Modulation of tonic immobility in guinea pig PAG by homocysteic acid, a glutamate agonist

Tonic immobility (TI) is an innate defensive behavior elicited by physical restriction and postural inversion, and is characterized by a profound and temporary state of motor inhibition. The participation of the periaqueductal gray matter (PAG) in TI modulation has previously been described. In addition, the excitatory amino acids (EAA) are important mediators involved in the adjustment of several defensive responses produced by PAG. In the present study, we investigated the effect of microinjection of the EAA agonist DL-homocysteic acid (DLH) and the N-methyl-D-aspartate (NMDA) receptor antagonist (MK-801) into the ventrolateral and dorsal PAG over the duration of TI in guinea pigs. Microinjection of 15 nmol/0.2 mu l of DLH into the ventrolateral PAG (vlPAG) and 30 nmol/0.2 mu l of DLH into the dorsal PAG (dPAG) promoted an increase and decrease in TI duration, respectively. These responses were blocked by prior microinjection of the NMDA receptor antagonist, MK-801 (3.6 nmol/0.2 mu l) at the same site. Microinjection of MK-801 alone into the APAG and dPAG did not alter the duration of TI episodes. These results suggest that NMDA receptors are involved in the modulation of TI in both the vlPAG and dPAG. In addition, PAC excitatory amino acids modulate the TI response via columnar organization of the PAC. In this manner, the vlPAG facilitates TI modulation whereas dPAG has an inhibitory role in TI. (C) 2008 Elsevier Inc. All rights reserved.

Development of edible films based on differently processed Atlantic halibut (Hippoglossus hippoglossus) skin gelatin

The gelatin prepared from the skins of the Atlantic halibut (Hippoglossus hippoglossus) was investigated for the development of edible films plasticized with 30g sorbitol/100g gelatin. Two types of dry gelatin preparations were obtained depending on whether an intermediate evaporation step at 60 degrees C in the drying procedure is included or not. The amino acid composition, molecular weight distribution (determined by SDS-polyacrylamide gel electrophoresis) and glass transition temperature (determined by differential scanning calorimetry) of the gelatins were determined and related to some physical properties of the resulting films. The gelatin extracted from the halibut skins showed a suitable filmogenic capacity, leading to transparent, weakly colored, water-soluble and highly extensible films. The intermediate evaporation step at 60 degrees C induced thermal protein degradation, causing the resulting films to be significantly less resistant and more extensible. No differences in water vapor permeability, viscoelasticity, glass transition or color properties were evidenced between the two gelatins tested. (C) 2007 Elsevier Ltd. All rights reserved.

Polyamines, amino acids, IAA and ABA contents during Ocotea catharinensis seed germination

The aim of this work was to Study biochemical variations of IAA (indole-3-acetic acid), ABA (abscisic acid). PAs (polyamines) and amino acids at endogenous levels, during seed germination in Ocotea catharinensis. Seeds were germinated in a vermiculite substratum (100%), samples being collected after 15, 30 and 60 days. Total amino acid levels decreased during the first 15 days. Followed by all increment at the end of germination. Among amino acids, higher concentration was observed in asparagine, this being the predominant amino acid during the whole germination period. Total PAs (free + conjugated) content increased during the first 15 days, followed by a decrease and stabilization between 30 and 60 days of germination. Among the PAs, free putrescine levels rose during the first 15 days, followed by a drop and Stabilization up to 60 days of germination, while spermidine and spermine (spm) contents diminished during the period. Only spin was detected in a conjugated form, with increasing concentrations starting from 30 days on. IAA levels increased during tire first 15 days. followed by a decrease and stabilization until the end of germination (60 days), while ABA contents dwindled during the first 15 days, with similar Values until the end of germination.

Amino acids and diabetes: implications for endocrine, metabolic and immune function

Aberrant alterations in glucose and lipid concentrations and their pathways of metabolism are a hallmark of diabetes. However, much less is known about alterations in concentrations of amino acids and their pathways of metabolism in diabetes. In this review we have attempted to highlight, integrate and discuss common alterations in amino acid metabolism in a wide variety of cells and tissues and relate these changes to alterations in endocrine, physiologic and immune function in diabetes.

CCP1/Nna1 functions in protein turnover in mouse brain: Implications for cell death in Purkinje cell degeneration mice

Purkinje cell degeneration (pcd) mice have a mutation within the gene encoding cytosolic carboxypeptidase 1 (CCP1/Nna1), which has homology to metallocarboxypeptidases. To assess the function of CCP1/Nna1, quantitative proteomics and peptidomics approaches were used to compare proteins and peptides in mutant and wild-type mice. Hundreds of peptides derived from cytosolic and mitochondrial proteins are greatly elevated in pcd mouse hypothalamus, amygdala, cortex, prefrontal cortex, and striatum. However, the major proteins detected on 2-D gel electrophoresis were present in mutant and wild-type mouse cortex and hypothalamus at comparable levels, and proteasome activity is normal in these brain regions of pcd mice, suggesting that the increase in cellular peptide levels in the pcd mice is due to reduced degradation of the peptides downstream of the proteasome. Both nondegenerating and degenerating regions of pcd mouse brain, but not wild-type mouse brain, show elevated autophagy, which can be triggered by a decrease in amino acid levels. Taken together with previous studies on CCP1/Nna1, these data suggest that CCP1/Nna1 plays a role in protein turnover by cleaving proteasome-generated peptides into amino acids and that decreased peptide turnover in the pcd mice leads to cell death.-Berezniuk, I., Sironi, J., Callaway, M. B., Castro, L. M., Hirata, I. Y., Ferro, E. S., Fricker, L. D. CCP1/Nna1 functions in protein turnover in mouse brain: Implications for cell death in Purkinje cell degeneration mice. FASEB J. 24, 1813-1823 (2010). www.fasebj.org

New alk genes detected in Antarctic marine sediments

Alkane monooxygenases (Alk) are the key enzymes for alkane degradation. In order to understand the dispersion and diversity of alk genes in Antarctic marine environments, this study analysed by clone libraries the presence and diversity of alk genes (alkB and alkM) in sediments from Admiralty Bay, King George Island, Peninsula Antarctica. The results show a differential distribution of alk genes between the sites, and the predominant presence of new alk genes, mainly in the pristine site. Sequences presented 53.10-69.60% nucleotide identity and 50.90-73.40% amino acid identity to alkB genes described in Silicibacter pomeroyi, Gordonia sp., Prauserella rugosa, Nocardioides sp., Rhodococcus sp., Nocardia farcinica, Pseudomonas putida, Acidisphaera sp., Alcanivorax borkumensis, and alkM described in Acinetobacter sp. This is the first time that the gene alkM was detected and described in Antarctic marine environments. The presence of a range of previously undescribed alk genes indicates the need for further studies in this environment.

Pyrodysinoic Acid Derivatives from the Marine Sponge Dysidea robusta

Three new nitrogen-containing terpenes related to pyrodysinoic acid (1) have been isolated from the sponge Dysidea robusta collected in Brazil. Isopyrodysinoic acid (2), 13-hydroxyisopyrodysinoic acid (3), and pyrodysinoic acid B (4) were obtained from the crude extract of D. robusta and identified by analysis of spectroscopic data. Pyrodysinoic acid B (4) is the first furodysin or furodysinin sesquiterpene derivative with a trans junction between the two six-membered rings of the 1,2,3,4,4a,7,8,8a-octahydro-1,1,6-trimethylnaphthalene moiety.

Determination of amino acids by capillary electrophoresis-electrospray ionization-mass spectrometry: An evaluation of different protein hydrolysis procedures

In this work, a CE equipment, online hyphenated to an IT MS analyzer by a linear sheath liquid interface promoting ESI, was used to develop a method for quantitative determination of amino acids. Under appropriate conditions (BGE composition, 0.8% HCOOH, 20% CH(3)OH; sheath liquid composition, 0.8% HCOOH, 60% methanol; V(ESI), +4.50 W), analytical curves of all amino acids from 3 to 80 mg/L were recorded presenting acceptable linearity (r > 0.99). LODs in the range of 16-172 mu mol/L were obtained. BSA, a model protein, was submitted to different hydrolysis procedures (classical acid and basic, and catalyzed by the H(+) form of a cation exchanger resin) and its amino acid profiles determined. In general, the resin-mediated hydrolysis yields were overall similar or better than those obtained by classical acid or basic hydrolysis. The resulting experimental-to-theoretical BSA concentration ratios served as correction factors for the quantitation of amino acids in Brazil nut resin generated hydrolysates.

A novel A2 allele found in Leishmania (Leishmania) infantum chagasi

Visceral leishmaniasis (VL) is a widely spread zoonotic disease. In Brazil the disease is caused by Leishmania (Leishmania) infantum chagasi. Peridomestic sandflies acquire the etiological agent by feeding on blood of infected reservoir animals, such as dogs or wildlife. The disease is endemic in Brazil and epidemic foci have been reported in densely populated cities all over the country. Many clinical features of Leishmania infection are related to the host-parasite relationship, and many candidate virulence factors in parasites that cause VL have been studied such as A2 genes. The A2 gene was first isolated in 1994 and then in 2005 three new alleles were described in Leishmania (Leishmania) infantum. In the present study we amplified by polymerase chain reaction (PCR) and sequenced the A2 gene from the genome of a clonal population of L. (L.) infantum chagasi VL parasites. The L. (L.) infantum chagasi A2 gene was amplified, cloned, and sequenced in. The amplified fragment showed approximately 90% similarity with another A2 allele amplified in Leishmania (Leishmania) donovani and in L.(L.) infantum described in literature. However, nucleotide translation shows differences in protein amino acid sequence, which may be essential to determine the variability of A2 genes in the species of the L. (L.) donovani complex and represents an additional tool to help understanding the role this gene family may have in establishing virulence and immunity in visceral leishmaniasis. This knowledge is important for the development of more accurate diagnostic tests and effective tools for disease control.

Composição química, valores de energia metabolizável e aminoácidos digestíveis de subprodutos do arroz para frangos de corte

Dois experimentos foram conduzidos para determinar a composição química, os valores de energia e os coeficientes de digestibilidade dos aminoácidos, do farelo de arroz integral (FAI) e da quirera de arroz (QA). No primeiro estudo, foram utilizadas 144 aves, com 21 dias de idade, machos, linhagem Cobb, que tiveram suas excretas totalmente coletadas para determinação da energia metabolizável aparente (EMA) e energia metabolizável aparente corrigida (EMAn). O delineamento experimental foi inteiramente casualizado, com três tratamentos e seis repetições, com oito aves cada. No segundo experimento, foi utilizado o método de alimentação forçada para a determinação dos coeficientes de digestibilidade dos aminoácidos. O delineamento foi inteiramente casualizado, com dois alimentos e um jejum e seis repetições com um galo cada. Os valores de MS, PB, EE, FB, EMA e EMAn foram, respectivamente, para FAI: 88,6%; 11,8%; 15,3%; 10,2%; 2968kcal kg-1 e 2804kcal kg-1 e para QA: 93,5%; 9,1%; 0,73%; 0,45%; 3338kcal kg-1 e 3239kcal kg-1. Os valores médios encontrados dos coeficientes de digestibilidade de aminoácidos essenciais e não essenciais foram, respectivamente, de 75,9% e 73,9%, para FAI, e 77,9% e 76,5%, para QA. Embora tenham apresentado níveis inferiores de energia, FAI e a QA podem ser utilizados nas rações de aves em substituição ao milho, uma vez que tiveram níveis maiores de proteína bruta e aminoácidos digestíveis.

Biodisponibilidade de fontes orgânicas e inorgânicas de zinco em ovinos

Compararam-se os efeitos de diferentes fontes e doses de zinco na dieta de ovinos Santa Inês sobre os níveis de zinco plasmático e de fosfatase alcalina. Foram utilizados 40 cordeiros, recém-desmamados, com média de peso de 18,4kg, distribuídos em 10 tratamentos: 1- dieta basal sem suplementação de zinco; 2- dieta basal + 200mg de Zn/kg de MS na forma de óxido de zinco; 3- dieta basal + 400mg de Zn/kg de MS na forma de óxido de zinco; 4- dieta basal + 600mg de Zn/kg de MS na forma de óxido de zinco; 5- dieta basal + 200mg de Zn/kg de MS na forma de zinco aminoácido; 6- dieta basal + 400mg de Zn/kg de MS na forma de zinco aminoácido; 7- dieta basal + 600mg de Zn/kg de MS na forma de zinco aminoácido; 8- dieta basal + 200mg de Zn/kg de MS na forma de zinco proteinato; 9- dieta basal + 400mg de Zn/kg de MS na forma de zinco proteinato; 10- dieta basal + 600mg de Zn/kg de MS na forma de zinco proteinato. A cada 28 dias, os animais foram pesados e tiveram seu sangue colhido para análise de zinco (Zn), análise de fosfatase alcalina e análises de imunoglobulinas G (IgG) e M (IgM). No final do experimento, foram coletadas amostras de fígado para estudo dos níveis de zinco hepático. Não houve diferença entre tratamentos nos níveis de fosfatase alcalina e de zinco hepático, e no ganho de peso (P>0,05), mas houve diferença (P<0,05) nos níveis de Zn plasmático e nos níveis de IgG e IgM. Baseando-se no acúmulo no fígado, a estimativa da biodisponibilidade de zinco, por intermédio das equações de regressão, mostrou que as fontes orgânicas e inorgânicas de zinco não diferiram entre si.

Dipeptide synthesis in biphasic medium: evaluating the use of commercial porcine pancreatic lipase preparations and the involvement of contaminant proteases

Dipeptide syntheses starting from Ac-L-Tyr-OEt or Z-L-X-OMe (X: Asp, Tyr, Phe, Arg, Lys or Thr) and glycine amide in biphasic reaction media were achieved using two commercially available porcine pancreatic lipase (PPL) preparations (crude (cPPL) and purified PPL (pPPL)). Under the mild conditions employed, α-chymotrypsin, a pancreatic protease that also presents esterase activity, catalyzed Ac-L-Tyr-Gly-NH2 synthesis with high productivity. Product hydrolysis also occurred in most of the syntheses studied. Polyacrylamide gel electrophoresis, enzymatic assays employing specific chromogenic substrates and size-exclusion chromatography revealed that cPPL and pPPL contain contaminant proteases and, therefore, exhibit esterase and amidase activities. Overall, these data indicate that those contaminants may be the main catalysts of peptide bond synthesis when Nα-blocked-L-amino acid esters and the commercial PPL preparations are used. On the other hand, such data do not contest the possibility of using such enzyme preparations as an inexpensive source of catalysts for dipeptide synthesis under soft conditions.

Characterization of the interdependency between residues that bind the substrate in a β-glycosidase

The manner by which effects of simultaneous mutations combine to change enzymatic activity is not easily predictable because these effects are not always additive in a linear manner. Hence, the characterization of the effects of simultaneous mutations of amino acid residues that bind the substrate can make a significant contribution to the understanding of the substrate specificity of enzymes. In the β-glycosidase from Spodoptera frugiperda (Sfβgly), both residues Q39 and E451 interact with the substrate and this is essential for defining substrate specificity. Double mutants of Sfβgly (A451E39, S451E39 and S451N39) were prepared by site-directed mutagenesis, expressed in bacteria and purified using affinity chromatography. These enzymes were characterized using p-nitrophenyl β-galactoside and p-nitrophenyl β-fucoside as substrates. The k cat/Km ratio for single and double mutants of Sfβgly containing site-directed mutations at positions Q39 and E451 was used to demonstrate that the effect on the free energy of ES‡ (enzyme-transition state complex) of the double mutations (∆∆G‡xy) is not the sum of the effects resulting from the single mutations (∆∆G‡x and ∆∆G‡y). This difference in ∆∆G‡ indicates that the effects of the single mutations partially overlap. Hence, this common effect counts only once in ∆∆G‡xy. Crystallographic data on β-glycosidases reveal the presence of a bidentate hydrogen bond involving residues Q39 and E451 and the same hydroxyl group of the substrate. Therefore, both thermodynamic and crystallographic data suggest that residues Q39 and E451 exert a mutual influence on their respective interactions with the substrate.

Aspectos atuais sobre aminoácidos de cadeia ramificada e exercício físico

Em humanos saudáveis, nove aminoácidos são considerados essenciais, uma vez que não podem ser sintetizados endogenamente e, portanto, devem ser ingeridos por meio da dieta. Dentre os aminoácidos essenciais, se incluem os três aminoácidos de cadeia ramificada, ou seja, leucina, valina e isoleucina. Esses aminoácidos participam da regulação do balanço protéico corporal além de serem fonte de nitrogênio para a síntese de alanina e glutamina. No tocante à regulação da síntese protéica muscular, verifica-se que a leucina age estimulando a fase de iniciação da tradução do RNA-mensageiro em proteína, por mecanismos tanto dependentes quanto independentes de insulina. No que concerne ao exercício físico, supõe-se que esses aminoácidos estejam envolvidos na fadiga central, no balanço protéico muscular, na secreção de insulina, na modulação da imunocompetência, no aumento da performance de indivíduos que se exercitam em ambientes quentes e na diminuição do grau de lesão muscular. Nesse contexto, essa revisão aborda os aspectos atuais do metabolismo e da suplementação de aminoácidos de cadeia ramificada no exercício físico.

Suplementação enteral e parenteral com glutamina em neonatos pré-termo e com baixo peso ao nascer

A glutamina é o aminoácido livre mais abundante no sangue e no músculo esquelético, bem como é o principal substrato energético para células de elevado turnover, como enterócitos e leucócitos. Adicionalmente, a glutamina representa o principal aminoácido transferido para o feto pela placenta e, juntamente com o glutamato, constituem os aminoácidos mais abundantes no leite materno. Todavia, bebês nascidos prematuramente sofrem interrupção abrupta do fornecimento placentário de glutamina, o que acarreta em dependência exclusiva da síntese endógena ou do fornecimento exógeno deste aminoácido. Aliado a isso, neonatos pré-termo (PT) e com baixo peso ao nascer (BPN), freqüentemente, recebem apenas nutrição parenteral total nas primeiras semanas de vida, a qual não contém glutamina. Cabe ainda destacar que esses bebês possuem pouca massa muscular e, portanto, seus estoques de glutamina são limitados. Uma vez que neonatos PT e com BPN estão sujeitos a intenso crescimento e a inúmeros estresses fisiológicos, é possível que a glutamina seja um nutriente condicionalmente essencial nessa fase da vida, fato que estimulou a realização de estudos com a finalidade de avaliar os possíveis benefícios clínicos da suplementação enteral e parenteral com glutamina em neonatos PT e com BPN.