Molecular characterization of the pig<Emphasis Type="Italic"> C3</Emphasis> gene and its association with complement activity

The complement system catalyzes direct lysis of micro-organisms and modulates phagocytosis, inflammation, humoral and cellular immune responses. Since the complement protein C3 is the central component within all pathways of complement activation, C3 is a candidate gene for complement activity and also for improved protection against many pathogens. The pig C3 gene was sequenced, screened for polymorphisms, and analyzed for association with hemolytic complement activity of the alternative and classical pathway (AH(50), CH(50)). C3c serum levels and haptoglobin (HP) serum concentrations were measured before and after vaccination against Mycoplasma hyopneumoniae, Aujeszky virus, and porcine reproductive and respiratory syndrome virus in F2 animals of a pig resource population based on crossbreeding of Duroc and Berlin Miniature Pig. The genomic C3 sequence covers 444 bp of promoter region, 41 exons and 40 introns, as well as 881 bp of the 3'-flanking region. The cDNA codes for a 1,661-amino acid precursor C3. Five polymorphic sites were detected in the 5'-UTR, intron 13, exon 15, exon 30, and the 3'-UTR. Within the resource population two haplotypes were found to segregate. Analysis of variance applying a repeated measures model revealed a significant effect of the interaction of C3 genotype and time of measurement relative to immunization on CH(50), AH(50,)and C3c that is likely to be due to variation of C3 expression. In contrast, the time course of the HP acute-phase reaction is not associated with C3 genomic variation. The association of C3 with complement activity indicates the importance of C3 as a candidate gene for natural resistance to micro-organisms, although the causative polymorphism modulating the expression of C3 remains to be delineated.     

An avian influenza H5N1 virus that binds to a human-type receptor

Avian influenza viruses preferentially recognize sialosugar chains terminating in sialic acid-alpha2,3-galactose (SAalpha2,3Gal), whereas human influenza viruses preferentially recognize SAalpha2,6Gal. A conversion to SAalpha2,6Gal specificity is believed to be one of the changes required for the introduction of new hemagglutinin (HA) subtypes to the human population, which can lead to pandemics. Avian influenza H5N1 virus is a major threat for the emergence of a pandemic virus. As of 12 June 2007, the virus has been reported in 45 countries, and 312 human cases with 190 deaths have been confirmed. We describe here substitutions at position 129 and 134 identified in a virus isolated from a fatal human case that could change the receptor-binding preference of HA of H5N1 virus from SAalpha2,3Gal to both SAalpha2,3Gal and SAalpha2,6Gal. Molecular modeling demonstrated that the mutation may stabilize SAalpha2,6Gal in its optimal cis conformation in the binding pocket. The mutation was found in approximately half of the viral sequences directly amplified from a respiratory specimen of the patient. Our data confirm the presence of H5N1 virus with the ability to bind to a human-type receptor in this patient and suggest the selection and expansion of the mutant with human-type receptor specificity in the human host environment.     

Proteome Changes in Thai Indigenous Chicken Muscle during Growth Period

Proteomic profiling of the pectoralis muscle of Thai indigenous chickens during growth period was analyzed using two-dimensional gel electrophoresis (2-DE) and matrix assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF/MS). A total of 259, 161, 120 and 107 protein spots were found to be expressed in the chicken pectoralis muscles at 0, 3, 6 and 18 weeks of age, respectively. From these expressed proteins, five distinct protein spots were significantly associated with chicken age. These protein spots were characterized and showed homology with phosphoglycerate mutase 1 (PGAM1), apolipoprotein A1 (APOA1), triosephosphate isomerase 1 (TPI1), heat shock protein 25 kDa (HSP25) and fatty acid binding protein 3 (FABP3). These five protein spots were categorized as follows: (i) the expression levels of PGAM1 and TPI1 proteins were positively correlated with chicken aging (p<0.05), (ii) the expression levels of APOA1 and FABP3 proteins were negatively correlated with chicken aging (p<0.05) and (iii) the expression levels of the HSP25 protein were up- and down-regulated during growth period. Moreover, the mRNA expression levels of the FABP3 and HSP25 genes were significantly decreased in muscle during the growth period (p<0.05), whereas no significant changes of the PGAM1, TPI1 and APOA1 gene expression from the chicken muscle was observed. The identified proteins were classified as metabolic and stress proteins. This demonstrates a difference in energy metabolism and stress proteins between age groups and shows that proteomics is a useful tool to uncover the molecular basis of physiological differences in muscle during the growth period.     

Quantification of DNA binding, uptake, transmission and expression in bovine sperm mediated gene transfer by RT-PCR: effect of transfection reagent and DNA architecture

In this study, we compared the transfection effectiveness of liposomes with the new transfection reagent FuGene 6 in bovine sperm mediated gene transfer (SMGT). Furthermore, we examined whether plasmid architecture affects overall efficiency by comparing two plasmids, one of them bearing an additional murine nontranscribed spacer (nts) insert (CMV-INF-tau-IRES-EGFP versus CMV-INF-tau-IRES-EGFP-nts). To accomplish that, we quantified plasmid binding and uptake to spermatozoon and transfer and expression of foreign DNA into embryos by real time PCR. More plasmids bound to spermatozoa when treated with FuGene 6 than with liposome treatment (p<0.05) reaching highest counts in plasmids bearing the nts sequence (p<0.05). Mean number of plasmids taken up was significantly (p<0.05) affected by transfection strategy (1-3 versus 15-81 versus 120-162) with plasmids bearing the nts sequence being 2-8 fold more effective (p<0.05). Culture of SMGT derived embryos up to day 9 did not result in any difference in terms of cleavage rate (64.2-84.2%) and development to blastocyst stage (18.8-26.3%) between different groups. Insert of the nts fragment significantly (p<0.05) affected mean number of transmitted plasmids to 4-cell stage embryos (44 versus 7) and relative INF-tau mRNA expression level in day 9 blastocysts (7-8 fold). However, only six blastocysts (3.6%) exhibited green fluorescence indicating low EGFP protein production. In conclusion, we were able to show effectiveness of sperm mediated gene transfer is significantly affected by choice of transfection reagent and by plasmid architecture.     

Megakaryocyte development is normal in mice with targeted disruption of Tescalcin

BackgroundTescalcin is an EF-hand calcium-binding protein that interacts with the Na+/H + exchanger 1 (NHE1). Levay and Slepak recently proposed a role for tescalcin in megakaryopoiesis that was independent of NHE1 activity. Their studies using K562 and HEL cell lines, and human CD34 + hematopoietic stem cells suggested an essential role for tescalcin in megakaryocyte differentiation.ObjectiveTo study the role of tescalcin in megakaryocyte development using a murine model of megakaryopoiesis.MethodsWe generated a mouse with targeted disruption of tescalcin and investigated megakaryocyte development.ResultsTescalcin-deficient mice had a normal number of megakaryocytes and platelets. The morphology, polyploidization profile, and expression of Fli-1 in bone marrow-derived megakaryocytes were also normal.ConclusionTescalcin does not appear to be necessary for normal megakaryocyte development.