Higher-order assemblies of oligomeric cargo receptor complexes form the membrane scaffold of the Cvt vesicle

Selective autophagy is the mechanism by which large cargos are specifically sequestered for degradation. The structural details of cargo and receptor assembly giving rise to autophagic vesicles remain to be elucidated. We utilize the yeast cytoplasm-to-vacuole targeting (Cvt) pathway, a prototype of selective autophagy, together with a multi-scale analysis approach to study the molecular structure of Cvt vesicles. We report the oligomeric nature of the major Cvt cargo Ape1 with a combined 2.8 Å X-ray and negative stain EM structure, as well as the secondary cargo Ams1 with a 6.3 Å cryo-EM structure. We show that the major dodecameric cargo prApe1 exhibits a tendency to form higher-order chain structures that are broken upon interaction with the receptor Atg19 in vitro The stoichiometry of these cargo-receptor complexes is key to maintaining the size of the Cvt aggregate in vivo Using correlative light and electron microscopy, we further visualize key stages of Cvt vesicle biogenesis. Our findings suggest that Atg19 interaction limits Ape1 aggregate size while serving as a vehicle for vacuolar delivery of tetrameric Ams1

Remarkable Reduction of MAP2 in the Brains of Scrapie-Infected Rodents and Human Prion Disease Possibly Correlated with the Increase of Calpain

Microtubule-associated protein 2 (MAP2) belongs to the family of heat stable MAPs, which takes part in neuronal morphogenesis, maintenance of cellular architecture and internal organization, cell division and cellular processes. To obtain insight into the possible alteration and the role of MAP2 in transmissible spongiform encephalopathies (TSEs), the MAP2 levels in the brain tissues of agent 263K-infected hamsters and human prion diseases were evaluated. Western blots and IHC revealed that at the terminal stages of the diseases, MAP2 levels in the brain tissues of scrapie infected hamsters, a patient with genetic Creutzfeldt-Jakob disease (G114V gCJD) and a patient with fatal familial insomnia (FFI) were almost undetectable. The decline of MAP2 was closely related with prolonged incubation time. Exposure of SK-N-SH neuroblastoma cell line to cytotoxic PrP106-126 peptide significantly down-regulated the cellular MAP2 level and remarkably disrupted the microtubule structure, but did not alter the level of tubulin. Moreover, the levels of calpain, which mediated the degradation of a broad of cytoskeletal proteins, were significantly increased in both PrP106-126 treated SK-N-SH cells and brain tissues of 263K prion-infected hamsters. Our data indicate that the decline of MAP2 is a common phenomenon in TSEs, which seems to occur at an early stage of incubation period. Markedly increased calpain level might contribute to the reduction of MAP2

Calpain Cleavage Prediction Using Multiple Kernel Learning

Calpain, an intracellular -dependent cysteine protease, is known to play a role in a wide range of metabolic pathways through limited proteolysis of its substrates. However, only a limited number of these substrates are currently known, with the exact mechanism of substrate recognition and cleavage by calpain still largely unknown. While previous research has successfully applied standard machine-learning algorithms to accurately predict substrate cleavage by other similar types of proteases, their approach does not extend well to calpain, possibly due to its particular mode of proteolytic action and limited amount of experimental data. Through the use of Multiple Kernel Learning, a recent extension to the classic Support Vector Machine framework, we were able to train complex models based on rich, heterogeneous feature sets, leading to significantly improved prediction quality (6% over highest AUC score produced by state-of-the-art methods). In addition to producing a stronger machine-learning model for the prediction of calpain cleavage, we were able to highlight the importance and role of each feature of substrate sequences in defining specificity: primary sequence, secondary structure and solvent accessibility. Most notably, we showed there existed significant specificity differences across calpain sub-types, despite previous assumption to the contrary. Prediction accuracy was further successfully validated using, as an unbiased test set, mutated sequences of calpastatin (endogenous inhibitor of calpain) modified to no longer block calpain's proteolytic action. An online implementation of our prediction tool is available at http://calpain.org

Heart Valve Tissue Engineering: Concepts, Approaches, Progress, and Challenges

Potential applications of tissue engineering in regenerative medicine range from structural tissues to organs with complex function. This review focuses on the engineering of heart valve tissue, a goal which involves a unique combination of biological, engineering, and technological hurdles. We emphasize basic concepts, approaches and methods, progress made, and remaining challenges. To provide a framework for understanding the enabling scientific principles, we first examine the elements and features of normal heart valve functional structure, biomechanics, development, maturation, remodeling, and response to injury. Following a discussion of the fundamental principles of tissue engineering applicable to heart valves, we examine three approaches to achieving the goal of an engineered tissue heart valve: (1) cell seeding of biodegradable synthetic scaffolds, (2) cell seeding of processed tissue scaffolds, and (3) in-vivo repopulation by circulating endogenous cells of implanted substrates without prior in-vitro cell seeding. Lastly, we analyze challenges to the field and suggest future directions for both preclinical and translational (clinical) studies that will be needed to address key regulatory issues for safety and efficacy of the application of tissue engineering and regenerative approaches to heart valves. Although modest progress has been made toward the goal of a clinically useful tissue engineered heart valve, further success and ultimate human benefit will be dependent upon advances in biodegradable polymers and other scaffolds, cellular manipulation, strategies for rebuilding the extracellular matrix, and techniques to characterize and potentially non-invasively assess the speed and quality of tissue healing and remodeling

DETECTION of IMMUNOGLOBULIN (IGG and IGA) ANTI-OUTER-MEMBRANE PROTEINS of ENTEROPATHOGENIC ESCHERICHIA-COLI (EPEC) in SALIVA, COLOSTRUM, BREAST-MILK, SERUM, CORD-BLOOD and AMNIOTIC-FLUID - STUDY of INHIBITION of LOCALIZED ADHERENCE of EPEC TO HELA-CELLS

ESCOLA PAULISTA MED,DEPT PEDIAT,DIV RHEUMATOL ALLERGY & CLIN IMMUNOL,BR-04025002 São Paulo,BRAZILESCOLA PAULISTA MED,DEPT MICROBIOL IMMUNOL & PARASITOL,São Paulo,BRAZILESCOLA PAULISTA MED,DEPT PEDIAT,DIV RHEUMATOL ALLERGY & CLIN IMMUNOL,BR-04025002 São Paulo,BRAZILESCOLA PAULISTA MED,DEPT MICROBIOL IMMUNOL & PARASITOL,São Paulo,BRAZILWeb of Scienc

Effect of pelleting and pellet size of a concentrate for intensively reared beef cattle on in vitro fermentation by two different approaches

Two in vitro experiments were performed to evaluate the effect of concentrate pelleting for intensively reared cattle on rumen microbial fermentation. In Experiment 1, a concentrate was incubated as meal (M) or pelleted to 3.5 (P3.5) and 10 (P10) mm diameter, either in their original form or re-ground to 1 mm particle size (MG, P3.5G and P10G) and their gas production pattern was studied. When the concentrate was incubated in its original form, gas produced with M was higher than with P10 at 1 h and from 3 to 6h of incubation (P<0.05). showing that pelleting to 10 mm diameter delayed microbial fermentation of the concentrate at the initial stages of incubation. When the concentrate was incubated ground, MG produced the lowest gas volume up to 8 h (P<0.05), but there were no differences with P10G afterwards. This indicates that, once avoided the limitation of microbial accessibility by pellet and particle size, fermentation increased because starch gelatinisation was promoted by processing. However, the magnitude of this effect was not different between the two pellet sizes. In Experiment 2, the original forms of concentrate (M, P3.5 and P10) were incubated for 24 h in a semicontinuous incubation system at pH 6.5 and 5.8, and concentration of volatile fatty acid (VFA) was monitored at 4 and 10 h. The pH reduction decreased gas production throughout the experimental period (P<0.001). Gas production from M was higher than P10 up to 4h (P<0.05), at 6h and from 20 to 24h (P<0.10). Total VFA concentration at 4h was lowest with P10 (P<0.01), but no differences were recorded at 10 h. Results from Experiment 2 support those from Experiment 1, even at pH 5.8 and at a liquid outflow rate of 0.10/h, that could be expected in intensively reared beef cattle. (C) 2010 Elsevier B.V. All rights reserved.Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES