Effect of the luteinizing hormone on embryo production in superovulated rabbit does

For most domestic animals, the responses to superovulation treatments are not controlled as a consequence of the lack of knowledge on exogenous gonadotrophins effects on the ovarian function. The role of luteinizing hormone (LH) on the number and quality of embryos produced was evaluated on rabbit does superovulated with porcine FSH (pFSH). Parameters of embryos recovery, in vitro and in vivo embryo development rates after freezing/thawing were compared. We used three experimental groups: (1) control group without superovulation treatment, (2) "pFSH+pLH" and (3) "pFSH" groups where females were treated with pFSH, respectively, with (20%) or without (0%) porcine LH supplementation. The number of corpora lutea and the number of embryos produced were significantly higher (p<0.001) in superovulated does than in control group (27.1, 26.7 versus 11.9 corpora lutea and 20.3, 21.2 versus 9.6 embryos produced for pFSH+pLH, pFSH and control group, respectively). However, both gonadotrophins administrations (groups 2 and 3) led to defaults of ovulation when compared with untreated does. No significant difference was observed between the number and quality of the embryos produced by does treated with pFSH+pLH or with pFSH alone. Moreover, we observed no significant difference between results of in vivo and in vitro viability assays after thawing. We concluded that pFSH alone seems to be sufficient to stimulate the follicles growth and that exogenous pLH administrated has no effect on the quantity and quality of embryos. Further studies are needed to evaluate the hormonal patterns before and after the gonadotrophins injections in the rabbit species.     

Evaluation of two library-independent microbial source tracking methods to identify sources of fecal contamination in French estuaries

In order to identify the origin of the fecal contamination observed in French estuaries, two library-independent microbial source tracking (MST) methods were selected: (i) Bacteroidales host-specific 16S rRNA gene markers and (ii) F-specific RNA bacteriophage genotyping. The specificity of the Bacteroidales markers was evaluated on human and animal (bovine, pig, sheep, and bird) feces. Two human-specific markers (HF183 and HF134), one ruminant-specific marker (CF193'), and one pig-specific marker (PF163) showed a high level of specificity (>90%). However, the data suggest that the proposed ruminant-specific CF128 marker would be better described as an animal marker, as it was observed in all bovine and sheep feces and 96% of pig feces. F RNA bacteriophages were detected in only 21% of individual fecal samples tested, in 60% of pig slurries, but in all sewage samples. Most detected F RNA bacteriophages were from genotypes II and III in sewage samples and from genotypes I and IV in bovine, pig, and bird feces and from pig slurries. Both MST methods were applied to 28 water samples collected from three watersheds at different times. Classification of water samples as subject to human, animal, or mixed fecal contamination was more frequent when using Bacteroidales markers (82.1% of water samples) than by bacteriophage genotyping (50%). The ability to classify a water sample increased with increasing Escherichia coli or enterococcus concentration. For the samples that could be classified by bacteriophage genotyping, 78% agreed with the classification obtained from Bacteroidales markers.     

Cutting Edge: IFN-gamma-producing CD4 T lymphocytes mediate spore-induced immunity to capsulated Bacillus anthracis

Virulent strains of Bacillus anthracis produce immunomodulating toxins and an antiphagocytic capsule. The toxin component-protective Ag is a key target of the antianthrax immune response that induces production of toxin-neutralizing Abs. Coimmunization with spores enhances the antitoxin vaccine, and inactivated spores alone confer measurable protection. We aimed to identify the mechanisms of protection induced in inactivated-spore immunized mice that function independently of the toxin/antitoxin vaccine system. This goal was addressed with humoral and CD4 T lymphocyte transfer, in vivo depletion of CD4 T lymphocytes and IFN-gamma, and Ab-deficient (muMT(-/-)) or IFN-gamma-insensitive (IFN-gammaR(-/-)) mice. We found that humoral immunity did not protect from nontoxinogenic capsulated bacteria, whereas a cellular immune response by IFN-gamma-producing CD4 T lymphocytes protected mice. These results are the first evidence of protective cellular immunity against capsulated B. anthracis and suggest that future antianthrax vaccines should strive to augment cellular adaptive immunity.     

Characterization of Saccharomyces cerevisiae Npa2p (Urb2p) reveals a low-molecular-mass complex containing Dbp6p, Npa1p (Urb1p), Nop8p, and Rsa3p involved in early steps of 60S ribosomal subunit biogenesis

We report the characterization of the yeast Npa2p (Urb2p) protein, which is essential for 60S ribosomal subunit biogenesis. We identified this protein in a synthetic lethal screening with the rsa3 null allele. Rsa3p is a genetic partner of the putative RNA helicase Dbp6p. Mutation or depletion of Npa2p leads to a net deficit in 60S subunits and a decrease in the levels all 27S pre-rRNAs and mature 25S and 5.8S rRNAs. This is likely due to instability of early pre-60S particles. Consistent with a role of Npa2p in 60S subunit biogenesis, green fluorescent protein-tagged Npa2p localizes predominantly to the nucleolus and TAP-tagged Npa2p sediments with large complexes in sucrose gradients and is associated mainly with 27SA(2) pre-rRNA-containing preribosomal particles. In addition, we reveal a genetic synthetic interaction between Npa2p, several factors required for early steps of 60S subunit biogenesis (Dbp6p, Dbp7p, Dbp9p, Npa1p, Nop8p, and Rsa3p), and the 60S protein Rpl3p. Furthermore, coimmunoprecipitation and gel filtration analyses demonstrated that at least Npa2p, Dbp6p, Npa1p, Nop8p, and Rsa3p are present together in a subcomplex of low molecular mass whose integrity is independent of RNA. Our results support the idea that these five factors work in concert during the early steps of 60S subunit biogenesis.     

Localisation of caveolin in mammary tissue depends on cell type

Caveolins, components of caveolae, are expressed in mammary tissue. In order to determine whether caveolins are present in different mammary cell types and whether their localisation depends on the physiological stage or species, cav-1 and cav-2 were characterised by immunoblotting in mammary tissues from the mouse, ewe and rabbit and localised, by immunofluorescence and electron microscopy, in mammary tissues from the mouse and ewe. At all the physiological stages studied, cav-1 and cav-2 were present in endothelial and myoepithelial cells in which flask-shaped caveolae were abundant. However, labelling of cav-1 and cav-2 associated with small vesiculo-tubular structures (including those close to lipid droplets) was low in epithelial cells. To study the possible association of cav-1 with lipid droplets, lactating ewe mammary fragments were treated in vitro with brefeldin A. This treatment did not modify the association of cav-1-labelled structures with lipid droplets. Finally, HC11 and MCF-10A mammary cell lines were treated with oleic acid. The total quantity of cav-1 was little affected by the treatment, although the lipid droplet labelling of cav-1 was amplified in MCF-10A cells. Thus, the synthesis and localisation of caveolins are mostly dependent upon the cell types of mammary tissue and upon their state of differentiation.     

Molecular Characterization of a Novel Bacteriocin and an Unusually Large Aggregation Factor of <Emphasis Type="Italic">Lactobacillus paracasei</Emphasis> subsp. <Emphasis Type="Italic">paracasei</Emphasis> BGSJ2-8, a Natural Isolate from Homemade Cheese

Screening the collection of natural isolates from semi-hard homemade cheese resulted in isolation and characterization of strain Lactobacillus paracasei subsp. paracasei BGSJ2-8. The strain BGSJ2-8 harbors several important phenotypes, such as bacteriocin production, aggregation phenomenon, and production of proteinase. Bacteriocin SJ was purified by three-step chromatography. Mass spectrometry established molecular mass of the active peptide at 5372 Da. The auto-aggregation phenotype of wild-type (WT) strain was mediated by secreted aggregation-promoting factor (protein of molecular mass > 200 kDa), probably acting in cooperation with other cell surface protein(s). Comparative study of WT and its spontaneous nonaggregating derivative revealed that aggregation factor was responsible for the observed differences in the bacteriocin and proteinase activities. Bacteriocin SJ activity and resistance to different stresses were higher in the presence of aggregating factor. In contrast, proteinase activity was stronger in the nonaggregating derivative.     

The cardiovascular paradox of pregnancy

Despite widespread accessibility to prenatal care, little is known on the mechanisms initiating early maternal adaptation to pregnancy. Moreover, preeclampsia and intrauterine growth retardation remain the most frequent and serious complications of pregnancy. Recent studies, both in humans and in laboratory animals, have shown that very early events in gestation may be important determinants for the continuation of healthy pregnancy. Certain of these early adaptations appear to be linked to the corpus luteum of pregnancy, as ovarian steroid hormones (especially progesterone) would set the basic hemodynamic conditions, more specifically, generalized vasodilation. This new hemodynamic setup initiates a vicious cycle in which the renin - angiotensin - aldosterone system is activated, together with the resetting of the control of antidiuretic hormone secretion relative to plasma osmolality. This leads to a gradual and substantial increase in plasma volume and a parallel increase in cardiac function (both heart rate and stroke volume) with the goal of maintaining blood pressure in the face of the generalised vasodilation. This includes the creation of a functional arterio-venous shunt represented by the utero-placental circulation. By the end of the first trimester, the decrease in peripheral vascular resistance is marked relative to the increase in cardiac output, resulting in a significant decrease in blood pressure that will be maintained until the third trimester. It is proposed that in preeclampsia, these very early events (vasodilation - increased plasma volume) fail to occur, resulting in an absence of the usual decrease in blood pressure, which is normally seen in the second trimester of pregnancy, and hypertension in the third trimester. Experimental animals, especially the rat, are suitable models to study this early maternal adaptation to pregnancy, since both endocrine and hemodynamic changes appear to be similar to humans.     

Incidence of deep vein thrombosis related to peripherally inserted central catheters in children and adolescents

Background

Peripherally inserted central catheters (PICC) in children and adolescents are being used with increasing frequency. We sought to determine the incidence and characterize risk factors of deep vein thrombosis associated with peripherally inserted central catheters in a pediatric population.

Methods

We conducted a prospective study involving consecutive patients referred to the radiology department of a tertiary care university-affiliated hospital for insertion of a peripherally inserted central catheter. We included patients aged 18 years or less who weighed more than 2.5 kg and had a peripherally inserted central catheter successfully inserted in his or her arm between June 2004 and November 2005. The primary outcome was the occurrence of partial or complete deep vein thrombosis evaluated by clinical examination, ultrasonography and venous angiography.

Results

A total of 214 patients (101 girls, 113 boys) were included in the study. Partial or complete deep vein thrombosis occurred in 20 patients, for an incidence of 93.5 per 1000 patients and 3.85 per 1000 catheter-days. Only 1 of the cases was symptomatic. In the univariable analyses, the only variable significantly associated with deep vein thrombosis was the presence of factor II mutation G20210A (odds ratio 7.08, 95% confidence interval 1.11–45.15, p = 0.04), a genetic mutation that increases the risk of a blood clot and that was present in 5 (2.3%) of the 214 patients.

Interpretation

The incidence of deep vein thrombosis related to peripherally inserted central catheters in our study was lower than the incidence related to centrally inserted venous catheters described in the pediatric literature (11%–50%).Most data available in the adult and pediatric literature on the incidence of deep vein thrombosis concern centrally inserted venous catheters, which are inserted directly in a central vein (jugular, subclavian or femoral). Typical symptoms of deep vein thrombosis are frequently absent in children and adolescents. Although the diagnosis of deep vein thrombosis is more reliable when based on Doppler ultrasonography or venous angiography,^1–10 in most studies these diagnostic tests were performed only when patients presented clinical symptoms of deep vein thrombosis or catheter dysfunction. In studies focused on the pediatric population, the frequency of deep vein thrombosis related to centrally inserted venous catheters has varied from 11% to 50%.^1,5,6,8,11In the past 10 years, peripherally inserted central catheters (PICC) have been used with increasing frequency in children and adolescents. The catheter is inserted percutaneously via a peripheral vein, with its tip residing in the superior vena cava. The main indications for this type of catheter insertion are difficult venous access, home intravenous antibiotic therapy, administration of chemotherapy or other hyperosmolar solution and long-term parenteral nutrition. The risk of deep vein thrombosis related to peripherally inserted central catheters could be greater among children and adolescents than among adults, given the size of the veins. Several studies have published complications related to peripherally inserted central catheters,^12–20 but few focused on the pediatric population.^13–21 Furthermore, in all of these studies, screening for deep vein thrombosis was not systematic. The real incidence of deep vein thrombosis related to peripherally inserted central catheters and their complications in the pediatric population are therefore unknown. We conducted this study to determine the incidence and characterize the risk factors of deep vein thrombosis related to peripherally inserted central catheters in children and adolescents in our institution.     

Contribution of two ionotropic purinergic receptors to ATP responses in submandibular gland ductal cells

The effect of extracellular ATP on salivary gland function was compared in wild-type (WT) and P2X(7) knockout (KO) mice. The increase in the intracellular concentration of calcium ([Ca(2+)](i)) in response to carbachol was similar in submandibular ductal cells of WT and KO mice. ATP and its analog, benzoyl-ATP, induced a sustained increase in the [Ca(2+)](i) in WT animals. In KO mice, ATP slightly and transiently increased the [Ca(2+)](i) and benzoyl-ATP had no effect. The response to ATP of WT but not KO mice was blocked by KN-62, Coomassie blue and magnesium. The small response of ATP observed in KO mice was completely blocked in the absence of extracellular calcium, unchanged by U73122 and potentiated by ivermectin indicating the probable involvement of a P2X(4) receptor. A RT-PCR and a Western blot confirmed the presence of these receptors in ducts of both WT and KO mice. ATP increased the permeability of the cells to ethidium bromide and stimulated a phospholipase A(2) activity in WT but not KO mice. Mice submandibular gland cells secreted IL-1beta but this secretion was not modified by ATP and was similar in both groups of animals. The volume of saliva provoked by pilocarpine and the concentration of proteins, sodium and chloride in this saliva was similar in both groups of animals. The concentration of potassium was higher in KO mice. We can conclude that the major purinergic receptors expressed in mice submandibular ductal cells are P2X(7) receptors but that P2X(4) receptors are also involved in some ATP effects.