Microdiálisis cerebral de alta resolución: Estudio del perfil funcional y estructural de las membranas de 100 kDa

La microdiàlisi és una tècnica de neuromonitoratge que permet el mostreig continu del contingut molecular i iònic de l’espai intersticial cerebral. Aquesta tècnica es basa en la implantació d’un catèter en el parènquima cerebral humà de manera mínimament invasiva. Actualment, la microdiàlisi s’ha implantat de manera rutinària en moltes unitats de cures intensives pel neuromonitoratge de pacients amb lesions cerebrals agudes. No obstant, l’estudi in vivo del perfil temporal del proteoma en aquestes lesions i la correcta avaluació de la concentració de les molècules d’interès en el líquid extracel•lular cerebral requereix la determinació prèvia in vitro del percentatge de recuperació relativa de les proteïnes d’estudi.

Proyecto kda.com : Un sitio donde compartir aficiones

Este proyecto consiste en un sitio web donde los usuarios pueden compartir sus aficiones y gestionar actividades, haciendo comentarios sobre las mismas.

Microdiàlisis cerebral aplicada a l'estudi del metabolisme energètic i a la resposta neuroinflamatòria dels pacients amb traumatisme cranioencefàlic

La tècnica de la microdiàlisis cerebral (MDC) és un instrument que proporciona informació rellevant en la monitorització del metabolisme cerebral en els pacients neurocrítics. El lactat i l’índex lactat-piruvat (ILP) són dos marcadors utilitzats per a la detecció de la hipòxia cerebral en pacients que han patit un traumatisme cranioencefàlic (TCE). Aquests dos marcadors poden estar anormalment elevats en circumstàncies que no cursen amb hipòxia tissular. Per una altra banda la recent aparició dels catèters de MDC amb porus de major mida denominats d’”alta resolució”, permet ampliar el rang de molècules que es poden detectar en el dialitzat. Objectius: 1) descriure les característiques del metabolisme energètic cerebral que s’observa en la fase aguda dels pacients que han patit un TCE en base als dos indicadors del metabolisme anaeròbic: lactat i ILP, i 2) determinar la recuperació relativa (RR) de les molècules implicades en la resposta neuroinflamatòria: de IL-1β, IL- 6, IL-8 i IL-10. Material i mètodes: Es van seleccionar 46 pacients d’una cohort de pacients amb TCE moderat o greu ingressats a la Unitat de Cures Intensives de l’Hospital Universitari de la Vall d’Hebron i monitoritzats amb MDC. Es van analitzar els nivells de lactat i ILP i es va correlacionar amb els nivells de PtiO2. Es van realitzar experiments in vitro per estudiar la recuperació de les membranes de 100 KDa per tal de poder interpretar posteriorment els nivells reals de les molècules estudiades en l’espai extracel•lular del teixit cerebral. Resultats: La concordança entre el lactat i l’índex LP per a determinar episodis de disfunció metabòlica va ser dèbil (índex de kappa = 0,36, IC 95%: 0,34-0,39). Més del 80% dels casos en què el lactat i l’índex LP es trobaven incrementats, els valors de la PtiO2 es van trobar dins els rangs de normalitat (PtiO2&15mmHg). La recuperació de les citoquines a través de la membrana de microdiàlisis va ser menor de l’esperat tenint en compte la mida dels porus de la membrana. Conclusions: el lactat i l’índex LP elevats va ser una troballa freqüent després d’un TCE i no es va relacionar, en la majoria de casos, amb episodis d’hipòxia tissular. Per un altra part la mida del porus de la membrana no és l’únic paràmetre indicador de la RR de macromolècules.

Changes in the enterocyte cytoskeleton in newborn rats exposed to ethanol in utero

BACKGROUND: Cytoskeletal changes after longterm exposure to ethanol have been described in a number of cell types in adult rat and humans. These changes can play a key part in the impairment of nutrient assimilation and postnatal growth retardation after prenatal damage of the intestinal epithelium produced by ethanol intake. AIMS: To determine, in the newborn rat, which cytoskeletal proteins are affected by longterm ethanol exposure in utero and to what extent. ANIMALS: The offspring of two experimental groups of female Wistar rats: ethanol treated group receiving up to 25% (w/v) of ethanol in the drinking fluid and control group receiving water as drinking fluid. METHODS: Single and double electron microscopy immunolocalisation and label density estimation of cytoskeletal proteins on sections of proximal small intestine incubated with monoclonal antibodies against actin, alpha-tubulin, cytokeratin (polypeptides 1, 5, 6, 7, 8, 10, 11, and 18), and with a polyclonal antibody anti-beta 1,4-galactosyl transferase as trans golgi (TG) or trans golgi network (TGN) marker, or both. SDS-PAGE technique was also performed on cytoskeletal enriched fractions from small intestine. Western blotting analysis was carried out by incubation with the same antibodies used for immunolocalisation. RESULTS: Intestinal epithelium of newborn rats from the ethanol treated group showed an overexpression of cytoskeletal polypeptides ranging from 39 to 54 kDa, affecting actin and some cytokeratins, but not tubulin. Furthermore, a cytokeratin related polypeptide of 28-29 kDa was identified together with an increase in free ubiquitin in the same group. It was noteworthy that actin and cytokeratin were abnormally located in the TG or the TGN, or both. CONCLUSIONS: Longterm exposure to ethanol in utero causes severe dysfunction in the cytoskeleton of the developing intestinal epithelium. Actin and cytokeratins, which are involved in cytoskeleton anchoring to plasma membrane and cell adhesion, are particularly affected, showing overexpression, impaired proteolysis, and mislocalisation.

Changes in the enterocyte cytoskeleton in newborn rats exposed to ethanol in utero

BACKGROUND: Cytoskeletal changes after longterm exposure to ethanol have been described in a number of cell types in adult rat and humans. These changes can play a key part in the impairment of nutrient assimilation and postnatal growth retardation after prenatal damage of the intestinal epithelium produced by ethanol intake. AIMS: To determine, in the newborn rat, which cytoskeletal proteins are affected by longterm ethanol exposure in utero and to what extent. ANIMALS: The offspring of two experimental groups of female Wistar rats: ethanol treated group receiving up to 25% (w/v) of ethanol in the drinking fluid and control group receiving water as drinking fluid. METHODS: Single and double electron microscopy immunolocalisation and label density estimation of cytoskeletal proteins on sections of proximal small intestine incubated with monoclonal antibodies against actin, alpha-tubulin, cytokeratin (polypeptides 1, 5, 6, 7, 8, 10, 11, and 18), and with a polyclonal antibody anti-beta 1,4-galactosyl transferase as trans golgi (TG) or trans golgi network (TGN) marker, or both. SDS-PAGE technique was also performed on cytoskeletal enriched fractions from small intestine. Western blotting analysis was carried out by incubation with the same antibodies used for immunolocalisation. RESULTS: Intestinal epithelium of newborn rats from the ethanol treated group showed an overexpression of cytoskeletal polypeptides ranging from 39 to 54 kDa, affecting actin and some cytokeratins, but not tubulin. Furthermore, a cytokeratin related polypeptide of 28-29 kDa was identified together with an increase in free ubiquitin in the same group. It was noteworthy that actin and cytokeratin were abnormally located in the TG or the TGN, or both. CONCLUSIONS: Longterm exposure to ethanol in utero causes severe dysfunction in the cytoskeleton of the developing intestinal epithelium. Actin and cytokeratins, which are involved in cytoskeleton anchoring to plasma membrane and cell adhesion, are particularly affected, showing overexpression, impaired proteolysis, and mislocalisation.

Isolation of a novel monkey adenovirus reveals a new phylogenetic clade in the evolutionary history of simian adenoviruses

Adenoviruses of primates include human (HAdV) and simian (SAdV) isolates classified into 8 species (Human Adenovirus A to G, and Simian Adenovirus A). In this study, a novel adenovirus was isolated from a colony of cynomolgus macaques (Macaca fascicularis) and subcultured in VERO cells. Its complete genome was purified and a region encompassing the hexon gene, the protease gene, the DNA binding protein (DBP) and the 100 kDa protein was amplified by PCR and sequenced by primer walking. Sequence analysis of these four genes showed that the new isolate had 80% identity to other primate adenoviruses and lacked recombination events. The study of the evolutionary relationships of this new monkey AdV based on the combined sequences of the four genes supported a close relationship to SAdV-3 and SAdV-6, lineages isolated from Rhesus monkeys. The clade formed by these three types is separated from the remaining clades and establishes a novel branch that is related to species HAdV-A, F and G. However, the genetic distance corresponding to the newly isolated monkey AdV considerably differs from these as to belong to a new, not yet established species. Results presented here widen our knowledge on SAdV and represents an important contribution to the understanding of the evolutionary history of primate adenoviruses.

Isolation of a novel monkey adenovirus reveals a new phylogenetic clade in the evolutionary history of simian adenoviruses

Adenoviruses of primates include human (HAdV) and simian (SAdV) isolates classified into 8 species (Human Adenovirus A to G, and Simian Adenovirus A). In this study, a novel adenovirus was isolated from a colony of cynomolgus macaques (Macaca fascicularis) and subcultured in VERO cells. Its complete genome was purified and a region encompassing the hexon gene, the protease gene, the DNA binding protein (DBP) and the 100 kDa protein was amplified by PCR and sequenced by primer walking. Sequence analysis of these four genes showed that the new isolate had 80% identity to other primate adenoviruses and lacked recombination events. The study of the evolutionary relationships of this new monkey AdV based on the combined sequences of the four genes supported a close relationship to SAdV-3 and SAdV-6, lineages isolated from Rhesus monkeys. The clade formed by these three types is separated from the remaining clades and establishes a novel branch that is related to species HAdV-A, F and G. However, the genetic distance corresponding to the newly isolated monkey AdV considerably differs from these as to belong to a new, not yet established species. Results presented here widen our knowledge on SAdV and represents an important contribution to the understanding of the evolutionary history of primate adenoviruses.

The glucose transporter 4 FQQI motif is necessary for Akt Substrate of 160-Kilodalton-dependent plasma membrane translocation but not Golgi-localized g-ear-containing Arf-binding protein-dependent entry into the insulin-responsive storage compartment.

Newly synthesized glucose transporter 4 (GLUT4) enters into the insulin-responsive storage compartment in a process that is Golgi-localized γ-ear-containing Arf-binding protein (GGA) dependent, whereas insulin-stimulated translocation is regulated by Akt substrate of 160 kDa (AS160). In the present study, using a variety of GLUT4/GLUT1 chimeras, we have analyzed the specific motifs of GLUT4 that are important for GGA and AS160 regulation of GLUT4 trafficking. Substitution of the amino terminus and the large intracellular loop of GLUT4 into GLUT1 (chimera 1-441) fully recapitulated the basal state retention, insulin-stimulated translocation, and GGA and AS160 sensitivity of wild-type GLUT4 (GLUT4-WT). GLUT4 point mutation (GLUT4-F5A) resulted in loss of GLUT4 intracellular retention in the basal state when coexpressed with both wild-type GGA and AS160. Nevertheless, similar to GLUT4-WT, the insulin-stimulated plasma membrane localization of GLUT4-F5A was significantly inhibited by coexpression of dominant-interfering GGA. In addition, coexpression with a dominant-interfering AS160 (AS160-4P) abolished insulin-stimulated GLUT4-WT but not GLUT4-F5A translocation. GLUT4 endocytosis and intracellular sequestration also required both the amino terminus and large cytoplasmic loop of GLUT4. Furthermore, both the FQQI and the SLL motifs participate in the initial endocytosis from the plasma membrane; however, once internalized, unlike the FQQI motif, the SLL motif is not responsible for intracellular recycling of GLUT4 back to the specialized compartment. Together, we have demonstrated that the FQQI motif within the amino terminus of GLUT4 is essential for GLUT4 endocytosis and AS160-dependent intracellular retention but not for the GGA-dependent sorting of GLUT4 into the insulin-responsive storage compartment.

Contribution of S6K1/MAPK signaling pathways in the response to oxidative stress: activation of RSK and MSK by hydrogen peroxide

Cells respond to different kind of stress through the coordinated activation of signaling pathways such as MAPK or p53. To find which molecular mechanisms are involved, we need to understand their cell adaptation. The ribosomal protein, S6 kinase 1 (S6K1), is a common downstream target of signaling by hormonal or nutritional stress. Here, we investigated the initial contribution of S6K1/MAPK signaling pathways in the cell response to oxidative stress produced by hydrogen peroxide (H2O2). To analyze S6K1 activation, we used the commercial anti-phospho-Thr389-S6K1 antibody most frequently mentioned in the bibliography. We found that this antibody detected an 80-90 kDa protein that was rapidly phosphorylated in response to H2O2 in several human cells. Unexpectedly, this phosphorylation was insensitive to both mTOR and PI3K inhibitors, and knock-down experiments showed that this protein was not S6K1. RSK and MSK proteins were candidate targets of this phosphorylation. We demonstrated that H2O2 stimulated phosphorylation of RSK and MSK kinases at residues that are homologous to Thr389 in S6K1. This phosphorylation required the activity of either p38 or ERK MAP kinases. Kinase assays showed activation of RSK and MSK by H2O2. Experiments with mouse embryonic fibroblasts from p38 animals" knockout confirmed these observations. Altogether, these findings show that the S6K1 signaling pathway is not activated under these conditions, clarify previous observations probably misinterpreted by non-specific detection of proteins RSK and MSK by the anti-phospho-Thr389-S6K1 antibody, and demonstrate the specific activation of MAPK signaling pathways through ERK/p38/RSK/MSK by H2O2.

DNA sequence of the gene encoding the Zc1 protein from Zea mays W64A

The Zein-2 component named Zc 1 corresponds to a storage protein of an apparent M.W. of 16 kDa present in maize endosperm.

DNA sequence of the gene encoding the Zc1 protein from Zea mays W64A

The Zein-2 component named Zc 1 corresponds to a storage protein of an apparent M.W. of 16 kDa present in maize endosperm.

Proteins influencing foam formation in wine and beer: the role of yeast

This review focuses on the role of proteins in the production and maintenance of foam in both sparkling wines and beer. The quality of the foam in beer but especially in sparkling wines depends, among other factors, on the presence of mannoproteins released from the yeast cell walls during autolysis. These proteins are hydrophobic, highly glycosylated, and their molecular masses range from 10 to 200 kDa characteristics that allow mannoproteins to surround and thus stabilize the gas bubbles of the foam. Both the production and stabilization of foam also depend on other proteins. In wine, these include grape-derived proteins such as vacuolar invertase; in beer, barley-derived proteins, such as LTP1, protein Z, and hordein-derived polypeptides, are even more important in this respect than mannoproteins

Seroprevalence of Angiostrongylus cantonensis in wild rodents from the Canary Islands

Background: Angiostrongylus cantonensis is a lungworm of rats (Muridae) that is the causative agent of human cerebral angiostrongyliasis. The life cycle of A. cantonensis involves rats and mollusks as the definitive and intermediate hosts, respectively. This study was designed to increase the knowledge about the occurrence and distribution of A. cantonensis in its definitive host in the Canary Islands, using parasitological and serological analysis in different areas and age groups.Methodology/Principal Findings: Between 2009 and 2010, 54 black rats (Rattus rattus) from Tenerife were captured from six human-inhabited areas and sera samples were obtained. The lung nematodes were identified by morphological and molecular tools as A. cantonensis. The 31-kDa glycoprotein antigen was purified from A. cantonensis adult worms by electrophoresis and electroelution. Of the 54 tested rodents, 30 showed IgG antibodies against A. cantonensis 31-kDa antigen by ELISA. Therefore, the overall seroprevalence was 55.6% (95% CI: 42.4-68). Seroprevalent rodents were found in all the 6 areas. This 31-kDa antigen was not recognized by some sera of rats infected by other helminth species (but not A. cantonensis). Seroprevalence of IgG antibodies against A. cantonensis and prevalence based on the presence of adult worms showed significant correlation (R2 = 0.954, p,0.05). Conclusions/Significance: The present results could indicate a high prevalence of A. cantonensisin Tenerife and suggest the inclusion of two new zones in the distribution area of the parasite. The commonness and wide distribution of A. cantonensis in rats implies the presence of intermediate hosts, indicating that humans may be at risk of getting infected.

Study on the removal of biodegradable NOM from seawater using biofiltration

Despite the low biodegradability of seawater NOM, problems associated with biofouling are common in facilities that handle seawater. In this work, a fixed-film aerobic biofilter is proposed as an effective unit for preventing biofouling in such facilities. A packed-bed biofilter with an EBCT = 6 - 11 min was employed. The results demonstrated that the DOC is reduced by 6% and the BOD7 is reduced up to 15%. The LC-OCD analysis revealed that biofiltration abates the LMW neutrals and biopolymer fractions by 33 and 17%, respectively. However, the fractionation with UF membrane showed that the biofiltration process is able to degrade the more biodegradable compounds that have molecular weights that are greater than 1 kDa and compounds with molecular weights of less than 1 kDa. After biofiltration, the biological activity measured in terms of ATP removal was reduced by 60%. Finally, a test to evaluate the biofilm formation capacity of a water sample revealed reductions of ~94% when comparing biofiltered and non-biofiltered seawater. Therefore, a fixed-film aerobic biofiltration process could be a useful treatment for the removal of biodegradable organic matter from seawater and for improving the water quality in terms of less biofilm formation capacity.