Purification and ligand binding of a soluble class I major histocompatibility complex molecule consisting of the first three domains of H-2Kd fused to beta 2-microglobulin expressed in the baculovirus-insect cell system.

A recombinant baculovirus encoding a single-chain murine major histocompatibility complex class I molecule in which the first three domains of H-2Kd are fused to beta 2-microglobulin (beta 2-m) via a 15-amino acid linker has been isolated and used to infect lepidopteran cells. A soluble, 391-amino acid single-chain H-2Kd (SC-Kd) molecule of 48 kDa was synthesized and glycosylated in insect cells and could be purified in the absence of detergents by affinity chromatography using the anti-H-2Kd monoclonal antibody SF1.1.1.1. We tested the ability of SC-Kd to bind antigenic peptides using a direct binding assay based on photoaffinity labeling. The photoreactive derivative was prepared from the H-2Kd-restricted Plasmodium berghei circumsporozoite protein (P.b. CS) peptide 253-260 (YIPSAEKI), a probe that we had previously shown to be unable to bind to the H-2Kd heavy chain in infected cells in the absence of co-expressed beta 2-microglobulin. SC-Kd expressed in insect cells did not require additional mouse beta 2-m to bind the photoprobe, indicating that the covalently attached beta 2-m could substitute for the free molecule. Similarly, binding of the P.b. CS photoaffinity probe to the purified SC-Kd molecule was unaffected by the addition of exogenous beta 2-m. This is in contrast to H-2KdQ10, a soluble H-2Kd molecule in which beta 2-m is noncovalently bound to the soluble heavy chain, whose ability to bind the photoaffinity probe is greatly enhanced in the presence of an excess of exogenous beta 2-m. The binding of the probe to SC-Kd was allele-specific, since labeling was selectively inhibited only by antigenic peptides known to be presented by the H-2Kd molecule.

Endogenous angiotensinergic system in neurons of rat and human trigeminal ganglia.

To clarify the role of Angiotensin II (Ang II) in the sensory system and especially in the trigeminal ganglia, we studied the expression of angiotensinogen (Ang-N)-, renin-, angiotensin converting enzyme (ACE)- and cathepsin D-mRNA, and the presence of Ang II and substance P in the rat and human trigeminal ganglia. The rat trigeminal ganglia expressed substantial amounts of Ang-N- and ACE mRNA as determined by quantitative real time PCR. Renin mRNA was untraceable in rat samples. Cathepsin D was detected in the rat trigeminal ganglia indicating the possibility of existence of pathways alternative to renin for Ang I formation. In situ hybridization in rat trigeminal ganglia revealed expression of Ang-N mRNA in the cytoplasm of numerous neurons. By using immunocytochemistry, a number of neurons and their processes in both the rat and human trigeminal ganglia were stained for Ang II. Post in situ hybridization immunocytochemistry reveals that in the rat trigeminal ganglia some, but not all Ang-N mRNA-positive neurons marked for Ang II. In some neurons Substance P was found colocalized with Ang II. Angiotensins from rat trigeminal ganglia were quantitated by radioimmunoassay with and without prior separation by high performance liquid chromatography. Immunoreactive angiotensin II (ir-Ang II) was consistently present and the sum of true Ang II (1-8) octapeptide and its specifically measured metabolites were found to account for it. Radioimmunological and immunocytochemical evidence of ir-Ang II in neuronal tissue is compatible with Ang II as a neurotransmitter. In conclusion, these results suggest that Ang II could be produced locally in the neurons of rat trigeminal ganglia. The localization and colocalization of neuronal Ang II with Substance P in the trigeminal ganglia neurons may be the basis for a participation and function of Ang II in the regulation of nociception and migraine pathology.

Effect on blood pressure and the renin-angiotensin system of repeated doses of the converting enzyme inhibitor CGS 14824A.

A new, orally active angiotensin converting enzyme (ACE) inhibitor, CGS 14824A, was evaluated in 12 healthy male volunteers. Two groups each of 6 volunteers were given 5 or 10 mg once daily p.o. for 8 days. Four hours after the first and the last morning doses, plasma angiotensin II, aldosterone and plasma converting enzyme activity had fallen, while blood angiotensin I and plasma renin activity had risen. Throughout the study, more than 90% inhibition of ACE was found immediately before giving either the 5 or 10 mg dose and 50% blockade was still present 72 h following the last dose. Based on the determination of ACE, there was no evidence of drug accumulation. No significant change in blood pressure or heart rate was observed during the course of the study. CGS 14824A was an effective, orally active, long-lasting and well tolerated converting enzyme inhibitor.

The impact of the reference price system on the pharmaceutical market: A theoretical approach

This article analyses the impact of the reference pricesystem on the price-setting strategies of thepharmaceutical firms and on the level of generic usage.This model is the first to take explicitly into accountthe impact of the reference price mechanism on the levelof competition between brand-name and generic drugs andnational pharmaceutical spending. We consider aduopolistic model with one firm producing the brand-namedrug, whose patent has already expired, and the otherproducing the corresponding generic version. We work ina partial equilibrium framework where firms set pricessequentially and consumers face heterogeneous switchingcosts.We show that brand producers compensate thedecline of profits by selling greater quantities insteadof charging higher prices, thus fostering pricecompetition in the pharmaceutical market. This result isa consequence of both the assumption of a verticallydifferentiated model and the introduction of thereference price system.

The crew-scheduling module in the GIST system

The public transportation is gaining importance every year basically duethe population growth, environmental policies and, route and streetcongestion. Too able an efficient management of all the resources relatedto public transportation, several techniques from different areas are beingapplied and several projects in Transportation Planning Systems, indifferent countries, are being developed. In this work, we present theGIST Planning Transportation Systems, a Portuguese project involving twouniversities and six public transportation companies. We describe indetail one of the most relevant modules of this project, the crew-scheduling module. The crew-scheduling module is based on the application of meta-heuristics, in particular GRASP, tabu search and geneticalgorithm to solve the bus-driver-scheduling problem. The metaheuristicshave been successfully incorporated in the GIST Planning TransportationSystems and are actually used by several companies.

Cost system design and cost management in the Spanish public sector

Cost systems have been shown to have developed considerably in recent years andactivity-based costing (ABC) has been shown to be a contribution to cost management,particularly in service businesses. The public sector is composed to a very great extentof service functions, yet considerably less has been reported of the use of ABC tosupport cost management in this sector.In Spain, cost systems are essential for city councils as they are obliged to calculate thecost of the services subject to taxation (eg. waste collection, etc). City councils musthave a cost system in place to calculate the cost of services, as they are legally requirednot to profit , from these services.This paper examines the development of systems to support cost management in theSpanish Public Sector. Through semi-structured interviews with 28 subjects within oneCity Council it contains a case study of cost management. The paper contains extractsfrom interviews and a number of factors are identified which contribute to thesuccessful development of the cost management system.Following the case study a number of other City Councils were identified where activity-based techniques had either failed or stalled. Based on the factors identified inthe single case study a further enquiry is reported. The paper includes a summary usingstatistical analysis which draws attention to change management, funding and politicalincentives as factors which had an influence on system success or failure.

Diversity and regional inequalities: Assessing the outcomes of the Spanish 'System of Health Care Services'

The consolidation of a universal health system coupled with a process of regionaldevolution characterise the institutional reforms of the National Health System(NHS) in Spain in the last two decades. However, scarce empirical evidence hasbeen reported on the effects of both changes in health inputs, outputs andoutcomes, both at the country and at the regional level. This paper examinesthe empirical evidence on regional diversity, efficiency and inequality ofthese changes in the Spanish NHS using cross-correlation, panel data andexpenditure decomposition analysis. Results suggest that besides significantheterogeneity, once we take into account region-specific needs there is evidenceof efficiency improvements whilst inequalities in inputs and outcomes, althoughmore visible , do not appear to have increased in the last decade. Therefore,the devolution process in the Spanish Health System offers an interesting casefor the experimentation of health reforms related to regional diversity butcompatible with the nature of a public NHS, with no sizeable regionalinequalitiest.

Optimal regulation of a fully insured deposit banking system

We analyze risk sensitive incentive compatible deposit insurancein the presence of private information when the market value of depositinsurance can be determined using Merton's (1997) formula. We show that,under the assumption that transferring funds from taxpayers to financialinstitutions has a social cost, the optimal regulation combines differentlevels of capital requirements combined with decreasing premia on depositinsurance. On the other hand, it is never efficient to require the banksto hold riskless assets, so that narrow banking is not efficient. Finally,chartering banks is necessary in order to decrease the cost of asymmetricinformation.

Utilisation en tomoscintigraphie d'anticorps monoclonaux radio-marqués pour la détection chez l'homme des cancers digestifs et des cancers médullaires de la thyroïde

Two 131-Iodine radiolabelled monoclonal antibodies were used to perform tomoscintigraphy in 42 patients: 11 patients bearing medullary thyroid cancers and 19 patients bearing gastrointestinal cancers received an antibody directed against carcino-embryonic antigen; 12 patients bearing gastro-intestinal cancers received an antibody directed against a non circulating antigen expressed by human colorectal cancers cell lines. Tomoscintigraphy is particularly useful for analysing the complex biodistribution of radiolabelled antibodies and the low contrast images encountered in immunoscintigraphy; the problems related to the true positive rate and to the clinical specificity of the method are discussed.

Aspects thérapeutiques de la vitamine D: quels taux cibler avec quels traitements [Therapeutic goal of vitamin D: optimal serum level and dose requirements].

Therapeutic goal of vitamin D: optimal serum level and dose requirements Results of randomized controlled trials and meta-analyses investigating the effect of vitamin D supplementation on falls and fractures are inconsistent. The optimal serum level 25(OH) vitamin D for musculoskeletal and global health is > or = 30 ng/ml (75 nmol/l) for some experts and 20 ng/ml (50 nmol/l) for some others. A daily dose of vitamin D is better than high intermittent doses to reach this goal. High dose once-yearly vitamin D therapy may increase the incidence of fractures and falls. High serum level of vitamin D is probably harmful for the musculoskeletal system and health at large. The optimal benefits for musculoskeletal health are obtained with an 800 UI daily dose and a serum level of near 30 ng/ml (75 nmol/l).

Characterization of functional role of α-ketoglutarate receptor in the kidney : role of the intrinsic circadian timing system in renin expressing cells

Le rein joue un rôle essential dans le maintien de l'homéostasie des fluides extracellulaires (FEC) et la pression artérielle. L'objectif de notre groupe est d'identifier de nouveaux mécanismes impliqués dans le contrôle de l'homéostasie des FEC et de la pression artérielle par le rein. Projet 1) Caractérisation du rôle fonctionnel du récepteur à l'a-cétogluatarate Oxgrl dans le rein Oxgrl est le récepteur spécifique de l'a-cétogluatarate, une moléule intermédiaire du cycle de l'acide citrique, filtrée par le rein et réabsorbée ou secrétée au niveau des tubules proximaux. Le rôle fonctionnel de ces deux récepteurs reste inconnu. Nos résultats montrent qu'Oxgrl est localisé au niveau des cellules intercalaires du tube collecteur (CCD). Des souris (Oxgrr/_) montrent une diminution du pH urinaire ,une augmentation de la concentration de l'acide urinaire titrable et une augmentation des niveaux d'a-cétoglutarate. Le traitement au Na-bicarbonate provoque une augmentation plus prononcée de l'alcalose métabolique chez les souris Oxgrl"7"' accompagnée d'une augmentation de la concentration de bicarbonate et une diminution du niveau de chlore plasmatique. En parallèle, des études de microperfusion ont montré que a-cétoglutarate stimule la réabsorption éléctroneutre de NaCl dans le CCD des souris de type sauvage mais pas des souris Oxgrl"7". En résumé, ces résultats montrent que l'a-cétoglutarate joue un rôle de molécule messagère du tubule proximal jusqu'au tube collecteur au niveau du rein et qu'Oxgrl pourrait être impliqué dans la régulation de l'échange Cl/bicarbonate et la réabsorption du NaCl dans les cellules intercalées. Projet 2) Rôle du système circadien dans les cellules productrices de rénine. Le système chronologique circadien est un mécanisme moléculaire ubiquitaire qui permet à l'organisme de coordonner ses fonctions principales en fonction du temps géophysique. Comme l'activité de la rénine plasmatique montre une rythmicité circadienne nette chez l'homme et la souris ; dans ce projet, nous avons abordé la question à savoir dans quelle mesure le système circadien est impliqué dans cette variabilité circadienne. Pour cela, le gène Bmall, élément principal de l'horloge moléculaire, a été perturbé dans les cellules granulaires productrices de rénine par le système Cre-LoxP. Nos résultats montrent que les souris Renld- Cre/Bmalllox/lox (cKO) présentent de faibles taux d'ARNm de Reni, altèrent la dynamique d'expression de la protéine rénine, mais il le niveau de concentration plasmatique de la rénine reste le même. Cependant, les souris cKO montrent une réduction significative de la concentration plasmatique de l'aldostérone. Nos analyses de l'urine récupérée dans des intervalles de temps de 24 et 1 heure montrent une augmentation du volume urinaire, une tendance à une hypercalciurie, ainsi qu'une altération de la dynamique d'excrétion urinaire de sodium chez les souris cKO. Plusieurs gènes impliqués dans la production/sécrétion de la rénine et dans le contrôle de la fonction rénale montrent une altération de l'expression circadienne d'ARNm. Par ailleurs, les souris cKO montrent une baisse significative de la pression artérielle. Nos résultats suggèrent que l'horloge intrinsèque des cellules productrices de la rénine joue un rôle important dans le control des FEC et l'homéostasie de la pression artérielle via régulation de la fonction rénale.

Racial Stereotypes in Fictions of Slavery: Uncle Tom´s Cabin by Harriet Beecher Stowe and O Escravo by José Evaristo D’ Almeida

Most stereotypes about Africans and their descendants started with colonialism in the fifteenth century. The encounter between Africans and Europeans facilitated the creation of myths and stereotypes about the colonized peoples, which were made effective through the naturalization of differences. The relationship between skin color and slavery developed to produce a racialized system of forced labor on which colonialism depended for its survival. Stereotypes functioned to legitimize colonial authority by building the notion that the colonizer ruled over the colonized because of an innate superiority. Therefore, stereotyping is an effective "discursive strategy" (Bhabha) based on fixity and repetition with the aim of controlling the other. Harriet Beecher Stowe’s Uncle Tom’s Cabin and José Evaristo D’Almeida O Escravo both denounced the evils of slavery in the United States of America and Cape Verde respectively, claiming for the end of the institution. However, they are both ambivalent towards slaves and blacks, being unable to envisage social equality for the two races. Both authors construct their black characters as stereotypical others, but they depict the light-skin characters as superior both culturally and physically. The bi-racial characters are portrayed as the ones who possess beauty and intelligence as an inheritance from their European ancestry, while blacks are relegated to the margins. We need to consider, however, that slavery in Cape Verde had different characteristics from its counterpart in the United States of America. In Cape Verde the Africans outnumbered the Europeans and that circumstance favored miscegenation and the emergence of forms of mixed culture, which came to be seen as positive and natural. In the United States of America miscegenation was regarded as a taboo since early. And even after Emancipation, “the one-drop rule” made the offspring of an African descendant black, however 'white' he or she might be.

Molecular determinants of vesicular storage and release of the gliotransmitter D-serine from cortical astrocytes

Neuron-astrocyte reciprocal communication at synapses has emerged as a novel signalling pathway in brain function. Astrocytes sense the level of synaptic activity and, in turn, influence its efficacy through the regulated release of 'gliotransmitters' such as glutamate, ATP or D-serine. A calcium-dependent exocytosis is proposed to drive the release of gliotransmitters but its existence is still debated. Over the last years, we have been studying the molecular determinants governing D-serine release from glia using different approaches. Using a novel bioassay for D-serine, we have been able to show that D-serine release occurs mainly through a calcium- and SNARE proteindependent mechanism just supporting the idea that this amino acid is released by exocytosis from glia. We next have pursued our exploration by confocal imaging and tracking of the exocytotic routes for Dserine- mediated gliotransmission and have shown that D-serine releasable pools are confined to synaptobrevin2/cellubrevin-bearing vesicles. To shed light onto the mechanisms controlling the storage and the release of gliotransmitters and namely D-serine, we have developed a new method for the immunoisolation of synaptobrevin 2- positive vesicles from rat cortical astrocytes in culture while preserving their content in gliotransmitters. The purified organelles are clear round shape vesicles of excellent purity with homogeneous size (40 nm) as judged by electron microscopy. Immunoblotting analysis revealed that isolated vesicles contain most of the major proteins already described for neuron-derived vesicles like synaptic vesicle protein 2 (SV2) and the proton pump H?-ATPase. In addition, we have analyzed the content for various amino acids of these vesicles by means of chiral capillary electrophoresis coupled to laser-induced fluorescence detection. The purified vesicles contain large amount of D-serine. We also detect peaks corresponding to unidentified compounds that may correspond to others amino acids. Postembedding immunogold labelling of the rat neocortex further revealed the expression of D-serine in astrocytes processes contacting excitatory synapses. Finally, we have examined the uptake properties for Dserine and glutamate inside the isolated glial vesicles. Our results provide significant support for the existence of an uptake system for D-serine in secretory glial vesicles and for the storage of chemical substances like D-serine and glutamate. 11th International Congress on Amino Acids, Peptides and Proteins 763 123