Molecular cloning and characterization of three phenylalanine ammonia-lyase genes from Schisandra chinensis / 中国天然药物

Phenylalanine ammonia-lyase (PAL), which catalyzes the conversion from L-phenylalanine to trans-cinnamic acid, is a well-known key enzyme and a connecting step between primary and secondary metabolisms in the phenylpropanoid biosynthetic pathway of plants and microbes. Schisandra chinensis, a woody vine plant belonging to the family of Magnoliaceae, is a rich source of dibenzocyclooctadiene lignans exhibiting potent activity. However, the functional role of PAL in the biosynthesis of lignan is relatively limited, compared with those in lignin and flavonoids biosynthesis. Therefore, it is essential to clone and characterize the PAL genes from this valuable medicinal plant. In this study, molecular cloning and characterization of three PAL genes (ScPAL1-3) from S. chinensis was carried out. ScPALs were cloned using RACE PCR. The sequence analysis of the three ScPALs was carried out to give basic characteristics followed by docking analysis. In order to determine their catalytic activity, recombinant protein was obtained by heterologous expression in pCold-TF vector in Escherichia coli (BL21-DE3), followed by Ni-affinity purification. The catalytic product of the purified recombinant proteins was verified using RP-HPLC through comparing with standard compounds. The optimal temperature, pH value and effects of different metal ions were determined. Vmax, Kcat and Km values were determined under the optimal conditions. The expression of three ScPALs in different tissues was also determined. Our work provided essential information for the function of ScPALs.

Effect of glycemic index on obesity control

Objective Evaluate the effect of glycemic index (GI) on biochemical parameters, food intake, energy metabolism, anthropometric measures and body composition in overweight subjects.Materials and methods Simple blind study, in which nineteen subjects were randomly assigned to consume in the laboratory two daily low GI (n = 10) or high GI (n = 9) meals, for forty-five consecutive days. Habitual food intake was assessed at baseline. Food intake, anthropometric measures and body composition were assessed at each 15 days. Energy metabolism and biochemical parameters were evaluated at baseline and the end of the study.Results Low GI meals increased fat oxidation, and reduced waist circumference and HOMA-IR, while high GI meals increased daily dietary fiber and energy intake compared to baseline. There was a higher reduction on waist circumference and body fat, and a higher increase on postprandial fat oxidation in response to the LGI meals than after high GI meals. High GI meals increased fasting respiratory coefficient compared to baseline and low GI meals.Conclusion The results of the present study showed that the consumption of two daily low GI meals for forty-five consecutive days has a positive effect on obesity control, whereas, the consumption of high GI meals result has the opposite effect. Arch Endocrinol Metab. 2015;59(3):245-51.

¿Qué debe saber el pediatra de las hiperfenilalaninemias? / What should the paediatrician know about hyperphenylalaninaemia?

Las hiperfenilalaninemias se definen por un nivel sanguíneo de fenilalanina sobre 2 mg/dl. La principal causa es una mutación en el gen que codifica la fenilalanina hidroxilasa que cataliza la reacción que transforma la fenilalanina en tirosina. Las hiperfenilalaninemias se clasifican en benignas o leves, y las fenilcetonurias en leves, moderadas y clásicas. Debido a que su detección más allá del periodo neonatal causa retardo mental severo, desde 1992 en Chile su detección, junto con la del hipotirodismo congénito, es parte del Programa Nacional de Pesquisa Neonatal. Este artículo pretende responder las preguntas más comunes que se puede hacer el pediatra cuando enfrenta a un paciente con hiperfenilalaninemias.

Hyperphenylalaninaemias are defined by a blood phenylalanine over 2 mg/dl. The main cause is due to a mutation in the gene that codes the phenylalanine hydroxylase that catalyses the reaction that converts phenylalanine into tyrosine. The hyperphenylalaninaemias are classified into benign or mild hyperphenylalaninaemias, or mild, moderate or classic phenylketonurias. Due to its delayed detection outside the neonatal period it causes severe mental retardation. Its detection along with congenital hypothyroidism has been part of the National Neonatal Screening Program since 1992 in Chile. This article aims to answer the most common questions asked by the paediatrician when faced with a patient with hyperphenylalaninaemias.

Evaluación del estado de salud en pacientes con fenilcetonuria / Assessment of the health status of patients with phenylketonuria

Introducción: la fenilcetonuria es el más conocido de todos los errores congénitos del metabolismo. Su detección precoz y una dieta baja en fenilalanina pueden prevenir el retraso mental en los pacientes. Objetivo: evaluar el impacto del programa de prevención precoz de fenilcetonuria en la provincia de Holguín, en el periodo de enero de 1986 a octubre de 2012. Métodos: se realizó un estudio descriptivo retrospectivo en los niños atendidos en la consulta de fenilcetonuria del Hospital Pediátrico de Holguín. El universo estuvo constituido por los 12 pacientes con el diagnóstico de fenilcetonuria. La información se obtuvo a través de las historias clínicas. Se evaluó el control metabólico mediante los valores de fenilalanina sérica registrados en las historias clínicas, el estado nutricional se valoró mediante las tasas de percentiles establecidas en Cuba y el coeficiente intelectual se analizó mediante test psicométricos. Resultados: el 66,6 por ciento(8 de los pacientes) presentó un control metabólico bueno, y 7 para un 58,3 por ciento un intelecto normal. De los pacientes diagnosticados antes de 1986, 3 presentan un retardo mental ligero (42,9 por ciento) y el 28,6 por ciento está incapacitado (2). El 100 por ciento de los diagnosticados por programa presentan un intelecto normal y 5 no presentan síntomas. Estudian 4 para un 80 por ciento. El 83,3 por ciento de los pacientes estudiados son eutróficos (10). Conclusiones: el programa de diagnóstico precoz ha permitido que el estado de salud de los pacientes fenilcetonúricos mejore en cuantía significativa, con una estabilidad en el control metabólico y una integración apreciable a la sociedad

Introduction: phenylketonuria is the most recognized congenital metabolic error. Its early detection and low phwnylalanine diet can prevent mental retardation in these patients. Objective: to evaluate the impact of the early phenylketonuria prevention program in Holguin province from January 1986 to October 2012. Methods: a retrospective descriptive study was made in children who were assisted at the phenylketonuria department of pediatric hospital in Holguin province. The universe of study was 12 patients diagnosed with phenylketonuria. The medical histories provided the necessary information. The metabolic control was evaluated through the serum phanylalanin figures recorded in the medical history, the nutritional status was assessed according to the percentile rates set in Cuba and the intellect coefficient was analyzed with psychometric tests. Results: in this group, 66.6 percent (8 patients) presented good metabolic control and 58.3 percent (7) showed normal intellect. Of the patients diagnosed with the disease before 1986, three (42.9 percent ) suffered mild mental retardation and two (28.6 percent) were disabled. All the patients diagnosed by this program showed normal intellect and 5 had no symptoms. Four were studying for 80 percent. Of the studied patients, 83.3 percent (10) were eutrophic. Conclusions: the early diagnosis program has allowed the significant improvement of the health status of the phenylketonuric patients, with stable metabolic control and noticeable integration to the society

Tissue-specific activation of mitogen-activated protein kinases for expression of transthyretin by phenylalanine and its metabolite, phenylpyruvic acid

Phenylketonuria is an autosomal recessive disorder caused by a deficiency of phenylalanine hydroxylase. Transthyretin has been implicated as an indicator of nutritional status in phenylketonuria patients. In this study, we report that phenylalanine and its metabolite, phenylpyruvic acid, affect MAPK, changing transthyretin expression in a cell- and tissue-specific manner. Treatment of HepG2 cells with phenylalanine or phenylpyruvic acid decreased transcription of the TTR gene and decreased the transcriptional activity of the TTR promoter site, which was partly mediated through HNF4alpha. Decreased levels of p38 MAPK were detected in the liver of phenylketonuria-affected mice compared with wild-type mice. In contrast, treatment with phenylalanine increased transthyretin expression and induced ERK1/2 activation in PC-12 cells; ERK1/2 activation was also elevated in the brainstem of phenylketonuria-affected mice. These findings may explain between-tissue differences in gene expression, including Ttr gene expression, in the phenylketonuria mouse model.

The homotrimeric structure of HtrA2 is indispensable for executing its serine protease activity

Serine protease activity of high temperature requrement 2 (HtrA2) is essential for promoting cell death, as well as for protecting against cellular stresses. An X-ray crystallographic study described the formation of a pyramid shaped homotrimer that is a proteolytically competent form of HtrA2; however, little is known about effects of the trimeric structure of HtrA2 on the natural substrates. In this study, we generated the HtrA2 protein that has a single point mutation at the homotrimerization motif to assess relationship between structure and the proteolytic activity of HtrA2 on its substrates. Using gel filtration, a native gel electrophoresis system, and a co-precipitation assay, we confirm that phenylalanine 149 in HtrA2 is a crucial determinant for the formation of the HtrA2 homotrimeric structure. Moreover, we described that the HtrA2 monomeric form abolished not only autoproteolytic activity, but also the proteolytic activity against XIAP (X-linked inhibitor of apoptosis protein) known as the HtrA2 substrate. Taken together, the results indicate that the homotrimeric structure of HtrA2 is required for executing its serine protease activity.

Estudo comparativo entre os efeitos da nateglinida e da rosiglitazona sobre os marcadores inflamatórios e funçào de células beta em portadores de diabetes tipo 2 / Comparative study on effects of the nateglinida and the rosiglitazone

INTRODUÇÃO tratamento intensivo dos diabéticos influiu pouco na progressão das complicações macrovasculares. Nateglinida e rosiglitazona podem modificar fatores relacionados. OBJETIVOS avaliar o perfil metabólico de DM tipo 2 em uso de rosiglitazona ou nateglinida. MÉTODOS 22 diabéticos, randomizados em 2 grupos para uso de nateglinida ou rosiglitazona por 4 meses seguidos de 8 semanas sem tratamento e cruzamento dos grupos. Foram avaliados com teste com dieta de 5 h glicose, insulina, pró-insulina, glucagon, GLP-1, triglicérides, AGL, HbA1c, colesterol total e frações, fibrinogênio, PCR, IL-6, TNF-beta e sICAM. Avaliamos 11 controles normais. CONCLUSÃO rosiglitazona foi mais efetiva que nateglinida no controle glicêmico, diminuiu a resistência insulínica, AGL, PCR e fibrinogênio / INTRODUCTION intensive treatment of diabetics improves little in macrovascular complications. Nateglinide and rosiglitazone could modify factors related to macrovascular alterations. OBJECTIVES to evaluate the metabolic profile of diabetics treated with rosiglitazone or nateglinide. METHODS 22 diabetics was randomized into 2 groups for treat with rosiglitazone or nateglinide for 4 months, washout of 8 weeks and cross over the groups. The patients were evaluated through oral test with 5 hours to dose glucose, insulin, proinsulin, glucagon, GLP-1, triglycerides and FFA and fasting levels of HbA1c, fractions and total cholesterol, fibrinogen, CRP, sICAM, IL6 and TNF-. 11 health people were availed. CONCLUSION: Rosiglitazona was more effective than nateglinida in total glycaemic, reducing both fasting and postprandial glucose levels, insulin resistance and FFA levels...

Phenylalanine transport in Aspergillus nidulans: demonstration of role of phenylalanine binding proteins.

Crude shock proteins extracted by two stage osmotic shock were further purified by affinity chromatography to obtain ligand (phenylalanine) specific binding protein (phebip) a component of phenylalanine (phe) transport system from wild type and a phe transport mutant fpaD11 of Aspergillus nidulans. A new eluent 0.1 M Tris-HCl containing 1.5 N NaCl and 0.5 N Na2CO3, pH 8 was used during the investigation. The elution profile of mutant phebip exhibited one simple and two compound peaks instead of three simple ones as exhibited by the wild type phebip. SDS-PAGE profile of mutant phebip showed faster electrophoretic mobility than that of wild type one. It is therefore evident that the mutant phebip has reduced molecular mass (M(r)) due to deletion of a segment that somehow has bearing on the binding capacity of the active site of phebip. The resultant erosion in the binding capacity of the mutant phebip is in turn responsible for its incapability to stimulate transport of ligand across the plasma membrane.