FSH up-regulates angiogenic factors in luteal cells of buffaloes

Follicle-stimulating hormone has been widely used to induce superovulation in buffaloes and cows and usually triggers functional and morphologic alterations in the corpus luteum (CL). Several studies have shown that FSH is involved in regulating vascular development and that adequate angiogenesis is essential for normal luteal development. Angiogenesis is regulated by many growth factors, of which vascular endothelial growth factor (VEGF) and fibroblast growth factor 2 (FGF2) have an established central role. Therefore, we have used a combination of in vitro and in vivo studies to assess the effects of FSH on the expression of VEGF and FGF2 and their receptors in buffalo luteal cells. The in vivo model consisted of 12 buffalo cows, divided into control (n = 6) and superovulated (n = 6) groups, and CL samples were collected on day 6 after ovulation. In this model, we analyzed the gene and protein expression of FGF2 and its receptors and the protein expression of VEGFA systems with the use of real-time PCR, Western blot analysis, and immunohistochemistry. In the in vitro model, granulosa cells were collected from small follicles (diameter, 4–6 mm) of buffaloes and cultured for 4 d in serum-free medium with or without FSH (10 ng/mL). To induce in vitro luteinization, LH (250 ng/mL) and fetal bovine serum (10%) were added to the medium, and granulosa cells were maintained in culture for 4 d more. The progesterone concentration in the medium was measured at days 4, 5, and 8 after the beginning of cell culture. Cells were collected at day 8 and subjected to real-time PCR, Western blot analysis, and immunofluorescence for assessment of the expression of FGF2, VEGF, and their receptors. To address the percentage of steroidogenic and growth factor-expressing cells in the culture, flow cytometry was performed. We observed that in superovulated buffalo CL, the FGF2 system mRNA expression was decreased even as protein expression was increased and that the VEGF protein was increased (P < 0.05). In vitro experiments with granulosa cells showed an increase in the mRNA expression of VEGF and FGF2 and its receptors 1 and 2 and protein expression of VEGF, kinase insert domain receptor, FGF receptor 2, and FGF receptor 3 in cells treated with FSH (P < 0.05), in contrast to the in vivo experiments. Moreover, the progesterone production by FSH-treated cells was elevated compared with untreated cells (P < 0.05). Our findings indicate that VEGF, FGF2, and their receptors were differentially regulated by FSH in vitro and in vivo in buffalo luteal cells, which points toward a role of CL environment in modulating cellular answers to gonadotropins.     

A Conus regularis Conotoxin with a Novel Eight-Cysteine Framework Inhibits CaV2.2 Channels and Displays an Anti-Nociceptive Activity

A novel peptide, RsXXIVA, was isolated from the venom duct of Conus regularis, a worm-hunting species collected in the Sea of Cortez, México. Its primary structure was determined by mass spectrometry and confirmed by automated Edman degradation. This conotoxin contains 40 amino acids and exhibits a novel arrangement of eight cysteine residues (C-C-C-C-CC-CC). Surprisingly, two loops of the novel peptide are highly identical to the amino acids sequence of ω-MVIIA. The total length and disulfide pairing of both peptides are quite different, although the two most important residues for the described function of ω-MVIIA (Lys2 and Tyr13) are also present in the peptide reported here. Electrophysiological analysis using superior cervical ganglion (SCG) neurons indicates that RsXXIVA inhibits Ca_V2.2 channel current in a dose-dependent manner with an EC₅₀ of 2.8 μM, whose effect is partially reversed after washing. Furthermore, RsXXIVA was tested in hot-plate assays to measure the potential anti-nociceptive effect to an acute thermal stimulus, showing an analgesic effect in acute thermal pain at 30 and 45 min post-injection. Also, the toxin shows an anti-nociceptive effect in a formalin chronic pain test. However, the low affinity for Ca_V2.2 suggests that the primary target of the peptide could be different from that of ω-MVIIA.     

Replication characteristics of swine influenza viruses in precision-cut lung slices reflect the virulence properties of the viruses

Precision-cut lung slices of pigs were infected with five swine influenza A viruses of different subtypes (A/sw/Potsdam/15/1981 H1N1, A/sw/Bad Griesbach/IDT5604/2006 H1N1, A/sw/Bakum/1832/2000 H1N2, A/sw/Damme/IDT5673/2006 H3N2, A/sw/Herford/IDT5932/2007 H3N2). The viruses were able to infect ciliated and mucus-producing cells. The infection of well-differentiated respiratory epithelial cells by swine influenza A viruses was analyzed with respect to the kinetics of virus release into the supernatant. The highest titres were determined for H3N2/2006 and H3N2/2007 viruses. H1N1/1981 and H1N2/2000 viruses replicated somewhat slower than the H3N2 viruses whereas a H1N1 strain from 2006 multiplied at significantly lower titres than the other strains. Regarding their ability to induce a ciliostatic effect, the two H3N2 strains were found to be most virulent. H1N1/1981 and H1N2/2000 were somewhat less virulent with respect to their effect on ciliary activity. The lowest ciliostatic effect was observed with H1N1/2006. In order to investigate whether this finding is associated with a corresponding virulence in the host, pigs were infected experimentally with H3N2/2006, H1N2/2000, H1N1/1981 and H1N1/2006 viruses. The H1N1/2006 virus was significantly less virulent than the other viruses in pigs which was in agreement with the results obtained by the in vitro-studies. These findings offer the possibility to develop an ex vivo-system that is able to assess virulence of swine influenza A viruses.     

Genotype by environment interaction and model comparison for growth traits of Santa Ines sheep

The objectives of the present study were to compare alternative models for the genetic evaluation and assess the importance of genotype by environment interaction (G×E) in the estimation of genetic parameters and genetic evaluation of birth weight (BW), weight at 60 days of age (W60) and weight at 180 days of age (W180) of Santa Ines sheep. Data comprise 7622 BW, 4673 W60 and 2830 W180 records from animals born in 44 Brazilian herds. Four models were used for the analyses: animal model (AM) with homogeneous residual variance (1), or heterogeneous residual variance (2), hierarchical reaction norms model (HRNM) with homogeneous (1) or heterogeneous residual variance (2). The models that best fit the BW, W60 and W180 data were AM2, HRNM1 and HRNM2 respectively. Thus, models for genetic evaluation that consider heterogeneity of variances are recommended to evaluate growth traits of sheep. The correlation between intercept and slope of the HRNM was higher than 0.70 for all traits studied, indicating that animals with higher average breeding values responded better to improvement in environmental conditions, a fact characterizing the scale effect of G×E. Therefore, G×E is an important factor to be considered in the estimation of genetic parameters and genetic evaluation of growth traits of sheep.     

Pseudomonas associated with Bursaphelenchus xylophilus,its insect vector and the host tree: A role in pine wilt disease?

In this study, we characterized the diversity of Pseudomonas associated with Bursaphelenchus xylophilus, its insect vector (Monochamus galloprovincialis) and its host (Pinus pinaster), by a culture‐independent approach using rpoD clone libraries. Clone libraries of Pseudomonas rpoD were obtained from B. xylophilus, M. galloprovincialis and infected P. pinaster. Most M. galloprovincialis and B. xylophilus sequences grouped together in the P. fluorescens group. Genes related to xenobiotics degradation and phenylacetate synthesis were present in the genomes of the type strains closely related to sequences retrieved from the nematode libraries. Results demonstrated that the nematode, during its life stages inside the tree, maintains a diverse Pseudomonas community that is closely related to the one associated with the insect vector. These bacteria might contribute to degradation of xenobiotics and tree weakening during the nematode tree infection.     

<Emphasis Type="Italic">Achromobacter insolitus</Emphasis> and <Emphasis Type="Italic">Zoogloea ramigera</Emphasis> associated with wheat plants (<Emphasis Type="Italic">Triticum aestivum</Emphasis>)

This study reports for the first time the presence of diazotrophic bacteria belonging to the genera Achromobacter and Zoogloea associated with wheat plants. These bacterial strains were identified by the analysis of 16S rDNA sequences. The bacterium IAC-AT-8 was identified as Azospirillum brasiliense, whereas isolates IAC-HT-11 and IAC-HT-12 were identified as Achromobacter insolitus and Zoogloea ramigera, respectively. A greenhouse experiment involving a non-sterilized soil was carried out with the aim to study the endophytic feature of these strains. After 40 days from inoculation, all the strains were in the inner of roots, but they were not detected in soil. In order to assess the location inside wheat plants, an experiment was conducted under axenic conditions. Fifteen days after inoculation, preparations of inoculated plants were observed by the scanning electron microscope, using the cryofracture technique, and by the transmission electron microscope. It was observed that all isolates were present on the external part of the roots and in the inner part at the elongation region, in cortex cells, but not in the endodermis or in the vascular bundle region. No colonizing bacterial cells were observed in wheat leaves.