Isolation and molecular characterization of Sul/01/09 avian infectious bronchitis virus, indicates the emergence of a new genotype in the Middle East.

Infectious bronchitis virus (IBV) was isolated from trachea and kidney tissues of eight broiler farms in Kurdistan region of North Iraq from 2008 to 2010. The birds were suffering from respiratory and nephropathological symptoms and lesions. A 1116 bp hyper mutable spike glycoprotein (S1) gene was amplified and sequenced using conventional RT-PCR. Sequence analysis and BLAST homology search in GenBank data base indicate that two of the farms were infected with the 4/91 strain, one with an unidentified IBV and five were infected with Sul/01/09. The birds in the latter five farms were suffering from nephropathogenic lesions, however, the virus was isolated from kidney but not from trachea in these cases. The birds were vaccinated regularly with 4/91 or MA5 vaccine. The deduced amino acid sequence of the isolated and amplified S1 subunit (372 aa) of Sul/01/09 was differed in 27-28% from that of all three vaccine strains (4/91, MA5, and H120) used in the region. This dissimilarity is most likely the cause of poor efficacy of vaccines used in the region, at least in five of these farms. Amino acid sequence comparison and phylogenetic tree analysis with other published IBV genotypes indicate that this newly isolated virus together with other regionally related and recently published isolates from Israel (IS/720/99, IS/885) and Egypt (egypt/Benisuef/01) belong to a new genotype. This is the first report of identification and genotyping of IBV isolate in Iraq, which indicate the circulation of 4/91 along with a new variant (Sul/01/09) of IBV in vaccinated broiler farms.

Duplication of the caspase-12 prodomain and inactivation of NLRC4/IPAF in the dog.

Caspases-1, 4, 5, and 12 and other proteins containing caspase recruitment domains (CARDs) play crucial roles in the induction of inflammatory processes. Recently, hybrid caspase-1/4 mRNAs encoding proteases with two CARDs were identified in cat and dog, indicating that the molecular machinery of caspase-dependent inflammation has an unconventional composition in members of the order Carnivora. Here we extended these studies and identified, both in cat and dog, splice variants of caspase-12, which also contained two CARDs. Comparative genomics analysis of the repertoire of canine CARD proteins revealed that the gene encoding NLRC4/IPAF, which is implicated in the inflammatory response to cytosolic flagellin, was inactivated by deleterious mutations in the dog. Our results demonstrate that the repertoires of CARD proteins in cat and dog differ significantly from that of humans and suggest the existence of uncharacterized pathways of inflammasome-mediated signaling in Carnivora.

Identification of novel mammalian caspases reveals an important role of gene loss in shaping the human caspase repertoire.

Proteases of the caspase family play central roles in apoptosis and inflammation. Recently, we have described a new gene encoding caspase-15 that has been inactivated independently in different mammalian lineages. To determine the dynamics of gene duplication and loss in the entire caspase gene family, we performed a comprehensive evolutionary analysis of mammalian caspases. By comparative genomics and reverse transcriptase-polymerase chain reaction analyses, we identified 3 novel mammalian caspase genes, which we tentatively named caspases-16 through -18. Caspase-16, which is most similar in sequence to caspase-14, has been conserved in marsupials and placental mammals, including humans. Caspase-17, which is most similar to caspase-3, has been conserved among fish, frog, chicken, lizard, and the platypus but is absent from marsupials and placental mammals. Caspase-18, which is most similar to caspase-8, has been conserved among chicken, platypus, and opossum but is absent from placental mammals. These gene distribution patterns suggest that, in the evolutionary lineage leading to humans, caspase-17 was lost after the split of protherian and therian mammals and caspase-18 was lost after the split of marsupials and placental mammals. In the canine genome, the number of caspases has been reduced by the fusion of the neighboring genes caspases-1 and -4, resulting in a single coding region. Further lineage-specific gene inactivations were found for caspase-10 in murine rodents and caspase-12 in humans, rabbit, and cow. Lineage-specific gene duplications were found for caspases-1, -3, and -12 in opossum and caspase-4 in primates. Other caspases were generally conserved in all mammalian species investigated. Using the positions of introns as stable characters during recent vertebrate evolution, we define 3 phylogenetic clades of caspase genes: caspases-1/-2/-4/-5/-9/-12/-14/-15/-16 (clade I), caspases-3/-6/-7/-17 (clade II), and caspases-8/-10/-18/CFLAR (clade III). We conclude that gene inactivations have occurred in each of the 3 caspase clades and that gene loss has been as critical as gene duplication in the evolution of the human repertoire of caspases.

Expression of BMI-1 in normal skin and inflammatory and neoplastic skin lesions.

BACKGROUND: BMI-1 is involved in the maintenance of stem cells and functions as an oncogene in both lymphomas and solid carcinomas, acting by downregulation of p16ink4a. We have investigated the expression profile of BMI-1 in normal and inflamed skin as well as in basal cell carcinomas (BCCs) and squamous cell carcinomas (SCCs). METHODS: BMI-1 expression was determined by immunohistochemistry and immunofluorescence, and evaluated semiquantitatively. RESULTS: BMI-1 was weakly expressed in nuclei of basal and sometimes suprabasal keratinocytes, in basal cells of sebaceous glands, weakly to moderately in the bulge area and the external root sheath of hair follicles, and strongly in sweat glands. Whereas BCCs showed strong and diffuse BMI-1 expression, SCCs expressed BMI-1 heterogeneously. Strong cytoplasmic expression of BMI-1 was found in dividing cells. CONCLUSIONS: BMI-1 expression marks stem cells within the hair follicle. As BMI-1 was also found in suprabasal keratinocytes and a variety of specialized cells, the distribution of BMI-1 only partly reflects the known distribution of stem cell compartments. BMI-1 is strongly overexpressed in BCCs, tumors linked to dysregulation of the sonic hedgehog pathway, which has been shown to upregulate BMI-1, suggesting a contribution of the BMI-1 oncogene in their pathogenesis.

Genome sequence comparison reveals independent inactivation of the caspase-15 gene in different evolutionary lineages of mammals.

We have recently demonstrated that placental mammalian species such as pig and dog express a novel proapoptotic protease, caspase-15, whereas mouse and humans lack this enzyme. Here we investigated the evolutionary fate of the caspase-15 gene in different mammalian lineages by analyzing whole-genome shotgun sequences of 30 mammalian species for the presence of caspase-15 orthologs. Caspase-15 gene sequences were found in representatives of all major mammalian clades except for the superorders Afrotheria (tenrec, rock hyrax, and elephant) and Euarchontoglires (rodents, rabbit, tree shrew, and primates), which either lacked any caspase-15-like sequences or contained mutated remnants of the caspase-15 gene. Polymerase chain reaction screenings confirmed the results of the database searches and showed that the caspase-15 gene is expressed not only in various placental mammals but also in the marsupial, Monodelphis domestica. The observed species distribution implies that caspase-15 has originated in an early ancestor of modern mammals and has been conserved, over more than 180 Myr, in marsupials and many placental mammals, whereas it was independently lost in 2 phylogenetically distant clades of placental mammals, that is, Afrotheria and Euarchontoglires. Our data suggest that the inactivation of the caspase-15 gene was not counteracted by, and may even have been driven by, evolutionary constraints in these clades, and therefore, caution against the uncritical use of gene absence for the inference of phylogenetic relationships.

Identification and characterization of a novel mammalian caspase with proapoptotic activity.

Caspases are essential proteases in programmed cell death and inflammation. Studies in murine and human cells have led to the characterization of 14 members of this enzyme family. Here we report the identification of caspase-15, a novel caspase that is expressed in various mammalian species including pig, dog, and cattle. The caspase-15 protein contains a catalytic domain with all amino acid residues critical for caspase activity and a prodomain that is predicted to fold into a pyrin domain structure, which is a unique feature among mammalian caspases. Recombinant porcine caspase-15 underwent autocatalytic processing into its subunits and cleaved both tetrapeptide caspase substrates and the apoptosis regulator protein Bid in vitro. Overexpression of caspase-15 in mammalian cells induced proenzyme maturation, cleavage of Bid, activation of caspase-3, and eventually cell death. Both the proteolytic and the pro-apoptotic activity of caspase-15 were abolished by mutation of the active site cysteine. Since a homolog of caspase-15 is absent in the human and the mouse genome, our results reveal an unexpected variability in the molecular apoptotic machinery of mammals.

Fluconazole upregulates sconC expression and inhibits sulphur metabolism in Microsporum canis.

Azole derivatives such as fluconazole are the mainstay of therapeutic agents for the treatment of fungal infections. Their mode of action involving alteration in the conversion of lanosterol to ergosterol is well established. Here we report the effect of fluconazole on the sulphur metabolism negative regulator gene (sconC) in Microsporum canis. Characterization of the M. canis sconC gene revealed that its ORF is comprised of 495bp interrupted by four introns of 47-70bp. Exposure of M. canis in suspension to fluconazole upregulates sconC mRNA level and protein expression as determined by Northern and Western blot analysis, respectively. Upregulation of sconC was accompanied by inhibition of sulphur metabolism of the fungus resulting in a greatly reduced incorporation of radioactive labelled sulphuric acid into fungal proteins. These data establish that in addition to its action on ergosterol synthesis, fluconazole acts on other biological pathways in fungal cells.

High-frequency of quinolone-resistant Neisseria gonorrhoeae in Austria with a common pattern of triple mutations in GyrA and ParC genes.

OBJECTIVE: Quinolones have a broad spectrum of antimicrobial activity and are widely used for the treatment of uncomplicated Neisseria gonorrhoeae infections. A dramatic increase in the number of reported N. gonorrhoeae infections as well as quinolone-resistant isolates in Vienna prompted us to investigate the pattern of mutations in these isolates. GOALS: The goal of this study was to investigate the pattern of mutations in GyrA and ParC genes in quinolone-resistant N. gonorrhoeae clinical isolates in Vienna from 1999 to 2002. STUDY: The antibiotic susceptibility of N. gonorrhoeae clinical isolates and point mutations of the GyrA and ParC genes of 104 clinical isolates were analyzed. RESULTS: Quinolone-resistant N. gonorrhoeae isolates increased from 3.9% (3 of 77) in 1999 to 59.4% (120 of 202) in 2002. As expected, none of the 46 N. gonorrhoeae quinolone-sensitive strains showed mutations at these positions of GyrA and ParC genes with the exception of 1 isolate, which had a single mutation at GyrA 91. Unlike what has been previously reported for other geographic areas, 96.6% (56 of 58) of the quinolone-resistant isolates harbored common triple mutations at Gyr 91, 95, and ParC 86. The majority of these isolates (76.8%) belong to the PPNG phenotype. CONCLUSIONS: Our data indicate that the pattern of mutations in GyrA and ParC subunits of N. gonorrhoeae in Austria differs from that reported from other geographic areas. The differences may either be the result of the difference in bacterial subtypes or various antibiotic regimens used in these regions.

Fluconazole downregulates metallothionein expression and increases copper cytotoxicity in Microsporum canis.

Azole antifungals are widely used to treat infections with dermatophyte fungi. Whereas it is well established that this class of drugs interferes with fungal ergosterol synthesis, little is known about its potential other biological effects. Here we report the isolation and structural organization of Microsporum canis metallothionein gene and demonstrate that fluconazole is able to downregulate the baseline as well as copper-induced expression of this gene. Since this effect occurred within 30 min after exposure of the fungus to fluconazole, it is unlikely that it is due to impaired ergosterol synthesis. Our additional demonstration that fluconazole enhances copper toxicity for M. canis suggests that inhibition of metallothionein expression by fluconazole is biologically relevant and may represent an important additional mode of the antifungal action of this drug. Therefore our data indicate that antifungal effects of azole derivatives might not only be due to interference with cell wall synthesis but may also affect other biological circuits within the fungal cells.