Leishmania antigens are presented to CD8+ T cells by a transporter associated with antigen processing-independent pathway in vitro and in vivo

CD8+ T cells are generated in response to Leishmania major (Lm) or Toxoplasma gondii parasitic infections, indicating that exogenously delivered Ag can be processed for presentation by MHC class I molecules. We show that presentation of Lm nucleotidase (NT)-OVA is TAP independent in vivo and in vitro, and is inhibited by chloroquine, but not by proteasome inhibitors. In contrast, the presentation of T. gondii P30-OVA relies on the TAP/proteasome pathway. Presentation of OVA- or rNT-OVA-coated beads also bypassed TAP requirement above a certain Ag threshold. TAP was also dispensable for the presentation of wild-type Lm Ags to primed CD8+ T cells in vitro. Finally, in vivo priming of CD8+ T cells involved in acquired resistance to Lm was not compromised in TAP-deficient mice. Thus, Leishmania Ags appear to be confined to an intraphagosomal processing pathway that requires higher concentrations of Ags, suggesting that these parasites may have evolved strategies to impair the efficient endoplasmic reticulum-based, TAP-dependent cross-presentation pathway to avoid or delay CD8+ T cell priming.     

Src-family kinases mediate an outside-in signal necessary for beta2 integrins to achieve full activation and sustain firm adhesion of polymorphonuclear leucocytes tethered on E-selectin

In cell suspensions subjected to high-shear rotatory motion, human PMN (polymorphonuclear cells) adhered to E-selectin-expressing CHO (Chinese-hamster ovary) cells (CHO-E), and formed homotypic aggregates when challenged by E-selectin-IgG fusion protein, by a mechanism that involved beta2 integrins. Both heterotypic and homotypic PMN adhesion was accompanied by tyrosine phosphorylation of a 110 kDa protein (P110). This event was prevented by blocking anti-(beta2 integrin) antibodies and by inhibitors of Src-family kinases, suggesting that it was part of an 'outside-in' signalling that was initiated by integrin engagement. Interestingly, Src-family kinase inhibitors prevented beta2-integrin-mediated (i) homotypic PMN adhesion triggered by E-selectin-IgG, (ii) heterotypic CHO-E/PMN adhesion in mixed-cell suspensions, and (iii) firm adhesion of PMN to CHO-E monolayers under physiological flow. Similarly to PMN treated with Src-family kinase inhibitors, PMN from hck-/-fgr-/- and hck-/-fgr-/-lyn-/- mice showed significant impairment of beta2-integrin-mediated adhesion to CHO-E. Moreover, the expression of beta2 integrin activation epitopes at the sites of cell-cell contact in CHO-E/PMN conjugates was abolished by Src-family kinase inhibitors. One component of P110 was identified as the FAK (focal adhesion kinase) Pyk2 (proline-rich tyrosine kinase 2), which was phosphorylated in a beta2 integrin- and Src-family-kinase-dependent manner. Thus, Src-family kinases, and perhaps Pyk2, mediate a signal necessary for beta2 integrin function in PMN tethered by E-selectin.     

High glucose increases B1-kinin receptor expression and signaling in endothelial cells

The loss of endothelial function is the initiating factor in the development of diabetic vascular disease. Kinins control endothelial function by the activation of two receptors: the B2 which is constitutively expressed, and the B1 which is highly induced in pathological conditions. In the present study, we observed that the levels of B1-receptor mRNA and protein are induced in endothelial cells incubated in high glucose. An increase in B1-receptor was also observed in the endothelial layer of aortas, from 4-week diabetic rats. When cells were grown in high glucose, the B1 agonist des-Arg9-BK increased nitrite levels, whereas in normal glucose nitrite levels were unchanged. Nitrite increase was blocked by L-NAME and 1400W indicating the participation of the inducible Nitric Oxide Synthase (iNOS). iNOS protein levels were also increased in high glucose. These results demonstrate the participation of the B1 receptor in the signaling pathways mediated by kinins in high glucose.     

αA-Crystallin and αB-Crystallin Reside in Separate Subcellular Compartments in the Developing Ocular Lens

αA-Crystallin (αA) and αB-crystallin (αB), the two prominent members of the small heat shock family of proteins are considered to be two subunits of one multimeric protein, α-crystallin, within the ocular lens. Outside of the ocular lens, however, αA and αB are known to be two independent proteins, with mutually exclusive expression in many tissues. This dichotomous view is buoyed by the high expression of αA and αB in the lens and their co-fractionation from lens extracts as one multimeric entity, α-crystallin. To understand the biological function(s) of each of these two proteins, it is important to investigate the biological basis of this perceived dichotomy; in this report, we address the question whether αA and αB exist as independent proteins in the ocular lens. Discontinuous sucrose density gradient fractionation and immunoconfocal localization reveal that in early developing rat lens αA is a membrane-associated small heat shock protein similar to αB but with remarkable differences. Employing an established protocol, we demonstrate that αB predominantly sediments with rough endoplasmic reticulum, whereas αA fractionates with smooth membranes. These biochemical observations were corroborated with immunogold labeling and transmission electron microscopy. Importantly, in the rat heart also, which does not contain αA, αB fractionates with rough endoplasmic reticulum, suggesting that αA has no influence on the distribution of αB. These data demonstrate presence of αA and αB in two separate subcellular membrane compartments, pointing to their independent existence in the developing ocular lens.     

Infant Mortality in Black-and-Gold Howlers (Alouatta caraya) Living in a Flooded Forest in Northeastern Argentina

Ecological and social factors have a significant effect on infant survivorship in nonhuman primates. We present 6293 group-months of infant birth and mortality data for 29 groups of Alouatta caraya inhabiting a flooded forest in northeastern Argentina, collected over 1.5–8?yr depending on the group. We tested whether infant mortality was a response to the effects of flooding on food availability and whether male takeovers resulted in greater opportunities for infanticide. During our study, 43 of 113 infants died at a mean age of 5?mo. In 24 cases the cause of death was unknown. In the remaining 19 cases infant deaths were attributed to periods of intense flooding (N?=?8), replacement of the breeding male (N?=?8), problems associated with birth (N?=?2), and injuries during an intergroup encounter (N?=?1). Flooding reduced the availability of mature leaves, which appeared to play an important role in the ability of mothers to nurse their offspring. Male replacements occurred in four social groups that contained only one fully adult male. Infant mortality was significantly higher in groups that experienced male replacement compared to groups without male replacement. These results indicate that infant mortality in Alouatta caraya is affected by several factors—natural disasters, maternal condition and food availability, infanticide after male replacement, and possibly disease and predation—each of which needs to be evaluated to understand the history and demography of this primate population.     

45th Discussion forum on LCA—environmentally extended input–output analysis and LCA, September 15, 2011, Berne, Switzerland

The discussion forum on life cycle assessment (LCA) on September 15, 2011, aimed at summarizing recent environmentally extended input?Coutput analysis (EE-IOA) and the combination with LCA for the computation of environmental impact of imports. Input?Coutput tables (IOT) represent the financial flows in a country or economic regions. Extending IOT with information on emissions and resource uses allows for the analysis of environmental impacts due to production and consumption activities in a country. This instrument is called EE-IOA. It enables the analysis of total environmental impacts of countries or economic regions. The combination with trade statistics and LCA was presented as an alternative to multiregional input?Coutput models for determining environmental impacts of imports over the whole life cycle. The 45th LCA forum gathered several international speakers who provided a broad and qualified view on the topic. The theoretical background, results for different countries and regions, uncertainties, and possible improvement options for EE-IOA were discussed. The following main conclusions were drawn at the end of the discussion forum: EE-IOA is a useful instrument for analyzing the total environmental impacts of countries and the main drivers of environmental impacts. As a next important step, the participants would like to see an increase in user friendliness of EE-IOA combined with LCA, e.g., by harmonizing data, data formats, and classifications.     

Chaenothecopsis quintralis, a new species of calicioid fungus

Chaenothecopsis quintralis from southwestern Argentina is described and illustrated as a new species in the family Mycocaliciaceae. It has been found in three localities in the Andean Patagonian temperate forests, growing strictly on dung of an endemic marsupial Dromiciops gliroides. The new species is distinguished by the hemispherical, black capitulum of ascoma, the presence of asci with croziers, one-celled brown ascospores, and its fimicolous habitat. Analysis of partial nuclear large subunit rDNA (LSU) sequences showed that this taxon is within Mycocaliciales.     

Flavodoxin displays dose-dependent effects on photosynthesis and stress tolerance when expressed in transgenic tobacco plants

Ferredoxins are iron–sulfur proteins involved in various one-electron transfer pathways. Ferredoxin levels decrease under adverse environmental conditions in photosynthetic organisms. In cyanobacteria, this decline is compensated by induction of flavodoxin, an isofunctional flavoprotein. Flavodoxin is not present in higher plants, but transgenic Nicotiana tabacum lines accumulating Anabaena flavodoxin in plastids display increased tolerance to different sources of environmental stress. As the degree of tolerance correlated with flavodoxin dosage in plastids of nuclear-transformed transgenic tobacco, we prepared plants expressing even higher levels of flavodoxin by direct plastid transformation. A suite of nuclear- and chloroplast-transformed lines expressing a wide range of flavodoxin levels, from 0.3 to 10.8?μmol?m^?2, did not exhibit any detectable growth phenotype relative to the wild type. In the absence of stress, the contents of both chlorophyll a and carotenoids, as well as the photosynthetic performance (photosystem II maximum efficiency, photosystem II operating efficiency, electron transport rates and carbon assimilation rates), displayed a moderate increase with flavodoxin concentrations up to 1.3–2.6?μmol flavodoxin m^?2, and then declined to wild-type levels. Stress tolerance, as estimated by the damage inflicted on exposure to the pro-oxidant methyl viologen, also exhibited a bell-shaped response, with a significant, dose-dependent increase in tolerance followed by a drop in the high-expressing lines. The results indicate that optimal photosynthetic performance and stress tolerance were observed at flavodoxin levels comparable to those of endogenous ferredoxin. Further increases in flavodoxin content become detrimental to plant fitness.     

Golgi complex fragmentation in G2/M transition: An organelle-based cell-cycle checkpoint

In mammalian cells, the Golgi complex is organized into a continuous membranous system known as the Golgi ribbon, which is formed by individual Golgi stacks that are laterally connected by tubular bridges. During mitosis, the Golgi ribbon undergoes extensive fragmentation through a multistage process that is required for its correct partitioning into the daughter cells. Importantly, inhibition of this Golgi disassembly results in cell-cycle arrest at the G2 stage, suggesting that accurate inheritance of the Golgi complex is monitored by a "Golgi mitotic checkpoint." Here, we discuss the mechanisms and regulation of the Golgi ribbon breakdown and briefly comment on how Golgi partitioning may inhibit G2/M transition.