Uterine and fetal asphyxia monitoring in parturient sows treated with oxytocin

Oxytocin is used to induce and control parturition, nevertheless, the increase of uterine contractions decreases blood flow and gaseous exchange through the womb predisposing to intra-partum mortality. The objective of the present study was to evaluate the effect of oxytocin on myometrial activity, fetal intrauterine hypoxia and postnatal asphyxia in sows during farrowing. Hybrid (n = 120) sows approaching the time of farrowing were randomly assigned in two groups of 60 animals each. Group I (G(1): control) was treated IM with saline solution and Group II (G(2)) was injected IM with oxytocin (1IU/6kg LW) as a single dose at birth of the first piglet. Both average number of myometrial contractions and intensity in G(2) were greater (P < 0.01) as compared with G(1). The mean of intra-partum stillbirths (IPS's) and those where fetal cardiac frequency (FCF) or heart beats, could not be detected after birth, were greater (P < 0.01) in G(2) as compared with G(1). The average decelerations of FCF known as dips II, which indicate severe hypoxia, was greater in G(2) (P < 0.01) as compared with that of G(1). There was a greater (P < 0.01) number of intra-partum stillbirths, stained with severe meconium in G(2) when compared with G(1). Oxytocin treatment increased (P < 0.01) the number of pigs born alive with ruptured umbilical cords and those with different grades of meconium staining on their skin. It was concluded that administration of oxytocin at the onset of parturition increased the myometrial activity, decreased fetal cardiac frequency, predisposed the rupture of umbilical cords and the degree of meconium staining, and increased intra-partum mortality.     

Identification and characterization of putative virulence genes and gene clusters in Aeromonas hydrophila PPD134/91

Aeromonas hydrophila is a gram-negative opportunistic pathogen of animals and humans. The pathogenesis of A. hydrophila is multifactorial. Genomic subtraction and markers of genomic islands (GIs) were used to identify putative virulence genes in A. hydrophila PPD134/91. Two rounds of genomic subtraction led to the identification of 22 unique DNA fragments encoding 19 putative virulence factors and seven new open reading frames, which are commonly present in the eight virulence strains examined. In addition, four GIs were found, including O-antigen, capsule, phage-associated, and type III secretion system (TTSS) gene clusters. These putative virulence genes and gene clusters were positioned on a physical map of A. hydrophila PPD134/91 to determine their genetic organization in this bacterium. Further in vivo study of insertion and deletion mutants showed that the TTSS may be one of the important virulence factors in A. hydrophila pathogenesis. Furthermore, deletions of multiple virulence factors such as S-layer, serine protease, and metalloprotease also increased the 50% lethal dose to the same level as the TTSS mutation (about 1 log) in a blue gourami infection model. This observation sheds light on the multifactorial and concerted nature of pathogenicity in A. hydrophila. The large number of putative virulence genes identified in this study will form the basis for further investigation of this emerging pathogen and help to develop effective vaccines, diagnostics, and novel therapeutics.     

Staphylococcus aureus develops an alternative, ica-independent biofilm in the absence of the arlRS two-component system

The biofilm formation capacity of Staphylococcus aureus clinical isolates is considered an important virulence factor for the establishment of chronic infections. Environmental conditions affect the biofilm formation capacity of S. aureus, indicating the existence of positive and negative regulators of the process. The majority of the screening procedures for identifying genes involved in biofilm development have been focused on genes whose presence is essential for the process. In this report, we have used random transposon mutagenesis and systematic disruption of all S. aureus two-component systems to identify negative regulators of S. aureus biofilm development in a chemically defined medium (Hussain-Hastings-White modified medium [HHWm]). The results of both approaches coincided in that they identified arlRS as a repressor of biofilm development under both steady-state and flow conditions. The arlRS mutant exhibited an increased initial attachment as well as increased accumulation of poly-N-acetylglucosamine (PNAG). However, the biofilm formation of the arlRS mutant was not affected when the icaADBC operon was deleted, indicating that PNAG is not an essential compound of the biofilm matrix produced in HHWm. Disruption of the major autolysin gene, atl, did not produce any effect on the biofilm phenotype of an arlRS mutant. Epistatic experiments with global regulators involved in staphylococcal-biofilm formation indicated that sarA deletion abolished, whereas agr deletion reinforced, the biofilm development promoted by the arlRS mutation.     

Diacylglycerol kinase alpha regulates the secretion of lethal exosomes bearing Fas ligand during activation-induced cell death of T lymphocytes

Fas ligand (FasL) mediates both apoptotic and inflammatory responses in the immune system. FasL function critically depends on the different forms of FasL; soluble Fas ligand lacking the transmembrane and cytoplasmic domains is a poor mediator of apoptosis, whereas full-length, membrane-associated FasL (mFasL) is pro-apoptotic. mFasL can be released from T lymphocytes, via the secretion of mFasL-bearing exosomes. mFasL in exosomes retains its activity in triggering Fas-dependent apoptosis, providing an alternative mechanism of cell death that does not necessarily imply cell-to-cell contact. Diacylglycerol kinase alpha (DGKalpha), a diacylglycerol (DAG)-consuming enzyme, is involved in the attenuation of DAG-derived responses initiated at the plasma membrane that lead to T lymphocyte activation. Here we studied the role of DGKalpha on activation-induced cell death on a T cell line and primary T lymphoblasts. The inhibition of DGKalpha increases the secretion of lethal exosomes bearing mFas ligand and subsequent apoptosis. On the contrary, the overactivation of the DGKalpha pathway inhibits exosome secretion and subsequent apoptosis. DGKalpha was found associated with the trans-Golgi network and late endosomal compartments. Our results support the hypothesis that the DGKalpha effect on apoptosis occurs via the regulation of the release of lethal exosomes by the exocytic pathway, and point out that the spatial orchestration of the different pools of DAG (plasma membrane and Golgi membranes) by DGKalpha is crucial for the control of cell activation and also for the regulation of the secretion of lethal exosomes, which in turn controls cell death.     

The incorporation of glucosamine into enterobacterial core lipopolysaccharide: two enzymatic steps are required

The core lipopolysaccharide (LPS) of Klebsiella pneumoniae is characterized by the presence of disaccharide alphaGlcN-(1,4)-alphaGalA attached by an alpha1,3 linkage to l-glycero-d-manno-heptopyranose II (ld-HeppII). Previously it has been shown that the WabH enzyme catalyzes the incorporation of GlcNAc from UDP-GlcNAc to outer core LPS. The presence of GlcNAc instead of GlcN and the lack of UDP-GlcN in bacteria indicate that an additional enzymatic step is required. In this work we identified a new gene (wabN) in the K. pneumoniae core LPS biosynthetic cluster. Chemical and structural analysis of K. pneumoniae non-polar wabN mutants showed truncated core LPS with GlcNAc instead of GlcN. In vitro assays using LPS truncated at the level of d-galacturonic acid (GalA) and cell-free extract containing WabH and WabN together led to the incorporation of GlcN, whereas none of them alone were able to do it. This result suggests that the later enzyme (WabN) catalyzes the deacetylation of the core LPS containing the GlcNAc residue. Thus, the incorporation of the GlcN residue to core LPS in K. pneumoniae requires two distinct enzymatic steps. WabN homologues are found in Serratia marcescens and some Proteus strains that show the same disaccharide alphaGlcN-(1,4)-alphaGalA attached by an alpha1,3 linkage to ld-HeppII.     

Indicators of physiological stress and the elaboration of sexual traits in the collared flycatcher

Stress may have consequences for the evolution of condition-dependentsexual traits. For example, stress may be related to sexualtraits through immune function, and sexual traits can reflecthow individuals bear the costs of stress-mediated immunosuppression.However, male traits may be directly associated with stress,and such traits would then indicate stress tolerance. Here,we present initial results for the relationship between physiologicalstress estimated by the levels of heat shock proteins (HSP60and HSP70) and heterophil/lymphocyte ratio and the elaborationof sexual traits, such as forehead and wing patch size and songfeatures in the collared flycatcher Ficedula albicollis. Malesproducing longer and more versatile songs had significantlyhigher levels of HSP70, but other traits were unrelated to stress.In general, effect sizes for the relationship between stressand sexual traits had broad confidence intervals and variedbetween being small and medium effects. Immunoglobulin levels,leukocyte abundance, haemoparasite prevalence, male age, anddate and time effects did not affect the relationship betweenstress and sexual traits. These preliminary results, servinga basis for further experimental studies indicate that the relationshipbetween sexual traits and stress does not seem to be strong,but stress may partially constrain the expression of some sexualtraits.     

Experimental evidence that egg color indicates female condition at laying in a songbird

The signaling hypothesis of eggshell coloration in birds isbased on the assumption that females of species with blue-greeneggs signal their phenotypic quality to their mates throughdeposition of the antioxidant biliverdin as pigment. Egg pigmentationmay be an expression of the condition of females at laying orof genetic linkages between egg color and female performancevariables. We have supplemented 16 pied flycatcher, Ficedulahypoleuca, females with mealworms before and during laying andcompared the mass and color of their eggs as measured on theday of laying to those of 16 control females with the same nestconstruction and laying dates and clutch sizes. Supplementedfemales laid significantly heavier and more intensely blue-greeneggs than control females. Egg blue-green chroma was significantlyassociated with the amount of biliverdin in eggshells. Egg color,and thus biliverdin content, is an expression of female conditionat laying.     

Stoichiometric model and metabolic flux analysis for Leptospirillum ferrooxidans

A metabolic model for Leptospirillum ferrooxidans was developed based on the genomic information of an analogous iron oxidizing bacteria and on the pathways of ferrous iron oxidation, nitrogen and CO₂ assimilation based on experimental evidence for L. ferrooxidans found in the literature. From this metabolic reconstruction, a stoichiometric model was built, which includes 86 reactions describing the main catabolic and anabolic aspects of its metabolism. The model obtained has 2 degrees of freedom, so two external fluxes were estimated to achieve a determined and observable system. By using the external oxygen consumption rate and the generation flux biomass as input data, a metabolic flux map with a distribution of internal fluxes was obtained. The results obtained were verified with experimental data from the literature, achieving a very good prediction of the metabolic behavior of this bacterium at steady state. Biotechnol. Bioeng. 2010;107:696–706. © 2010 Wiley Periodicals, Inc.     

Polar flagellum biogenesis in Aeromonas hydrophila

Mesophilic Aeromonas spp. constitutively express a single polar flagellum that helps the bacteria move to more favorable environments and is an important virulence and colonization factor. Certain strains can also produce multiple lateral flagella in semisolid media or over surfaces. We have previously reported 16 genes (flgN to flgL) that constitute region 1 of the Aeromonas hydrophila AH-3 polar flagellum biogenesis gene clusters. We identified 39 new polar flagellum genes distributed in four noncontiguous chromosome regions (regions 2 to 5). Region 2 contained six genes (flaA to maf-1), including a modification accessory factor gene (maf-1) that has not been previously reported and is thought to be involved in glycosylation of polar flagellum filament. Region 3 contained 29 genes (fliE to orf29), most of which are involved in flagellum basal body formation and chemotaxis. Region 4 contained a single gene involved in the motor stator formation (motX), and region 5 contained the three master regulatory genes for the A. hydrophila polar flagella (flrA to flrC). Mutations in the flaH, maf-1, fliM, flhA, fliA, and flrC genes, as well as the double mutant flaA flaB, all caused loss of polar flagella and reduction in adherence and biofilm formation. A defined mutation in the pomB stator gene did not affect polar flagellum motility, in contrast to the motX mutant, which was unable to swim even though it expressed a polar flagellum. Mutations in all of these genes did not affect lateral flagellum synthesis or swarming motility, showing that both A. hydrophila flagellum systems are entirely distinct.     

Characterization of the last step of lignin biosynthesis in Zinnia elegans suspension cell cultures

The last step of lignin biosynthesis in Zinnia elegans suspension cell cultures (SCCs) catalyzed by peroxidase (ZePrx) has been characterized. The k(3) values shown by ZePrx for the three monolignols revealed that sinapyl alcohol was the best substrate, and were proportional to their oxido/reduction potentials, signifying that these reactions are driven exclusively by redox thermodynamic forces. Feeding experiments demonstrate that cell wall lignification in SCCs is controlled by the rate of supply of H(2)O(2). The results also showed that sites for monolignol beta-O-4 cross-coupling in cell walls may be saturated, suggesting that the growth of the lineal lignin macromolecule is not infinite.