Analysis of Intracellular Substrates and Products of Thimet Oligopeptidase in Human Embryonic Kidney 293 Cells

Thimet oligopeptidase (EC 3.4.24.15; EP24.15) is an intracellular enzyme that has been proposed to metabolize peptides within cells, thereby affecting antigen presentation and G protein-coupled receptor signal transduction. However, only a small number of intracellular substrates of EP24.15 have been reported previously. Here we have identified over 100 peptides in human embryonic kidney 293 (HEK293) cells that are derived from intracellular proteins; many but not all of these peptides are substrates or products of EP24.15. First, cellular peptides were extracted from HEK293 cells and incubated in vitro with purified EP24.15. Then the peptides were labeled with isotopic tags and analyzed by mass spectrometry to obtain quantitative data on the extent of cleavage. A related series of experiments tested the effect of overexpression of EP24.15 on the cellular levels of peptides in HEK293 cells. Finally, synthetic peptides that corresponded to 10 of the cellular peptides were incubated with purified EP24.15 in vitro, and the cleavage was monitored by high pressure liquid chromatography and mass spectrometry. Many of the EP24.15 substrates identified by these approaches are 9-11 amino acids in length, supporting the proposal that EP24.15 can function in the degradation of peptides that could be used for antigen presentation. However, EP24.15 also converts some peptides into products that are 8-10 amino acids, thus contributing to the formation of peptides for antigen presentation. In addition, the intracellular peptides described here are potential candidates to regulate protein interactions within cells.

Análise estática não linear de um edifício em betão armado

Trabalho Final de Mestrado para obtenção do grau de Mestre em Engenharia Civil

Recent clinical trials with omapatrilat: new developments.

Omapatrilat belongs to the vasopeptidase inhibitors, ie, drugs that possess the ability to inhibit simultaneously the membrane-bound zinc metalloproteases, angiotensin-converting enzyme (ACE), and the neutral endopeptidase EC 3.4.24.11 (NEP). Omapatrilat was targeted to treat patients with hypertension and congestive heart failure. The preclinical and early clinical studies conducted with omapatrilat were very promising. Indeed, omapatrilat appeared to be a very potent antihypertensive agent with very favorable effects on cardiac function in heart failure patients. In contrast to these early studies, the large clinical trials were more disappointing. The results of the OCTAVE trial confirmed the antihypertensive efficacy of omapatrilat, but at the price of an angioedema rate more than threefold higher than that of an ACE inhibitor in the overall population (2.17% vs 0.68%), and close to fourfold higher in the black population. In OVERTURE, a large randomized control trial in heart failure, angioedema was also more common with omapatrilat, but the incidence was much lower (0.8% with omapatrilat vs 0.5% with enalapril). However, omapatrilat was not convincingly superior to the ACE inhibitor. Because angioedema is probably a class side effect of vasopeptidase inhibitors, the higher incidence of this potentially life-threatening complication with omapatrilat has likely stopped the development of this new class of agents. The future of vasopeptidase inhibitors will depend on the ability to improve the risk/benefit ratio either by developing agents that produce less angioedema, or by defining more precisely a high-risk population that could take advantage of dual ACE/NEP inhibition.

Neutral endopeptidase versus angiotensin converting enzyme inhibition in essential hypertension.

OBJECTIVE: To evaluate the antihypertensive efficacy of sinorphan, an orally active inhibitor of neutral endopeptidase EC 3.4.24.11. DESIGN: The ability of sinorphan (100 mg twice a day) to lower blood pressure was compared with that of the angiotensin converting enzyme (ACE) inhibitor captopril (25 mg twice a day) using a randomized-sequence, double-blind crossover design in 16 patients with essential hypertension. Each treatment was administered for 4 weeks and treatments were separated by a 3-week placebo period. At the end of the last phase of treatment sinorphan was combined with captopril for a further 4-week period. The changes in systolic (SBP) and diastolic blood pressure (DBP) were monitored using repeated ambulatory blood pressure monitoring. RESULTS: When given as monotherapy for 4 weeks, neither sinorphan nor captopril significantly reduced the 24-h or the 14-h daytime mean SBP or DBP. However, a significant decrease in DBP was observed during the first 6 h after the morning administration of captopril. With sinorphan only a significant decrease in night-time SBP was found. With the combined therapy of sinorphan and captopril, significant decreases both in SBP and in DBP were observed, which were sustained over 24 h. After 4 weeks of sinorphan alone or in combination with captopril, no change in plasma atrial natriuretic peptide level was found. However, urinary cyclic GMP excretion increased transiently after administration of the neutral endopeptidase inhibitor. CONCLUSIONS: Neutral endopeptidase inhibition with sinorphan has a limited effect on blood pressure in hypertensive patients when given alone. However, simultaneous neutral endopeptidase and ACE inhibition induces a synergistic effect, and might therefore represent an interesting new therapeutic approach to the treatment of essential hypertension.

Percutaneous drainage versus emergency cholecystectomy for the treatment of acute cholecystitis in critically ill patients: does it matter?

Background The aim if this study was to compare percutaneous drainage (PD) of the gallbladder to emergency cholecystectomy (EC) in a well-defined patient group with sepsis related to acute calculous/acalculous cholecystitis (ACC/AAC).Methods Between 2001 and 2007, all consecutive patients of our ICU treated by either PD or EC were retrospectively analyzed. Cases were collected from a prospective database. Percutaneous drainage was performed by a transhepatic route and EC by open or laparoscopic approach. Patients' general condition and organ dysfunction were assessed by two validated scoring systems (SAPS II and SOFA, respectively). Morbidity, mortality, and long-term outcome were systematically reviewed and analyzed in both groups.Results Forty-two patients [median age = 65.5 years (range = 32-94)] were included; 45% underwent EC (ten laparoscopic, nine open) and 55% PD (n = 23). Both patient groups had similar preoperative characteristics. Percutaneous drainage and EC were successful in 91 and 100% of patients, respectively. Organ dysfunctions were similarly improved by the third postoperative/postdrainage days. Despite undergoing PD, two patients required EC due to gangrenous cholecystitis. The conversion rate after laparoscopy was 20%. Overall morbidity was 8.7% after PD and 47% after EC (P = 0.011). Major morbidity was 0% after PD and 21% after EC (P = 0.034). The mortality rate was not different (13% after PD and 16% after EC, P = 1.0) and the deaths were all related to the patients' preexisting disease. Hospital and ICU stays were not different. Recurrent symptoms (17%) occurred only after ACC in the PD group.Conclusions In high-risk patients, PD and EC are both efficient in the resolution of acute cholecystitis sepsis. However, EC is associated with a higher procedure-related morbidity and the laparoscopic approach is not always possible. Percutaneous drainage represents a valuable intervention, but secondary cholecystectomy is mandatory in cases of acute calculous cholecystitis.

Quinoa BRS Piabiru: alternativa para diversificar os sistemas de produção de grãos

A diversificação da agricultura possibilita implementar a renda, reduzir custos, disponibilizar nutrientes, proteger o solo, reduzir impacto ambiental negativo e ofertar alimentos. A quinoa (Chenopodium quinoa Willd.), uma Chenopodiaceae originária dos Andes, destaca-se por tolerância à seca, elevada qualidade da proteína, baixo colesterol, ausência de glúten (útil a pacientes celíacos), e uso na alimentação animal. A espécie apresenta diversidade, com ciclo variável entre 80 e 150 dias no Brasil central. Os frutos, do tipo aquênio, são pequenos, achatados e sem dormência. BRS Piabiru, primeira recomendação de quinoa ao cultivo no Brasil, originou-se da linhagem EC 3, selecionada em uma população procedente de Quito, Equador. Após dois anos de ensaios, foi uniformizada em suas características agronômicas a partir de 1998. Em sucessão à soja (safrinha) e na entressafra, sob irrigação, apresentou produção média de 2,8 t/ha de grãos, com 145 dias da emergência à maturação. Constitui um potencial componente do sistema plantio direto.

Enxertia de maracujazeiro-azedo em estacas herbáceas enraizadas de espécies de passifloras nativas

As doenças provocadas por patógenos do solo em maracujazeiro constituem-se em um dos principais problemas para essa cultura no Brasil. Uma das alternativas de controle dessas doenças seria a utilização de porta-enxertos resistentes. Várias espécies de passifloras nativas vêm apresentando resistência a essas doenças, mas a utilização destas como porta-enxertos oriundos de sementes tem sido dificultada pelas diferenças de diâmetro entre o porta-enxerto e o enxerto da espécie comercial, o que não aconteceria caso fossem utilizadas as estacas herbáceas como porta-enxerto. No presente experimento, utilizaram-se estacas herbáceas retiradas da parte mediana de ramos de plantas de Passiflora setacea (acesso EC-PS 1), P. nitida (acesso EC-PN 1), P. caerulea (acesso EC-PC 1), P. actinia (acesso EC-PA 1) e de um híbrido F1 entre P. setacea x P. edulis f. flavicarpa comercial e tratadas com ácido naftaleno acético (ANA) a 500 mg/L e mantidas em câmaras de nebulização. As enxertias do tipo "garfagem lateral no topo" foram efetuadas aos 40; 55 e 70 dias após a coleta e plantio das estacas, utilizando garfos de uma única planta de maracujazeiro-azedo. As avaliações foram efetuadas aos 145 e 150 dias após o plantio das estacas, determinando-se a porcentagem de pegamento da enxertia e de enxertos brotados e o comprimento do broto do enxerto. A produção de mudas por enxertia em estacas herbáceas enraizadas de Passiflora nitida e do híbrido F1 (P. setacea x P. edulis f. flavicarpa) foi tecnicamente viável.

Reação a doenças e produtividade de um clone de maracujazeiro-azedo propagado por estaquia e enxertia em estacas herbáceas de Passiflora silvestre

As doenças causadas por patógenos do solo provocam perdas expressivas em maracujazeiros comerciais. Uma das alternativas de controle dessas doenças seria a utilização de porta-enxertos resistentes. Passiflora nitida é uma das espécies de Passifloraceae que apresentam resistência a essas doenças, mas suas sementes geram porta-enxertos com caules finos e, portanto, incompatíveis com o diâmetro dos garfos da espécie comercial. Este problema pode ser resolvido pela utilização da enxertia em estacas enraizadas ou pela enxertia hipocotiledonar, mas, no Brasil, dados sobre o desenvolvimento e produtividade, em campo, de maracujazeiros enxertados, ainda são escassos. Este trabalho teve como objetivo avaliar a reação a doenças, produtividade e características físicas de frutos de um clone de maracujazeiro-azedo (Passiflora edulis f. flavicarpa) comercial propagado por estaquia, enxertia em estacas herbáceas enraizadas de P. nitida (acesso EC-PN 1) e por sementes. As plantas foram cultivadas em um Latossolo Vermelho-Amarelo, onde foram conduzidas em espaldeiras verticais com 1,90 metro em altura, com irrigação por gotejamento. As colheitas foram efetuadas semanalmente, durante 14 meses, e as avaliações das doenças foram efetuadas aos 17; 18 e 19 meses após o plantio. A produtividade das plantas propagadas por estaquia foi o dobro das enxertadas e das propagadas por sementes. Plantas enxertadas e propagadas por estaquia foram menos afetadas pelas doenças.

Características físico-químicas de cinco genótipos de maracujazeiro-azedo cultivados no distrito federal

O objetivo deste trabalho foi de avaliar as características físico-químicas de cinco genótipos de maracujazeiro-azedo cultivado no Distrito Federal nas épocas de fevereiro, março e abril de 2005. O experimentofoi conduzido na área experimental da Fazenda Água Limpa da UnB, Brasília-DF. Utilizou-se o delineamento em blocos casualizados, com quatro repetições, cinco genótipos, três épocas de avaliação e sete plantas úteis por parcela. Foram avaliados os cinco genótipos: Rubi-Gigante, EC-3-0, EC-L-7, Redondão e Gigante-Amarelo. Os frutos produzidos em abril/2005 apresentam maior comprimento, massa fresca do fruto, da polpa, do suco, acidez titulável, pH e número de sementes. O genótipo Rubi-Gigante apresentou frutos com menor comprimento e maior teor de sólidos solúveis. Os genótipos Redondão e Rubi- Gigante apresentaram as menores relações comprimento/diâmetro. Os genótipos Gigante- Amarelo e Redondão apresentam frutos com menor espessura da casca e maior cavidade ovariana e, consequentemente, maior quantidade de polpa. Este último genótipo também obteve frutos com maior diâmetro, teor de sólidos solúveis e menor pH, sendo esta última característica importante para o processamento, pois frutos com elevada acidez conferem uma diminuição na adição de acidificantes no suco. O formato alongado predomina nos frutos dos cinco genótipos, nas três épocas analisadas.

Dipeptidyl peptidase IV (CD26) activity in the hematopoietic system: differences between the membrane-anchored and the released enzyme activity

Dipeptidyl peptidase IV (DPP-IV; CD26) (EC 3.4.14.5) is a membrane-anchored ectoenzyme with N-terminal exopeptidase activity that preferentially cleaves X-Pro-dipeptides. It can also be spontaneously released to act in the extracellular environment or associated with the extracellular matrix. Many hematopoietic cytokines and chemokines contain DPP-IV-susceptible N-terminal sequences. We monitored DPP-IV expression and activity in murine bone marrow and liver stroma cells which sustain hematopoiesis, myeloid precursors, skin fibroblasts, and myoblasts. RT-PCR analysis showed that all these cells produced mRNA for DPP-IV. Partially purified protein reacted with a commercial antibody to CD26. The K M values for Gly-Pro-p-nitroanilide ranged from 0.43 to 0.98 mM for the membrane-associated enzyme of connective tissue stromas, and from 6.76 to 8.86 mM for the enzyme released from the membrane, corresponding to a ten-fold difference, but only a two-fold difference in K M was found in myoblasts. K M of the released soluble enzyme decreased in the presence of glycosaminoglycans, nonsulfated polysaccharide polymers (0.8-10 µg/ml) or simple sugars (320-350 µg/ml). Purified membrane lipid rafts contained nearly 3/4 of the total cell enzyme activity, whose K M was three-fold decreased as compared to the total cell membrane pool, indicating that, in the hematopoietic environment, DPP-IV activity is essentially located in the lipid rafts. This is compatible with membrane-associated events and direct cell-cell interactions, whilst the long-range activity depending upon soluble enzyme is less probable in view of the low affinity of this form.

GABA accumulation and the hypersensitive response in isolated mesophyll cells treated with the G protein activator mastoparan

GABA (y-amino butyric acid) is a non-protein amino acid synthesized through the a-decarboxylation of L-glutamate. This reaction is catalyzed by L-glutamate decarboxylase (EC 4.1.1.15), a cytosolic Ca2+/calmodulin-stimulated enzyme. The purpose of this study is to determine whether or not GABA accumulation is associated with the hypersensitive response of isolated Asparagus sprengeri mesophyll cells. The addition of 25 J.lM mastoparan, a G protein activator, to suspensions of isolated asparagus mesophyll cells significantly increased GABA synthesis and cell death. Cell death was assessed using Evan's blue dye and fluorescein diacetate tests for cell viability. In addition, mastoparan stimulated pH-dependent alkalinization of the external medium, and a rapid and large 02 consumption followed by a loss of photosynthetic activity. The rate of 02 consumption and the net decrease in 02 in the dark was enhanced by light. The inactive mastoparan analogue Mas17 was ineffective in stimulating GABA accumulation, medium alkalinization, 02 uptake and cell death. Accumulation of H202 in response tomastoparan was not detected, however, mastoparan caused the cell-dependent degradation of added H202. The pH dependence of mastoparan-stimulated alkalinization suggests cellular electrolyte leakage, while the consumption of 02 corresponds to the oxidative burst in which 02 at the cell surface is reduced to form various active oxygen species. The results are indicative of the "hypersensitive response" of plants to pathogen attack, namely, the death of cells in the locality of pathogen invasion. The data are compatible with a model in which mastoparan triggers G protein activity, subsequent intracellular signal transduction pathway/s, and the hypersensitive response. It is postulated that the physiological elicitation of the hypersensitive response involves G protein signal transduction. The synthesis of GABA during the hypersensitive response has not been documented previously; however the role/s of GABA synthesis in the hypersensitive response, if any, remain unclear.

L-Glutaminase production by Marine Bacteria

L-glutaminases (L—glutamine amidohydrolase EC.3.5.l.2) is proposed as a prospective candidate for enzyme therapy cnf cancer and also as zui important additive during enzymatic digestion of shoyu koji since it could enhance glutamate content of soysauce. Commercial production of glutaminase could make possible its wide application in these areas, which would demand availability of potential sources and suitable fermentation techniques. The ‘present investigation highlighted marine environment as a potential source of efficient glutaminase producing bacteria mainly species of pseudomonas, aeromonas ,vibrio,alcaligenes, acinetobacter bacillus and planococci.Among them pseudomonas fluorescens ACMR 267 and v.cholerae ACMR 347 were chosen as the ideal strains for glutaminase production.Extracellular glutaminase fraction from all strains were in higher titres than intracellular enzymes during growth in mineral media, nutrient broth and nutrient broth added with glutamine.Glutaminase from all strains were purified employing (NH4)2SO4 fractionation followed tnr dialysis and ion exchange chromatography. The purified glutaminase from all strains were observed to be active and stable over a wide range of gfii and temperature.Optimization studies cflf environmental variables that normally influence time yiehi of glutaminase indicated that the optimal requirements of these bacteria for maximal glutaminase production remained stable irrespective of the medium, they are provided with for enzyme production. However, solid state fermentation technique was observed to be the most suitable process for the production of Glutaminase.

Cambios en la Litosfera.

Los títulos de los cuadernillos son: I.Cambios bruscos: volcanes y terremotos - II.Cambios lentos: tectónica de placas

Formamos lectores y escritores activos.

Anexo Memoria en C-Innov. 47 del ejemplar existente en el CIDE

La investigación sobre profesorado (II) : 1993-1997.

Este trabajo es una investigación publicada