Increased mobility of major histocompatibility complex I-peptide complexes decreases the sensitivity of antigen recognition

CD8(+) cytotoxic T lymphocytes (CTL) can recognize and kill target cells expressing only a few cognate major histocompatibility complex (MHC) I-peptide complexes. This high sensitivity requires efficient scanning of a vast number of highly diverse MHC I-peptide complexes by the T cell receptor in the contact site of transient conjugates formed mainly by nonspecific interactions of ICAM-1 and LFA-1. Tracking of single H-2K(d) molecules loaded with fluorescent peptides on target cells and nascent conjugates with CTL showed dynamic transitions between states of free diffusion and immobility. The immobilizations were explained by association of MHC I-peptide complexes with ICAM-1 and strongly increased their local concentration in cell adhesion sites and hence their scanning by T cell receptor. In nascent immunological synapses cognate complexes became immobile, whereas noncognate ones diffused out again. Interfering with this mobility modulation-based concentration and sorting of MHC I-peptide complexes strongly impaired the sensitivity of antigen recognition by CTL, demonstrating that it constitutes a new basic aspect of antigen presentation by MHC I molecules.     

The significance of fire intensity in creating local patchiness in the Chilean matorral

The hypothesis that high and low intensity human-made fires can produce ecologically different effects in the Chilean matorral is examined. We compared the abundance of naturally established seedlings under shrubs burned by low and high intensity fires on five north and five south facing slopes. On south facing slopes, we found 54 shrubs burned by low intensity fires and only 4 shrubs burned by high intensity fires. In contrast, north facing slopes had approximately the same number of shrubs burned by low and high fire intensity fires (24 versus 19, respectively). We only found seedlings under shrubs burned by low intensity fires and most of them were of Muehlenbeckia hastulata and Trevoa trinervis. Also viable seeds were only found under shrubs burned by low intensity fires. Results indicate that fire intensity can be an important factor determining species distribution patterns in the matorral.     

Heritability of running economy: a study made on twin brothers

Running economy (RE), defined as the steady-state of oxygen uptake (V˙O₂) for a given running velocity, is a factor of sports performance the genetic component of which has seldom been reported to date. We studied this component using a heritability index (HI) in a group of 32 male twins, 8 monozygotic (MZ) and 8 dizygotic (DZ) pairs, all sportsmen with similar perinatal and environmental backgrounds. Zygocity was determined by the identity of erythrocytic antigenic, protein and enzymatic polymorphism, and human leucocyte antigen serologic types between co-twins. The subjects exercised twice on a treadmill, once until exhaustion and again at submaximal intensities. Pulmonary gas exchange was measured continuously using an automatic analyser system during both tests. Blood samples were obtained during the recovery period to determine lactate concentrations. No significant differences were observed between MZ and DZ, in respect of RE at any speed or in maximal V˙O₂ relative to body mass. Nevertheless, significant HI (P < 0.05) was found in maximal lactate concentrations (HI = 0.75) and in respiratory equivalent for oxygen at two speeds, 7 km · h⁻¹ (HI = 0.71) and 8 km · h⁻¹ (HI = 0.79), differences which probably suggest that there are differences in RE. In conclusion, we did not detect a genetic component in RE or in maximal oxygen uptake, but a genetic component for markers of anaerobic metabolism was present. Accepted: 5 November 1997     

Putative Glycosyltransferases and Other Plant Golgi Apparatus Proteins Are Revealed by LOPIT Proteomics

The Golgi apparatus is the central organelle in the secretory pathway and plays key roles in glycosylation, protein sorting, and secretion in plants. Enzymes involved in the biosynthesis of complex polysaccharides, glycoproteins, and glycolipids are located in this organelle, but the majority of them remain uncharacterized. Here, we studied the Arabidopsis (Arabidopsis thaliana) membrane proteome with a focus on the Golgi apparatus using localization of organelle proteins by isotope tagging. By applying multivariate data analysis to a combined data set of two new and two previously published localization of organelle proteins by isotope tagging experiments, we identified the subcellular localization of 1,110 proteins with high confidence. These include 197 Golgi apparatus proteins, 79 of which have not been localized previously by a high-confidence method, as well as the localization of 304 endoplasmic reticulum and 208 plasma membrane proteins. Comparison of the hydrophobic domains of the localized proteins showed that the single-span transmembrane domains have unique properties in each organelle. Many of the novel Golgi-localized proteins belong to uncharacterized protein families. Structure-based homology analysis identified 12 putative Golgi glycosyltransferase (GT) families that have no functionally characterized members and, therefore, are not yet assigned to a Carbohydrate-Active Enzymes database GT family. The substantial numbers of these putative GTs lead us to estimate that the true number of plant Golgi GTs might be one-third above those currently annotated. Other newly identified proteins are likely to be involved in the transport and interconversion of nucleotide sugar substrates as well as polysaccharide and protein modification.The Golgi apparatus is the central organelle in the secretory pathway, and in higher plants it is involved in the biosynthesis and transport of cell wall matrix polysaccharides, glycoproteins, proteoglycans, and glycolipids as well as in protein trafficking to different subcellular compartments. The last decade has produced substantial findings on the function of the Golgi apparatus: insights into the protein trafficking at the endoplasmic reticulum (ER)/Golgi interface, Golgi structural maintenance, its involvement in endocytosis, and its behavior during cell division (for review, see Faso et al., 2009). However, despite its importance, only a small proportion of the Golgi proteome has been studied: relatively few Golgi proteins have been localized, and even fewer have been functionally characterized.The Golgi apparatus is thought to contain a large and diverse group of membrane-bound glycosyltransferases (GTs). The current view is that different GT activities are required for synthesis of the linkage between different donor and acceptor sugars. Having in mind the diversity of linkage types found in cell wall polysaccharides, the number of different GTs involved is likely to be very large. For instance, it has been estimated that for the biosynthesis of pectin alone, the action of 65 different enzymatic activities is needed (Caffall and Mohnen, 2009). By the end of the year 2011, 468 Arabidopsis (Arabidopsis thaliana) sequences had been annotated in the Carbohydrate-Active EnZymes (CAZy) GT database (Cantarel et al., 2009; http://www.cazy.org). We estimate that two-thirds of these CAZy-classified GTs may be targeted to the Golgi. The remaining one-third are cytosolic or plastidic enzymes involved in processes including, secondary metabolism or starch synthesis. The reported sequences are classified into 43 CAZy families based on amino acid sequence similarities within which at least one member has been biochemically characterized. Each family is likely to have a common structural fold, and three-dimensional (3-D) structures have been resolved for 20 of these 43 families. These are divided mostly into two structural classes, having either a GT-A fold or a GT-B fold (Unligil and Rini, 2000; Bourne and Henrissat, 2001). Moreover, most of the structurally uncharacterized GT families are predicted to adopt either the GT-A or GT-B fold based on 3-D structural homology modeling (Coutinho et al., 2003; Lairson et al., 2008). Despite this conserved 3-D structure, different GT families have very low or undetectable sequence similarities. Consequently, predicting novel GTs based solely on their amino acid sequence similarities is not always achievable, and structural homology searches have also proven useful (Hansen et al., 2009).The length and properties of the transmembrane domain (TMD) of endomembrane proteins appear to play a role in protein sorting and location within the secretory pathway and can be used to predict protein localization (Hanton et al., 2005; Sharpe et al., 2010). In order to perform such predictions, a high number of experimentally localized proteins is required, but only limited data sets have been available for plants to date.In order to identify the most abundant CAZy-classified GTs as well as novel putative GTs, in this work we rigorously extended our proteomic studies of the Golgi apparatus. We have previously developed a high-throughput mass spectrometry (MS)-based quantitative proteomics technique for localization of organelle proteins by isotope tagging (LOPIT; Dunkley et al., 2004, 2006). Here, we report new LOPIT data sets and apply a new method of combining them with published LOPIT data sets, localizing an unprecedented number of plant organelle proteins. We have analyzed the TMD properties of the proteins assigned to the ER, Golgi, and plasma membrane (PM) and determined the organelle-specific features. Structural prediction analysis of the Golgi-localized proteins with unknown functions assessed the protein sequences for the potential to fold similarly to known GT structures. We found that the Golgi contains a substantial number of candidate GT families that have no characterized functions. These results yield a broader understanding of the Golgi function and its biochemical properties.     

Effect of prior ingestion of glucose or fructose on the performance of exercise of intermediate duration

The metabolic responses induced by the ingestion of a beverage containing glucose (G), fructose (F) or placebo (W) 30 min before exercise of high intensity and intermediate duration have been investigated; in these conditions the energy processes are mostly dependent on aerobic reactions. A group of 11 male recreational sportsmen ran on a treadmill, at an intensity corresponding to 82% of peak oxygen consumption, until exhaustion on three different occasions (after ingestion of a beverage containing 75 g of G, 75 g of F or W). Plasma glucose, insulin, and lactic acid concentrations were determined just prior to the ingestion of the beverages, 30 min afterwards and 10 and 30 min after completion of the exercise. The mean endurance time was 644 (SD 261) s after the ingestion of G, 611 (SD 227) s after the ingestion of F and 584 (SD 189) s after the ingestion of the W (P < 0.05 between G and W). No differences in the oxygen uptake, respiratory quotient or lactate concentrations between the three trials were observed. Both plasma glucose and insulin concentrations determined in samples obtained immediately before the onset of exercise were higher when G was ingested than when F (P < 0.05 andP < 0.05, respectively) or W (P < 0.001 and P < 0.005, respectively) were ingested. These findings would suggest that the ingestion of G prior to an effort of intermediate duration may improve physical performance.     

The GATC-binding protein SeqA is required for bile resistance and virulence in Salmonella enterica serovar typhimurium

Disruption of the seqA gene of Salmonella enterica serovar Typhimurium causes defects similar to those described in E. coli: filament formation, aberrant nucleoid segregation, induction of the SOS response, envelope instability, and increased sensitivity to membrane-damaging agents. Differences between SeqA⁻ mutants of E. coli and S. enterica, however, are found. SeqA⁻ mutants of S. enterica form normal colonies and do not exhibit alterations in phage plaquing morphology. Lack of SeqA causes attenuation of S. enterica virulence by the oral route but not by the intraperitoneal route, suggesting a virulence defect in the intestinal stage of infection. However, SeqA⁻ mutants are fully proficient in the invasion of epithelial cells. We hypothesize that attenuation of SeqA⁻ mutants by the oral route may be caused by bile sensitivity, which in turn may be a consequence of envelope instability.     

Survey of Argentine Health Researchers on the Use of Evidence in Policymaking

Objective

In this study, Argentine health researchers were surveyed regarding their perceptions of facilitators and barriers to evidence-based policymaking in Argentina, as well as their publication activities, and research environment satisfaction.

Methods

A self-administered online survey was sent to health researchers in Argentina. The survey questions were based on a preceding qualitative study of Argentine health researchers, as well as the scientific literature.

Results

Of the 647 researchers that were reached, 226 accessed the survey, for a response rate of 34.9%. Over 80% of researchers surveyed had never been involved in or contributed to decision-making, while over 90% of researchers indicated they would like to be involved in the decision-making process. Decision-maker self-interest was perceived to be the driving factor in the development of health and healthcare policies. Research conducted by a research leader was seen to be the most influential factor in influencing health policy, followed by policy relevance of the research. With respect to their occupational environment, researchers rated highest and most favourably the opportunities available to present, discuss and publish research results and their ability to further their education and training. Argentine researchers surveyed demonstrated a strong interest and willingness to contribute their work and expertise to inform Argentine health policy development.

Conclusion

Despite Argentina’s long scientific tradition, there are relatively few institutionalized linkages between health research results and health policymaking. Based on the results of this study, the disconnect between political decision-making and the health research system, coupled with fewer opportunities for formalized or informal researcher/decision-maker interaction, contribute to the challenges in evidence informing health policymaking in Argentina. Improving personal contact and the building of relationships between researchers and policymakers in Argentina will require taking into account researcher perceptions of policymakers, as highlighted in this study.     

PSEUDO-NITZSCHIA PUNGENS AND P. MULTISENES (BACILLARIOPHYCEAE): NUCLEAR RIBOSOMAL DNAS AND SPECIES DIFFERENCES1

The region of the nuclear ribosomal DNA (rDNA) operon containing the small subunit (SSU), internal transcribed spacer 1 (ITS1), and a portion of the 5.8s rDNA gene was sequenced in one isolate each of Pseudo-nitzschia multiseries (Hasle) Hasle and Pseudo-nitzschia pungens (Grunow in Cleve & Möller) Hasle. The SSUs of these two species were highly similar, differing only in 14 point mutations and one insertion/deletion in 1774 bp. The ITS1 sequences were more variable, with 57 point mutations and three insertion/deletions in 257 bp. There were no differences in 44 bp of the 5.8S sequences. Restriction fragment patterns (RFPs) for the restriction endonucleases HaeIII, Hha1, and Rsa1 for 13 isolates of P. multiseries from the Atlantic, Pacific, and Gulf coasts of the United States and 16 isolates of P. pungens from the three coasts of the United States, in addition to Japan and China, were compared. There were differences between the RFPs of P. multiseries and P. pungens that corresponded to sites mapped by the DNA sequences, but no infraspecific variation in RFPs was observed for either species. The differences in RFPs correlate with morphological, immunological, and other rDNA differences and support the recognition of these taxa as separate species.     

The initiation factor eIF3-f is a major target for atrogin1/MAFbx function in skeletal muscle atrophy

In response to cancer, AIDS, sepsis and other systemic diseases inducing muscle atrophy, the E3 ubiquitin ligase Atrogin1/MAFbx (MAFbx) is dramatically upregulated and this response is necessary for rapid atrophy. However, the precise function of MAFbx in muscle wasting has been questioned. Here, we present evidence that during muscle atrophy MAFbx targets the eukaryotic initiation factor 3 subunit 5 (eIF3-f) for ubiquitination and degradation by the proteasome. Ectopic expression of MAFbx in myotubes induces atrophy and degradation of eIF3-f. Conversely, blockade of MAFbx expression by small hairpin RNA interference prevents eIF3-f degradation in myotubes undergoing atrophy. Furthermore, genetic activation of eIF3-f is sufficient to cause hypertrophy and to block atrophy in myotubes, whereas genetic blockade of eIF3-f expression induces atrophy in myotubes. Finally, eIF3-f induces increasing expression of muscle structural proteins and hypertrophy in both myotubes and mouse skeletal muscle. We conclude that eIF3-f is a key target that accounts for MAFbx function during muscle atrophy and has a major role in skeletal muscle hypertrophy. Thus, eIF3-f seems to be an attractive therapeutic target.