Regions Important for Hemagglutination Activity and Serotypes of Avibacterium paragallinarum HMTp210 Protein.

Avibacterium paragallinarum is an important respiratory pathogen of domestic chickens. Avibacterium paragallinarum has been subtyped into three serogroups and nine serovars according to the Page and revised Kume schemes. The major hemagglutinin antigen of A. paragallinarum is HMTp210, which is a large protein of about 2000 amino acids (aa), including a 70-aa signal peptide at its N-terminal end. However, the regions important for the hemagglutination (HA) activity and serotypes of HMTp210 remain unclear. In this study we constructed a series of A. paragallinarum strains expressing HMTp210 in-frame deletion mutants and determined their HA titers to identify the regions important for the HA activity and serotypes of HMTp210. Two distinct types of HA activities were found in HMTp210. The type 1 HA activity resided in the region spanning the full-length HA (aa 71-2084), whereas the type 2 resided in the region spanning aa 1003-2084. The putative ligand binding of the type 1 HA activity was located at aa 176-360, which had a structure similar to YadA of Yersinia enterocolitica. The putative ligand binding site of the type 2 HA activity was located at aa 1003-1125, which had a structure similar to UspA1 from Moraxella catarrhalis. The type 1 HA activity appeared to be Page serogroup specific, whereas type 2 appeared to be Kume serovar specific. Finally, sequence analyses of the regions spanning aa 1-400 and aa 1100-1600 of HMTp210 could be useful for the molecular serotyping (the Page and revised Kume schemes) of A. paragallinarum isolates.

Regiones importantes para la actividad de hemaglutinación y serotipos de la proteína HMTp210 de Avibacterium paragallinarum. La bacteria Avibacterium paragallinarum es un patógeno respiratorio importante de los pollos domésticos. Avibacterium paragallinarum se subtipificó en tres serogrupos y nueve serovares de acuerdo con los esquemas revisados de Page y Kume. El principal antígeno de la hemaglutinina de A. paragallinarum es la proteína HMTp210, que es una proteína grande de unos 2000 aminoácidos (aa), que incluye un péptido señal de 70 aminoácidos en su extremo N-terminal. Sin embargo, las regiones importantes para la actividad de hemaglutinación (HA) y de los serotipos de la proteína HMTp210 siguen sin estar determinados. En este estudio, se construyó una serie de cepas de A. paragallinarum que expresaban mutantes de deleción en marco de lectura de HMTp210 y se determinaron sus títulos de hemaglutinación para identificar las regiones importantes para la actividad de hemaglutinación y de los serotipos de HMTp210. Se encontraron dos tipos distintos de actividades hemaglutinación en la proteína HMTp210. La actividad de hemaglutinación de tipo 1 residía en la región que abarcaba la longitud completa (aminoácidos 71­2084), mientras que la de tipo 2 residía en la región que abarcaba entre los aminoácidos 1003­2084. El sitio supuesto de unión al ligando de la actividad de hemaglutinación tipo 1 se ubicó entre los aminoácidos 176­360, que tenía una estructura similar a la proteína YadA de Yersinia enterocolitica. El supuesto sitio de unión del ligando de la actividad de hemaglutinación tipo 2 se ubicó entre los aminoácidos 1003­1125, que tenía una estructura similar a la proteína UspA1 de Moraxella catarrhalis. La actividad de hemaglutinación tipo 1 parecía ser específica del serogrupo Page, mientras que la hemaglutinación tipo 2 parecía ser específica del serovar Kume. Finalmente, los análisis de secuencias de las regiones que abarcan los aminácidos 1­400 y aminoácidos 1100­1600 de HMTp210 podrían ser útiles para la serotipificación molecular (por el esquema revisado de Page y Kume revisado) de aislamientos de A. paragallinarum.

Mechanical injury of bovine cartilage explants induces depth-dependent, transient changes in MAP kinase activity associated with apoptosis.

OBJECTIVE: To characterize mitogen activated protein (MAP) kinase activity and chondrocyte apoptosis in an in vitro model of cartilage mechanical injury as a function of tissue depth and time post-injury. DESIGN: Mechanically injured osteochondral explants were assessed for cell viability, MAP kinase and caspase-3 activity over 15 days using immunofluorescence microscopy and Western blot. Zonal distributions of cell viability and apoptosis were quantified in the presence of specific mitogen activated protein kinase inhibitors. RESULTS: Viability rapidly decreased post-injury, most significantly in the superficial zone, with some involvement of the middle and deep zones, which correlated with increased caspase-3 activity. Transient and significant increases in extracellular-regulated protein kinase (ERK) activity were observed in middle and deep zones at 1 and 6 days post-injury, while c-Jun-amino terminal protein kinase activity increased in the deep zone at 1 and 6 days compared to uninjured controls. Changes in p38 activity were particularly pronounced, with significant increases in all three zones 30 min post-injury, but only in the middle and deep zones after 1 and 6 days. Inhibition of ERK and p38 increased chondrocyte viability which correlated with decreased apoptosis. CONCLUSIONS: Spatiotemporal patterns of MAP kinase signalling in cartilage after mechanical injury strongly correlate with changes in cell viability and chondrocyte apoptosis. Importantly, these signals may be pro-survival or pro-apoptotic depending on zonal location and time post-injury. These data yield mechanistic insights which may improve the diagnosis and treatment of cartilage injuries.

Nucleotide sequences of goose circovirus isolated in Taiwan.

We report the complete nucleotide (nt) sequences of eleven goose circovirus (GoCV) isolated in Taiwan. Nine out of the eleven isolates had a genome size of 1821 nt, whereas the remaining two isolates have a size of 1820 nt. Sequence comparisons of the eleven Taiwanese GoCV isolates and a German isolate revealed that these viruses could be divided into three distinct genetic groups. Group I contains the German isolate, group II contains three Taiwanese isolates, and group III contains eight Taiwanese isolates. Nucleotide differences between viruses of different genetic groups ranged from 7.0-7.7%, whereas the differences within the same group were only 0.2-1.0%. The most diversified sequences were found at a region between nt 27-72 of the viral genome, which corresponded to the right one-third of the 5' intergenic region. Open reading frame analysis shows that the genome of all Taiwanese GoCV isolates could encode four proteins: V1 (Rep, 293 amino acids), V2 (37 amino acids), C1 (capsid, 250 amino acids), and C2 (99 amino acids). The sizes of V1, C1 and C2 proteins of all Taiwanese isolates and the German GoCV isolates were identical. However, the size of V2 protein (37 amino acids), although identical in all Taiwanese isolates, was much smaller than that of the German isolate (120 amino acids). Moreover, the initiation codon of the V2 ORF of three Taiwanese isolates was ATA rather than ATG. Our result indicates that GoCV of multiple genetic groups might have been circulating in Europe and Asia, and these viruses differ in their nucleotide sequences, sizes of the genome, and sizes of the V2 ORFs.