Metabolismo de triptofano em melanomas: o que dizem as células do microambiente? / Tryptophan metabolism in melanomas: what do microenvironment cells can say?

O melanoma é composto por células malignas e também por um estroma de sustentação que inclui fibroblastos, células imunológicas, endoteliais, matriz extracelular, dentre outros fatores. Assim, os tumores não são entidades independentes, eles interagem ativamente com o microambiente adjacente de forma bidirecional através de sinais moleculares que modulam o fenótipo maligno. Um dos sinais bioquímicos para desenvolvimento desse fenótipo se dá pelo catabolismo de Trp pela via das quinureninas, que gera compostos com diversas atividades biológicas, que no tumor estão envolvidas com tolerância e imunoescape e, logo, com prognóstico ruim para os pacientes. Até o presente momento apenas o consumo de Trp e a formação de um único metabólito, a quinurenina (KYN), tem sido associada a malignidade dos melanomas. A fim de ampliar e elucidar os mecanismos bioquímicos do metabolismo desse aminoácido em melanomas, estudamos mais de quinze compostos de todas as rotas catabólicas de Trp em células da pele, células imunológicas, linhagens tumorais e amostras clínicas de melanoma. De forma inédita pudemos observar que as células da pele tem maior habilidade de sintetizar KYN quando comparadas às linhagens tumorais, demonstrando que o catabolismo de Trp peritumoral pode ser responsável pelos fenômenos de imunotolerância e escape. Além disso, o metabolismo de Trp pode estar envolvido nos mecanismos de homeostasia da pele, já que especificamente essas células produzem compostos com atividade biológica nesse órgão. As células imunológicas possuem um perfil metabólico completamente diferente umas das outras: monócitos, macrófagos e dendríticas possuem maior ativação da via KYN enquanto linfócitos e neutrófilos possuem maior indução da rota que gera serotonina e melatonina. Mesmo nos diferentes fenótipos de macrófagos, M1 e M2a, foram observadas marcações especificas de metabolismo, que podem estar relacionadas às atividades anti- ou pró-tumoral dessas células no microambiente. Em amostras clínicas, apesar da principal diferença entre nevos e melanomas ser a concentração de KYN, diversas outras alterações no metabolismo de tiptofano foram observadas, o que mostra a complexa magnitude deste metabolismo na fisiopatologia da pele

Melanoma is composed of malignant cells and also by a stromal support that includes fibroblasts, immune cells, endothelial cells, extracellular matrix, among other factors. Thus, tumors are not separate entities; they actively interact with the surrounding microenvironment bi-directionally through molecular signals that modulate the malignant phenotype. One of biochemical signals for the development of this phenotype occurs by Trp catabolism through kynurenine pathway, that generates compounds with diverse biological activities, which in tumors are involved with tolerance and imunoescape and therefore with poor prognosis for patients. To date only the consumption of Trp and formation of a single metabolite, kynurenine (KYN), has been associated with malignant melanomas. In order to enlarge and clarify the biochemical mechanisms of this amino acid metabolism in melanomas, we have studied more than fifteen compounds of all catabolic routes of Trp in skin cells, immune cells, tumor cell lines and clinical samples of melanoma. In an unique way we could observe that the skin cells has superior ability to synthesize KYN when compared to tumor cell lines, demonstrating that the peritumoral catabolism of Trp may be responsible for the phenomena of immune tolerance and escape. Furthermore, the Trp metabolism may be involved in skin homeostasis mechanisms, since these cells produce specific compounds with biological activity in this organ. The immune cells have a completely different metabolic profile among them: monocytes, macrophages and dendritic cells have greater KYN pathway activation, and lymphocytes and neutrophils possess greater induction of the route that generates serotonin and melatonin. Even in different macrophages phenotypes, M1 and M2a, we observed specific metabolic marks, which may be related to the anti- or pro-tumoral activity of these cells in the tumor microenvironment. In clinical samples, although the main difference between nevi and melanomas is the concentration of KYN, a range of other changes in Trp metabolism were observed, which shows the complex magnitude of this metabolism in the skin pathophysiology

Síntesis de serotonina y presencia de hidroxilasa de triptófano en linfocitos de pacientes con depresión mayor tratados con fluoxetina y ácido fólico / Synthesis of serotonin and tryptophan hydroxylase in lymphocytes from patients with major depression treated with fluoxetine and folic acid

El ácido fólico ha sido utilizado como coadyuvante antidepresivo, y se han reportado bajos niveles séricos en deprimidos. Debido al papel del ácido fólico y a la relevancia del sistema serotonérgico linfocitario en la depresión, el objetivo de este estudio es determinar la capacidad de producción de serotonina y la presencia de la hidroxilasa del triptófano en linfocitos de pacientes deprimidos tratados con fluoxetina y ácido fólico. Los pacientes fueron diagnosticados con los criterios del Manual Diagnóstico y Estadístico de la Asociación Psiquiátrica Americana, la intensidad del episodio depresivo se determinó mediante la Escala de Hamilton para Depresión. Veintisiete pacientes (21-58 años) fueron seleccionados, no presentaban otra patología ni riesgo suicida. Se distribuyeron en forma aleatoria en dos grupos experimentales, unos (14) recibieron fluoxetina, 20 mg/d, más ácido fólico, 10 mg/d y otros (13) fluoxetina más placebo. El grupo control fue constituido por 15 sujetos aparentemente sanos (26-49 años). Se tomaron muestras de sangre al principio y después de seis semanas. Diez pacientes de cada grupo experimental culminaron el estudio. La homocisteína plasmática disminuyó con la administración de ácido fólico. Los linfocitos fueron aislados por gradientes de densidades con Ficoll/Hypaque y adhesión diferencial al plástico. Las concentraciones de serotonina en linfocitos se determinaron por cromatografía líquida de alta resolución con detector electroquímico y no difirieron entre los dos grupos ni en relación al control, pero fueron bajas en los que recibieron el tratamiento. La síntesis de serotonina a partir de triptófano fue menor en los pacientes en relación a controles, y disminuyó después de los dos tratamientos. El número de linfocitos con la enzima fue menor en los pacientes y decreció después del ácido fólico.

Current concepts of the relationship between cholesterol fed rats and endogenous amino acids

The effect of long term feeding of cholesterol 100 mg/kg/day for 20 weeks on free amino acid patterns in plasma and liver contents in rats was investigated using LKB-amino acid analyzer [Biochrom Ltd., Cambridge, England] and correlated these results with their effects on plasma levels of cholesterol, triglyceride, lipoprotein and Gamma Glutamyl Transferase [yGT]. indicate that all amino acid contents in the liver were significantly decreased in the cholesterol feed animal group. The amplitude of reductions varied between 40-100%. Glutamine and tryptophan were not detected in the liver of cholesterol feed group. The plasma concentrations of taurine, glutamate, alanine, valine and phenylalanine were elevated whose mean percentage increases were 48 +/- 4, 40 +/- 3, 25 +/- 3 and 35 +/- 4 respectively. These increases were associated with significant decrease in the concentration of ornithine [37%]. Meanwhile proline was not detected in the plasma of treated animals. Also, plasma cholesterol, triglyceride, lipoproteins and yGT were determined by colorimetric methods using Kits from Boehringer Mannheim [GmbH]. Results indicated that feeding cholesterol significantly increased the plasma yGT activity. In these experimental conditions the chronic intake of cholesterol had no significant effects on plasma cholesterol or other plasma lipids parameters tested except plasma triglyceride which was significantly increased, these results indicated that there are interactions between cholesterol intake and hepatic glutamine and tryptophan as well as plasma proline and these interaction mechanisms may be considered the factor generating metabolic events, which play physiological functions in the regulation of plasma cholesterol. Thus, under pathological conditions there is an imbalance between these interaction mechanisms which cause an increase in the circulating levels of cholesterol, leading to pathological processes such as hyperlipidemias, atherosclerosis and bile stones

Avaliaçäo da composiçäo em aminoácidos de Pleurotus spp. cultivados em folha de bananeira / Amino acid composition evaluation of Pleurotus spp. cultivated in banana leaves

A qualidade da proteína de cogumelos, além de ser específica para a espécie/linhagem, pode variar também com o substrato de cultivo. Esse estudo teve por objetivo determinar a composiçao em aminoácidos da proteína dos cogumelos comestíveis Pleurotus sp. "Florida" (L1), P. ostreatoroseus (L2) e P. sajor-caju (L3), cultivados em folha de bananeira (PB) unicamente e, esta misturada ao bagaço de cana (PBBC). Foram avaliados os aminoácidos totais, cistina e triptofano; calculou-se o escore químico e o PDCAAS- "protein digestibility-correct amino acid scoring". As espécies estudadas apresentaram todos os aminoácidos essenciais, de ambos substratos; os aminoácidos presentes em maiores quantidades foram, em ordem decrescente, ácido glutâmico, ácido aspártico, leucina e lisina. O escore químico da proteína de L1 foi de 90,4, com limitaçao em sulfurados e aromáticos no substrato PB e, no PBBC, o escore químico foi de 88,7 com limitaçao em aromáticos. O escore químico de L2 e L3 foi igual a 100, independente do substrato de cultivo. O PDCAAS calculado, considerando-se 90 por cento de digestibilidade recomendado, variou entre 80,0 a 96 por cento. As proteínas de L1 apresentaram-se limitantes em sulfurados e aromáticos e com menor valor de PDCAAS (approximatelly 80,0) nos dois substratos empregados; a proteína de L3 apresentou limitaçao em aromáticos, sulfurados e triptofano dependendo do substrato de cultivo; as proteínas de L2 apresentaram o maior valor de PDCAAS (96 por cento) e limitaçao em aromáticos e/ou sulfurados, dependendo do substrato de cultivo. Considerando-se as condiçoes desse estudo, a proteína das espécies estudadas é incompleta porém de alto valor biológico, comparáveis às da carne.

Thermal stabilization of multimeric proteins: a case study with alpha-globulin.

Preferential interaction parameters of multisubunit protein, alpha-globulin and monomeric protein human serum albumin (HSA) were determined in different cosolvents using precision densitymetry. The apparent partial specific volumes were determined under both isomolal and isopotential conditions for alpha-globulin in 0.02 M glycine-NaOH buffer at pH 10 and the values were 0.692+/-0.002 and 0.688+/-0.001, ml/g, respectively, at 20.00+/-0.01 degrees C. From the partial specific volume data with cosolvents the preferential interaction parameter (xi3) and other thermodynamic parameters were calculated at different solvent concentrations. The (xi3) values increased with an increase in the solvent concentration up to 30% and reached a maximum with the values of-0.111+/-0.018 g/g and -0.076+/-0.012 g/g in sucrose and sorbitol, respectively. In glycerol the (xi3) values decreased with an increase in solvent concentration. The above data is further supported by thermal denaturation profiles in which the apparent thermal denaturation temperature (apparent Tm) of alpha-globulin shows an increase from 63 degrees C to higher temperatures in the order of sucrose, sorbitol and glycerol. Alpha-globulin showed coagulation due to protein interaction at temperatures above 50 degree C. The apparent Tm of 63 degrees C for control protein was increased significantly up to 75 degrees C in 40% sorbitol with two fold increase in the delta(S) values showing the increased structural stability of alpha-globulin. At high solvent concentration the protein gets dissociated and the resultant monomers are hydrated which was evident by fluorescence data and the difference spectral results with a 6nm red shift in the emission maximum and 2 nm blue shift in UV-absorption maximum arising out of perturbation of aromatic chromophores. The studies were performed both at native pH of 7.9 where the protein is in its oligomeric form and at pH of 10 where it is dissociated form and the results compared. The data showed that the solvent is excluded more from the protein vicinity in the dissociated state.

Separación de aminoácidos mediante cromatografía líquida de alta resolución / Amino acid analysis by high-performance liquid chromatography

Se presenta una compilación de la aplicación de la cromatografía líquida de alta resolución en el análisis de aminoácidos. Se analizan las formas de derivatización, se comparan los distintos derivados según sus alcances y limitaciones, la forma de detección y los distintos modos cromatográficos: cromatografía de intercambio iónico, cromatografía en fase normal, cromatografía en fase reversa y cromatografía en fase quiral. También se presentan los métodos automatizados comerciales más recientes para este tipo de análisis. Se dan ejemplos de aplicación de interés en el campo clínico-bioquímico, con el análisis de muestras de líquido cafalorraquídeo, de orina y de plasma/sangre o manchas de sangre seca. El análisis de los aminoácidos de hidrolizados de proteínas y de péptidos resulta más complejo y las condiciones de hidrólisis juegan un rol muy importante en los resultados, que son tratados en esta compilación. Se incluyen, además, aspectos referidos al análisis de los aminoácidos lábiles en proteínas, la preparación de las muestras, las recomendaciones prácticas al hacer HPLC y la influencia de los buffers

Harina de frijol endurecido (Phaseolus vulgaris L.) en la preparación de pan / Flour of hard bean (Phaseolus vulgaris) in the bread preparation

Con el propósito de utilizar el frijol endurecido, se propuso la búsqueda del método indicado para obtener harinas de frijol, determinar su valor nutritivo y elaborar panes con mezclas de harina de frijol y trigo de buena calidad industrial, nutricional, y sensorial. Se elaboraron dos tipos de harina, ensayándose cuatro temperaturas de remojo (22, 30, 40 y 50§C) y dos métodos de separación de testa (húmedo y seco). A nivel de laboratorio la separación de testa en seco proporcionó los mejores rendimientos harineros (x = 85.8%) y el más alto contenido proteínico (x = 23.7%). La comparación entre las temperaturas de remojo de 30 y 50§C respecto al rendimiento de harina no fueron significativas (* = 0.05). A nivel de planta piloto, con el remojo de 50§C los rendimientos harineros fueron de 58.0% para la separación de testa en húmedo (H1) y 74.0% para la separación en seco (H2), con porcentajes de proteína de 22.6% y 23.0% para H2. Para elaborar el pan de caja se adicionaron 5, 10 y 15% de harinas H1 y H2 a la de trigo, encontrando que la mezcla con 5.0% de H1 presentaba una buena calidad panadera, proteínica y sensorial, semejante al pan control, de ...

Essential tryptophan residues of ribulose 1,5-bisphosphate carboxylase.

Ribulose 1,5-bisphosphate carboxylase [3-phospho-D-glyceratecarboxy-lyase (dimerizing), EC 4.1.1.39] is rapidly and irreversibly inactivated by micromolar concentrations of dimethyl (2-hydroxy-5-nitrobenzyl) sulphonium bromide (DMHNB), a tryptophan selective reagent, after reversible protection of the reactive sulphydryl groups. The inactivation followed pseudo-first-order reaction kinetics. Replots of the kinetic data indicated that no reversible enzyme-inhibitor complex was formed prior to irreversible modification. Kinetic analysis and the correlation of the spectral data at 410 nm with enzyme activity indicated that inactivation by DMHNB resulted from modification of on an average one tryptophan per 67 kDa combination of large and small subunits. Several competitive inhibitors and substrate RuBP offered strong protection against inhibition. The k1/2 (protection) for RuBP was 1.3 mM, indicating that the tryptophan residues may be located at or near the substrate binding site. Free and total sulphydryl groups were not affected by the reagent. The modified enzyme exhibited significantly reduced intrinsic fluorescence, indicating that the microenvironment of the tryptophans at the active site is significantly perturbed. Tryptic peptide profiles and CD spectral analyses suggested that inactivation may not be due to the extensive conformational changes in the enzyme molecule during modification.