In silico analysis identifies a C3HC4-RING finger domain of a putative E3 ubiquitin-protein ligase located at the C-terminus of a polyglutamine-containing protein

Almost identical polyglutamine-containing proteins with unknown structures have been found in human, mouse and rat genomes (GenBank AJ277365, AF525300, AY879229). We infer that an identical new gene (RING) finger domain of real interest is located in each C-terminal segment. A three-dimensional (3-D) model was generated by remote homology modeling and the functional implications are discussed. The model consists of 65 residues from terminal position 707 to 772 of the human protein with a total length of 796 residues. The 3-D model predicts a ubiquitin-protein ligase (E3) as a binding site for ubiquitin-conjugating enzyme (E2). Both enzymes are part of the ubiquitin pathway to label unwanted proteins for subsequent enzymatic degradation. The molecular contact specificities are suggested for both the substrate recognition and the residues at the possible E2-binding surface. The predicted structure, of a ubiquitin-protein ligase (E3, enzyme class number 6.3.2.19, CATH code 3.30.40.10.4) may contribute to explain the process of ubiquitination. The 3-D model supports the idea of a C3HC4-RING finger with a partially new pattern. The putative E2-binding site is formed by a shallow hydrophobic groove on the surface adjacent to the helix and one zinc finger (L722, C739, P740, P741, R744). Solvent-exposed hydrophobic amino acids lie around both zinc fingers (I717, L722, F738, or P765, L766, V767, V733, P734). The 3-D structure was deposited in the protein databank theoretical model repository (2B9G, RCSB Protein Data Bank, NJ).

Glucocorticoids, master modulators of the thymic catecholaminergic system?

There is evidence that the major mediators of stress, i.e., catecholamines and glucocorticoids, play an important role in modulating thymopoiesis and consequently immune responses. Furthermore, there are data suggesting that glucocorticoids influence catecholamine action. Therefore, to assess the putative relevance of glucocorticoid-catecholamine interplay in the modulation of thymopoiesis we analyzed thymocyte differentiation/maturation in non-adrenalectomized and andrenalectomized rats subjected to treatment with propranolol (0.4 mg·100 g body weight-1·day-1) for 4 days. The effects of β-adrenoceptor blockade on thymopoiesis in non-adrenalectomized rats differed not only quantitatively but also qualitatively from those in adrenalectomized rats. In adrenalectomized rats, besides a more efficient thymopoiesis [judged by a more pronounced increase in the relative proportion of the most mature single-positive TCRαβhigh thymocytes as revealed by two-way ANOVA; for CD4+CD8- F (1,20) = 10.92, P < 0.01; for CD4-CD8+ F (1,20) = 7.47, P < 0.05], a skewed thymocyte maturation towards the CD4-CD8+ phenotype, and consequently a diminished CD4+CD8-/CD4-CD8+ mature TCRαβhigh thymocyte ratio (3.41 ± 0.21 in non-adrenalectomized rats vs 2.90 ± 0.31 in adrenalectomized rats, P < 0.05) were found. Therefore, we assumed that catecholaminergic modulation of thymopoiesis exhibits a substantial degree of glucocorticoid-dependent plasticity. Given that glucocorticoids, apart from catecholamine synthesis, influence adrenoceptor expression, we also hypothesized that the lack of adrenal glucocorticoids affected not only β-adrenoceptor- but also α-adrenoceptor-mediated modulation of thymopoiesis.

Antifungal activity of the naphthoquinone beta-lapachone against disseminated infection with Cryptococcus neoformans var. neoformans in dexamethasone-immunosuppressed Swiss mice

The in vivo antifungal activity of the naphthoquinone beta-lapachone against disseminated infection by Cryptococcus neoformans was investigated. Swiss mice were immunosuppressed daily with dexamethasone (0.5 mg per mouse) intraperitoneally for 3 days, the procedure was repeated 4 days later, and the animals were then challenged intravenously with C. neoformans (10(6) CFU/mL) 1 week later. Seven days after infection, the mice were divided into groups and treated daily with beta-lapachone (10 mg/kg, iv) for 7 (N = 6) and 14 days (N = 10). Amphotericin B (0.5 mg/kg) was used as comparator drug and an additional group received PBS. Treatment with beta-lapachone cleared the yeast from the spleen and liver, and the fungal burden decreased approximately 10(4) times in the lungs and brain 14 days after infection when compared to the PBS group (P < 0.05). This result was similar to that of the amphotericin B-treated group. Protection was suggestively due to in vivo antifungal activity of this drug and apparently not influenced by activation of the immune response, due to similar leukocyte cell counts among all groups. This study highlights the prospective use of beta-lapachone for treatment of disseminated cryptococcosis.

Antipyretic and antioxidant activities of 5-trifluoromethyl-4,5-dihydro-1H-pyrazoles in rats

The objective of this study was to determine the effect of eight 5-hydroxy-5-trifluoromethyl-4,5-dihydro-1H-1-carboxyamidepyrazoles (TFDPs) on rat body temperature and baker’s yeast-induced fever. TFDPs or vehicle (5% Tween 80 in 0.9% NaCl, 5 mL/kg) were injected subcutaneously and rectal temperature was measured as a function of time in 28-day-old male Wistar rats (N = 5-12 per group). Antipyretic activity was determined in feverish animals injected with baker’s yeast (Saccharomyces cerevisiae suspension, 0.135 mg/kg, 10 mL/kg, ip). 3-Ethyl- and 3-propyl-TFDP (140 and 200 μmol/kg, respectively, 4 h after yeast injection) attenuated baker’s yeast-induced fever by 61 and 82%, respectively. These two effective antipyretics were selected for subsequent analysis of putative mechanisms of action. We then determined the effects on cyclooxygenase-1 and -2 (COX-1 and COX-2) activities on 1,1-diphenyl-2-picrylhydrazyl (DPPH) oxidation in vitro, on tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) levels and on leukocyte counts in the washes of peritoneal cavities of rats injected with baker’s yeast. While 3-ethyl- and 3-propyl-TFDP did not reduce baker’s yeast-induced increases of IL-1β or TNF-α levels, 3-ethyl-TFDP caused a 42% reduction in peritoneal leukocyte count. 3-Ethyl- and 3-propyl-TFDP did not alter COX-1 or COX-2 activities in vitro, but presented antioxidant activity in the DPPH assay with an IC50 of 39 mM (25-62) and 163 mM (136-196), respectively. The data indicate that mechanisms of action of these two novel antipyretic pyrazole derivatives do not involve the classic inhibition of the COX pathway or pyrogenic cytokine release.

Effect of selective β-adrenoceptor blockade and surgical resection of the celiac-superior mesenteric ganglion complex on delayed liquid gastric emptying induced by dipyrone, 4-aminoantipyrine, and antipyrine in rats

There is evidence for participation of peripheral β-adrenoceptors in delayed liquid gastric emptying (GE) induced in rats by dipyrone (Dp), 4-aminoantipyrine (AA), and antipyrine (At). The present study aimed to determine whether β-adrenoceptors are involved in delayed GE induced by phenylpyrazole derivatives and the role of the prevertebral sympathetic nervous system in this condition. Male Wistar rats weighing 220-280 g were used in the study. In the first experiment rats were intravenously pretreated with vehicle (V), atenolol 30 mg/kg (ATE, β1-adrenergic antagonist), or butoxamine 25 mg/kg (BUT, β2-adrenergic antagonist). In the second experiment, rats were pretreated with V or SR59230A 2 mg/kg (SRA, β3-adrenergic antagonist). In the third experiment, rats were subjected to surgical resection of the celiac-superior mesenteric ganglion complex or to sham surgery. The groups were intravenously treated with saline (S), 240 µmol/kg Dp, AA, or At, 15 min after pretreatment with the antagonists or V and nine days after surgery. GE was determined 10 min later by measuring the percentage of gastric retention (%GR) of saline labeled with phenol red 10 min after gavage. The %GR (means±SE, n=6) values indicated that BUT abolished the effect of Dp (BUT+Dp vs V+Dp: 35.0%±5.1% vs 56.4%±2.7%) and At (BUT+At vs V+At: 33.5%±4.7% vs 52.9%±2.6%) on GE, and significantly reduced (P<0.05) the effect of AA (BUT+AA vs V+AA: 48.0%±5.0% vs 65.2%±3.8%). ATE, SRA, and sympathectomy did not modify the effects of treatments. These results suggest that β2-adrenoceptor activation occurred in delayed liquid gastric emptying induced by the phenylpyrazole derivatives dipyrone, 4-aminoantipyrine, and antipyrine. Additionally, the released neurotransmitter did not originate in the celiac-superior mesenteric ganglion complex.

Variação no conteúdo de beta-glucanas em cultivares brasileiros de aveia

Com o crescente interesse em alimentos funcionais e nutracêuticos, a aveia (Avena sativa L.) tem se destacado, devido ao seu teor de fibras alimentares e principalmente às beta-glucanas. As (1,3)(1,4)-beta-D-glucanas, fibras alimentares na maioria solúveis, atuam na redução do colesterol em indivíduos com hipercolesterolemia. Existem estudos para determinar as causas de variação do teor desta fibra em aveia, porém, pouco se sabe sobre a aveia cultivada no Brasil. O objetivo deste trabalho foi verificar se existem diferenças no conteúdo de beta-glucanas entre cultivares brasileiros e se há variação na porcentagem desta fibra devido ao ano de cultivo. Os cultivares IAC7, UFRGS14, UPF16 e UPF17 (3 amostras de cada), e ainda três amostras do cultivar IAC7 para cada ano de cultivo (97 e 98), foram analisados segundo os métodos da AACC (American Association of Cereal Chemists). Os teores médios (peso seco) de beta-glucanas foram 6,50% (IAC7), 4,30% (UFRGS14), 3,51% (UPF16) e 3,78% (UPF17), com erro padrão de ±0,084 e coeficiente de variação de 7,89 %. Observou-se efeito significativo dos cultivares (p=0,03) e grande variabilidade entre as amostras (p=0,0001). O cultivar IAC7 apresentou média de beta-glucanas de 5,11% em 97 e 6,50 % em 98 (erro padrão ±0,14; CV=10,53%) e observou-se efeito significativo do ano de cultivo.

Composição centesimal e teor de beta-glucanas em cereais e derivados

Foi utilizado o método enzimático recomendado pela AOAC para determinação de beta-glucanas em cereais e alimentos que os contém. O método, utiliza liquenase (EC 3.2.1.73) e beta-glucosidase (EC 3.2.1.21) para hidrólise debeta-glucanas, é rápido, fácil de executar e específico para beta-glucanas com ligações beta(1->3) e beta(1->4). As sementes analisadas foram subministradas pelo Instituto Agronômico de Campinas (IAC) e os alimentos adquiridos nos supermercados. Aveia e cevada são os grãos com maior conteúdo de beta-glucanas. Na aveia os teores determinados foram 6,48 e 5,94%. Nos 10 cultivares de cevada os teores de beta-glucanas oscilaram entre 2,04 e 9,68%. Trigo e triticale apresentaram teores de b-glucanas menores que 1%. Nos produtos comerciais o teor de beta-glucanas estava relacionado ao tipo de cereal da fórmula. O produto comercial de maior conteúdo de beta-glucanas é o farelo de aveia. As beta-glucanas são ingredientes funcionais em potencial e a conveniência ou não de estimular sua incorporação em alimentos deve ser mais estudada. Quanto à composição centesimal dos grãos de cereais, o teor de proteínas foi o que apresentou a maior variação e isso se reflete na composição dos produtos comerciais.

Determinação da concentração de beta-glucano em cogumelo Agaricus blazei Murill por método enzimático

Cogumelos comestíveis contêm importantes propriedades funcionais. Em particular, beta-glucanos, homo- e hetero-glucanos com ligações glicosídicas beta(1->3), beta(1->4) e beta(1->6), supostamente responsáveis por algumas propriedades benéficas à saúde humana, como atividade imunomodulatória, antioxidante, antiinflamatória e anticancerígena. Neste trabalho, a quantidade de beta-glucano presente em cogumelo Agaricus blazei Murill, coletados de três diferentes locais, foi analisada utilizando-se método enzimático. As amostras (em base seca) foram tratadas com alfa-amilase, protease bacteriana e com glicoamilase fúngica. beta-glucano foi quantificado após hidrólise ácida e determinação da glicose liberada. Foi verificado que amostras de A. blazei Murill cultivadas em estufas apresentaram menor concentração de b-glucano (8,4±0,9 e 7,6±2,8g/100g) quando comparado com amostras cultivadas em campo aberto (10,1±2,1g/100g). Observou-se ainda que, mesmo sendo cultivado em condições semelhantes, porém em locais diferentes, as amostras apresentaram diferenças significativas (7,6±2,8 e 8,4±0,9g/100g).

Estudo da produção de beta-galactosidase por fermentação de soro de queijo com Kluyveromyces marxianus

A hidrólise enzimática da lactose por beta-galactosidase desempenha importante papel no processamento de produtos lácteos, como na obtenção de leite com baixo teor de lactose para consumo por indivíduos intolerantes à mesma e na prevenção da cristalização em produtos de laticínio. Neste trabalho, a enzima beta-galactosidase foi produzida pelo cultivo do microrganismo Kluyveromyces marxianus, em meio de cultura à base de soro de queijo em diferentes concentrações iniciais de lactose e extrato de levedura, de acordo com um planejamento fatorial. As fermentações foram conduzidas em incubador rotativo a 150rpm, a 30°C e pH inicial 5,5. A concentração celular inicial foi de 10(7) células/mL. Para a extração da enzima beta-galactosidase, foi realizada autólise das células utilizando como solvente o clorofórmio em tampão fosfato. No meio de cultura enriquecido com (NH4)2SO4, KH2PO4 e MgSO4, nas concentrações iniciais de lactose e de extrato de levedura iguais a 50g/L e 12g/L, respectivamente, obteve-se uma atividade de 28,0UGl/mL e uma concentração celular máxima de 5,3g/L.

Comportamento da beta-ciclodextrina adicionada ao leite de cabra submetido ao processo de desidratação por "spray-dryer"

Este trabalho avaliou o comportamento do agente encapsulante beta -ciclodextrina ( beta-CD) adicionado ao leite de cabra submetido ao processo de desidratação por "spray-dryer", através de análise termogravimétrica e de cromatografia gasosa. Após a desidratação, a amostra adicionada de beta-CD apresentou um rendimento real de 10,59% com taxa de perda de 0,04% (em relação ao valor teórico esperado 10,6% ); enquanto na amostra sem adição do agente encapsulante o rendimento real foi de 9,57%, com taxa de perda de 4,27% (valor teórico esperado 10% ). Através da análise termogravimétrica (TGA), verificou-se que são volatilizados 44% e 21% dos ácidos comerciais C8 e C10 , respectivamente. Os resultados cromatográficos mostraram uma perda de aproximadamente 30% dos ácidos C8 e 20% dos ácidos C10 , nas amostras de leite de cabra sem beta -CD em relação às amostras com beta-CD. Tais porcentagens estão de acordo com os valores estimados para os ácidos comerciais. Com base nos parâmetros estudados, podemos inferir que há menor perda dos ácidos graxos C8 e C10 na amostra de leite de cabra com beta-CD, provavelmente devido ao efeito encapsulante, aumentando a estabilidade térmica dos ácidos.

Influência da temperatura na solubilidade de beta-caroteno em solventes orgânicos à pressão ambiente

O presente trabalho reporta dados experimentais de solubilidade em pressão ambiente de beta-caroteno em solventes orgânicos puros (etanol, acetona, acetato de etila e diclorometano) e em misturas de tais solventes no intervalo de temperatura de 10 a 60 °C. Para este fim, adotou-se o método gravimétrico para a determinação da solubilidade, utilizando células encamisadas de equilíbrio. Os resultados mostraram que valores mais elevados de solubilidade são obtidos quando foram empregados solventes com parâmetros de solubilidade mais próximos daquele do soluto. Verificou-se que o aumento da temperatura, tanto para solventes puros, como para as misturas de solventes, acarretou num aumento da solubilidade do beta-caroteno para todas as condições experimentais. Observou-se ainda, que nas condições experimentais investigadas, não houve sinergismo significativo para as misturas de solventes quando comparadas aos valores de solubilidade obtidos para os solventes puros. O modelo UNIFAC se mostrou útil na previsão qualitativa dos resultados de solubilidade.

Effect of processing on antioxidant potential and total phenolics content in beet (Beta vulgaris L.)

The antioxidant capacity of beet is associated with non-nutritive constituents, such as phenolic compounds. The purpose of this research was to evaluate the effect of two different heat-processing techniques (drying and canned) on the antioxidant potential (ABTS) and phenolics content of beets. A forced air circulation dehydrator was used for the drying. Drying at high temperatures (100 + 90 °C/5.6 hours; 90 °C/6 hours) increased the antioxidant potential of the processed products while mild drying conditions decreased it (80 °C/6 hours; 100 + 70 °C/6 hours) or had no effect on it (70 °C/7 hours; 100 + 80 °C/6 hours). For the canned products, the antioxidant potential did not differ according to the pH (4.2 to 3.8) for any of the four acids tested. Some processing methods influenced the antioxidant potential of the processed products, and this was also dependent on changes in the total phenolics content.

In-house method validation and occurrence of alpha-, beta-endosulfan, endosulfan sulphate, lambda-cyhalothrin, procymidone and trifluralin residues in strawberry

A method for determination of organohalogen pesticides in strawberry by gas chromatography with electron capture detection was validated and applied in a monitoring program. Linearity, matrix effects, and day effect were evaluated for the analytes alpha-endosulfan, beta-endosulfan, endosulfan sulphate, lambda-cyhalothrin, procymidone, and trifluralin. The linear range varied according to the chromatographic response of the analyte. Significant matrix effects were observed. The mean recoveries ranged from 74.6 to 115.4%, with repeatability standard deviations between 1.6 and 21.0% and intermediate precision between 5.9 and 21.0%. Detection, quantification and decision limit, and detection capacity ranged from 0.003 to 0.007 mg/kg, 0.005 to 0.013 mg/kg; 0.003 to 3.128 mg/kg; and 0.005 to 3.266 mg/kg, respectively. The method was fit for the purpose of monitoring organohalogen residues in strawberries. Residues of these pesticides were detected in 124 of the 186 samples analyzed between 2009 and 2011 in the state of Minas Gerais. Nine of them did not comply with the current legislation requirements; among them, seven (3.8%) had residues of unauthorized pesticide for the culture of strawberry, one (0.5%) had residues above the maximum residue limit, and another one (0.5%) exhibited both non-conformities.

The application of the structural correlation method to determine the reaction coordinate for a putative ring closure reaction

The cr ystal structure of the compound 2-benzoylethylidene-3-(2,4- dibromophenyl)-2,3-dihydro-5-phenyl-l,3,4-thiadiazole* C23H16Br2NZOS (BRMEO) has been determined by using three dimensiona l x-ray diffraction data. The crys tal form is monoclinic, space group P21/c, a = 17.492(4), o -.t' 0 R 0 b =: 16.979(1), c = 14.962(1) A, "X. =o= 90 ',= 106.46(1) , z = 8, graphite-monochromatized Mo~ rad iation, Jl= 0.710J3~, D = 1.62g/cc and o D = 1.65g/cc. The data were col lected on ~ Nonius CAD-4 c diffractometer. The following atoms were made anisotropic: Br, S, N, 0, C7, and C14-C16 for each i ndependent molecu le ; the rest were left isotropic. For 3112 independent refl ec tions with F > 6G\F), R == 0.057. The compound has two independent molecules within the asymmetric unit. Two different conformers were observed which pack well together. /l The S---O interaction distances of 2.493(6) and 2 . 478(7) A were observed for molecules A and B respectively. These values are consistent with earlier findings for 2-benzoylmethylene-3-(2,4-dibromophenyl)- ~~ 2,3-dihydro-5-phenyl-l,3,4-thiadiazole C22H14Br2N20S (BRPHO) and 2-benzoylpropylidene-3-(2,4-dibromophenyl)-2,3-dihydroiii ,'r 5-phenyl-l,3,4-thiadiazole C24H18Br2N20S (BRPETO ) where S---O distances are l ess than the van der Waals (3.251\) but greater than those expected for () a single bond (1.50A). From the results and the literature it appears obvious that the energy/reaction coordinate pathway has a minimum between the end structures (the mono- and bicyclic compounds). * See reference (21) for nomenclature.

Alternative splicing of DNA polymerase beta in vertebrates

Alternative splicing (AS) is the predominant mechanism responsible for increasing eukaryotic transcriptome and proteome complexity. In this phenomenon, numerous mRNA transcripts are produced from a single pre-mRNA sequence. AS is reported to occur in 95% of human multi-exon genes; one specific gene that undergoes AS is DNA polymerase beta (POLB). POLB is the main DNA repair gene which performs short patch base excision repair (BER). In primate untransformed primary fibroblast cell lines, it was determined that the splice variant (SV) frequency of POLB correlates positively with species lifespan. To date, AS patterns of POLB have only been examined in mammals primarily through the use of cell lines. However, little attention has been devoted to investigating if such a relationship exists in non-mammals and whether cell lines reflect what is observed in vertebrate tissues. This idea was explored through cloning and characterization of 1,214 POLB transcripts from four non-mammalian species (Gallus gallus domesticus, Larus glaucescens, Xenopus laevis, and Pogona vitticeps) and two mammalian species (Sylvilagus floridanus and Homo sapiens) in two tissue types, liver and brain. POLB SV frequency occurred at low frequencies, < 3.2%, in non-mammalian tissues relative to mammalian (>20%). The highest POLB SV frequency was found in H. sapiens liver and brain tissues, occurring at 65.4% and 91.7%, respectively. Tissue specific AS of POLB was observed in L. glaucescens, P. vitticeps, and H. sapiens, but not G. gallus domesticus, X. laevis and S. floridanus.The AS patterns of a second gene, transient receptor potential cation channel subfamily V member 1 (TRPV1), were compared to those of POLB in liver and brain tissues of G. gallus domesticus, X. laevis and H. sapiens. This comparison was performed to investigate if any changes (either increase or decrease) observed in the AS of POLB were gene specific or if they were tissue specific, in which case similar changes in AS would be seen in POLB and TRPV1. Analysis did not reveal an increase or decrease in both the AS of POLB and TRPV1 in either the liver or brain tissues of G. gallus domesticus and H. sapiens. This result suggested that the AS patterns of POLB were not influenced by tissue specific rates of AS. Interestingly, an increase in the AS of both genes was only observed in X. laevis brain tissue. This result suggests that AS in general may be increased in the X. laevis brain as compared to liver tissue. No positive correlation between POLB SV frequency and species lifespan was found in non-mammalian tissues. The AS patterns of POLB in human primary untransformed fibroblast cell lines were representative of those seen in human liver tissue but not in brain tissue. Altogether, the AS patterns of POLB from vertebrate tissues and primate cell lines revealed a positive correlation between POLB SV frequency and lifespan in mammals, but not in non-mammals. It appears that this positive correlation does not exist in vertebrate species as a whole.