibitory effect. Moreover, relatively lower doses of IL-13 also significantly increased the growth of tracheal epithelial cells through a distinct signaling pathway from that of IL-17A. These findings provide the first evidence that IL-17A stimulates the growth of airway epithelial cells through the ERK MAP kinase pathway.     

The selective continued linkage of centromeres from mitosis to interphase in the absence of mammalian separase

Separase is an evolutionarily conserved protease that is essential for chromosome segregation and cleaves cohesin Scc1/Rad21, which joins the sister chromatids together. Although mammalian separase also functions in chromosome segregation, our understanding of this process in mammals is still incomplete. We generated separase knockout mice, reporting an essential function for mammalian separase. Separase-deficient mouse embryonic fibroblasts exhibited severely restrained increases in cell number, polyploid chromosomes, and amplified centrosomes. Chromosome spreads demonstrated that multiple chromosomes connected to a centromeric region. Live observation demonstrated that the chromosomes of separase-deficient cells condensed, but failed to segregate, although subsequent cytokinesis and chromosome decondensation proceeded normally. These results establish that mammalian separase is essential for the separation of centromeres, but not of the arm regions of chromosomes. Other cell cycle events, such as mitotic exit, DNA replication, and centrosome duplication appear to occur normally. We also demonstrated that heterozygous separase-deficient cells exhibited severely restrained increases in cell number with apparently normal mitosis in the absence of securin, which is an inhibitory partner of separase.     

6-Amino-6-deoxy-chitosan. Sequential chemical modifications at the C-6 positions of N-phthaloyl-chitosan and evaluation as a gene carrier

The C-6 positions of chitosan were successively modified in a highly regioselective manner. The starting material, N-phthaloyl-chitosan, was successfully converted into the corresponding 6-deoxy-6-halo derivatives by reaction with N-halosuccinimides and triphenylphosphine in N-methyl-2-pyrrolidone. The resulting chloride and bromide derivatives were then substituted with azido groups by reaction with sodium azide at 120 and 80 degrees C, respectively. The azido groups were then reduced to amines via formation of the triphenylphosphinimine intermediate followed by hydrolysis using aqueous hydrazine, which also led to the removal of the N-phthaloyl groups at the C-2 positions. This sequence gave 6-amino-6-deoxy-chitosan, which, unlike chitosan, is soluble in water at neutral pH. The synthesized 6-amino-6-deoxy-chitosan derivative was evaluated as a gene carrier, and the transfection efficiency for COS-1 cells was shown to be superior to chitosan. In addition, the cytotoxicity was similar to chitosan.     

PAR3 is essential for cyst-mediated epicardial development by establishing apical cortical domains

Epithelial cysts are one of the fundamental architectures for mammalian organogenesis. Although in vitro studies using cultured epithelial cells have revealed proteins required for cyst formation, the mechanisms that orchestrate the functions of these proteins in vivo remain to be clarified. We show that the targeted disruption of the mouse Par3 gene results in midgestational embryonic lethality with defective epicardial development. The epicardium is mainly derived from epicardial cysts and essential for cardiomyocyte proliferation during cardiac morphogenesis. PAR3-deficient epicardial progenitor (EPP) cells do not form cell cysts and show defects in the establishment of apical cortical domains, but not in basolateral domains. In PAR3-deficient EPP cells, the localizations of aPKC, PAR6beta and ezrin to the apical cortical domains are disturbed. By contrast, ZO1 and alpha4/beta1 integrins normally localize to cell-cell junctions and basal domains, respectively. Our observations indicate that EPP cell cyst formation requires PAR3 to interpret the polarity cues from cell-cell and cell-extracellular matrix interactions so that each EPP cell establishes apical cortical domains. These results also provide a clear example of the proper organization of epithelial tissues through the regulation of individual cell polarity.     

Inhibition of MHC class II expression and immune responses by c-MIR

We previously reported a novel E3 ubiquitin ligase (E3), designated as c-MIR, which targets B7-2 to lysosomal degradation and down-regulates the B7-2 surface expression through ubiquitination of its cytoplasmic tail. B7-2 is well known as a costimulatory molecule for Ag presentation, suggesting that the manipulation of c-MIR expression modulates immune responses in vivo. To examine this hypothesis, we generated genetically modified mice in which c-MIR was expressed under an invariant chain (Ii) promoter. Dendritic cells derived from genetically engineered mice showed low ability to present Ags. In addition, these mice showed resistance to the onset of experimental autoimmune encephalomyelitis and an impaired development of CD4 T cells in the thymus and the periphery. These findings led us to conclude that MHC class II (MHC II) is an additional target for c-MIR. Indeed, forced expression of c-MIR in several B cell lines down-regulated the surface expression of MHC II, and down-regulation was found to depend on the presence of a single lysine residue in the cytoplasmic tail of the I-A beta-chain. In a reconstitution system using 293T cells, we found that the lysine residue at position 225 in the I-A beta-chain was ubiquitinated by c-MIR. To our knowledge, c-MIR is the first example of an E3 that is capable of inhibiting MHC II expression. Our findings suggest that c-MIR might potently regulate immune responses in vivo.     

Ex vivo characterization of allo-MHC-restricted T cells specific for a single MHC-peptide complex

Alloreactive T cells are thought to be a potentially rich source of high-avidity T cells with therapeutic potential since tolerance to self-Ags is restricted to self-MHC recognition. Given the particularly high frequency of alloreactive T cells in the peripheral immune system, we used numerous MHC class I multimers to directly visualize and isolate viral and tumor Ag-specific alloreactive CD8 T cells. In fact, all but one specificities screened were undetectable in ex vivo labeling. In this study, we report the occurrence of CD8 T cells specifically labeled with allo-HLA-A*0201/Melan-A/MART-1(26-35) multimers at frequencies that are in the range of 10(-4) CD8 T cells and are thus detectable ex vivo by flow cytometry. We report the thymic generation and shaping of tumor Ag-specific, alloreactive T cells as well as their fate once seeded in the periphery. We show that these cells resemble their counterparts in HLA-A*0201-positive individuals, based on their structural and functional attributes.     

Silencing of vanilloid receptor TRPV1 by RNAi reduces neuropathic and visceral pain in vivo

RNA interference (RNAi) has proven to be a powerful technique to study the function of genes by producing knock-down phenotypes. Here, we report that intrathecal injection of an siRNA against the transient receptor potential vanilloid receptor 1 (TRPV1) reduced cold allodynia of mononeuropathic rats by more than 50% over a time period of approximately 5 days. A second siRNA targeted to a different region of the TRPV1 gene was employed and confirmed the analgesic action of a TRPV1 knock-down. Furthermore, siRNA treatment diminished spontaneous visceral pain behavior induced by capsaicin application to the rectum of mice. The analgesic effect of siRNA-mediated knockdown of TRPV1 in the visceral pain model was comparable to that of the low-molecular weight receptor antagonist BCTC. Our data demonstrate that TRPV1 antagonists, including TRPV1 siRNAs, have potential in the treatment of both, neuropathic and visceral pain.     

Double-stranded RNA is internalized by scavenger receptor-mediated endocytosis in Drosophila S2 cells

Double-stranded RNA (dsRNA) fragments are readily internalized and processed by Drosophila S2 cells, making these cells a widely used tool for the analysis of gene function by gene silencing through RNA interference (RNAi). The underlying mechanisms are insufficiently understood. To identify components of the RNAi pathway in S2 cells, we developed a screen based on rescue from RNAi-induced lethality. We identified Argonaute 2, a core component of the RNAi machinery, and three gene products previously unknown to be involved in RNAi in Drosophila: DEAD-box RNA helicase Belle, 26 S proteasome regulatory subunit 8 (Pros45), and clathrin heavy chain, a component of the endocytic machinery. Blocking endocytosis in S2 cells impaired RNAi, suggesting that dsRNA fragments are internalized by receptor-mediated endocytosis. Indeed, using a candidate gene approach, we identified two Drosophila scavenger receptors, SR-CI and Eater, which together accounted for more than 90% of the dsRNA uptake into S2 cells. When expressed in mammalian cells, SR-CI was sufficient to mediate internalization of dsRNA fragments. Our data provide insight into the mechanism of dsRNA internalization by Drosophila cells. These results have implications for dsRNA delivery into mammalian cells.     

Large-scale Identification of N-Glycosylated Proteins of Mouse Tissues and Construction of a Glycoprotein Database, GlycoProtDB

Protein glycosylation is a common post-translational modification that plays important roles in terms of protein function. However, analyzing the relationship between glycosylation and protein function remains technically challenging. This problem arises from the fact that the attached glycans possess diverse and heterogeneous structures. We believe that the first step to elucidate glycan function is to systematically determine the status of protein glycosylation under physiological conditions. Such studies involve analyzing differences in glycan structure on cell type (tissue), sex, and age, as well as changes associated with perturbations as a result of gene knockout of glycan biosynthesis-related enzyme, disease and drug treatment. Therefore, we analyzed a series of glycoproteomes in several mouse tissues to identify glycosylated proteins and their glycosylation sites. Comprehensive analysis was performed by lectin- or HILIC-capture of glycopeptide subsets followed by enzymatic deglycosylation in stable isotope-labeled water (H(2)(18)O, IGOT) and finally LC-MS analyses. In total, 5060 peptides derived from 2556 glycoproteins were identified. We then constructed a glycoprotein database, GlycoProtDB, using our experimental-based information to facilitate future studies in glycobiology.