Anaesthetic impairment of immune function is mediated via GABA(A) receptors.

BACKGROUND: GABA(A) receptors are members of the Cys-loop family of neurotransmitter receptors, proteins which are responsible for fast synaptic transmission, and are the site of action of wide range of drugs. Recent work has shown that Cys-loop receptors are present on immune cells, but their physiological roles and the effects of drugs that modify their function in the innate immune system are currently unclear. We are interested in how and why anaesthetics increase infections in intensive care patients; a serious problem as more than 50% of patients with severe sepsis will die. As many anaesthetics act via GABA(A) receptors, the aim of this study was to determine if these receptors are present on immune cells, and could play a role in immunocompromising patients. PRINCIPAL FINDINGS: We demonstrate, using RT-PCR, that monocytes express GABA(A) receptors constructed of α1, α4, β2, γ1 and/or δ subunits. Whole cell patch clamp electrophysiological studies show that GABA can activate these receptors, resulting in the opening of a chloride-selective channel; activation is inhibited by the GABA(A) receptor antagonists bicuculline and picrotoxin, but not enhanced by the positive modulator diazepam. The anaesthetic drugs propofol and thiopental, which can act via GABA(A) receptors, impaired monocyte function in classic immunological chemotaxis and phagocytosis assays, an effect reversed by bicuculline and picrotoxin. SIGNIFICANCE: Our results show that functional GABA(A) receptors are present on monocytes with properties similar to CNS GABA(A) receptors. The functional data provide a possible explanation as to why chronic propofol and thiopental administration can increase the risk of infection in critically ill patients: their action on GABA(A) receptors inhibits normal monocyte behaviour. The data also suggest a potential solution: monocyte GABA(A) receptors are insensitive to diazepam, thus the use of benzodiazepines as an alternative anesthetising agent may be advantageous where infection is a life threatening problem.

Micro-systems for Optical Protein-Chip

Protein-Chip as micro-assays for the determination of protein interaction, the analysis, the identification and the purification of proteins has large potential applications. The Optical Protein-Chip is able to detect the multi-interaction of proteins and multi-bio-activities of molecules directly and simultaneously with no labeling. The chip is a small matrix on solid substrate containing multi-micro-area prepared by microfabrication with photolithography or soft lithography for surface patterning, and processed with surface modification which includes the physical, chemical, and bio-chemical modifications, etc. The ligand immobilization, such as protein immobilization, especially the oriented immobilization with low steric hindrance and high bio-specific binding activity between ligand and receptor is used to form a sensing surface. Each area of the pattern is corresponding to only one bioactivity. The interval between the areas is non-bioactive and optically extinctive. The affinity between proteins is used to realize non-labeling microassays for the determination of protein identification and protein interaction. The sampling of the chip is non-disturbing, performed with imaging ellipsometry and image processing on a database of proteins.

Probing biomolecular interaction forces using an anharmonic acoustic technique for selective detection of bacterial spores.

Receptor-based detection of pathogens often suffers from non-specific interactions, and as most detection techniques cannot distinguish between affinities of interactions, false positive responses remain a plaguing reality. Here, we report an anharmonic acoustic based method of detection that addresses the inherent weakness of current ligand dependant assays. Spores of Bacillus subtilis (Bacillus anthracis simulant) were immobilized on a thickness-shear mode AT-cut quartz crystal functionalized with anti-spore antibody and the sensor was driven by a pure sinusoidal oscillation at increasing amplitude. Biomolecular interaction forces between the coupled spores and the accelerating surface caused a nonlinear modulation of the acoustic response of the crystal. In particular, the deviation in the third harmonic of the transduced electrical response versus oscillation amplitude of the sensor (signal) was found to be significant. Signals from the specifically-bound spores were clearly distinguishable in shape from those of the physisorbed streptavidin-coated polystyrene microbeads. The analytical model presented here enables estimation of the biomolecular interaction forces from the measured response. Thus, probing biomolecular interaction forces using the described technique can quantitatively detect pathogens and distinguish specific from non-specific interactions, with potential applicability to rapid point-of-care detection. This also serves as a potential tool for rapid force-spectroscopy, affinity-based biomolecular screening and mapping of molecular interaction networks.

Comportamiento prooxidante y antioxidante de hierbas aromáticas en queso

Los quesos de cabra, con mejores cualidades funcionales provienen de leche de cabras en pastoreo, esto obedece al mayor contenido de ácidos grasos ω-3 y ω-6 en una relación óptima para la salud, sumado a un menor contenido de grasa y colesterol. Saborizar quesos con especias, otorga beneficios sensoriales y saludables al producto. Las especias son usadas para realzar el flavor de los alimentos además presentan beneficios antioxidantes. Sin embargo, se reportaron evidencias del doble papel de los flavonoides como antioxidantes o prooxidantes en función de la concentración de uso. El objetivo del trabajo es evaluar el efecto de distintas concentraciones de especias aromáticas con capacidad antioxidante, frente a las reacciones de deterioro que ocurren en el almacenamiento de quesos de cabra. Se estudió la actividad antirradicalaria (AAR) de 18 especias. Para evaluar la actividad antioxidante (AA0) se usó la técnica de desaparición de un radical libre estable el 2,2-difenil-1-picril hidrazilo (DPPH) y se calculó la AAR porcentual. Definido el queso base estándar (control), se ensayaron tres variedades con especies, elegidas de entre las de mayor AAR (orégano, romero y tomillo) y se adicionaron con las concentraciones 0,4 – 1 % p/p. Estos, fueron madurados 30 días y almacenados 7 meses a 4 °C. Se analizaron cada 4 semanas. Se realizaron pruebas sensoriales de aceptabilidad con 60 consumidores de queso (condición), usando planillas, con escala hedónica. Se trabajó sobre los lípidos extraídos de los quesos. El rancidez y deterioro fueron evaluados con análisis de acidez; índice peróxido y sustancias reactivas al ácido TBA. Los resultados se compararon con el comportamiento sensorial del producto en el tiempo de almacenamiento. En los quesos adicionados al 1 %, la acidez se incrementó con el almacenamiento. El máximo valor corresponde al queso con orégano (3,72 % de ac. láctico a los 126 días). En el control, la acidez inicial fue inferior y alcanzó 1,05 g de ác. láctico % a los 166 días. El IP más alto corresponde a los quesos con orégano al 1% , resultado que se contrapone a la actividad antirradicalaria de esta especia. Al 0,4 %, tanto el IP como el número de TBA son menores en quesos adicionados, respecto del control. En los quesos de cabra analizados las especias agregadas al 1% ejercieron acción prooxidante, mientras que al ser agregadas al 0,4% la acción es antioxidante, lo que indica la importancia de la selección de concentraciones adecuadas.

Accuracy in Copy Number Calling by qPCR and PRT : A Matter of DNA

7 p.

Monitoring hydrilla using two RAPD procedures and the Nonindigenous Aquatic Species database

Hydrilla ( Hydrilla verticillata (L.f.) Royle), an invasive aquatic weed, continues to spread to new regions in the United States. Two biotypes, one a female dioecious and the other monoecious have been identified. Management of the spread of hydrilla requires understanding the mechanisms of introduction and transport, an ability to map and make available information on distribution, and tools to distinguish the known U.S. biotypes as well as potential new introductions. Review of the literature and discussions with aquatic scientists and resource managers point to the aquarium and water garden plant trades as the primary past mechanism for the regional dispersal of hydrilla while local dispersal is primarily carried out by other mechanisms such as boat traffic, intentional introductions, and waterfowl. The Nonindigenous Aquatic Species (NAS) database is presented as a tool for assembling, geo-referencing, and making available information on the distribution of hydrilla. A map of the current range of dioecious and monoecious hydrilla by drainage is presented. Four hydrilla samples, taken from three discrete, non-contiguous regions (Pennsylvania, Connecticut, and Washington State) were examined using two RAPD assays. The first, generated using primer Operon G17, and capable of distinguishing the dioecious and monoecious U.S. biotypes, indicated all four samples were of the monoecious biotype. Results of the second assay using the Stoffel fragment and 5 primers, produced 111 markers, indicated that these samples do not represent new foreign introductions. The differences in the monoecious and dioecious growth habits and management are discussed.

Wide Screening of Phage-Displayed Libraries Identifies Immune Targets in Planta

Microbe-Associated Molecular Patterns and virulence effectors are recognized by plants as a first step to mount a defence response against potential pathogens. This recognition involves a large family of extracellular membrane receptors and other immune proteins located in different sub-cellular compartments. We have used phage-display technology to express and select for Arabidopsis proteins able to bind bacterial pathogens. To rapidly identify microbe-bound phage, we developed a monitoring method based on microarrays. This combined strategy allowed for a genome-wide screening of plant proteins involved in pathogen perception. Two phage libraries for high-throughput selection were constructed from cDNA of plants infected with Pseudomonas aeruginosa PA14, or from combined samples of the virulent isolate DC3000 of Pseudomonas syringae pv. tomato and its avirulent variant avrRpt2. These three pathosystems represent different degrees in the specificity of plant-microbe interactions. Libraries cover up to 26107 different plant transcripts that can be displayed as functional proteins on the surface of T7 bacteriophage. A number of these were selected in a bio-panning assay for binding to Pseudomonas cells. Among the selected clones we isolated the ethylene response factor ATERF-1, which was able to bind the three bacterial strains in competition assays. ATERF-1 was rapidly exported from the nucleus upon infiltration of either alive or heat-killed Pseudomonas. Moreover, aterf-1 mutants exhibited enhanced susceptibility to infection. These findings suggest that ATERF-1 contains a microbe-recognition domain with a role in plant defence. To identify other putative pathogen-binding proteins on a genome-wide scale, the copy number of selected-vs.-total clones was compared by hybridizing phage cDNAs with Arabidopsis microarrays. Microarray analysis revealed a set of 472 candidates with significant fold change. Within this set defence-related genes, including well-known targets of bacterial effectors, are over-represented. Other genes non-previously related to defence can be associated through this study with general or strain-specific recognition of Pseudomonas.

Extent and toxicity of contaminated marine sediments in Southeastern Florida

Thirty sites were sampled in southern Biscayne Bay and Manatee Bay in December 1999 to determine the extent of toxicity in sediments. Analyses and assays included: pesticides and phenols in seawater; chemical contaminants in sediment; amphipod mortality, HRGS P450, sea urchin sperm fertilization and embryology, MicrotoxTM, MutatoxTM, grass shrimp AChE and juvenile clam mortality assays; sea urchin sperm, amphipod and oyster DNA damage; and benthic community assessment. Sediment sites near the mouth of canals showed evidence of contamination. Contaminant plumes and associated toxicity do not appear to extend seaward of the mouth of the canals in an appreciable manner. Concentrations of contaminants in the sediments in open areas of Biscayne and Manatee Bays are generally low. (PDF contains 140 pages)

SNP Discovery in European Anchovy (Engraulis encrasicolus, L) by High-Throughput Transcriptome and Genome Sequencing

12 p.

A Computational Module Assembled from Different Protease Family Motifs Identifies PI PLC from Bacillus cereus as a Putative Prolyl Peptidase with a Serine Protease Scaffold

Proteolytic enzymes have evolved several mechanisms to cleave peptide bonds. These distinct types have been systematically categorized in the MEROPS database. While a BLAST search on these proteases identifies homologous proteins, sequence alignment methods often fail to identify relationships arising from convergent evolution, exon shuffling, and modular reuse of catalytic units. We have previously established a computational method to detect functions in proteins based on the spatial and electrostatic properties of the catalytic residues (CLASP). CLASP identified a promiscuous serine protease scaffold in alkaline phosphatases (AP) and a scaffold recognizing a beta-lactam (imipenem) in a cold-active Vibrio AP. Subsequently, we defined a methodology to quantify promiscuous activities in a wide range of proteins. Here, we assemble a module which encapsulates the multifarious motifs used by protease families listed in the MEROPS database. Since APs and proteases are an integral component of outer membrane vesicles (OMV), we sought to query other OMV proteins, like phospholipase C (PLC), using this search module. Our analysis indicated that phosphoinositide-specific PLC from Bacillus cereus is a serine protease. This was validated by protease assays, mass spectrometry and by inhibition of the native phospholipase activity of PI-PLC by the well-known serine protease inhibitor AEBSF (IC50 = 0.018 mM). Edman degradation analysis linked the specificity of the protease activity to a proline in the amino terminal, suggesting that the PI-PLC is a prolyl peptidase. Thus, we propose a computational method of extending protein families based on the spatial and electrostatic congruence of active site residues.

Role of G protein coupled inwardly rectifier K+ channels (GIRK) on the neurobiology depression

353 págs.

Prognostic Role of Host Cyclooxygenase and Cytokine Genotypes in a Caucasian Cohort of Patients with Gastric Adenocarcinoma

11 p.

Expression and activity profiles of DPP IV/CD26 and NEP/CD10 glycoproteins in the human renal cancer are tumor-type dependent

Background: Cell-surface glycoproteins play critical roles in cell-to-cell recognition, signal transduction and regulation, thus being crucial in cell proliferation and cancer etiogenesis and development. DPP IV and NEP are ubiquitous glycopeptidases closely linked to tumor pathogenesis and development, and they are used as markers in some cancers. In the present study, the activity and protein and mRNA expression of these glycoproteins were analysed in a subset of clear-cell (CCRCC) and chromophobe (ChRCC) renal cell carcinomas, and in renal oncocytomas (RO). Methods: Peptidase activities were measured by conventional enzymatic assays with fluorogen-derived substrates. Gene expression was quantitatively determined by qRT-PCR and membrane-bound protein expression and distribution analysis was performed by specific immunostaining. Results: The activity of both glycoproteins was sharply decreased in the three histological types of renal tumors. Protein and mRNA expression was strongly downregulated in tumors from distal nephron (ChRCC and RO). Moreover, soluble DPP IV activity positively correlated with the aggressiveness of CCRCCs (higher activities in high grade tumors). Conclusions: These results support the pivotal role for DPP IV and NEP in the malignant transformation pathways and point to these peptidases as potential diagnostic markers.

Food composition and digestive enzymes in the gut of the African electric catfish Malapterurus electricus (Gmelin 1789) (Malapteruridae)

Analyses of stomach contents of 330 Malapterurus electricus (standard length, 10.1-30.5 cm) in Mahin Lagoon (southwestern Nigeria) established it as a bottom feeder. There was a preponderance of insects accounting for >80% occurrence and >25% of total volume in stomachs of specimens, suggesting a stenophagous predatory habit. Qualitative and quantitative assays of digestive enzymes in the different regions of the gut (oesophagus, stomach, duodenum, ileum, rectum) were investigated. Carbohydrases (amylase, maltase), chitinase, proteases (pepsin, chymotrypsin, trypsin) and lipases were detected in different gut regions with different activity. The pattern of distribution and relative activity of the enzymes correlated with the predatory diet

rhegf-Loaded Plga-Alginate Microspheres Enhance The Healing Of Full-Thickness Excisional Wounds In Diabetised Wistar Rats

[EN] Diabetic foot ulcers (DFUs) represent a major clinical challenge in the ageing population. To address this problem, rhEGF-loaded Poly-Lactic-co-Glycolic-Acid (PLGA)-Alginate microspheres (MS) were prepared by a modified w/o/w-doubleemulsion/ solvent evaporation method. Different formulations were evaluated with the aim of optimising MSs properties by adding NaCl to the surfactant solution and/or the solvent removal phase and adding alginate as a second polymer. The characterization of the developed MS showed that alginate incorporation increased the encapsulation efficiency (EE) and NaCl besides increasing the EE also became the particle surface smooth and regular. Once the MS were optimised, the target loading of rhEGF was increased to 1% (PLGA-Alginate MS), and particles were sterilised by gamma radiation to provide the correct dosage for in vivo studies. In vitro cell culture assays demonstrated that neither the microencapsulation nor the sterilisation process affected rhEGF bioactivity or rhEGF wound contraction. Finally, the MS were evaluated in vivo for treatment of the full-thickness wound model in diabetised Wistar rats. rhEGF MS treated animals showed a statistically significant decrease of the wound area by days 7 and 11, a complete re-epithelisation by day 11 and an earlier resolution of the inflammatory process. Overall, these findings demonstrate the promising potential of rhEGF-loaded MS (PLGA-Alginate MS) to promote faster and more effective wound healing, and suggest its possible application in DFU treatment.