Purificação, caracterização e avaliação da atividade antiproliferativa de um inibidor de quimotripsina tipo kunitz de semente de Eryhrina velutina

A chymotrypsin inhibitor was purified from Erythrina velutina seeds by ammonium sulphate fractionation, affinities chromatographies on Trypsin-Sepharose, Quimotrypsin-Sepharose and reversed phase C-18 FPLC/AKTA system. The inhibitor, named EvCI, shown molecular mass of 17 kDa, as determined by SDSPAGE. 2D-PAGE showed four isoinhibitors with pI values of 4,42, 4,63, 4,83 and 5,06, with molecular mass of 17 kDa each. The aminoacid sequence of EvCI was determined by MALDI-TOF-MS and showed a high similarity with other Kunitz-type inhibitor of Erythrina variegata. EvCI competitively inhibited chymotrypsin, with Ki of 4 x10-8 M, but did not inhibited trypsin, pancreatic elastase, bromelain and papain. The inhibitory activity of EvCI was stable over wide pH and temperature ranges. In the presence of DTT 100 mM for 120 min, EvCI lost 50 % of activity. Cytotoxicity was studied in HeLa, MDA, HepG2, K562 and PC3 cells after 72-h incubation period. EvCl inhibited HeLa cells growth with an IC50 value of 50 μg/ml. Subsequent studies in HeLa cells analysis of cell death by annexin V/PI double-staining and cell cycle, using flow cytometry. The results provide evidence for a cytostatic activity of EvCl and support further studies on potential application of this inhibitors as an antiproliferative agent in combined therapy against cervical cancer

Um novo inibidor de tripsina multifuncional de sementes de Erythrina velutina: caracterizacao e propriedades farmacologicas

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior

Parâmetros eletrocardiográficos e valores séricos e urinários de sódio e potássio em cães tratados com furosemida

The serum and urine Na+ and K+ levels and their relationship with electrocardiography characteristics,were studied in 15 male adult mongrel dogs, treated twice-a-day,with 2, 4, 8mg/kg of live weight doses of furosemide and furosemide associated with KCl during 35 days. Urine samples were analyzed weekly in order to determine the Na+ and K+ concentrations and to evaluate the urinary excretion of both cations. Electrocardiography was performed simultaneously, using limb derivations, speed 50mm/sec and calibration of 1 cm corresponding to 1mV. Data showed decreased seric concentrations of Na+ and Ki, increase in Na+ fractional excretion between days 7 and 21 of the treatment, slower heart rate and longer PR, QRS and QT intervals in the ECG. Briefly 2mg/kg furosemide associated with KCl was the most suitable treatment since it induced lesser side effects.

Características de latossolos roxos desenvolvidos de rochas alcalinas e básicas de Jaboticabal, SP

Comparou-se as características químicas, físicas e mineralógicas de Latossolos Roxos desenvolvidos de rocha alcalina (LRe) e de rocha eruptiva básica (LRd), da região de Jaboticabal, SP. Os resultados indicaram que o LRe apresenta-se num estádio de desenvolvimento, comparativamente, menos evoluído que o LRd, enquanto esse se apresenta com tendência para a maturidade. Os menores teores de argila e ferro total e maiores teores de saturação por bases, Ki e CTC da fração argila apresentados pelo LRe comprovam esta tendência. Conclui-se que a ocorrência de Latossolo Roxo eutrófico no campus da UNESP-Jaboticabal, pode estar associada a participação de rochas alcalinas na sua pedogênese.

Modelos para estimativa do subfator cobertura-manejo (CiII) relativo à erosão entressulcos

A análise de um modelo de erosão é um crítico passo no desenvolvimento de uma ferramenta de predição da erosão aplicável e válida; isso é crucial para avaliar o desempenho dos modelos existentes para assegurar que as estimativas de um modelo condizem com a realidade. O objetivo do presente trabalho foi avaliar modelos para a predição do subfator cobertura e manejo (CiII) relativo à erosão entressulcos. Um experimento fatorial completo foi conduzido com cinco doses de resíduo de milho (0; 0,05; 0,15; 0,40 e 0,80 kg m-2), quatro declives e duas repetições, sob condições de prévio umedecimento para determinar as taxas de erosão entressulcos (Di) e enxurrada (R). Num primeiro experimento, foi avaliada a erodibilidade entressulcos (Ki) e o subfator cobertura e manejo (CiII), em parcelas experimentais de 0,5 x 0,75 m, em solo recentemente preparado. Num segundo experimento, foram avaliados Di, R, Ki e CiII, também em parcelas de 0,5 x 0,75 m, em solo recém- preparado. Os valores de Di, R, Ki e CiII, obtidos no segundo experimento, foram utilizados na avaliação dos modelos testados. Os modelos CiII = e-2,50 CS/100 e CiII = e -2,238 CS/100 apresentaram boas estimativas para o subfator CiII.

Inorganic pyrophosphate-phosphohydrolytic activity associated with rat osseous plate alkaline phosphatase

Purified membrane-bound alkaline phosphatase from rat osseous plate hydrolyzed pyrophosphate in the presence of magnesium ions, with a specific activity of 92.7 U/mg. Optimal apparent pH for pyrophosphatase activity was 8.0 and it remained unchanged on increasing the pyrophosphate concentration. In the absence of magnesium ions the enzyme had a K-m = 88 mu M and V = 36.7 U/mg for pyrophosphate and no inhibition by excess substrate was observed. Pyrophosphatase activity was rapidly destroyed at temperatures above 40 degrees C, but magnesium ions apparently protected the enzyme against danaturation. Sodium metavanadate (Ki = 1.0 mM) was a competitive inhibitor of pyrophosphatase activity, while levamisole (Ki = 8.2 mM) and theophylline (Ki = 7.4 mM) were uncompetitive inhibitors. Magnesium ions (K-0.5 = 1.7 mu M) stimulated pyrophosphatase activity, while cobalt (Ki = 48.5 mu M) and zinc (Ki = 22.0 mu M) ions were non-competitive inhibitors. Manganese and calcium ions had no effect on pyrophosphatase activity. The M-w of the pyrophosphatase: protein was 130 kDa by gel filtration, but a value of 65 kDa was obtained by dissociative gel electrophoresis, suggesting that it was a dimer of apparently identical subunits. These results suggested that pyrophosphatase activity stems from the membrane-bound osseous plate alkaline phosphatase and not from a different protein.

POLYPHENOLOXIDASE FROM ATEMOYA FRUIT (ANNONA CHERIMOLA MILL. ANNONA SQUAMOSA L.)

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Immobilization of streptavidin in sol-gel films: Application on the diagnosis of hepatitis C virus

Recent advances have accelerated the development of biosensors for the analysis of specific gene sequences. In this kind of biosensor, a DNA probe is immobilized on a transducer and the hybridization with the target DNA is monitored by suitable methodology. In the present work, the streptavidin (STA) was encapsulated in thin films siloxane-poly(propylene oxide) hybrids prepared by sol-gel method and deposited on the graphite electrode surface by dip-coating process. Biotinylated 18-mer probes were immobilized through STA and a novel amperometric DNA biosensor for the detection and genotyping of the hepatitis C virus (genotypes 1, 2A/C, 2B and 3) is described. The HCV RNA from serum was submitted to reverse transcriptase-linked polymerase chain reaction (RT-PCR) and biotin-labeled cDNA was obtained. Thus, the cDNA was hybridized to the target-specific oligonucleotide probe immobilized on the graphite electrode surface and following the avidin-peroxidase conjugate was added. The enzymatic response was investigated by constant potential amperometry at -0.45 V versus Ag/AgCl using H2O2 and KI solutions. HCV RNA negative and positive controls and positive samples of sera patients were analyzed and the results were compared to commercial kit. The proposed methodology appeared to be suitable and convenient tool for streptavidin immobilization and diagnose of HCV disease. (c) 2006 Elsevier B.V. All rights reserved.

Ionically bound peroxidase from peach fruit

Soluble, ionically bound peroxidase (POD) and polyphenoloxidase (PPO) were extracted from the pulp of peach fruit during ripening at 20degreesC Ionically bound form was purified 6.1 -fold by DEAE-cellulose and Sephadex G-100 chromatography. The purified enzyme showed only one peak of activity on Sephadex G-100 and PAGE revealed that the enzyme was purified by the procedures adopted. The purified enzyme showed a molecular weight of 29000 Da, maximum activity at pH 5.0 and at 40degreesC the calculated apparent activation energy (Ea) for the reaction was 10.04 kcal/mol. The enzyme was heat-labile in the temperature range of 60 to 75degreesC with a fast inactivation at 75degreesC Measurement of residual activity showed a stabilizing effect of sucrose at various temperature/sugar concentrations (0, 10, 20 %, w/w), with an activation energy (Ea) for inactivation increasing with sucrose concentration from 0 to 20% (w/w). The Km and V-max values were 9.35 and 15.38 mM for O-dianisidine and H2O2, respectively. The bound enzyme was inhibited competitively by (.)ferulic, caffeic and protocatechuic acids with different values of Ki,. L-cysteine, p-coumaric and indolacetic acid and Fe++ also inhibited the enzyme but at a lower grade. N-ethylmaleimide and p-CMB were not effective to inhibit the enzyme demonstrating the non-essentiality of SH groups.

Some Biochemical properties of polyphenoloxidase from spearmint (Mentha arvensis)

Polifenoloxidase (PPO, EC 1.14.18.1) extraída de folhas de Mentha arvensis foi isolada por precipitação com (NH4)2SO4 e diálise extensiva. Seu pH e temperatura ótimos variaram com o substrato. A PPO apresentou atividade com vários difenóis. Valores de Km foram 0,825; 0,928 e 7,41 mM para ácido caféico, 4-metilcatecol e catecol, respectivamente. Na inativação térmica, 50% da enzima foi inativada após 60 e 15 segundos a 70 e 75ºC, respectivamente. A medida de atividade residual mostrou um efeito estabilizante de sacarose a várias temperaturas e uma energia de ativação (Ea) para inativação aumentando com a concentração de sacarose de 0 a 40% (p/p). Valores de energias de ativação de 78,13; 80,37; 82,79 and 81,00 kJ/Mol foram encontradas para 0, 15, 30 e 40% de sacarose, respectivamente. A PPO foi inibida pelos ácidos ascórbico, benzóico, cinamico, ferulico, p-cumárico, protocatéquico, além de metabisulfito de sódio, resorcinol e pirogalol. Os valores de Ki mostram que o ácido ascórbico foi o mais efetivo inibidor. O tipo de inibição foi determinado para cada inibidor.

Expressão imunoistoquímica de Glut-1 e marcadores de proliferação e apoptose em anomalias vasculares orais

Vascular anomalies constitute a distinct group of lesions, but they may present similar clinical and histopatological characteristics, which can lead to diagnostic mistakes. This study aimed by histopathology and immunohistochemical expression of human glucose transporter protein (GLUT-1), correctly identify and classify oral vascular anomalies, besides analyzing the immunoexpression of markers proliferation and apoptosis (Ki-67 and Bcl-2). All cases diagnosed as "oral hemangiomas" belonging to the archives of the Service of Pathological Anatomy from the subject of Oral Pathology of the Department of Dentistry (DOD), of the Federal University of Rio Grande do Norte (UFRN) were reviewed, totalizing 77 cases. Immunohistochemical analysis for GLUT-1 showed that only 26 (33.8%) of the specimens were true infantile hemangiomas (IHs). The 51 (66.2%%) GLUT-1 negative specimens were then reclassified as pyogenic granulomas (PGs) and vascular malformations (VMs) from their histopathologic characteristics,totalizing 26 (33.8%) cases of IHs, 20 (26.0%) of PGs and 31 (40.2) cases of oral VMs. The cases analyzed by the marker Ki-67 showed different median IH (13,85), PG (33,70) and VM (4.55) with statistically significant differences between them (p <0.001). In relation to the protein Bcl-2, the groups also showed different median of the established scores IH (1.00), PG (1.50), VMs (0.0) demonstrating statistically significant differences between them (p<0,001). No statistically significant correlation between the indexes of positivity for Ki-67 and the scores of immunoexpression of Bcl-2 were observed in any group. Thus, we can conclude that it is necessary a careful and parameterized review of cases of vascular anomalies making use of auxiliary tools such as GLUT-1, since the histopathological findings alone, sometimes, are not sufficient to differentiate some anomalies. Furthermore, analysis of the expressions of markers involved in the levels of proliferation of lesions is important for a better understanding of its biological behavior aspect

Hidróxidos duplos lamelares aplicados à obtenção de biodiesel

In this paper, the Layered Double Hydroxides (LDH s) type hydrotalcite were synthesized, characterized and tested as basic heterogeneous catalysts for the production of biodiesel by transesterification of sunflower oil with methanol. The synthesis of materials Layered Double Hydroxides (LDH s) by co-precipitation method from nitrates of magnesium and aluminum, and sodium carbonate. The materials were submitted to the variation in chemical composition, which is the amount of Mg2+ ions replaced by Al3+. This variation affects the characteristic physico-chemical and reaction the solid. The molar ratio varied in the range of 1:1 and 3:1 magnesium / aluminum, and their values between 0.2 and 0.33. This study aims to evaluate the influence of variation of molar ratio of mixed oxides derived from LDH s and the influence of impregnation of a material with catalytic activity, the KI, the rate of conversion of sunflower oil into methyl esters (biodiesel) through transesterification by heterogeneous catalysis. .The catalysts were calcined at 550 ° C and characterized by X-ray diffraction (XRD), scanning electron microscopy and energy dispersive spectroscopy of X-ray (SEM / EDS), thermogravimetric analysis (TG) and test basicity. The transesterification reaction was performed for reflux is a mixture of sunflower oil and methanol with a molar ratio of 15:1, a reaction time of 4h and a catalyst concentration of 2% by weight. The physical-chemical characterization of sunflower oil and biodiesel obtained by the route methyl submitted according NBR, EN, ASTM. Subsequently, it was with the chromatographic and thermogravimetric characterizations of oils. The results of chromatographic analysis showed that the catalysts were effective in converting vegetable oil into biodiesel, in particular the type hydrotalcite KI-HDL-R1, with a conversion of 99.2%, indicating the strong influence of the chemical composition of the material, in special due to presence of potassium in the structure of the catalyst

Ensaio comparativo de cultivares de soja em época convencional em Selvíria, MS: características agronômicas e produtividade

The performance evaluation it's an important procedure for recommendation of a cultivar to an specific region, because the better adapted will present superior development and bigger level of productivity. The work aimed evaluates the behavior of soybean cultivars IAC 15-1, IAC 18, IAC 20, BRS 133, FT-2000, KI-S 602 RCH, MT/BRS-55 (Uirapuru), MT/BR-45 (Paiaguas), MT/BRS-51 (Xingu), KI-S 801, MSOY 7701, MT/BR-49 (Pioneer), Empresa Brasileira de Pesquisa Agropecuária (EMBRAPA) 65 (Itapoty), Empresa Brasileira de Pesquisa Agropecuária (EMBRAPA) 64 (Ponta Pora), Empresa Brasileira de Pesquisa Agropecuária (EMBRAPA) 61, IAC 8-2, IAC 19, Crystalline FT RCH, KI-S 702, IAC Foscarin 31, IAC-PL1 and MT/BRS-46 (Conquista), related to agronomical characteristics of plants (cycle, height, height of first beans insertion and stand) and productivity of grains, when sowed in conventional time. The experimental design was randomized blocks with four replicates, and the averages compared with Tukey Test (5%). It is viable soybean production at Selviria, MS, because practically all cultivars evaluated are adapted to the soil and climate conditions of that area. To cultivar KI-S 801, the use of smaller stand to the observed in that work it can avoid lodging. Caution should be had to use cultivar MT/BRS-55 (Uirapuru).

High volume peritoneal dialysis vs daily hemodialysis: A randomized, controlled trial in patients with acute kidney injury

There is no consensus in the literature on the best renal replacement therapy (RRT) in acute kidney injury (AKI), with both hemodialysis (HD) and peritoneal dialysis (PD) being used as AKI therapy. However, there are concerns about the inadequacy of PD as well as about the intermittency of HD complicated by hemodynamic instability. Recently, continuous replacement renal therapy (CRRT) have become the most commonly used dialysis method for AKI around the world. A prospective randomized controlled trial was performed to compare the effect of high volume peritoneal dialysis (HVPD) with daily hemodialysis (DHD) on AKI patient survival. A total of 120 patients with acute tubular necrosis (ATN) were assigned to HVPD or DHD in a tertiary-care university hospital. The primary end points were hospital survival rate and renal function recovery, with metabolic control as the secondary end point. Sixty patients were treated with HVPD and 60 with DHD. The HVPD and DHD groups were similar for age ( 64.2 +/- 19.8 and 62.5 +/- 21.2 years), gender ( male: 72 and 66%), sepsis ( 42 and 47%), hemodynamic instability ( 61 and 63%), severity of AKI ( Acute Tubular Necrosis-Index Specific Score (ATN-ISS): 0.68 +/- 0.2 and 0.66 +/- 0.2), Acute Physiology, Age, and Chronic Health Evaluation Score (APACHE II) (26.9 +/- 8.9 and 24.1 +/- 8.2), pre-dialysis BUN (116.4 +/- 33.6 and 112.6 +/- 36.8mg per 100 ml), and creatinine ( 5.8 +/- 1.9 and 5.9 +/- 1.4 mg per 100 ml). Weekly delivered Kt/V was 3.6 +/- 0.6 in HVPD and 4.7 +/- 0.6 in DHD ( P<0.01). Metabolic control, mortality rate ( 58 and 53%), and renal function recovery ( 28 and 26%) were similar in both groups, whereas HVPD was associated with a significantly shorter time to the recovery of renal function. In conclusion, HVPD and DHD can be considered as alternative forms of RRT in AKI.