Molecular cloning and characterization of Rab6 gene in duck.

Rab (ras-like in rat brain) proteins are small GTP-binding proteins that belong to largest subfamily in the small G protein, which are important for molecular modulation of membrane in the vesicular trafficking pathways. We have cloned and sequenced full length cDNA of Rab6 gene in duck. The cDNA sequence consists of 761 nucleotides and contains a complete open reading frame (ORF) of 627 nucleotides; the putative protein includes 208 amino acids. The CDS of duck Rab6 gene shares 86.1-90.0% homology with house mouse, silurana tropicalis, dog, human and orangutan, which indicates the Rab6 gene is high evolutional conservation in above animals.     

Molecular cloning and characterization of avian N-methyl-d-aspartate receptor type1 (NMDA-R1) gene

Birds have several advantages in the study of memory formation, as imprinting and passive avoidance behaviors in chick are often used as model systems. However, the primary structure of the bird N-methyl-d-aspartate (NMDA) responsive glutamate receptor, which is assumed to play a critical role in memory formation, has not been determined. In this report we describe the cDNA cloning of a subunit of NMDA receptors (NMDA-R1) from duck and analysis of its structure and distribution in the brain. The N-terminal 898 amino acids of the NMDA-R1 were well conserved between duck and mammals, but the homology was completely lost in the C-terminus. In situ hybridization showed that the duck NMDA-R1 gene was expressed throughout the brain as it is in mammals.Special issue dedicated to Dr. Bernard W. Agranoff.     

Molecular cloning and characterization of the human NIMA-related protein kinase 3 gene (NEK3)

NEKs (NIMA-related kinases) are a group of protein kinases sharing high amino acid sequence identities with NIMA (never in mitosis gene a) which control mitosis in Aspergillus nidulans. We have cloned a cDNA for human NEK3, a novel human gene structurally related to NIMA, by RT-PCR. Its open reading frame encodes a protein of 489 amino acid residues with the calculated molecular mass of 56.0 kDa and a predicted pI of 6.58. Phylogenetic analysis suggests that mouse and human NEK3s constitute a subfamily within the NIMA family of protein kinases. The expression pattern of NEK3 was studied by RT-PCR and a high level of expression was detected in testis, ovary, and brain, with low-level expression being detected in most of the tissues studied. NEK3 mRNA was detected in all the proliferating cell lines studied, and the amount did not change during the cell cycle. The human NEK3 gene was assigned to human chromosome 13 by somatic cell hybrids and 13q14.2 by radiation hybrid mapping.     

Molecular cloning, sequence characterization, SNP detection, and tissue expression analysis of duck FMO3 gene

Flavin-containing monooxygenase 3 (FMO3) is an important monooxygenase for catalytic oxygenation of many harmful xenobiotics. Mutations in the FMO3 gene have been identified as causing trimethylaminuria in human and fishy off-flavor in cow milk and chicken eggs. In this study, the full-length cDNA sequence of Pekin duck FMO3 gene was cloned, sequenced, and characterized. The full-length cDNA sequence consisted of 1,846 bp and contained a 1,599 bp open-reading frame encoding 532 amino acids. Duck FMO3 gene shared a similar nine exon–eight intron structure with chicken and human. The duck FMO3 putative protein sequence showed high identity with that of chicken (82 %), and relative low identity with those of mammals (61–66 %). We also found that the duck FMO3 gene was dramatically expressed in liver, lung, and kidney compared to that in other tissues in the ducks, indicating the possible roles the FMO3 gene could play in the three tissues. By bidirectional sequencing, we also found one nonsense mutation, 5 nonsynonymous, and 21 synonymous mutations in the coding region of the FMO3 gene in 11 duck breeds and some of them were predicted to be potentially associated with the activities of FMO3 protein.     

Molecular cloning and characterization of Toll-like receptor 14 in Japanese flounder,Paralichthys olivaceus

Toll-like receptors (TLRs) are essential for activation of the innate immune system in response to invading pathogens. TLR14, which is unique to fish, has been identified in several fish species, but its function is unclear. In this study, Japanese flounder (Paralichthys olivaceus) TLR14 gene (JfTLR14) was cloned and its expression profiles were analyzed after infection with viral hemorrhagic septicemia virus, gram-positive Streptococcus iniae and gram-negative Edwardsiella tarda. The coding region of JfTLR14 cDNA was 2,607 bp, encoding 878 amino acid residues. JfTLR14 was highly expressed in head kidney of healthy flounder. In response to infection with VHSV and S. iniae, the JfTLR14 gene was up-regulated at only 1 day post-infection (dpi). However, E. tarda infection increased JfTLR14 gene expression from 1 to 6 dpi. These results imply that JfTLR14 participates more in the immune response against E. tarda infection than in the immune responses to other pathogen infections.     

Toll-like receptor (TLR) 3 as a surrogate sensor of retroviral infection in human cells

The toll-like receptor (TLR)-7 has been shown to sense the retroviral infection. However, a surrogate sensor has been implicated. We examined whether retrovirus serves as a TLR3 ligand in human cells by utilizing cell lines LNCaP and PC-3 lacking TLR7, and the xenotropic murine leukemia virus-relamoted virus (XMRV) insensitive to human tripartite motif-containing (TRIM) 5, a newly characterized pattern recognition receptor (PRR). A dominant-negative TLR3 or a chemical inhibitor of TLR3 attenuated the XMRV-induced IP-10/CXCL10 expression, a marker of TLR3 response. These data clearly indicated that retroviral infection exemplified by XMRV activates the TLR3 signal in human cells.     

Molecular cloning, expression profile, polymorphism and the genetic effects of the dopamine D1 receptor gene on duck reproductive traits

The dopamine D1 receptor (DRD1), a member of the dopamine receptor (DR) gene family, participates in the regulation of reproductive behaviors in birds. In this study, a 1,390 bp fragment covering the complete coding region (CDS) of duck DRD1 gene was obtained. The cDNA (GenBank: JQ346726) contains a 1,353 bp CDS and a 37 bp 3'- UTR including a TGA termination codon (nucleotides 1,354-1,356 bp). The duck DRD1 shares about 76-96 % nucleic acid identity and 82-98 % amino acid identity with their counterparts in other species. A phylogenetic tree based on amino acid sequences displays that duck DRD1 protein is closely related with those of chicken and zebra finch. The quantitative real-time PCR analysis indicates that the DRD1 mRNA is widely expressed in all examined tissues. Five single nucleotide polymorphisms (SNPs) (c.189A > T, c.507C > T, c.681C > T, c.765A > T, c.1044A > G) in the CDS of duck DRD1 gene were indentified, c.681C > T and c.765A > T were genotyped and analyzed in a two generations duck population by using of PCR-RFLP. Association analysis demonstrated that the c.681C > T genotypes were significantly associated with body weight at sexual maturity (when laying their first egg) (P < 0.01), egg production within 360 days (P < 0.05) and 420 days (P < 0.01); the c.765A > T genotypes were significantly associated with egg shape index and egg shell strength (P < 0.05). Those results suggest that the DRD1 gene may be a potential genetic marker to improve some reproductive traits in ducks.     

Toll-like receptor 4 mediates innate immune responses to Haemophilus influenzae infection in mouse lung.

Toll-like receptors (TLRs) have been implicated in the regulation of host responses to microbial Ags. This study characterizes the role of TLR4 in the innate immune response to intrapulmonary administration of Haemophilus influenzae in the mouse. Two different strains of mice efficiently cleared aerosolized H. influenzae concurrent with a brisk elaboration of IL-1beta, IL-6, TNF-alpha, macrophage-inflammatory protein (MIP)-1alpha, and MIP-2 in bronchoalveolar lavage and a corresponding mobilization of intrapulmonary neutrophils. Congenic strains of mice deficient in TLR4 demonstrated a substantial delay in clearance of H. influenzae with diminished IL-1beta, IL-6, TNF-alpha, MIP-1alpha, and MIP-2 in bronchoalveolar lavage and a notable absence of intrapulmonary neutrophils. In TLR4-expressing animals, but not TLR4-deficient animals, TNF-alpha and MIP-1alpha expression was up-regulated in epithelial cells of the conducting airway in response to H. influenzae which was preceded by an apparent activation of the NF-kappaB pathway in these cells based on the findings of decreased overall IkappaB and an increase in its phosphorylated form. This study demonstrates a critical role of TLR4 in mediating an effective innate immune response to H. influenzae in the lung. This suggests that the airway epithelia might contribute to sensing of H. influenzae infection and signaling the innate immune response.