Antitumoural effect of an L-amino acid oxidase isolated from Bothrops jararaca snake venom

An L-amino acid oxidase (BjarLAAO-I) from Bothrops jararaca snake venom was highly purified using a stepwise sequential chromatography on Sephadex G-75, Benzamidine Sepharose and Phenyl Sepharose. Purified BjarLAAO-I showed a molecular weight around 60,000 under reducing conditions and about 125,000 in the native form, when analysed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration, respectively. BjarLAAO-I is a homodimeric acidic glycoprotein, pI similar to 5.0, and N-terminal sequence showing close structural homology with other snake venom LAAOs. The purified enzyme catalysed the oxidative deamination of L-amino acids, the most specific substrate being L-Phe. Five amino acids, L-Ser, L-Pro, L-Gly, L-Thr and L-Cys were not oxidized, clearly indicating a significant specificity. BjarLAAO-I significantly inhibited Ehrlich ascites tumour growth and induced an influx of polymorphonuclear cells, as well as spontaneous liberation of H(2)O(2) from peritoneal macrophages. Later, BjarLAAO-I induced mononuclear influx and peritoneal macrophage spreading. Animals treated with BjarLAAO-I showed higher survival time.

Annexin 1 localisation in tissue eosinophils as detected by electron microscopy

BACKGROUND: Human and rodent leukocytes express high levels of the glucocorticoid-inducible protein annexin 1 ( ANXA1) ( previously referred to as lipocortin 1). Neutrophils and monocytes have abundant ANXA1 levels.Aim: We have investigated, for the first time, ANXA1 ultrastructural expression in rat eosinophils and compared it with that of extravasated neutrophils. The effect of inflammation ( carrageenin peritonitis) was also monitored.Methods: Electron microscopy was used to define the sub-cellular localisation of ANXA1 in rat eosinophils and neutrophils extravasated in the mesenteric tissue. A pair of antibodies raised against the ANXA1 N-terminus (i.e. able to recognise intact ANXA1, termed LCPS1) or the whole protein ( termed LCS3) was used to perform the ultrastructural analysis.Results: the majority of ANXA1 was localised in the eosinophil cytosol (similar to 60%) and nucleus (30-40%), whereas a small percentage was found on the plasma membrane (< 10%). Within the cytosol, the protein was equally distributed in the matrix and in the granules, including those containing the typical crystalloid. The two anti-ANXA1 antibodies gave similar results, with the exception that LCPS1 gave a lower degree of immunoreactivity in the plasma membrane. Inflammation (i.e. carrageenin injection) produced a modest increase in eosinophil-associated ANXA1 reactivity ( significant only in the cytoplasm compartment). Extravasated neutrophils, used for comparative purposes, displayed a much higher degree of immunoreactivity for the protein.Conclusion: We describe for the first time ANXA1 distribution in rat eosinophil by ultrastructural analysis, and report a different protein mobilisation from extravasated neutrophils, at least in this acute model of peritonitis.

Cell localization of the anti-inflammatory protein annexin 1 during experimental inflammatory response

The localization of the glucocorticoid-inducible protein annexin 1 (ANX-1) in leukocytes during the process of experimental inflammation has been studied using immunocytochemistry. ANX-1 immunoreactivity was detected in extravasated neutrophils and eosinophils as well as in resident tissue mast cells. Following injection of carrageenin, the mesenteric tissue was highly inflamed with large presence of leukocytes (predominantly neutrophils with a small percentage of eosinophils) adherent to post-capillary venules and extravasated in the perivascular tissue. ANX-1 immunoreactivity was detected in the cytosol of neutrophils and eosinophils mainly associated with granules and/or vesicles. A good degree of localization in the endosomes was observed in the neutrophils, In both cell types, some ANX-1 immunoreactivity in the nucleus and in the plasma membrane was also detected. Resident mast cells were also activated. Mast cells were positive for ANX-1, without apparent changes in protein content in relation to their activation status. Degranulated mast cells still presented ANX-1 associated with the granule matrix. In conclusion, this study demonstrated the presence of ANX-1 in leukocytes that play a central role in the host inflammatory response. These are the extravasating polymorphonuclear cells, or the resident mast cells. These data provide morphological support to the notion that endogenous and exogenous ANX-1 are able to modulate the reactivity of these cell types, and more generally, of the experimental inflammatory reaction.

Pharmacological modulation of allergic inflammation in the rat airways and association with mast cell heterogeneity

Administration of ovalbumin by aerosol to sensitised rats produced a rapid (15 min) protein exudation in different airway tissues, as determined by Evans blue staining. This was associated with marked mast cell degranulation determined by histological examination, with there being no difference between mucosal and connective tissue mast cells. A 5-day administration regimen with compound 48/80 selectively depleted connective tissue mast cell (Positive to berberine staining) without modifying ovalbumin-induced plasma protein extravasation. Treatment of rats with dexamethasone (1 mg/kg, - 12 h) or nor-dihydroguaiaretic acid (30 mg/kg i.p., - 30 min) significantly reduced ovalbumin-induced protein extravasation and preserved mucosal mast cell morphology. Indomethacin (4 mg/kg i.v., - 30 min) exerted no effect on either parameter. In conclusion, we propose the mucosal mast cell as a target cell responsible at least partly for the inhibitory actions of known anti-inflammatory drugs. We suggest an involvement of endogenous leukotriene(s), but not prostanoid(s), in mucosal mast cell activation/degranulation. (C) 2001 Published by Elsevier B.V. B.V.

The effect of alpha-tocopherol on the oxidative cleavage of beta-carotene

Two cleavage pathways of beta-carotene have been proposed, one by central cleavage and the other by random (excentric) cleavage. The central cleavage pathway involves the metabolism of beta-carotene at the central double bond (15, 15') to produce retinal by beta-carotene 15, 15'-dioxygenase (E.C.888990988). The random cleavage of beta-carotene produces beta-apo-carotenoids, but the mechanism is not clear. To understand the various mechanisms of beta-carotene cleavage, beta-carotene was incubated with the intestinal postmitochondrial fractions of 10-week-old male rats for 1 h and cleavage products of beta-carotene were analyzed using reverse-phase, high-performance liquid chromatography (HPLC). We also studied the effects of alpha-tocopherol and NAD(+)/NADH on beta-carotene cleavage. In addition to beta-carotene, we used retinal and beta-apo-14'-carotenoic acid as substrates in these incubations. Beta-apo-14'-carotenoic acid is the two-carbon longer homologue of retinoic acid. In the presence of alpha-tocopherol, beta-carotene was converted exclusively to retinal, whereas in the absence of alpha-tocopherol, both retinal and beta-apo-carotenoids were formed. Retinoic acid was produced from both retinal and beta-apo-14'-carotenoic acid incubations only in the presence of NAD(+). Our data suggest that in the presence of an antioxidant such as alpha-tocopherol, beta-carotene is converted exclusively to retinal by central cleavage. In the absence of an antioxidant, beta-carotene is cleaved randomly by enzyme-related radicals to produce beta-apo-carotenoids, and these beta-apo-carotenoids can be oxidized further to retinoic acid via retinal. (C) 2000 Elsevier B.V.

Correlation between carotenoid concentrations in serum and normal breast adipose tissue of women with benign breast tumor or breast cancer

To evaluate the relationship between carotenoid concentrations in serum and breast tissue, we measured serum carotenoid concentrations and endogenous carotenoid levels in breast adipose tissue of women with benign breast tumor (n = 46) or breast cancer (n = 44). Before extraction, serum was digested with lipase and cholesterol esterase, and breast adipose tissue was saponified. Serum and tissue carotenoids were extracted with ether/hexane and measured by using HPLC with a C30 column. Serum retinoic acid was extracted with chloroform/methanol and measured using HPLC with a C18 column. There were no significant differences in serum carotenoids [lutein, zeaxanthin, cryptoxanthin (both α- and β-), α-carotene, all-trans β-carotene, 13-cis β-carotene and lycopene], retinoids (retinol, all-trans and 13-cis retinoic acids), and α- and -γ- tocopherol concentrations between benign breast tumor patients and breast cancer patients. A substantial amount of 9-cis β- carotene was present in adipose tissue and was the only carotenoid that had a significantly lower level in benign breast tumor patients than in breast cancer patients. Correlations between carotenoid concentrations in serum and in breast adipose tissue were determined by combining the data of the two groups. Concentrations of the major serum carotenoids except cryptoxanthin showed significant correlations with breast adipose tissue carotenoid levels. When the concentrations of serum carotenoids were adjusted for serum triglycerides or LDL, correlations between serum carotenoid concentrations and breast adipose tissue carotenoid levels markedly increased, including that of cryptoxanthin (P <0.001). The strong correlation between serum carotenoid concentrations and endogenous breast adipose tissue carotenoid levels indicate that dietary intake influences adipose tissue carotenoid levels as well as serum concentrations, and that adipose tissue is a dynamic reservoir of fat-soluble nutrients.

Detection of classical 17p11.2 deletions, an atypical deletion and RAI1 alterations in patients with features suggestive of Smith-Magenis syndrome

Smith-Magenis syndrome (SMS) is a complex disorder whose clinical features include mild to severe intellectual disability with speech delay, growth failure, brachycephaly, flat midface, short broad hands, and behavioral problems. SMS is typically caused by a large deletion on 17p11.2 that encompasses multiple genes including the retinoic acid induced 1, RAI1, gene or a mutation in the RAI1 gene. Here we have evaluated 30 patients with suspected SMS and identified SMS-associated classical 17p11.2 deletions in six patients, an atypical deletion of ∼139 kb that partially deletes the RAI1 gene in one patient, and RAI1 gene nonsynonymous alterations of unknown significance in two unrelated patients. The RAI1 mutant proteins showed no significant alterations in molecular weight, subcellular localization and transcriptional activity. Clinical features of patients with or without 17p11.2 deletions and mutations involving the RAI1 gene were compared to identify phenotypes that may be useful in diagnosing patients with SMS. © 2012 Macmillan Publishers Limited All rights reserved.

Efeito do paclobutrazol e de ácido giberélico na indução floral do maracujazeiro-amarelo em condições de entressafra

Objetivou-se estudar os efeitos de ácido giberélico (GA3) (i.a.), nas concentrações de 50 e 100 mg L-1, acrescido do espalhante adesivo Extravon® a 0,05%, em duas aplicações via foliar e do paclobutrazol (PBZ) (i.a.), em doses de 2 e 4 g por planta, aplicado via solo, em uma única aplicação, na antecipação da floração do maracujazeiro-amarelo, em Araguari-MG. O delineamento experimental empregado foi em parcelas subdivididas, com cinco tratamentos (parcelas) e dois tratamentos (subparcelas), com quatro repetições e quatro plantas por parcela. Foram identificados 12 ramos terciários por parcela, com seis ramos expostos à luminosidade predominante pela manhã e seis com luminosidade predominante à tarde. Avaliaram-se os dois lados da espaldeira aos 103 dias após a primeira aplicação dos tratamentos, observando-se o comprimento dos ramos e de entrenós, número de nós e de botões florais. Os reguladores não promoveram respostas significativas para o comprimento dos entrenós e o número de botões florais. Porém, observaram-se indícios de maior número de botões florais com PBZ 2 g. O maior comprimento dos ramos foi obtido com GA3 100 mg L-1, sob luminosidade, pela manhã. Ao se compararem os tratamentos nas duas condições de luminosidade, o PBZ 4 g, independentemente da condição de luminosidade, proporcionou o menor comprimento dos ramos.

Formulação de rações para poedeiras com base em aminoácidos totais e digestíveis utilizando diferentes estimativas da composição de aminoácidos em alimentos

Foram utilizadas poedeiras comerciais com 27 semanas de idade, distribuídas em delineamento inteiramente ao acaso, em esquema fatorial 3 x 3, com três repetições de seis aves por tratamento. Os fatores consistiram de três métodos de estimativa da composição de aminoácidos em ingredientes (tabelas brasileiras, equações de predição e fator para correção de aminoácido em função do teor de proteína do ingrediente) e três recomendações de aminoácidos, sendo duas de aminoácidos digestíveis e uma de aminoácidos totais. Os métodos de estimativa da composição de aminoácidos nos ingredientes afetaram apenas a conversão alimentar e a espessura de casca, que apresentaram os melhores resultados com a utilização das tabelas brasileiras. Embora as recomendações de aminoácidos tenham determinado diferenças em todos os parâmetros de desempenho, não afetaram a qualidade dos ovos. O desempenho das aves foi prejudicado pelos níveis de aminoácidos digestíveis, entretanto, ambas as recomendações promoveram desempenho semelhante e inferior ao de aminoácidos totais. O pior desempenho das aves alimentadas com as rações formuladas com aminoácidos digestíveis pode ser atribuído à deficiência em nitrogênio para a síntese de aminoácidos não-essenciais, visto que o nível protéico foi reduzido (12,5% PB), ou ainda à deficiência nos aminoácidos arginina, histidina, isoleucina, leucina e valina, cujos requerimentos mínimos não foram considerados na formulação das rações.

Efeito de diferentes doses de ácido retinoico sobre a resistência óssea de ratos jovens

OBJETIVO: Avaliar os efeitos da suplementação de diferentes doses de todo-trans ácido retinóico sobre a resistência óssea, por meio de ensaio biomecânico de flexão, em tíbia de ratos jovens. MÉTODOS: Foram estudados 58 ratos jovens, com quatro diferentes doses de vitamina A em suas dietas, sendo divididos em 4 grupos: grupo-controle (n=15), sem acréscimo de todo-trans ácido retinoico; grupo com acréscimo de 0,3mg de todo-trans ácido retinoico por kg de ração (n=13); grupo com 10mg de todo-trans ácido retinoico por kg de ração (n=15); e grupo com 50mg de todo-trans ácido retinoico por kg de ração (n=15). O estudo durou 30 dias. Após o sacrifício dos animais, suas patas esquerdas foram congeladas, dissecadas e as tíbias submetidas ao ensaio de flexão. Foram avaliados a carga máxima e o coeficiente de rigidez. Foi aplicada análise de variância one-way. O nível de significância estatístico adotado foi p<0,05. RESULTADOS: Os valores médios de carga máxima (em Newton) foram: grupo-controle =37,94, DP=4,76; grupo todo-trans ácido retinoico 0,3=36,49, DP= 4,38; grupo todo-trans ácido retinoico 10=40,12, DP=6,03; grupo todo-trans ácido retinoico 50=35,68, DP=5,22 (p=0,107). Os valores médios de coeficiente de rigidez (em Newton/milímetros) foram: grupo-controle =31,84 DP=6,75; grupo todo-trans ácido retinoico 0,3=29,18, DP=4,35; grupo todo-trans ácido retinoico 10=35,48, DP=8,14; grupo todo-trans ácido retinoico 50=30,31, DP=7,14 (p=0,85). CONCLUSÃO: Conclui-se que a exposição a diferentes doses de todo-trans ácido retinoico, em ratos, durante 30 dias, não exerce efeito sobre a resistência óssea, quando avaliada por ensaios biomecânicos.

DNA methylation patterns in bladder cancer and washing cell sediments: a perspective for tumor recurrence detection

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Associations between LEP, DGAT1 and FABP4 gene polymorphisms and carcass and meat traits in Nelore and crossbred beef cattle

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Redução da proteína da dieta com base no conceito de proteína ideal para tilápias-do-nilo (Oreochromis niloticus) criadas em tanques-rede

Avaliou-se o desempenho de tilápias-do-nilo (Oreochromis niloticus) criadas em tanques-rede e alimentadas com dietas contendo 27,0 (controle); 25,2; 24,3 e 22,7% de proteína digestível. Aminoácidos cristalinos (L-lisina, DL-metionina e L-treonina) foram adicionados à dieta considerando o conceito de proteína ideal e simulando o perfil de aminoácidos da dieta controle. Os peixes (34,63±19 g) foram alimentados manualmente com dietas isoenergéticas (3.075 kcal de energia digestível/kg de dieta) até saciedade aparente, três vezes ao dia, durante 91 dias. Utilizou-se um delineamento inteiramente casualizado, com quatro tratamentos, três repetições e 25 peixes/unidade experimental. Não foram observados efeitos dos níveis de proteína digestível sobre o ganho de peso, a conversão alimentar, a taxa de eficiência protéica, o peso da carcaça eviscerada, o rendimento de carcaça, o peso e o rendimento de filé, a sobrevivência e o hematócrito. Houve efeito quadrático dos níveis de proteína digestível sobre o consumo; o maior valor foi estimado para a dieta contendo 24,41% de proteína digestível e excreção de nitrogênio, na qual o melhor resultado estimado foi obtido com peixes que receberam a dieta contendo 24,92% de PD. Com a redução nos níveis de proteína digestível, observou-se aumento linear na retenção de nitrogênio. É possível reduzir o nível de proteína digestível, de 27 (29,1% de PB) para 24,3% (26,6% de PB), em dietas para tilápias-do-nilo criadas em tanques-rede. Essa redução deve ser feita por meio da suplementação de aminoácidos (com base no conceito de proteína ideal), considerando o desempenho e o custo da dieta/kg ganho em filé.

Potencial de transdiferenciação neural das células-tronco mesenquimais da medula óssea de equino

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Expression of the Interleukin-10 Signaling Pathway Genes in Individuals With Down Syndrome and Periodontitis

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)