C-terminal sequences in R-Ras are involved in integrin regulation and in plasma membrane microdomain distribution

The small GTPases R-Ras and H-Ras are highly homologous proteins with contrasting biological properties, for example, they differentially modulate integrin affinity: H-Ras suppresses integrin activation in fibroblasts whereas R-Ras can reverse this effect of H-Ras. To gain insight into the sequences directing this divergent phenotype, we investigated a panel of H-Ras/R-Ras chimeras and found that sequences in the R-Ras hypervariable C-terminal region including amino acids 175-203 are required for the R-Ras ability to increase integrin activation in CHO cells; however, the proline-rich site in this region, previously reported to bind the adaptor protein Nck, was not essential for this effect. In addition, we found that the GTPase TC21 behaved similarly to R-Ras. Because the C-termini of Ras proteins can control their subcellular localization, we compared the localization of H-Ras and R-Ras. In contrast to H-Ras, which migrates out of lipid rafts upon activation, we found that activated R-Ras remained localized to lipid rafts. However, functionally distinct H-Ras/R-Ras chimeras containing different C-terminal R-Ras segments localized to lipid rafts irrespective of their integrin phenotype. (C) 2003 Elsevier Inc. All rights reserved.

14-3-3 acts as an intramolecular bridge to regulate cdc25B localization and activity

One of the major regulators of mitosis in somatic cells is cdc25B. cdc25B is tightly regulated at multiple levels. The final activation step involves the regulated binding of 14-3-3 proteins. Previous studies have demonstrated that Ser-323 is a primary 14-3-3 binding site in cdc25B, which influences its activity and cellular localization. 14-3-3 binding to this site appeared to interact with the N-terminal domain of cdc25B to regulate its activity. The presence of consensus 14-3-3 binding sites in the N-terminal domain suggested that the interaction is through direct binding of the 14-3-3 dimer to sites in the N-terminal domain. We have identified Ser-151 and Ser-230 in the N-terminal domain as functional 14-3-3 binding sites utilized by cdc25B in vivo. These low affinity sites cooperate to bind the 14-3-3 dimer bound to the high affinity Ser-323 site, thus forming an intramolecular bridge that constrains cdc25B structure to prevent access of the catalytic site. Loss of 14-3-3 binding to either N-terminal site relaxes cdc25B structure sufficiently to permit access to the catalytic site, and the nuclear export sequence located in the N-terminal domain. Mutation of the Ser-323 site was functionally equivalent to the mutation of all three sites, resulting in the complete loss of 14-3-3 binding, increased access of the catalytic site, and access to nuclear localization sequence.

The development of chromatography for the characterization of protein interactions: a personal perspective

This article reviews the progress of a personal endeavour to develop chromatography as a quantitative procedure for the determination of reaction stoichiometries and equilibrium constants governing protein interactions. As well as affording insight into an aspect of chromatography with which many protein chemists are unfamiliar, it shows the way in which minor adaptations of conventional chromatographic practices have rendered the technique one of the most powerful methods available for the characterization of interactions. That pathway towards quantification is followed from the introduction of frontal gel filtration for the study of protein self-association to the characterization of ligand binding by the biosensor variant of quantitative affinity chromatography.

Dietary interactions influence the effects of bovine folate-binding protein on the bioavailability of tetrahydrofolates in rats

The newborns of mammals have a high folate demand, yet obtain adequate folate nutrition solely from their mothers' milk despite its low folate content. Milk folate is entirely bound by an excess of folate-binding protein (FBP), prompting speculation that FBP may affect the bioavailability of the limited folate supply. Previous research has shown that FBP-bound folic acid is more gradually absorbed, thereby reducing the peak plasma folate concentration and preventing loss into the urine. Natural folates are reduced derivatives of folic acid, with milk predominantly containing 5-methyltetrahydrofolate, yet little research has been carried out to determine the role of FBP in the bioavailability of reduced folates. We studied the effect of FBP on folate nutrition of rats in both single-dose and 4-wk feeding experiments. The effect of FBP was influenced by the presence of other milk components. FBP increased bioavailability of dietary folate when it was consumed with other whey proteins or with soluble casein. However, in the presence of acid-precipitated casein or a whey preparation enriched in lipids, bioavailability was decreased. These results highlight the difficulties of extrapolating from experimental results obtained using purified diets alone and of studying interactions among dietary components. They suggest that the addition of FBP-rich foods to folate-rich foods could enhance the bioavailability of natural folates, but that the outcome of such a combination would depend on interactions with other components of the diet.

Greater replication and differentiation of preadipocytes in inherited corticosteroid-binding globulin deficiency

Glucocorticoids are pivotal for adipose tissue development. Rodent studies suggest that corticosteroid-binding globulin (CBG) modulates glucocorticoid action in adipose tissue. In humans, both genetic CBG deficiency and suppressed CBG concentrations in hyperinsulinemic states are associated with obesity. We hypothesized that CBG deficiency in humans modulates the response of human preadipocytes to glucocorticoids, predisposing them to obesity. We compared normal preadipocytes with subcultured preadipocytes from an individual with the first ever described complete deficiency of CBG due to a homozygous null mutation. CBG-negative preadipocytes proliferated more rapidly and showed greater peroxisome proliferator-activated receptor-gamma-mediated differentiation than normal preadipocytes. CBG was not expressed in normal human preadipocytes. Glucocorticoid receptor number and binding characteristics and 11beta-hydroxysteroid dehydrogenase activity were similar for CBG-negative and normal preadipocytes. We propose that the increased proliferation and enhanced differentiation of CBG-negative preadipocytes may promote adipose tissue deposition and explain the obesity seen in individuals with genetic CBG deficiency. Furthermore, these observations may be relevant to obesity occurring with suppressed CBG concentrations associated with hyperinsulinemia.

Identification and characterization of the hepatic stellate cell transferrin receptor

Activated hepatic stellate cells have been implicated in the fibrogenic process associated with iron overload, both in animal models and in human hemochromatosis. Previous studies have evaluated the role of ferritin/ferritin receptor interactions in the activation of stellate cells and subsequent fibrogenesis; however, the role of transferrin in hepatic stellate cell biology is unknown. This study was designed to identify and characterize the stellate cell transferrin receptor and to evaluate the influence of transferrin on stellate cell activation. Identification and characterization of the stellate cell transferrin receptor was determined by competitive displacement assays. The effect of transferrin on stellate cell activation was assessed using western blot analysis for alpha-smooth muscle actin expression, [H-3]Thymidine incorporation, and real-time RT-PCR for procollagen 1(I) mRNA expression. A specific receptor for rat transferrin was observed on activated but not quiescent stellate cells. Transferrin significantly increased the expression of alpha-smooth muscle actin, but caused a decrease in proliferation. Transferrin induced a significant increase in procollagen alpha1(I) mRNA expression. In conclusion, this study has demonstrated for the first time a specific, high affinity receptor for rat transferrin on activated hepatic stellate cells, which via interaction with transferrin regulates stellate cell activation. This suggests that transferrin may be an important factor in the activation of hepatic stellate cells in conditions of iron overload.

Purification of post-translationally modified proteins from bacteria: homologous expression and purification of histidine-tagged pilin from Neisseria meningitidis

Until recently, glycosylation of proteins in prokaryotes was regarded as uncommon and thought to be limited to special cases such as S-layer proteins and some archeal outer membrane proteins. Now, there are an increasing number of reports of bacterial proteins that are glycosylated. Pilin of pathogenic Neisseria is one of the best characterised post-translation ally modified bacterial proteins, with four different types of modifications reported, including a novel glycosylation. Pilin monomers assemble to form pilus fibres, which are long protein filaments that protrude from the surface of bacterial cells and are key virulence factors. To aid in the investigation of these modifications, pure pilin is required. A number of pilin purification methods have been published, but none are appropriate for the routine purification of pilin from many different isolates. This study describes a novel, rapid, and simple method of pilin purification from Neisseria meningitidis C311#3, which facilitates the production of consistent quantities of pure, native pilin. A 6 x histidine tag was fused to the C-terminus of the pilin subunit structural gene, pilE, via homologous recombination placing the 6 x histidine-tagged allele in the chromosome of N. meningitidis C311#3. Pilin was purified under non-denaturing conditions via a two-step process using immobilised metal affinity chromatography (IMAC), followed by dye affinity chromatography. Analysis of the purified pilin confirmed that it retained both of the post-translational modifications examined. This novel approach may prove to be a generally applicable method for purification and analysis of post-translationally modified proteins in bacteria. (C) 2003 Elsevier Science (USA). All rights reserved.

Recombinant expression of Munc18c in a baculovirus system and interaction with syntaxin4

Two protein families that are critical for vesicle transport are the Syntaxin and Munc18/Sec1. families of proteins. These two molecules form a high affinity complex and play an essential role in vesicle docking and fusion. Munc18c was expressed as an N-terminally His-tagged fusion protein from recombinant baculovirus in Sf9 insect cells. His-tagged Munc18c was purified to homogeneity using both cobalt-chelating affinity chromatography and gel filtration chromatography. With this simple two-step protocol, 3.5 mg of purified Munc18c was obtained from a 1 L culture. Further, the N-terminal His-tag could be removed by thrombin cleavage while the tagged protein was bound to metal affinity resin. Recombinant Munc18c produced in this way is functional, in that it forms a stable complex with the SNARE interacting partner, syntaxin4. Thus we have developed a method for producing and purifying large amounts of functional Munc18c-both tagged and detagged-from a baculovirus expression system. We have also developed a method to purify the Munc18c:syntaxin4 complex. These methods will be employed for future functional and structural studies. Crown copyright (C) 2003 Published by Elsevier Inc. All rights reserved.

Práticas de militância nômades : experimentações rebeldes e novas estéticas de luta no contemporâneo

A presente dissertação, realizada no âmbito da experiência de militância deste pesquisador em três coletivos distintos, Brigada Indígena (2005-2008), Assembléia Popular (2009) e Fórum Municipal dos Psicólogos da Secretaria de Assistência Social da Prefeitura de Vitória (2009-2011), se propõem a discutir os modos de funcionamento e as formas de organização dos movimentos sociais na invenção de estéticas de luta problematizadoras das redes de poder e resistência instituídas no contemporâneo. Por meio da narrativa dessas experiências, procura-se evidenciar, no interior dessas movimentações, a gestação de práticas de militância (pré) ocupadas não apenas com a concretização de projetos, mas principalmente, com a experimentação de novos modos de existência avessos à lógica de funcionamento do aparelho estatal. Vamos, ao longo do texto, mostrar como esses coletivos caracterizam-se pela insubmissão às condutas regradas de espaços institucionalizados, exercitando levantes provisórios, anônimos, situados e descentralizados, instituídos por e instituinte de ecossistemas afetivos-políticossociais autônomos. As análises aqui empreendidas se fizeram no entrecruzamento dos aportes teóricos advindos dos trabalhos de Foucault, Lourau, Lapassade, Deleuze e Guattari, Hakim Bey, Michel Hardt e Toni Negri. Ao final, a pesquisa sinaliza a possibilidade de pensarmos uma antropologia rebelde, no qual grupos de afinidade com padrão horizontalizado de funcionamento interpelam relações hierárquicas e centralizadoras que marcam o modo de funcionamento da máquina administrativa do Estado e dos próprios movimentos sociais, potencializando agenciamentos nômades implicados na construção de espaços auto-gestionados.

UN Security Council decision-making: testing the bribery hypothesis

Liberal-Institutionalism and Structural Realism expectations about international organizations are confronted by looking at if and how US-controlled international aid is granted, and particularly if it is related or not to political affinity and to United Nations Security Council (UNSC) non-permanent membership. A preliminary assessment suggests that these relations only hold for the period of the Cold War, and, even then, only when UNSC non-permanent membership is in years in which the Security Council was deemed very important.

Surface plasmon resonance as a tool in the functional analisys of an immunodominant site in foot-and-mouth disease virus

A fast and direct surface plasmon resonance (SPR) method for the kinetic analysis of the interactions between peptide antigens and immobilised monoclonal antibodies (mAb) has been established. Protocols have been developed to overcome the problems posed by the small size of the analytes (< 1600 Da). The interactions were well described by a simple 1:1 bimolecular interaction and the rate constants were self-consistent and reproducible. The key features for the accuracy of the kinetic constants measured were high buffer flow rates, medium antibody surface densities and high peptide concentrations. The method was applied to an extensive analysis of over 40 peptide analogues towards two distinct anti-FMDV antibodies, providing data in total agreement with previous competition ELISA experiments. Eleven linear 15-residue synthetic peptides, reproducing all possible combinations of the four replacements found in foot-and-mouth disease virus (FMDV) field isolate C-S30, were evaluated. The direct kinetic SPR analysis of the interactions between these peptides and three anti-site A mAbs suggested additivity in all combinations of the four relevant mutations, which was confirmed by parallel ELISA analysis. The four-point mutant peptide (A15S30) reproducing site A from the C-S30 strain was the least antigenic of the set, in disagreement with previously reported studies with the virus isolate. Increasing peptide size from 15 to 21 residues did not significantly improve antigenicity. Overnight incubation of A15S30 with mAb 4C4 in solution showed a marked increase in peptide antigenicity not observed for other peptide analogues, suggesting that conformational rearrangement could lead to a stable peptide-antibody complex. In fact, peptide cyclization clearly improved antigenicity, confirming an antigenic reversion in a multiply substituted peptide. Solution NMR studies of both linear and cyclic versions of the antigenic loop of FMDV C-S30 showed that structural features previously correlated with antigenicity were more pronounced in the cyclic peptide. Twenty-six synthetic peptides, corresponding to all possible combinations of five single-point antigenicity-enhancing replacements in the GH loop of FMDV C-S8c1, were also studied. SPR kinetic screening of these peptides was not possible due to problems mainly related to the high mAb affinities displayed by these synthetic antigens. Solution affinity SPR analysis was employed and affinities displayed were generally comparable to or even higher than those corresponding to the C-S8c1 reference peptide A15. The NMR characterisation of one of these multiple mutants in solution showed that it had a conformational behaviour quite similar to that of the native sequence A15 and the X-ray diffraction crystallographic analysis of the peptide ? mAb 4C4 complex showed paratope ? epitope interactions identical to all FMDV peptide ? mAb complexes studied so far. Key residues for these interactions are those directly involved in epitope ? paratope contacts (141Arg, 143Asp, 146His) as well as residues able to stabilise a particular peptide global folding. A quasi-cyclic conformation is held up by a hydrophobic cavity defined by residues 138, 144 and 147 and by other key intrapeptide hydrogen bonds, delineating an open turn at positions 141, 142 and 143 (corresponding to the Arg-Gly-Asp motif).

Description of Lutzomyia (Pifanomyia) robusta n. sp. (Diptera, Psychodidae, Phlebotominae) from Peruvian Equadorean interandean areas

Description of Lutzomyia robusta, n. sp. (Diptera, Psychodidae, Phlebotominae) from interandean areas of Peru and Equador. Lutzomyia robusta, n. sp., probable vector of human bartonellosis and cutaneous leishmaniasis, is described and illustrated. This species presents strong affinity with L. serrana (Damasceno & Arouck, 1949) but they can be distinguished by variance analysis of four male characteristics and only one female characteristic. In the variance analysis, populations of L. serrana, of Amazonian areas of Brazil, Peru and Bolivia, the coast of Equador and other areas of Brazil were studied. The synonymy of Lutzomyia guayasi (Rodriguez) and L. serrana was corroborated.

Caracterização do papel da adrenalina na maturação dos adrenorreceptores B2

As respostas pós-juncionais mediadas por adrenorreceptores β2 (ARβ2), responsáveis pelo relaxamento do músculo liso, na veia safena do cão, estão ausentes à nascença. Pelo contrário, no rato recém-nascido já se verifica a estimulação da adenilil ciclase pela activação dos ARβ2. Não existem ainda estudos no coelho recém-nascido. O principal objectivo deste trabalho é avaliar as respostas pós-juncionais mediadas pelos ARβ2 em coelhos recém-nascidos e jovens e relacionar essas respostas com a adrenalina produzida nas glândulas supra-renais. Traçaram-se curvas de dose-resposta à isoprenalina (agonista β) utilizando-se anéis de aorta montados em banho de órgãos isolados ligado a um transdutor de força isométrica. As catecolaminas das supra-renais foram quantificadas por RP-HPLC-ED. Em aortas pré-contraídas com fenilefrina (agonista α1), a isoprenalina causou relaxamento total apenas em coelhos recém-nascidos (n=10). O relaxamento máximo nos coelhos jovens foi de 21±4% (n=23). A potência da isoprenalina foi maior nos recém-nascidos (EC50=1.15×10-8±7.2×10-10 M, n=10) do que nos coelhos jovens (EC50=1.29×10-7 ±4.7×10-9 M, n=23). O relaxamento máximo com isoprenalina, em aortas pré-contraídas com prostaglandina F2α (PGF2α), no grupo de coelhos recém-nascidos foi de 95±3.6% (n=16). O relaxamento máximo nos coelhos jovens foi de 43.7±8.6% (n=9). Na pré-contracção com PGF2α a potência da isoprenalina registou-se maior nos recémnascidos (EC50=9.59×10-9±4.0×10-10 M, n=16) do que nos coelhos jovens (EC50=2.13×10- 8±3.8×10-9 M, n=9), estando concordante com os resultados da pré-contracção com fenilefrina. Nas supra-renais dos recém-nascidos, o conteúdo de noradrenalina foi de 586±128 nmol/mg e da adrenalina foi de 1915±356 nmol/mg (n=4) e nos coelhos jovens foi de 112±12 nmol/mg e de 3644±403 nmol/mg (n=6), respectivamente. As respostas mediadas por ARβ2 no coelho desenvolvem-se mais cedo do que no cão, pois já estão presentes no nascimento. Tal como no rato, no coelho a adrenalina é já a catecolamina em maior quantidade à nascença, enquanto no cão é vestigial. Há uma relação temporal entre a síntese da adrenalina, a única catecolamina biogénica com alta afinidade para os ARβ2 e a maturação das respostas pós-juncionais mediadas por esses receptores. Um protocolo para experiências futuras destinadas a testar esta hipótese, com base no knockdown da Feniletanolamina-N-metiltransferase por RNAi foi elaborado e incluído neste documento.

Bioconversion of D-glucose into D-glucosone by Glucose 2-oxidase from Coriolus versicolor at Moderate Pressures

Glucose 2-oxidase (pyranose oxidase, pyranose: oxygen-2-oxidoreductase, EC 1.1.3.10) from Coriolus versicolor catalyses the oxidation of D-glucose at carbon 2 in the presence of molecular O(2) producing D-glucosone (2-keto-glucose and D-arabino-2-hexosulose) and H(2)O(2). It was used to convert D-glucose into D-glucosone at moderate pressures (i.e. up to 150 bar) with compressed air in a modified commercial batch reactor. Several parameters affecting biocatalysis at moderate pressures were investigated as follows: pressure, [enzyme], [glucose], pH, temperature, nature of fluid and the presence of catalase. Glucose 2-oxidase was purified by immobilized metal affinity chromatography on epoxy-activated Sepharose 6B-IDA-Cu(II) column at pH 6.0. The rate of bioconversion of D-glucose increased with the pressure since an increase in the pressure with compressed air resulted in higher rates of conversion. On the other hand, the presence of catalase increased the rate of reaction which strongly suggests that H(2)O(2) acted as inhibitor for this reaction. The rate of bioconversion of D-glucose by glucose 2-oxidase in the presence of either nitrogen or supercritical CO(2) at 110 bar was very low compared with the use of compressed air at the same pressure. The optimum temperature (55 degrees C) and pH (5.0) of D-glucose bioconversion as well as kinetic parameters for this enzyme were determined under moderate pressure. The activation energy (E(a)) was 32.08 kJmol(-1) and kinetic parameters (V(max), K(m), K(cat) and K(cat)/K(m)) for this bioconversion were 8.8 Umg(-1) protein, 2.95 mM, 30.81 s(-1) and 10,444.06 s(-1)M(-1), respectively. The biomass of C. versicolor as well as the cell-free extract containing glucose 2-oxidase activity were also useful for bioconversion of D-glucose at moderate pressures. The enzyme was apparently stable at moderate pressures since such pressures did not affect significantly the enzyme activity.