Next Generation Semiconductor Based-Sequencing of a Nutrigenetics Target Gene (GPR120) and Association with Growth Rate in Italian Large White Pigs

The GPR120 gene (also known as FFAR4 or O3FAR1) encodes for a functional omega-3 fatty acid receptor/sensor that mediates potent insulin sensitizing effects by repressing macrophage-induced tissue inflammation. For its functional role, GPR120 could be considered a potential target gene in animal nutrigenetics. In this work we resequenced the porcine GPR120 gene by high throughput Ion Torrent semiconductor sequencing of amplified fragments obtained from 8 DNA pools derived, on the whole, from 153 pigs of different breeds/populations (two Italian Large White pools, Italian Duroc, Italian Landrace, Casertana, Pietrain, Meishan, and wild boars). Three single nucleotide polymorphisms (SNPs), two synonymous substitutions and one in the putative 3′-untranslated region (g.114765469C > T), were identified and their allele frequencies were estimated by sequencing reads count. The g.114765469C > T SNP was also genotyped by PCR-RFLP confirming estimated frequency in Italian Large White pools. Then, this SNP was analyzed in two Italian Large White cohorts using a selective genotyping approach based on extreme and divergent pigs for back fat thickness (BFT) estimated breeding value (EBV) and average daily gain (ADG) EBV. Significant differences of allele and genotype frequencies distribution was observed between the extreme ADG-EBV groups (P < 0.001) whereas this marker was not associated with BFT-EBV.     

Inheritance of a mutant histocompatibility gene and a new mammary tumor virus genome in the B6.KH-84 mouse strain

The potential association between integration or deletion of mouse mammary tumor virus (MMTV) retroviral sequences and the appearance of non-H-2 histocompatibility (H) antigen mutations was investigated. Genomic blots from inbred strains carrying 22 loss, gain-loss, and gain mutations on the BALB/c and C57BL/6 backgrounds were hybridized with probes homologous to the long terminal repeat (LTR) and envelope (env) regions of MMTV. Twenty-one mutants were identical in restriction patterns to the respective background strains with all tested restriction enzymes and both probes. However, genomic blots of one gain mutant, B6.C-KH-84, exhibited restriction fragments which were not exhibited by either of the parental strains, C57BL/6 or BALB/c. An additional 5.5 kb Eco RI fragment was observed with the env probe and additional 9.2 kb and 5.5 kb fragments were observed with the LTR probe. These observations were substantiated by hybridization of these two probes with genomic blots generated with additional restriction enzymes. Assuming that the new provirus contains a single, internal Eco RI site as has been observed for other MMTV proviral sequences, it is presumed that the new provirus includes both 5 and 3 LTRs in addition to the env region. Based on the unique sizes of the observed restriction fragments relative to other identified MMTV proviral sequences, this provirus has been designated Mtv-22. The potential role of Mtv-22 in the genesis of the gained histocompatibility antigen in B6.C-KH-84 is discussed.On leave of absence from Istituto Nazionale per to Studio e la Cura dei Tumori, Milano, Italy     

Special Issue Dedicated to Dr. Héctor S. Barra

Dedication

Special Issue Dedicated to Dr. Héctor S. Barra     

Complement factor H-binding protein, a putative virulence determinant of Borrelia hermsii, is an antigenic target for protective B1b lymphocytes

Vaccination is the most effective way to control infectious diseases. A variety of microbial pathogens use antigenic variation, an immune evasion strategy that poses a challenge for vaccine development. To understand protective immune responses against such pathogens, we have been studying Borrelia hermsii, a bacterium that causes recurrent bacteremia due to antigenic variation. An IgM response is necessary and sufficient to control B. hermsii infection. We have recently found a selective expansion of B1b cells concurrent with the resolution of B. hermsii bacteremia. B1b cells from convalescent but not naive mice confer long-lasting immunity, but the Ag(s) driving the protective IgM responses is unknown. Herein we demonstrate that convalescent B1b cell-derived IgM recognizes complement factor H-binding protein (FhbA), a B. hermsii outer-surface protein and putative virulence factor that does not undergo antigenic variation and is expressed by all clinical isolates. A progressive increase in the IgM response to FhbA correlated with the kinetics of B1b cell expansion, diminished the severity of bacteremic episodes, and led to the eventual resolution of the infection. These data indicate that FhbA is a specific target for protective B1b cell responses. Ags recognized by B1b cells may be considered as an important component in vaccination strategies.     

Gonadal development and sex-specific demographics of the shovelnose sturgeon in the Middle Mississippi River

Harvest of the shovelnose sturgeon, Scaphirhynchus platorynchus for caviar has increased. To determine whether populations can withstand increased harvest, detailed information regarding sexual demographics is needed. We describe gender and reproductive development of 306 shovelnose sturgeon from the Middle Mississippi River (River km 0–322) during September 2001 through December 2003. Using dissection and histology, we identified three of the four gonadal stages described previously for male lake sturgeon and all seven stages for females. Males reached maturity at a smaller size than did females. Gonads can be rapidly inspected for sex and stage of development for the shovelnose sturgeon. The sex ratio was not different from 1 : 1. Seven intersexual fish occurred. Female fecundity was positively related to body weight (number of eggs = 30.24 × body weight − 8392; P = 0.013; r² = 0.45) and weakly related to fork length (number of eggs = 146.37 × fork length − 66 176, P = 0.053, r² = 0.23).     

Elucidation of the opening of the epoxidic ring of the 3beta-acetoxy-14alpha,15alpha-epoxy-5alpha-cholest-8-en-7-one by methanol, using NMR techniques assisted by a conformational study through theoretical calculations

This paper demonstrates that the crystallization of 3beta-acetoxy-14alpha,15alpha-epoxy-5alpha-cholest-8-en-7-one from methanol affords the 3beta-acetoxy-9alpha-methoxy-15alpha-hydroxycholest-8(14)-en-7-one. The structure of this steroid, which shows an apparently anomalous UV absorption maximum, is determined by high field NMR experiments, supporting the coupling constant values assignments and the NOE contacts by a conformational study through theoretical calculations at the B3LYP/6-31G* level. The computational study also justifies the observed UV absorption of the steroid, thus demonstrating the usefulness of computer chemistry in providing support for the identification of unknown compounds.     

Synaptophysin I selectively specifies the exocytic pathway of synaptobrevin 2/VAMP2

Biogenesis and recycling of synaptic vesicles are accompanied by sorting processes that preserve the molecular composition of the compartments involved. In the present study, we have addressed the targeting of synaptobrevin 2/VAMP2 (vesicle-associated membrane protein 2), a critical component of the synaptic vesicle--fusion machinery, in a heterotypic context where its sorting is not confounded by the presence of other neuron-specific molecules. Ectopically expressed synaptophysin I interacts with VAMP2 and alters its default surface targeting to a prominent vesicular distribution, with no effect on the targeting of other membrane proteins. Protein-protein interaction is not sufficient for the control of VAMP2 sorting, which is mediated by the C-terminal domain of synaptophysin I. Synaptophysin I directs the sorting of VAMP2 to vesicles before surface delivery, without influencing VAMP2 endocytosis. Consistent with this, dynamin and alpha-SNAP (soluble N-ethylmaleimide-sensitive fusion protein-attachment protein) mutants which block trafficking at the plasma membrane do not abrogate the effect of synaptophysin I on VAMP2 sorting. These results indicate that the sorting determinants of synaptic vesicle proteins can operate independently of a neuronal context and implicate the association of VAMP2 with synaptophysin I in the specification of the pathway of synaptic vesicle biogenesis.     

A full conformational characterization of 13-ethylprogestogens through theoretical calculations and nuclear magnetic resonance spectroscopy

The conformational preferences of a group of 13-ethylsteroids and related estranes have been determined through theoretical calculations at the B3LYP/6-31G(*) level in order to ascertain differences and similarities in the conformational behavior which might, in principle, influence the activity. Attention was focussed on two geometrical features usually related to the progestational activity of this class of compounds, namely, the inversion of the A ring and the orientation of the 13-ethyl group. The present calculations show that the absence of the C10 methyl group, like in levonorgestrel, 3-ketodesogesterel, and gestodene, makes the inversion of the A ring easier than in norethisterone and ethisterone even if in any case the 1alpha,2beta-half-chair conformation remains preferred. The preference of the ethyl group for the trans orientation is maintained by all the 13-ethylprogestogens though in gestodene also the g(+)gauche conformation is significantly populated. This preference was experimentally supported through a high field NMR spectroscopy study of the ethylsteroids.     

Long-term study on symptomless human metapneumovirus infection in hematopoietic stem cell transplant recipients

From October 2004 through October 2006 a study was performed to evaluate the prevalence of human Metapneumovirus (hMPV) infection in adult hematopoietic stem cell transplant (HSCT) recipients. Sequential nasopharyngeal aspirates (NPA) were collected independently from respiratory symptoms and evaluated for hMPV-RNA by polymerase chain reaction (PCR) and sequence analysis. Results indicate epidemiological and molecular differences between the 2004-2005 and 2005-2006 periods and that hMPV seems not to symptomatically affect HSCT patients or cause late respiratory sequelae. In addition, data collected suggest a hospital origin of hMPV infection in most HSCT patients during the 2004-2005 period.