REEVALUATION OF THE NITROGEN FIXATION BEHAVIOR IN THE MARINE NON‐HETEROCYSTOUS CYANOBACTERIUM LYNGBYA MAJUSCULA1

Lyngbya majuscula Harvey ex Gomont is a common marine cyanobacterium in tropical and subtropical near‐shore waters. A few reports have indicated that L. majuscula fixes nitrogen only in the light. Because this feature is uncommon among non‐heterocystous cyanobacteria, we attempted a reevaluation. Nitrogenase activity, regulation, and localization were examined over diel cycles on natural populations of L. majuscula growing in subtidal zones off Zanzibar in the western Indian Ocean. The data show that L. majuscula fixed nitrogen and synthesized nitrogenase in all cells during the dark phase of a diel cycle. During the light phase, nitrogenase was degraded to undetectable levels.     

Distance between south-European and south-west Asiatic refugial areas involved morphological differentiation: Pinus sylvestris case study

The phenotypic differentiation of relic P. sylvestris in southern Europe and southwestern Asia was verified using thirty-two populations sampled from the Iberian Peninsula, Massif Central, Balkan Peninsula, Crimea and Anatolia. Twenty-one morphological and anatomical needle traits and 18 cone morphological characteristics were examined to describe the population diversity and differentiation. The needle characters were not correlated to those of cone. The differences between regions were significant based on 12 needle and 9 cone characteristics, suggesting spatial isolation. The differentiation between the Iberian and Anatolian populations was the highest, which indicates the isolation by distance. The high level of morphological differentiation was also found among Iberian populations, supporting the already known complex history of the species in that region. Populations within other regions were differentiated at lower levels; however, the West Anatolian populations differed morphologically from the eastern ones. The described pattern of morphological differentiation supports the idea of the long-lasting existence of P. sylvestris in the south-European and Anatolian mountain regions. To conserve this variation, seed transfer between regions in the forest economy should be restricted.     

Interaction of the HOPS complex with Syntaxin 17 mediates autophagosome clearance in Drosophila

Homotypic fusion and vacuole protein sorting (HOPS) is a tethering complex required for trafficking to the vacuole/lysosome in yeast. Specific interaction of HOPS with certain SNARE (soluble NSF attachment protein receptor) proteins ensures the fusion of appropriate vesicles. HOPS function is less well characterized in metazoans. We show that all six HOPS subunits (Vps11 [vacuolar protein sorting 11]/CG32350, Vps18/Dor, Vps16A, Vps33A/Car, Vps39/CG7146, and Vps41/Lt) are required for fusion of autophagosomes with lysosomes in Drosophila. Loss of these genes results in large-scale accumulation of autophagosomes and blocks autophagic degradation under basal, starvation-induced, and developmental conditions. We find that HOPS colocalizes and interacts with Syntaxin 17 (Syx17), the recently identified autophagosomal SNARE required for fusion in Drosophila and mammals, suggesting their association is critical during tethering and fusion of autophagosomes with lysosomes. HOPS, but not Syx17, is also required for endocytic down-regulation of Notch and Boss in developing eyes and for proper trafficking to lysosomes and eye pigment granules. We also show that the formation of autophagosomes and their fusion with lysosomes is largely unaffected in null mutants of Vps38/UVRAG (UV radiation resistance associated), a suggested binding partner of HOPS in mammals, while endocytic breakdown and lysosome biogenesis is perturbed. Our results establish the role of HOPS and its likely mechanism of action during autophagy in metazoans.     

Inhibition of Staphylococcus aureus cysteine proteases by human serpin potentially limits staphylococcal virulence

Bacterial proteases are considered virulence factors and it is presumed that by abrogating their activity, host endogenous protease inhibitors play a role in host defense against invading pathogens. Here we present data showing that Staphylococcus aureus cysteine proteases (staphopains) are efficiently inhibited by Squamous Cell Carcinoma Antigen 1 (SCCA1), an epithelial-derived serpin. The high association rate constant (k(ass)) for inhibitory complex formation (1.9×10(4) m/s and 5.8×10(4) m/s for staphopain A and staphopain B interaction with SCCA1, respectively), strongly suggests that SCCA1 can regulate staphopain activity in vivo at epithelial surfaces infected/colonized by S. aureus. The mechanism of staphopain inhibition by SCCA1 is apparently the same for serpin interaction with target serine proteases whereby the formation of a covalent complex result in cleavage of the inhibitory reactive site peptide bond and associated release of the C-terminal serpin fragment. Interestingly, the SCCA1 reactive site closely resembles a motif in the reactive site loop of native S. aureus-derived inhibitors of the staphopains (staphostatins). Given that S. aureus is a major pathogen of epithelial surfaces, we suggest that SCCA1 functions to temper the virulence of this bacterium by inhibiting the staphopains.     

Basic energetic parameters of Acanthamoeba castellanii mitochondria and their resistance to oxidative stress

The purpose of this study was establishing the basic energetic parameters of amoeba Acanthamoeba castellanii mitochondria respiring with malate and their response to oxidative stress caused by hydrogen peroxide in the presence of Fe(2+) ions. It appeared that, contrary to a previous report (Trocha LK, Stobienia O (2007) Acta Biochim Polon 54: 797), H(2)O(2)-treated mitochondria of A. castellanii did not display any substantial impairment. No marked changes in cytochrome pathway activity were found, as in the presence of an inhibitor of alternative oxidase no effects were observed on the rates of uncoupled and phosphorylating respiration and on coupling parameters. Only in the absence of the alternative oxidase inhibitor, non-phosphorylating respiration progressively decreased with increasing concentration of H(2)O(2), while the coupling parameters (respiratory control ratio and ADP/O ratio) slightly improved, which may indicate some inactivation of the alternative oxidase. Moreover, our results show no change in membrane potential, Ca(2+) uptake and accumulation ability, mitochondrial outer membrane integrity and cytochrome c release for 0.5-25 mM H(2)O(2)-treated versus control (H(2)O(2)-untreated) mitochondria. These results indicate that short (5 min) incubation of A. castellanii mitochondria with H(2)O(2) in the presence of Fe(2+) does not damage their basic energetics.     

Relationships of morphological and phototextural attributes of presumptive ovine zygotes and early embryos to their developmental competence in vitro: a preliminary assessment using time-lapse imaging

The assessment of morphology and digital image opacity may provide valuable information on the present embryo quality. Time-lapse imaging has been employed in research to establish a means of monitoring the dynamic nature of preimplantation embryo development. The aim of present study was to use time-lapse imaging for assessing various prospective morphometric and phototextural markers of the developmental potential of in vitro-derived ovine embryos. Oocytes were obtained by scarification of ovaries from nine Polish Longwool ewes. After in vitro maturation (IVM) and fertilization (IVF) of oocytes with fresh ram semen, the development of embryos to the blastocyst stage was monitored and evaluated using Primo Vision time-lapse imaging technology. Commercially available Image-Pro^® Plus software was used to measure zona pellucida thickness, embryo diameter, total area of the perivitelline space, cellular grey-scale pixel intensity and cellular pixel heterogeneity. Statistical assessment of all attributes was done at various time points during embryo development (i.e., presumptive zygote stage: t(0); first cleavage detected at t(2) or t(3); and second cleavage detected at t(4) or t(6)). Out of thirty-seven zygotes analyzed in this study, five did not divide, 26 arrested before and six developed to the blastocyst stage. Our present results indicate that most parameters analyzed did not differ among embryos varying in their developmental fate except for the perivitelline space area that was greater (P<0.05) for non-dividing zygotes than future blastocysts at the presumptive zygote stage (4040±1850 vs. 857±262 µm², respectively; means±SEM). Consequently, the measurement of perivitelline space at t(0) can potentially be used to prognosticate developmental potential of in vitro-produced ovine embryos albeit further confirmational studies are needed.     

Gene Expression Profiles of Chicken Embryo Fibroblasts in Response to Salmonella Enteritidis Infection

The response of chicken to non-typhoidal Salmonella infection is becoming well characterised but the role of particular cell types in this response is still far from being understood. Therefore, in this study we characterised the response of chicken embryo fibroblasts (CEFs) to infection with two different S. Enteritidis strains by microarray analysis. The expression of chicken genes identified as significantly up- or down-regulated (≥3-fold) by microarray analysis was verified by real-time PCR followed by functional classification of the genes and prediction of interactions between the proteins using Gene Ontology and STRING Database. Finally the expression of the newly identified genes was tested in HD11 macrophages and in vivo in chickens. Altogether 19 genes were induced in CEFs after S. Enteritidis infection. Twelve of them were also induced in HD11 macrophages and thirteen in the caecum of orally infected chickens. The majority of these genes were assigned different functions in the immune response, however five of them (LOC101750351, K123, BU460569, MOBKL2C and G0S2) have not been associated with the response of chicken to Salmonella infection so far. K123 and G0S2 were the only ’non-immune’ genes inducible by S. Enteritidis in fibroblasts, HD11 macrophages and in the caecum after oral infection. The function of K123 is unknown but G0S2 is involved in lipid metabolism and in β-oxidation of fatty acids in mitochondria.