Spontaneous fibrillation of the native neuropeptide hormone Somatostatin-14

Natural Somatostatin-14 is a small cyclic neuropeptide hormone with broad inhibitory effects on endocrine secretions. Here we show that natural Somatostatin-14 spontaneously self-assembles in water and in 150 mM NaCl into liquid crystalline nanofibrils, which follow characteristic structural features of amyloid fibrils. These non-covalent highly stable structures are based on the Somatostatin native backbone conformation and are formed under non-denaturing conditions. Our results support the hypothesis that self-assembly into amyloid fibrils is a generic property of the polypeptide chain under appropriate conditions. Given recent advances on the mechanisms of biological storage and sorting modes of peptide/protein hormones into secretory granules, we propose that Somatostatin-14 fibrillation could be relevant to the regulated secretion pathway of this neuropeptide hormone. Such a hypothesis is consistent with the emerging concept of the existence of non-disease related but functional amyloids.     

Proteomic analysis of the secretome of human umbilical vein endothelial cells using a combination of free‐flow electrophoresis and nanoflow LC‐MS/MS

Human umbilical vein endothelial cells are the most widely used in vitro model for endothelial cells. Their secreted proteins, however, have not been comprehensively analysed so far. In this study, we accomplished to map the secretome of human umbilical vein endothelial cells by combining free‐flow electrophoresis with nanoflow LC‐MS/MS. This comprehensive analysis provides a basis for future comparative studies of protein secretion by endothelial cells in response to cardiovascular risk factors and is available on our website http://www.vascular‐proteomics.com .     

A double residue substitution in the coenzyme-binding site accounts for the different kinetic properties between yeast and human formaldehyde dehydrogenases

Glutathione-dependent formaldehyde dehydrogenase (FALDH) is the main enzymatic system for formaldehyde detoxification in all eukaryotic and many prokaryotic organisms. The enzyme of yeasts and some bacteria exhibits about 10-fold higher k(cat) and K(m) values than those of the enzyme from animals and plants. Typically Thr-269 and Glu-267 are found in the coenzyme-binding site of yeast FALDH, but Ile-269 and Asp-267 are present in the FALDH of animals. By site-directed mutagenesis we have prepared the T269I and the D267E mutants and the D267E/T269I double mutant of Saccharomyces cerevisiae FALDH with the aim of investigating the role of these residues in the kinetics. The T269I and the D267E mutants have identical kinetic properties as compared with the wild-type enzyme, although T269I is highly unstable. In contrast, the D267E/T269I double mutant is stable and shows low K(m) (2.5 microM) and low k(cat) (285 min(-1)) values with S-hydroxymethylglutathione, similar to those of the human enzyme. Therefore, the simultaneous exchange at both residues is the structural basis of the two distinct FALDH kinetic types. The local structural perturbations imposed by the substitutions are suggested by molecular modeling studies. Finally, we have studied the effect of FALDH deletion and overexpression on the growth of S. cerevisiae. It is concluded that the FALDH gene is not essential but enhances the resistance against formaldehyde (0.3-1 mM). Moreover, the wild-type enzyme (with high k(cat) and K(m)) provides more resistance than the double mutant (with low k(cat) and K(m)).     

Localization to the proteasome is sufficient for degradation

The majority of unstable proteins in eukaryotic cells are targeted for degradation through the ubiquitin-proteasome pathway. Substrates for degradation are recognized by the E1, E2, and E3 ubiquitin conjugation machinery and tagged with polyubiquitin chains, which are thought to promote the proteolytic process through their binding with the proteasome. We describe a method to bypass the ubiquitination step artificially both in vivo and in a purified in vitro system. Seven proteasome subunits were tagged with Fpr1, and fusion reporter constructs were created with the Fpr1-rapamycin binding domain of Tor1. Reporter proteins were localized to the proteasome by the addition of rapamycin, a drug that heterodimerizes Fpr1 and Tor1. Degradation of reporter proteins was observed with proteasomes that had either Rpn10 or Pre10 subunits tagged with Fpr1. Our experiments resolved a simple but central problem concerning the design of the ubiquitin-proteasome pathway. We conclude that localization to the proteasome is sufficient for degradation and, therefore, any added functions polyubiquitin chains possess beyond tethering substrates to the proteasome are not strictly necessary for proteolysis.     

How Do Astronomers Share Data? Reliability and Persistence of Datasets Linked in AAS Publications and a Qualitative Study of Data Practices among US Astronomers

We analyze data sharing practices of astronomers over the past fifteen years. An analysis of URL links embedded in papers published by the American Astronomical Society reveals that the total number of links included in the literature rose dramatically from 1997 until 2005, when it leveled off at around 1500 per year. The analysis also shows that the availability of linked material decays with time: in 2011, 44% of links published a decade earlier, in 2001, were broken. A rough analysis of link types reveals that links to data hosted on astronomers'' personal websites become unreachable much faster than links to datasets on curated institutional sites. To gauge astronomers'' current data sharing practices and preferences further, we performed in-depth interviews with 12 scientists and online surveys with 173 scientists, all at a large astrophysical research institute in the United States: the Harvard-Smithsonian Center for Astrophysics, in Cambridge, MA. Both the in-depth interviews and the online survey indicate that, in principle, there is no philosophical objection to data-sharing among astronomers at this institution. Key reasons that more data are not presently shared more efficiently in astronomy include: the difficulty of sharing large data sets; over reliance on non-robust, non-reproducible mechanisms for sharing data (e.g. emailing it); unfamiliarity with options that make data-sharing easier (faster) and/or more robust; and, lastly, a sense that other researchers would not want the data to be shared. We conclude with a short discussion of a new effort to implement an easy-to-use, robust, system for data sharing in astronomy, at theastrodata.org, and we analyze the uptake of that system to-date.     

Cellular transplants in amyotrophic lateral sclerosis patients: an observational study

Background aimsCytotherapy is a promising option for neurodegenerative disease treatment. Because of the fatal prognosis and imperative need for effective treatment, amyotrophic lateral sclerosis (ALS) patients request this therapy before its effectiveness has been verified. The increase in clinics offering cytotherapies but providing little scientific information has prompted considerable medical tourism. We present an observational study of Spanish ALS patients receiving cytotherapy, analyzing the experiences arising from the treatment (TX) and considering two progression markers, FVC and ALSFRS-R.MethodsTwelve ALS patients with a mean age of 48.6 years (SD 12.8) received cytotherapy 26.9 months (SD 15.8) after clinical onset. ALSFRS-R and FVC at TX were 32.3 (SD 6.8) and 63.4% (SD 15.3), respectively. TX involved transplants of olfactory ensheathing cells in three patients, and autologous mesenchymal stromal cells in the remainder.ResultsOne patient died 33 months post-TX after surviving for 49 months. Five required mechanical non-invasive home ventilation 7.4 months post-TX. Two required invasive ventilation 13 months post-TX. Five patients needed gastrostomy feeding 23.3 months post-TX. Survival between clinical onset and the study end date was 50 months (SD 17.2). No significant adverse events or changes in the decline of FVC and ALSFRS-R compared with the disease's natural history were observed.ConclusionsOur observations suggest that these therapies do not halt the course of the disease. Cytotherapy cannot yet be considered a curative treatment for ALS.     

Ultrastructure and cytochemistry of the integument of Modiolicola gracilis,parasitic copepod in mussel gills (Mytilus galloprovincialis and Mytilus edulis)

Of mussels taken from the Ebro Delta River (E. Spain), 3% have a nonmodified copepod, Modiolicola gracilis, in the gill tissues. The cuticle of different segments of the body has an epicuticle with two layers, which show external microvilli-like projections. Weakly positive reactivity to the PTA technique has been detected in the external region. The procuticle has the helicoidal architecture of the chitinous tegument in arthropods, whereas the cuticle shows discontinuities in the regions of ducts in tegumental glands. The integument is comprised of three types of cells. Epidermal cells are flat with numerous mitochondria. Muscle cells show well-developed mitochondria with several longitudinally distributed cristae. A third and secretory cell shows a well-developed rough endoplasmic reticulum and Golgi complex in the basal zone. Its apical portion is full of secretory granules. Through the cuticle, these integumental glands open directly to the cuticular surface via a short duct coated by epicuticle. The composition and specializations of this complex cuticular architecture differ markedly from those shown by an endoparasitic copepod detected in the digestive gland of the mussel. It does not appear that the specializations detected in the cuticle of M. gracilis lead to any histopathological alteration in host tissues. © 1994 Wiley-Liss, Inc.     

Fate and composition of cytopiasmic droplet of hamster epididymal spermatozoa

Observations on sperm maturation in the hamster (Mesocricetus auratus) epididymis revealed that the cytoplasmic droplet migrates from the proximal position of the sperm flagellum to the end of the midpiece. This process begins in the testis or vas efferens and ends in the cauda region of the epididymis. A study of different regions of the epididymis and vas deferens demonstrated that the cytoplasmic droplet is not released from the sperm into the luminal fluid. An ultrastructural study of the cytoplasmic droplet demonstrated changes in its morphology as its position moved distally along the midpiece. Membranous components called saccules or vesicles, believed to be remnants of the Golgi apparatus present in the cytoplasmic droplet, changed their morphology during the migration process. Inclusion bodies within vesicles were released to the lumen at the levels of the cauda epididymis and the vas deferens. © 1994 Wiley-Liss, Inc.