Prediction of culture performance of juvenile Litopenaeus vannamei by in vitro (pH-stat) degree of feed protein hydrolysis with species-specific enzymes

Rapid in vitro methods for measuring digestibility may be useful in analysing aqua feeds if the extent and limits of their application are clearly defined. The pH-stat protein digestibility routine with shrimp hepatopancreas enzymes was previously related to apparent protein digestibility with juvenile Litopenaeus vannamei fed diets containing different protein ingredients. The potential of the method to predict culture performance of shrimp fed six commercial feeds (T3, T4, T5, T6, T7 and T8) with 350 g kg(-1) declared crude-protein content was assessed. The consistency of results obtained using hepatopancreas enzyme extracts from either pond or clear water-raised shrimp was further verified in terms of reproducibility and possible diet history effects upon in vitro outputs. Shrimps were previously acclimated and then maintained over 56 days (initial mean weight 3.28 g) on each diet in 500-L tanks at 114 ind m(-2), clear water closed system with continuous renewal and mechanical filtering (50 mu m), with four replicates per treatment. Feeds were offered four times daily (six days a week) delivered in trays at feeding rates ranging from 4.0% to 7.0% of stocked shrimp biomass. Feed was accessible to shrimp 4 h daily for 1-h feeding period after which uneaten feed was recovered. Growth and survival were determined every 14 days from a sample of 16 individuals per tank. Water quality was monitored daily (pH, temperature and salinity) and managed by water back flushing filter cleaning every 7-10 days. Feeds were analysed for crude protein, gross energy, amino acids and pepsin digestibility. In vitro pH-stat degree of protein hydrolysis (DH%) was determined for each feed using hepatopancreas enzyme extracts from experimental (clear water) or pond-raised shrimp. Feeds resulted in significant differences in shrimp performance (P < 0.05) as seen by the differences in growth rates (0.56-0.98 g week(-1)), final weight and feed conversion ratio (FCR). Shrimp performance and in vitro DH% with pond-raised shrimp enzymes showed significant correlation (P < 0.05) for yield (R-2 = 0.72), growth rates (R-2 = 0.72-0.80) and FCR (R-2 = -0.67). Other feed attributes (protein : energy ratio, amino acids, true protein, non-protein nitrogen contents and in vitro pepsin digestibility) showed none or limited correlation with shrimp culture performance. Additional correlations were found between growth rates and methionine (R-2 = 0.73), FCR and histidine (R-2 = -0.60), and DH% and methionine or methionine+cystine feed contents (R-2 = 0.67-0.92). pH-stat assays with shrimp enzymes generated reproducible DH% results with either pond (CV <= 6.5%) or clear water (CV <= 8.5%) hepatopancreas enzyme sources. Moreover, correlations between shrimp growth rates and feed DH% were significant regardless of the enzyme origin (pond or clear water-raised shrimp) and showed consistent R-2 values. Results suggest the feasibility of using standardized hepatopancreas enzyme extracts for in vitro protein digestibility.

Batroxase, a new metalloproteinase from B. atrox snake venom with strong fibrinolytic activity

The structures and functional activities of metalloproteinases from snake venoms have been widely studied because of the importance of these molecules in envenomation. Batroxase, which is a metalloproteinase isolated from Bothrops atrox (Para) snake venom, was obtained by gel filtration and anion exchange chromatography. The enzyme is a single protein chain composed of 202 amino acid residues with a molecular mass of 22.9 kDa, as determined by mass spectrometry analysis, showing an isoelectric point of 7.5. The primary sequence analysis indicates that the proteinase contains a zinc ligand motif (HELGHNLGISH) and a sequence C164I165M166 motif that is associated with a "Met-turn" structure. The protein lacks N-glycosylation sites and contains seven half cystine residues, six of which are conserved as pairs to form disulfide bridges. The three-dimensional structure of Batroxase was modeled based on the crystal structure of BmooMP alpha-I from Bothrops moojeni. The model revealed that the zinc binding site has a high structural similarity to the binding site of other metalloproteinases. Batroxase presented weak hemorrhagic activity, with a MHD of 10 mu g, and was able to hydrolyze extracellular matrix components, such as type IV collagen and fibronectin. The toxin cleaves both a and beta-chains of the fibrinogen molecule, and it can be inhibited by EDTA. EGTA and beta-mercaptoethanol. Batroxase was able to dissolve fibrin clots independently of plasminogen activation. These results demonstrate that Batroxase is a zinc-dependent hemorrhagic metalloproteinase with fibrin(ogen)olytic and thrombolytic activity. Published by Elsevier Ltd.

CSTX-1, a toxin from the venom of the hunting spider Cupiennius salei, is a selective blocker of L-type calcium channels in mammalian neurons

The inhibitor cystine-knot motif identified in the structure of CSTX-1 from Cupiennius salei venom suggests that this toxin may act as a blocker of ion channels. Whole-cell patch-clamp experiments performed on cockroach neurons revealed that CSTX-1 produced a slow voltage-independent block of both mid/low- (M-LVA) and high-voltage-activated (HVA) insect Ca(v) channels. Since C. salei venom affects both insect as well as rodent species, we investigated whether Ca(v) channel currents of rat neurons are also inhibited by CSTX-1. CSTX-1 blocked rat neuronal L-type, but no other types of HVA Ca(v) channels, and failed to modulate LVA Ca(v) channel currents. Using neuroendocrine GH3 and GH4 cells, CSTX-1 produced a rapid voltage-independent block of L-type Ca(v) channel currents. The concentration-response curve was biphasic in GH4 neurons and the subnanomolar IC(50) values were at least 1000-fold lower than in GH3 cells. L-type Ca(v) channel currents of skeletal muscle myoballs and other voltage-gated ion currents of rat neurons, such as I(Na(v)) or I(K(v)) were not affected by CSTX-1. The high potency and selectivity of CSTX-1 for a subset of L-type channels in mammalian neurons may enable the toxin to be used as a molecular tool for the investigation of this family of Ca(v) channels.

ROLE OF GSH METABOLISM IN MEDIATING STROMAL-LEUKEMIA INTERACTION AND PROMOTING CELL SURVIVAL AND DRUG RESISTANCE IN CHRONIC LYMPHOCYTIC LEUKEMIA

Chronic lymphocytic leukemia (CLL) is the most common adult leukemia in the western countries. The interaction between CLL cells and the bone marrow stromal environment is thought to play a major role in promoting the leukemia cell survival and drug resistance. My dissertation works proved a novel biochemical mechanism by which the bone marrow stromal cells exert a profound influence on the redox status of primary CLL cells and enhance their ability to sustain oxidative stress and drug treatment. Fresh leukemia cells isolated from the peripheral blood of CLL patients exhibited two major redox alterations when they were cultured alone: a significant decrease in cellular glutathione (GSH) and an increase in basal ROS levels. However, when cultured in the presence of bone marrow stromal cells, CLL cells restored their redox balance with an increased synthesis of GSH, a decrease in spontaneous apoptosis, and an improved cell survival. Further study showed that CLL cells were under intrinsic ROS stress and highly dependent on GSH for survival, and that the bone marrow stromal cells promoted GSH synthesis in CLL cells through a novel biochemical mechanism. Cysteine is a limiting substrate for GSH synthesis and is chemically unstable. Cells normally obtain cysteine by uptaking the more stable and abundant precursor cystine from the tissue environment and convert it to cysteine intracellularly. I showed that CLL cells had limited ability to take up extracellular cystine for GSH synthesis due to their low expression of the transporter Xc-, but had normal ability to uptake cysteine. In the co-culture system, the bone marrow stromal cells effectively took up cystine and reduced it to cysteine for secretion into the tissue microenvironment to be taken up by CLL cells for GSH synthesis. The elevated GSH in CLL cells in the presence of bone marrow stromal cells significantly protected the leukemia cells from stress-induced apoptosis, and rendered them resistant to standard therapeutic agents such as fludarabine and oxaliplatin. Importantly, disabling of this protective mechanism by depletion of cellular GSH using a pharmacological approach potently sensitized CLL cells to drug treatment, and effectively enhanced the cytotoxic action of fludarabine and oxaliplatin against CLL in the presence of stromal cells. This study reveals a key biochemical mechanism of leukemia-stromal cells interaction, and identifies a new therapeutic strategy to overcome drug resistance in vivo.

Prognostic significance of xCT polymorphisms and expression in patients with advanced pancreatic cancer treated with chemotherapy

The plasma membrane xc- cystine/glutamate transporter mediates cellular uptake of cystine in exchange for intracellular glutamate and is highly expressed by pancreatic cancer cells. The xCT gene, encoding the cystine-specific xCT protein subunit of xc-, is important in regulating intracellular glutathione (GSH) levels, critical for cancer cell protection against oxidative stress, tumor growth and resistance to chemotherapeutic agents including platinum. We examined 4 single nucleotide polymorphisms (SNPs) of the xCT gene in 269 advanced pancreatic cancer patients who received first line gemcitabine with or without cisplatin or oxaliplatin. Genotyping was performed using Taqman real-time PCR assays. A statistically significant correlation was noted between the 3' untranslated region (UTR) xCT SNP rs7674870 and overall survival (OS): Median survival time (MST) was 10.9 and 13.6 months, respectively, for the TT and TC/CC genotypes (p = 0.027). Stratified analysis showed the genotype effect was significant in patients receiving gemcitabine in combination with platinum therapy (n = 145): MST was 10.5 versus 14.1 months for the TT and TC/CC genotypes, respectively (p = 0.013). The 3' UTR xCT SNP rs7674870 may correlate with OS in pancreatic cancer patients receiving gemcitabine and platinum combination therapy. Paraffin-embedded core and surgical biopsy tumor specimens from 98 patients with metastatic pancreatic adenocarcinoma were analyzed by immunohistochemistry using an xCT specific antibody. xCT protein IHC expression scores were analyzed in relation to overall survival in 86 patients and genotype in 12 patients and no statistically significant association was found between the level of xCT IHC expression score and overall survival (p = 0.514). When xCT expression was analyzed in terms of treatment response, no statistically significant associations could be determined (p = 0.908). These data suggest that polymorphic variants of xCT may have predictive value, and that the xc- transporter may represent an important target for therapy in pancreatic cancer.

Estrategias de desarrollo sostenible de la arquitectura del oasis de M’hamid, desierto del Sahara

Esta investigación se plantea con la hipótesis radical de cómo habitar el desierto de forma sostenible, desde una actitud pragmática y experimental basada en el progreso. La justificación se basa en primer lugar en los 2.000 millones de personas en el mundo que viven en entornos desérticos, el 80% de ellas, en países en desarrollo, porque el 40% de la superficie terrestre está bajo amenaza de desertificación afectando al 37% de la población mundial, con 12 millones de hectáreas al año perdidas por esa causa, y por último, porque se considera el desierto como un entorno de gran atractivo y potencial. El contenido de la investigación se estructura en tres movimientos: posicionamiento, mirada y acción: Desde el posicionamiento se define en primer lugar la sostenibilidad, aportando un nuevo diagrama donde se incorpora el ámbito arquitectónico como uno de los pilares principales, y, posteriormente, se establecen los criterios de evaluación de la sostenibilidad, aportando un sistema de indicadores donde se incorporan parámetros adecuados a las circunstancias del oasis. Del mismo modo, se estudian y analizan metodologías de actuación y proyectos de desarrollo sostenible existentes que enmarcan el estado del arte, constatando la dificultad de adaptación de los mismos a las condiciones de los oasis, por lo que se elabora una metodología propia donde se modifica la dinámica estratégica, de forma que el impulso se plantea desde la acción social, a través de hipótesis de estrategias basadas en sistemas low-cost, autoconstruidas, asumibles económicamente y de implantación factible. El caso de estudio específico radica en la situación extrema de las condiciones en el oasis de M’hamid, donde se evidencia un proceso de desintegración y abandono. Esto es debido a una acumulación de circunstancias externas e internas, de múltiples factores: naturales y antrópicos que afectan al oasis, llevando al extremo las condiciones climáticas y la escasez de recursos, naturales y artificiales. Factores como el cambio climático, la sequía, los cambios en las políticas del agua, la amenaza de desertificación, los conflictos sociales, el desequilibrio ecológico, la escasez económica, la crisis energética, la obsolescencia arquitectónica, el patrimonio construido prácticamente destruido, y la malentendida nueva arquitectura. Es importante constatar la escasa documentación gráfica existente sobre la zona de actuación lo que ha conllevado un amplio trabajo de documentación, tanto cartográfica como de observación directa, aportada a la tesis como investigación de elaboración propia. La mirada analítica al caso de estudio permite conocer los recursos disponibles y las potencialidades latentes del oasis de M’hamid, que permitirán actuar para subvertir la dinámica involutiva imperante, de forma que los dibujos iniciales de apropiación contextual y análisis críticos derivan en mapas de acción diagramados conformados por un sistema de objetos y la definición de estrategias transversales, deconstruyendo el pasado y reconstruyendo el futuro, incorporando sistemas alternativos que se definen en 7 líneas estratégicas de acción formuladas desde los 3 ámbitos relacionados con el ecosistema: ecológico, socio- económico y arquitectónico. Así, la tesis defiende la acción arquitectónica como impulsora del desarrollo sostenible, apoyada en 3 elementos: - la creación de objetos “tecnoartesanos”, para el aprovechamiento de los recursos energéticos - las transformaciones arquitectónicas, para reformular el hábitat desde la eficiencia energética y el progreso - y el impulso de acciones cotidianas, que redefinan las relaciones sociales, creando entornos cooperativos y colaborativos. En el ámbito ecológico se proponen actuaciones anti desertificación mediante incubadoras de árboles; sistemas alternativos de gestión del agua, como la lluvia sólida; estrategias de potenciación de la producción agrícola; la construcción de mecanismos de obtención de energía a partir de residuos, como los paneles solares con botellas PET. En el ámbito socioeconómico se plantean nuevas formas de acción social y de reactivación económica. Por último, en el ámbito urbano-arquitectónico, se incorporan modificaciones morfológicas a la arquitectura existente y una relectura contemporánea de la tierra, como material que permite nuevas geometrías, obteniendo arena petrificada por procesos microbiológicos, y potenciando la tierra como recurso artístico. Esta tesis es un punto de partida, recoge sistemas, estrategias y experiencias, para funcionar como un estímulo o impulso dinamizador del futuro desarrollo sostenible del oasis, abriendo vías de investigación y experimentación. ABSTRACT This research puts forth the radical hypothesis of how to inhabit the desert in a sustainable way, using a pragmatic and experimental approach based on progress. The justification for this resides in the fact that there are 2,000 million people in the world living in desert environments, 80% of them in developing countries. Forty percent of the earth’s surface is under threat of desertification, affecting 37% of the world population and with 12 million hectares being lost each year. And finally, the desert is considered as an attractive environment and therefore, with great potential. The content of the research is structured in three main sections: positioning, observation and action: As a point of departure, sustainability is defined, proposing a new framework where architecture is incorporated as one of the main pillars. Then, the criteria for evaluating sustainability are established. These provide a system of indicators, which incorporate parameters based on the specific circumstances of the oasis. Methodologies and existing sustainable development projects that represent the state-of-the-art are analyzed, discussing the difficulty of adapting them to conditions of oases. A methodology that modifies strategic concepts is developed, whereby the catalyst is social action, and strategies are developed based on low-cost, self-built, and feasible implementation systems. The specific case study lies in the extreme conditions in the oasis of M'hamid, where a process of decay and neglect is evident. This deterioration is due to an accumulation of external and internal circumstances, and of natural and anthropogenic factors that affect the oasis, leading to extreme weather conditions and a shortage of both natural and artificial resources. Factors include; climate change, drought, changes in water policies, the threat of desertification, social conflicts, ecological imbalance, economic shortage, the energy crisis, architectural obsolescence, destruction of built heritage, and misunderstood new architecture. It is important to note the extremely limited graphic information about the area has led me to produce an extensive archive of maps and drawings, many developed by direct observation, that contribute to the research. The case study analysis of the oasis of M'hamid examines the resources available and the latent potential to slow the prevailing trend towards deterioration. The initial drawings of contextual appropriation and critical analysis result in maps and diagrams of action, which are formed by a system of objects and the definition of strategies. These can be thought of as understanding or “deconstructing” the past to reconstruct the future. Alternative approaches defined in seven strategies for action are based on three fields related to the ecosystem: ecological, socioeconomic and architectural. Thus, the thesis defends architectural action to promote sustainable development, based on three elements: - The creation of "techno-artisans", to make use of energy resources - Architectural changes, to reformulate habitat in terms of energy efficiency and progress - And the promotion of everyday actions, to redefine social relations, creating cooperative and collaborative environments. In the ecological field, I propose anti-desertification actions such as; tree incubators, alternative water management systems(such as solid rain),; strategies to empower the agricultural production, energy from low-cost systems made out from recycled materials(such as solar panels from PET bottles or wind turbine from bicycle wheels). In the socioeconomic sphere, I propose to implement new forms of social action and economic regeneration. Finally, within the urban and architectural field, I propose morphological changes to the existing architecture and a contemporary reinterpretation of the earth as a material that allows new geometries, creating petrified sand by microbiological processes or enhancing nature as an artistic and energy resource. This thesis is a starting point. It collects systems, strategies and experiences to serve as a stimulus or dynamic momentum for future sustainable development of the oasis, opening new avenues of research and experimentation. RÉSUMÉ Cette recherche part d'une hypothèse radicale : comment habiter le désert de façon durable, et ce à partir d'une approche pragmatique et expérimentale basée sur le progrès. Cette hypothèse se justifie en raison des 2 milliards de personnes qui dans le monde habitent des environnements désertiques, 80% d'entre eux dans des pays en voie de développement, mais aussi parce que 40% de la surface de la planète est sous menace de désertification, un phénomène affectant 37% de la population mondiale et qui cause la perte de 12 millions d'hectares par an; et enfin parce que le désert est considéré comme un environnement très attrayant et fort d’un grand potentiel. Le contenu de la recherche se divise en trois mouvements: le positionnement, le regard et l'action : Du point de vue du positionnement on définit tout d'abord la durabilité, présentant un nouveau schéma où le domaine de l'architecture devient un des principaux piliers, et, par la suite, des critères d'évaluation de la durabilité sont établis, en fournissant un système d’indicateurs qui intègre les paramètres appropriés aux circonstances de l'oasis. De même, des méthodologies et des projets de développement durable existants sont étudiés et analysés, ce qui encadre l'état de l'art, remarquant la difficulté de les adapter aux conditions des oasis. De cette difficulté découle l'élaboration d'une méthodologie qui modifie la dynamique stratégique, de sorte que l'impulsion provient de l'action sociale, à travers des hypothèses de stratégie basées sur des systèmes low-cost, auto-construits, et de mise en oeuvre économiquement viable. Le cas d'étude spécifique réside en la situation extrême des conditions de l'oasis de M’hamid, où un processus de décadence et de négligence est évident. Cela est dû à une accumulation de circonstances externes et internes, de multiples facteurs: les facteurs naturels et anthropiques qui affectent l'oasis, menant à l'extrême les conditions météorologiques et la pénurie de ressources, autant naturelles qu'artificielles. Des facteurs tels que le changement climatique, la sécheresse, les changements dans les politiques de l'eau, la menace de la désertification, les conflits sociaux, le déséquilibre écologique, la pénurie économique, la crise de l'énergie, l'obsolescence architecturale, le patrimoine bâti pratiquement détruit et une mauvais compréhensif de la nouvelle architecture. Il est important de de faire remarquer le peu d'informations graphiques du domaine d'action, ce qui a conduit à un vaste travail de documentation, autant cartographique que relative à l'observation directe. Cette documentation s'ajoute à la thèse en tant que recherche propre. Le regard analytique sur le cas d'étude permet de connaître les ressources disponibles et le potentiel latent de l'oasis de M’hamid, qui agiront pour renverser la dynamique d'involution en vigueur. Ainsi, les premiers dessins d'appropriation contextuelle et analyse critique deviennent des cartes d'action schématisées formées par un système d'objets et la définition de stratégies transversales, qui déconstruisent le passé et reconstruisent l'avenir, en incorporant des systèmes alternatifs qui se définissent sur 7 lignes stratégiques d'action formulées à partir des 3 domaines en relation avec l’écosystème: l’écologique, le socio-économique et l'architectural. Ainsi, la thèse défend l'action architecturale en tant que promotrice du développement durable, et ce basé sur 3 éléments: - la création d'objets "technoartisans" pour l'exploitation des ressources énergétiques - les modifications architecturales, pour reformuler l'habitat du point de vue de l'efficacité énergétique et le progrès - et la promotion des actions quotidiennes, pour redéfinir les relations sociales, et la création d'environnements de coopération et collaboration. Dans le domaine de l'écologie des actions de lutte contre la désertification sont proposées à travers des pépinières d'arbres, des systèmes alternatifs de gestion de l'eau comme par exemple la pluie solide, des stratégies de mise en valeur de la production agricole, la construction de mécanismes de production d'énergie à partir de résidus, tels que les panneaux solaires ou les bouteilles en PET. Dans le domaine socio-économique, l'on propose de nouvelles formes d'action sociale et de reprise économique. Enfin, dans le domaine de l'urbain et de l'architectural, on incorpore des changements morphologiques à l'architecture existante et une relecture contemporaine de la terre, comme matériau qui permet de nouvelles géométries, en obtenant du sable pétrifié par des procédés microbiologiques et en mettant en valeur la terre comme une ressource artistique. Cette thèse n'est qu'un point de départ. Elle recueille des systèmes, des stratégies et des expériences pour servir de stimulus ou d'impulsion dynamisatrice du futur développement durable de l'oasis, en ouvrant des voies de recherche et d'expérimentation.

Phosphatidylcholine-specific phospholipase C regulates glutamate-induced nerve cell death

Phosphatidylcholine-specific phospholipase C (PC-PLC) is a necessary intermediate in transducing apoptotic signals for tumor necrosis factor and Fas/Apo-1 ligands in nonneuronal cells. The data presented here show that PC-PLC also is required in oxidative glutamate-induced programmed cell death of both immature cortical neurons and a hippocampal nerve cell line, HT22. In oxidative glutamate toxicity, which is distinct from excitotoxicity, glutamate interferes with cystine uptake by blocking the cystine/glutamate antiporter, indirectly causing a depletion of intracellular glutathione. A PC-PLC inhibitor blocks oxidative glutamate toxicity, and exogenous PC-PLC potentiates glutamate toxicity. The inhibition of PC-PLC uncouples the cystine uptake from glutamate inhibition, allowing the maintenance of glutathione synthesis and cell viability. These data suggest that PC-PLC modulates neuronal cell death through a mechanism that is distinct from that involved in nonneuronal apoptosis.

Vascular endothelial growth factor: Crystal structure and functional mapping of the kinase domain receptor binding site

Vascular endothelial growth factor (VEGF) is a homodimeric member of the cystine knot family of growth factors, with limited sequence homology to platelet-derived growth factor (PDGF) and transforming growth factor β2 (TGF-β). We have determined its crystal structure at a resolution of 2.5 Å, and identified its kinase domain receptor (KDR) binding site using mutational analysis. Overall, the VEGF monomer resembles that of PDGF, but its N-terminal segment is helical rather than extended. The dimerization mode of VEGF is similar to that of PDGF and very different from that of TGF-β. Mutational analysis of VEGF reveals that symmetrical binding sites for KDR are located at each pole of the VEGF homodimer. Each site contains two functional “hot spots” composed of binding determinants presented across the subunit interface. The two most important determinants are located within the largest hot spot on a short, three-stranded sheet that is conserved in PDGF and TGF-β. Functional analysis of the binding epitopes for two receptor-blocking antibodies reveal different binding determinants near each of the KDR binding hot spots.

Posttranslational modification of the glycosylation inhibiting factor (GIF) gene product generates bioactive GIF

Glycosylation inhibiting factor (GIF) and macrophage migration inhibitory factor (MIF) share an identical structure gene. Here we unravel two steps of posttranslational modifications in GIF/MIF molecules in human suppressor T (Ts) cell hybridomas. Peptide mapping and MS analysis of the affinity-purified GIF from the Ts cells revealed that one modification is cysteinylation at Cys-60, and the other is phosphorylation at Ser-91. Cysteinylated GIF, but not the wild-type GIF/MIF, possessed immunosuppressive effects on the in vitro IgE antibody response and had high affinity for GIF receptors on the T helper hybridoma cells. In vitro treatment of wild-type recombinant human GIF/MIF with cystine resulted in preferential cysteinylation of Cys-60 in the molecules. The cysteinylated recombinant human GIF and the Ts hybridoma-derived cysteinylated GIF were comparable both in the affinity for the receptors and in the immunosuppressive activity. Polyclonal antibodies specific for a stretch of the amino acid sequence in α2-helix of GIF bound bioactive cysteinylated GIF but failed to bind wild-type GIF/MIF. These results strongly suggest that cysteinylation of Cys-60 and consequent conformational changes in the GIF/MIF molecules are responsible for the generation of GIF bioactivity.

Sulfur K-edge x-ray absorption spectroscopy: A spectroscopic tool to examine the redox state of S-containing metabolites in vivo

The sulfur K-edge x-ray absorption spectra for the amino acids cysteine and methionine and their corresponding oxidized forms cystine and methionine sulfoxide are presented. Distinct differences in the shape of the edge and the inflection point energy for cysteine and cystine are observed. For methionine sulfoxide the inflection point energy is 2.8 eV higher compared with methionine. Glutathione, the most abundant thiol in animal cells, also has been investigated. The x-ray absorption near-edge structure spectrum of reduced glutathione resembles that of cysteine, whereas the spectrum of oxidized glutathione resembles that of cystine. The characteristic differences between the thiol and disulfide spectra enable one to determine the redox status (thiol to disulfide ratio) in intact biological systems, such as unbroken cells, where glutathione and cyst(e)ine are the two major sulfur-containing components. The sulfur K-edge spectra for whole human blood, plasma, and erythrocytes are shown. The erythrocyte sulfur K-edge spectrum is similar to that of fully reduced glutathione. Simulation of the plasma spectrum indicated 32% thiol and 68% disulfide sulfur. The whole blood spectrum can be simulated by a combination of 46% disulfide and 54% thiol sulfur.

The role of distinct p185neu extracellular subdomains for dimerization with the epidermal growth factor (EGF) receptor and EGF-mediated signaling

The extracellular domain of p185c-neu can be viewed as a complex structure of four subdomains, two of which are cysteine-rich subdomains. We have investigated the contribution of these distinct p185c-neu extracellular subdomains to p185/epidermal growth factor receptor (EGFR) heteromer formation and EGF-induced heteromeric signaling. Our studies indicate that at least two separate p185 subdomains, a region spanning subdomains I and II and subdomain IV are involved in association of p185 with the EGFR. We also demonstrated that subdomain IV reduced the heteromeric signaling and transforming activities induced by EGF after associating with EGFR. When 126 aa were deleted from subdomain IV, this small subdomain IV-derived fragment could still lead to heterodimers with EGFR and suppress EGF-induced mitogen-activated protein kinase activation and subsequent transformation abilities. These data provide information about trans-inhibitory mechanisms of mutant p185 species and also indicate that both the entire and a part of subdomain IV may represent a therapeutic target for erbB-overexpressing tumors. Finally, these studies define a basic feature of receptor-receptor associations that are determined by cystine-knot containing subdomains.

Purification and Characterization of a Galactose-Rich Basic Glycoprotein in Tobacco1

We found a galactose-rich basic glycoprotein (GBGP) in the cell walls of cultured tobacco (Nicotiana tabacum) cells. GBGP and extensin were isolated as the major components of basic, salt-extracted cell wall glycoproteins. GBGP and extensin were separated by gel filtration in 6 m guanidine hydrochloride as 49- and 90-kD peaks, respectively, and further purified with reverse-phase chromatography. The protein moiety of GBGP constitutes about one-half of the molecule (w/w) and contains lysine (16%), proline (12%), hydroxyproline (10%), tyrosine (4%), alanine (7%), leucine (6%), and cystine (1.4%). Galactose accounted for 72% of the sugar moiety, arabinose content was low (17%), and a significant amount of mannose (7%) was found. No immunological cross-reaction was detected between GBGP and extensin. The antibody against native GBGP with sugar chains reacted with other glycoproteins on the gel blots, whereas the antibodies against deglycosylated GBGP and native extensin were highly specific. Immunolocalization analysis in tobacco stems showed that GBGP is specific to parenchyma tissue and that extensin localizes in the epidermis. This tissue-specific and exclusive distribution suggests important functions of these basic glycoproteins.

Active site topology and reaction mechanism of GTP cyclohydrolase I.

GTP cyclohydrolase I of Escherichia coli is a torus-shaped homodecamer with D5 symmetry and catalyzes a complex ring expansion reaction conducive to the formation of dihydroneopterin triphosphate from GTP. The x-ray structure of a complex of the enzyme with the substrate analog, dGTP, bound at the active site was determined at a resolution of 3 A. In the decamer, 10 equivalent active sites are present, each of which contains a 10-A deep pocket formed by surface areas of 3 adjacent subunits. The substrate forms a complex hydrogen bond network with the protein. Active site residues were modified by site-directed mutagenesis, and enzyme activities of the mutant proteins were measured. On this basis, a mechanism of the enzyme-catalyzed reaction is proposed. Cleavage of the imidazole ring is initiated by protonation of N7 by His-179 followed by the attack of water at C8 of the purine system. Cystine Cys-110 Cys-181 may be involved in this reaction step. Opening of the imidazole ring may be in concert with cleavage of the furanose ring to generate a Schiff's base from the glycoside. The gamma-phosphate of GTP may be involved in the subsequent Amadori rearrangement of the carbohydrate side chain by activating the hydroxyl group of Ser-135.

Complex flexibility of the transforming growth factor beta superfamily.

The transforming growth factors beta (TGF-beta s) are important modulators of growth and differentiation. They are intermolecular disulfide-bonded homodimeric molecules. The monomer fold has a conserved cystine knot and lacks a hydrophobic core. The biological specificity of a given member of the family is believed to be determined by the conformational flexibility of the variable loop regions of the monomer. The monomer subunit assembly in the dimer is stabilized mainly by hydrophobic contacts and a few hydrogen bonds. Since these interactions are nondirectional, we examined subunit assemblies of TGF-beta by using conformational analysis. The different subunit assemblies in TGF-beta 2 dimer were characterized in terms of the intersubunit disulfide torsion. Our analyses show that the subunit assemblies fall into two states: the crystallographically observed gauche+conformation and the previously not reported gauche--conformation, both having almost identical interaction energies. Furthermore, there is significant flexibility in the subunit assembly within the gauche+ and the gauche- states of the disulfide bond. The monomer subunit assembly is independent of the variations about the loop regions. The variations in the loop regions, coupled with flexibility in the monomer assembly, lead to a complex flexibility in the dimer of the TGF-beta superfamily. For the TGF-beta superfamily, the cystine knot acts as a scaffold and complex flexibility provides for biological selectivity. Complex flexibility might provide an explanation for the diverse range of biological activities that these important molecules display.

Cistinose : a propósito de uma doença renal de longa evolução com repercussões sobre o globo ocular

Trabalho Final do Curso de Mestrado Integrado em Medicina, Faculdade de Medicina, Universidade de Lisboa, 2014