Bactericidal activity of peripheral blood neutrophils during the oestrous cycle and early pregnancy in the mare

The oestrous cycles of 20 mixed-breed mares were synchronized with daily injections of 10 mg oestradiol-17 beta and 150 mg progesterone given i.m. for 10 days. On the 10th day, 10-15 mg prostaglandin F-2 alpha was administered i.m. to induce oestrus. Neutrophils were isolated from jugular blood on the 2nd or 3rd day of oestrus, Days 5 and 7 after ovulation or during early pregnancy (Days 18-34 of pregnancy). Neutrophils were challenged with Staphylococcus aureus and their bactericidal activity examined after 30 and 120 min of incubation for a reduction of colony forming units. Bactericidal activity increased with the time of incubation (P less than 0.01) but did not differ for the oestrous cycle or pregnancy (P greater than 0.05).     

Use of an immediate, qualitative progesterone assay for determination of day of ovulation in an equine embryo transfer program

An immediate, qualitative enzyme-linked immunosorbent assay (ELISA) for progesterone was evaluated for use in determining the day of ovulation in an equine embryo transfer program. Plasma samples were collected from 27 mares from the third day of estrus to the second day of diestrus for 50 cycles. Ovulation was detected by ultrasound examination per rectum. Plasma progesterone concentrations were estimated using the qualitative assay to detect the time of the rise in progesterone after ovulation. Qualitative scores were compared to progesterone concentrations for the same samples as measured by a quantitative ELISA; the correlation between the two methods, expressed as a contingency coefficient, was 0.56. The accuracy of determining day of ovulation using qualitative progesterone results was compared to that achieved using the quantitative assay or detection of the first day of diestrus by teasing. Accuracy in determining day of ovulation +/- 1 d using the three methods was qualitative, 36/50 (72%); quantitative, 44/50 (88%); and teasing, 43/50 (86%). There was a significant difference in accuracy between the qualitative and quantitative progesterone assays (P<0.05).     

Equine granulosa-theca cell tumors express inhibin alpha- and beta A-subunit messenger ribonucleic acids and proteins

The association of equine granulosa-theca cell tumors with atrophied contralateral ovaries and abnormal estrous cycles suggests that these tumors produce hormones that affect pituitary gonadotropin production. Because inhibin, a heterodimer protein secreted by granulosa cells, decreases FSH production, we examined the presence of inhibin alpha- and beta A-subunits and their mRNAs in ovarian tumors obtained from three mares. These tumors contained neoplastic cords and nodules, multiple fluid-filled cysts, and a predominance of neoplastic granulosa cells. Reduced proteins from tumor-conditioned media were analyzed by electrophoresis and immunoblotting using antibodies directed against peptide fragments of the alpha- and beta A-chains of porcine inhibin. Specific bands at 50-kDa and 36-kDa for the inhibin alpha-subunit and at 44 kDa and 13 kDa for the inhibin beta A-subunit were observed in these tumors. Northern blot hybridization of 32P-labeled rat inhibin alpha- and beta A-subunit complementary RNAs to total RNA from each tumor revealed predominant bands of activity in all three tumors at 1.5 and 7 kb for the alpha- and beta A-subunit mRNAs, respectively. These results demonstrate that equine granulosa-theca cell tumors express the mRNAs for inhibin alpha- and beta A-subunits and also secrete inhibin subunits that could potentially affect gonadotropin production in afflicted mares. Furthermore, cells derived from these tumors may provide a useful model for understanding inhibin gene regulation and ovarian tumorigenesis.     

A cryptic invasion within an invasion and widespread introgression in the European water frog complex: consequences of uncontrolled commercial trade and weak international legislation

In Western Europe, many pond owners introduce amphibians for ornamental purposes. Although indigenous amphibians are legally protected in most European countries, retailers are circumventing national and international legislation by selling exotic nonprotected sibling species. We investigated to what extent non‐native species of the European water frog complex (genus Pelophylax) have become established in Belgium, using morphological, mitochondrial and nuclear genetic markers. A survey of 87 sampling sites showed the presence of non‐native water frogs at 47 locations, mostly Marsh frogs (Pelophylax ridibundus). Surprisingly, at least 19% of all these locations also harboured individuals with mitochondrial haplotypes characteristic of Anatolian water frogs (Pelophylax cf. bedriagae). Nuclear genotyping indicated widespread hybridization and introgression between P. ridibundus and P. cf. bedriagae. In addition, water frogs of Turkish origin obtained through a licensed retailer, also contained P. ridibundus and P. cf. bedriagae, with identical haplotypes to the wild Belgian populations. Although P. ridibundus might have invaded Belgium by natural range expansion from neighbouring countries, our results suggest that its invasion was at least partly enhanced by commercial trade, with origins as far as the Middle East. Also the invasion and rapid spread of Anatolian lineages, masked by their high morphological similarity to P. ridibundus, is likely the result of unregulated commercial trade. We expect that Anatolian frogs will further invade the exotic as well as the native range of P. ridibundus and other Pelophylax species elsewhere in Western and Central Europe, with risks of large‐scale hybridization and introgression.     

Coupling of M3 Acetylcholine Receptor to Gq16 Activates a Natriuretic Peptide Receptor Guanylyl Cyclase

Muscarinic activation of tracheal smooth muscle (TSM) involves a M₃AChR/heterotrimeric-G protein/NPR-GC coupling mechanism. G protein activators Mastoparan (MAS) and Mastoparan-7 stimulated 4- and 10-fold the NPR-GC respectively, being insensitive to PTX and antibodies against Gα_i/o subfamily. Muscarinic and MAS stimulation of NPR-GC was blocked by antibodies against C-terminal of Gα_q16, whose expression was confirmed by RT-PCR. However, synthetic peptides from C-terminal of Gα_q15/16 stimulated the NPR-GC. Coupling of α_q16 to M₃AChR is supported by MAS decreased [³H]QNB binding, being abolished after M₃AChR-4-DAMP-alkylation. Anti-i₃M₃AChR antibodies blocked the muscarinic activation of NPR-GC, and synthetic peptide from i₃M₃AChR (M₃P) was more potent than MAS increasing GTPγ [³⁵S] and decreasing the [³H]QNB activities. Coupling between NPR-GC and Gα_q16 was evaluated by using trypsin-solubilized-fraction from TSM membranes, which displayed a MAS-sensitive-NPR-GC activity, being immunoprecipitated with anti-Gα_q16, also showing an immunoreactive heterotrimeric-G-β -subunit. These data support the existence of a novel transducing cascade, involving Gα_q16β γ coupling M₃AChR to NPR-GC.     

Sperm ultrastructure, morphometry, and abnormal morphology in American black bears (Ursus americanus)

The objective of this study was to describe sperm ultrastructure, morphometry, and abnormal morphology in American black bears. Electroejaculation was successful in 53.8% (7/13) of the attempts, but urine contamination was common. Epididymal sperm samples were also obtained from five bears. Sperm had a paddle-like head shape and the ultrastructure was similar to that of most other mammals. The most striking particularity of black bear sperm ultrastructure was a tightening of the nucleus in the equatorial region. Although the differences were not significant in all bears, the overall decrease in sperm nucleus dimensions during transport from the caput epididymis to the cauda suggested increasing compaction of the nucleus during maturation. For ejaculated sperm, nucleus length, width, and base width were 4.9, 3.7, and 1.8 μm, respectively, whereas sperm head length, width, and base width were 6.6, 4.8, and 2.3 μm, and midpiece, tail (including midpiece), and total sperm lengths were 9.8, 68.8, and 75.3 μm. Evaluation of sperm cytoplasmic droplets in the epididymis revealed that proximal droplets start migrating toward a distal position in the caput epididymis and that the process was mostly completed by the time sperm reached the cauda epididymis. The proportion of morphologically normal sperm in the ejaculate was 35.6%; the most prevalent sperm defects were distal cytoplasmic droplets and bent/coiled tails. The morphology of abnormal sperm and the underlying ultrastructural defects were similar to that in other large domestic animals thus suggesting similar underlying pathogenesis of specific sperm defects and similar effects on fertility.     

Intramembrane client recognition potentiates the chaperone functions of calnexin

One‐third of the human proteome is comprised of membrane proteins, which are particularly vulnerable to misfolding and often require folding assistance by molecular chaperones. Calnexin (CNX), which engages client proteins via its sugar‐binding lectin domain, is one of the most abundant ER chaperones, and plays an important role in membrane protein biogenesis. Based on mass spectrometric analyses, we here show that calnexin interacts with a large number of nonglycosylated membrane proteins, indicative of additional nonlectin binding modes. We find that calnexin preferentially bind misfolded membrane proteins and that it uses its single transmembrane domain (TMD) for client recognition. Combining experimental and computational approaches, we systematically dissect signatures for intramembrane client recognition by calnexin, and identify sequence motifs within the calnexin TMD region that mediate client binding. Building on this, we show that intramembrane client binding potentiates the chaperone functions of calnexin. Together, these data reveal a widespread role of calnexin client recognition in the lipid bilayer, which synergizes with its established lectin‐based substrate binding. Molecular chaperones thus can combine different interaction modes to support the biogenesis of the diverse eukaryotic membrane proteome.     

Sensitivity of methods for estimating breeding values using genetic markers to the number of QTL and distribution of QTL variance

The objective of this simulation study was to compare the effect of the number of QTL and distribution of QTL variance on the accuracy of breeding values estimated with genomewide markers (MEBV). Three distinct methods were used to calculate MEBV: a Bayesian Method (BM), Least Angle Regression (LARS) and Partial Least Square Regression (PLSR). The accuracy of MEBV calculated with BM and LARS decreased when the number of simulated QTL increased. The accuracy decreased more when QTL had different variance values than when all QTL had an equal variance. The accuracy of MEBV calculated with PLSR was affected neither by the number of QTL nor by the distribution of QTL variance. Additional simulations and analyses showed that these conclusions were not affected by the number of individuals in the training population, by the number of markers and by the heritability of the trait. Results of this study show that the effect of the number of QTL and distribution of QTL variance on the accuracy of MEBV depends on the method that is used to calculate MEBV.     

Production of Diamino propionic acid ammonia lyase by a new strain of Salmonella typhimurium PU011

Background  

Seeds of the legume plant Lathyrus sativus, which is grown in arid and semi arid tropical regions, contain Diamino Propionic acid (DAP). DAP is a neurotoxin, which, when consumed, causes a disease called Lathyrism. Lathryrism may manifest as Neurolathyrism or Osteolathyrism, in which the nervous system, and bone formation respectively, are affected. DAP ammonia lyase is produced by a few microorganisms such as Salmonella typhi, Salmonella typhimurium and Pseudomonas, and is capable of detoxifying DAP.