Assay of amino acids in leaves of Eucommia ulmoides under arbor forest mode and leaf-oriented cultivation mode by pre-column derivatization HPLC / 中国中药杂志

This study aims to develop the pre-column derivatization high performance liquid chromatography(HPLC) method for the determination of 16 kinds of amino acids in Eucommia ulmoides leaves, and compare the content of amino acids in the leaves harvested at different time and under leaf-oriented cultivation mode(LCM) and arbor forest mode(AFM). The HPLC conditions are as below: phenyl isothiocyanate(PITC) as pre-column derivatization agent, Agilent ZORBAX C_(18 )column(4.6 mm×250 mm, 5 μm), mobile phase A of acetonitrile-water(80∶20), mobile phase B of 0.1 mol·L~(-1) sodium acetate solution-acetonitrile(94∶6), gradient elution, flow rate of 1.0 mL·min~(-1), injection volume of 5 μL, column temperature of 40 ℃, and detection wavelength of 254 nm. The HPLC profile indicated well separation of 16 kinds of amino acids and the amino acid content in E. ulmoides leaves was up to 16.26%. In addition, the amino acid content in leaves of E. ulmoides under LCM was higher than under AFM. The amino acid content varied with the harvesting time. Through orthogonal partial least squares discriminant analysis, the amino acids of E. ulmoides under LCM and AFM were compared, which can distinguish the leaves under LCM from those under AFM. Principal component analysis was applied to comprehensively score the amino acids of E. ulmoides leaves. The results showed that the score of leaves under LCM was higher than that under AFM. Nutritional evaluation results indicated that the proteins in E. ulmoides leaves belonged to high-quality vegetable proteins. The established method for the determination of amino acid content is reliable. With the amino acid content as index, the leaf quality of E. ulmoides under LCM is better than that under AFM. This study lays a theoretical basis for the promotion of LCM for E. ulmoides and the development of medicinal and edible products from E. ulmoides leaves.

Modification of C20 oxidase in tanshinone biosynthesis pathway / 中国中药杂志

Tanshinones are one of the main effective components of Salvia miltiorrhiza, which play important roles in the treatment of cardiovascular diseases. Microbial heterogony production of tanshinones can provide a large number of raw materials for the production of traditional Chinese medicine(TCM) preparations containing S. miltiorrhiza, reduce the extraction cost, and relieve the pressure of clinical medication. The biosynthetic pathway of tanshinones contains multiple P450 enzymes, and the catalytic element with high efficiency is the basis of microbial production of tanshinones. In this study, the protein modification of CYP76AK1, a key P450-C20 hydroxylase in tanshinone pathway, was researched. The protein modeling methods SWISS-MODEL, Robetta, and AlphaFold2 were used, and the protein model was analyzed to obtain the reliable protein structure. The semi-rational design of mutant protein was carried out by molecular docking and homologous alignment. The key amino acid sites affecting the oxidation activity of CYP76AK1 were identified by molecular docking. The function of the obtained mutations was studied with yeast expression system, and the CYP76AK1 mutations with continuous oxidation function to 11-hydroxysugiol were obtained. Four key amino acid sites that affected the oxidation acti-vity were analyzed, and the reliability of three protein modeling methods was analyzed according to the mutation results. The effective protein modification sites of CYP76AK1 were reported for the first time in this study, which provides a catalytic element for different oxidation activities at C20 site for the study of the synthetic biology of tanshinones and lays a foundation for the analysis of the conti-nuous oxidation mechanism of P450-C20 modification.

Identification and verification of key salt-tolerant amino acid sites of banana MaNHX5 / 生物工程学报

In order to improve the salt tolerance of banana NHX genes, we cloned a MaNHX5 gene from Musa acuminata L. AAA group and predicted the key salt-tolerant amino acid sites and mutant protein structure changes of MaNHX5 by using bioinformatics tools. The 276-position serine (S) of MaNHX5 protein was successfully mutated to aspartic acid (D) by site-directed mutagenesis, and the AXT3 salt-sensitive mutant yeast was used for a functional complementation test. The results showed that after the mutated MaNHX5 gene was transferred to AXT3 salt-sensitive mutant yeast, the salt tolerance of the mutant yeast was significantly improved under 200 mmol/L NaCl treatment. It is hypothesized that Ser276 of MaNHX5 protein plays an important role in the transport of Na+ across the tonoplast.

Advances in stress tolerance mechanisms and synthetic biology for the industrial robustness of Corynebacterium glutamicum / 生物工程学报

As a model industrial host and microorganism with the generally regarded as safe (GRAS) status, Corynebacterium glutamicum not only produces amino acids on a large scale in the fermentation industry, but also has the potential to produce various new products. C. glutamicum usually encounters various stresses in the process of producing compounds, which severely affect cell viability and production performance. The development of synthetic biology provides new technical means for improving the robustness of C. glutamicum. In this review, we discuss the tolerance mechanisms of C. glutamicum to various stresses in the fermentation process. At the same time, we highlight new synthetic biology strategies for boosting C. glutamicum robustness, including discovering new stress-resistant elements, modifying transcription factors, and using adaptive evolution strategies to mine stress-resistant functional modules. Finally, prospects of improving the robustness of engineered C. glutamicum strains ware provided, with an emphasis on biosensor, screening and design of transcription factors, and utilizing the multiple regulatory elements.

Solid state fermentation to obtain vegetable products bio-enriched with isoflavone aglycones using lactic cultures / Fermentación en sustrato sólido utilizando cultivos lácticos para la obtención de productos vegetales bioenriquecidos con isoflavonas agliconas

The consumption of soybean isoflavones (IS) is associated with several beneficial properties on human health. Some lactic acid bacteria possess ß-glucosidase enzyme, that allows to obtain the active form of IS (aglycone). The solid state fermentation (SSF) has received great attention in the last years in order to obtain several valuable compounds. SSF, using soybean as substrate and Lactobacillus rhamnosus CRL 981 as starter, was studied in the present work. Sucrose was added into soybean paste to study the effect on the behavior of the selected strain. The development of L. rhamnosus CRL 981 through pH and recount measures, sugar intake, organic acid production, ß-glucosidase activity and IS conversion were analyzed. No significant differences in growth and acidity were observed between soybean pastes with and without sucrose added, but the production of lactic acid was higher in the latter paste. The ß-glucosidase activity was detected in both pastes and the complete hydrolysis of IS at 12 h of fermentation was observed. Also, this strain was able to increase the free amino acids in soybean paste. SSF, using soybean as substrate and L. rhamnosus CRL 981 as starter culture, is an alternative process to obtain a soybean product bio-enriched in active IS with attractive nutritional characteristics.

El consumo de isoflavonas de soja (IS) está asociado a diversos beneficios para la salud humana. Ciertas bacterias lácticas poseen la enzima ß-glucosidasa, que permite obtener la forma bioactiva (agliconas) de las IS. La fermentación en sustrato sólido (FSS) ha recibido gran atención en los últimos anos debido a sus numerosas ventajas, y permite la obtención de productos con valor agregado. En el presente trabajo se estudió la FSS utilizando soja como sustrato y Lactobacillus rhamnosus CRL981 como cultivo iniciador. Con el fin de estudiar el efecto de una fuente de carbono externa sobre el comportamiento de la cepa seleccionada, se adicionó sacarosa a la pasta de soja. Se evaluó el crecimiento de L. rhamnosus CRL 981 a través de medidas de pH y recuento en placa. Además, se analizó el consumo de azúcares, producción de ácidos orgánicos, actividad ß-glucosidasa y conversión de IS. No se observaron diferencias significativas en el crecimiento y acidez entre las pastas de soja sin adición de sacarosa y con ella, sin embargo, la producción de ácido láctico fue mayor en esta última. La actividad de ß-glucosidasa se detectó en ambas pastas y se observó la hidrólisis completa de IS a las 12 h de fermentación. Además, esta cepa fue capaz de aumentar los aminoácidos libres en la pasta de soja. La FSS, utilizando soja como sustrato y L. rhamnosus CRL 981 como cultivo iniciador, es un proceso alternativo para obtener un producto de soja bioenriquecido en IS bioactivas con características nutricionales atractivas.

Changes induced by co-inoculation in nitrogen-carbon metabolism in cowpea under salinity stress

ABSTRACT To mitigate the deleterious effects of abiotic stress, the use of plant growth-promoting bacteria along with diazotrophic bacteria has been increasing. The objectives of this study were to investigate the key enzymes related to nitrogen and carbon metabolism in the biological nitrogen fixation process and to elucidate the activities of these enzymes by the synergistic interaction between Bradyrhizobium and plant growth-promoting bacteria in the absence and presence of salt stress. Cowpea plants were cultivated under axenic conditions, inoculated with Bradyrhizobium and co-inoculated with Bradyrhizobium sp. and Actinomadura sp., Bradyrhizobium sp. and Bacillus sp., Bradyrhizobium sp. and Paenibacillus graminis, and Bradyrhizobium sp. and Streptomycessp.; the plants were also maintained in the absence (control) and presence of salt stress (50 mmolL-1 NaCl). Salinity reduced the amino acids, free ammonia, ureides, proteins and total nitrogen content in nodules and increased the levels of sucrose and soluble sugars. The co-inoculations responded differently to the activity of glutamine synthetase enzymes under salt stress, as well as glutamate synthase, glutamate dehydrogenase aminating, and acid invertase in the control and salt stress. Considering the development conditions of this experiment, co-inoculation with Bradyrhizobium sp. and Bacillus sp. in cowpea provided better symbiotic performance, mitigating the deleterious effects of salt stress.

Removing the by-products acetic acid and NH4 + from the L-tryptophan broth by vacuum thin film evaporation during L-tryptophan production

Background: During L-tryptophan production by Escherichia coli, the by-products, acetic acid and NH4 +, accumulate in the fermentation broth, resulting in inhibited cell growth and activity and decreased L-tryptophan production. To improve the L-tryptophan yield and glucose conversion rate, acetic acid and NH4 + were removed under low-temperature vacuum conditions by vacuum scraper concentrator evaporation; the fermentation broth after evaporation was pressed into another fermenter to continue fermentation. To increase the volatilisation rate of acetic acid and NH4 + and reduce damage to bacteria during evaporation, different vacuum evaporation conditions were studied. Results: The optimum operating conditions were as follows: vacuum degree, 720 mm Hg; concentration ratio, 10%; temperature, 60°C; and feeding rate, 300 mL/min. The biomass yield of the control fermentation (CF) and fermentation by vacuum evaporation (VEF) broths was 55.1 g/L and 58.3 g/L at 38 h, respectively, (an increase of 5.8%); the living biomass yield increased from 8.9 (CF) to 10.2 pF (VEF; an increase of 14.6%). L-tryptophan production increased from 50.2 g/L (CF) to 60.2 g/L (VEF) (an increase of 19.9%), and glucose conversion increased from 18.2% (CF) to 19.5% (VEF; an increase of 7.1%). The acetic acid concentrations were 2.74 g/L and 6.70 g/L, and the NH4 + concentrations were 85.3 mmol/L and 130.9 mmol/L in VEF and CF broths, respectively. Conclusions: The acetic acid and NH4 + in the fermentation broth were quickly removed using the vacuum scraper concentrator, which reduced bacterial inhibition, enhanced bacterial activity, and improved the production of L-tryptophan and glucose conversion rate.

Mannheimia haemolytica growth and leukotoxin production for vaccine manufacturing: a bioprocess review

Mannheimia haemolytica leukotoxin (LKT) is a known cause of bovine respiratory disease (BRD) which results in severe economic losses in the cattle industry (up to USD 1 billion per year in the USA). Vaccines based on LKT offer the most promising measure to contain BRD outbreaks and are already commercially available. However, insufficient LKT yields, predominantly reflecting a lack of knowledge about the LKT expression process, remain a significant engineering problem and further bioprocess optimization is required to increase process efficiency. Most previous investigations have focused on LKT activity and cell growth, but neither of these parameters defines reliable criteria for the improvement of LKT yields. In this article, we review the most important process conditions and operational parameters (temperature, pH, substrate concentration, dissolved oxygen level, medium composition and the presence of metabolites) from a bioprocess engineering perspective, in order to maximize LKT yields.

Effects of monosulfuron-ester on metabolic processes of nitrogen-fixing cyanobacteria Anabaena flos-aquae and Anabaena azotica

Abstract Presence of the relatively new sulfonylurea herbicide monosulfuron-ester at 0.03-300 nmol/L affected the growth of two non-target nitrogen-fixing cyanobacteria (Anabaena flos-aquae and Anabaena azotica) and substantially inhibited in vitro Acetolactate synthase activity, with IC50 of 3.3 and 101.3 nmol/L for A. flos-aquae and A. azotica, respectively. Presenting in 30-300 nmol/L, it inhibited protein synthesis of the cyanobacteria with less amino acids produced as its concentration increased. Our findings support the view that monosulfuron-ester toxicity in both nitrogen-fixing cyanobacteria is due to its interference with protein metabolism via inhibition of branch-chain amino acid biosynthesis, and particularly Acetolactate synthase activity.

Impact of dexmedetomidine on amino acid contents and the cerebral ultrastructure of rats with cerebral ischemia-reperfusion injury

Abstract Purpose: To investigate the effects of dexmedetomidine (DEX) on amino acid contents and the cerebral ultrastructure of rats with cerebral ischemia-reperfusion injury (I/R). Methods: Thirty-six, male, Wistar rats were randomly divided into three groups: the sham operation group (group C), the ischemia-reperfusion group (group I/R), and the DEX group (group D). The middle cerebral artery occlusion model was prepared by the modified Longa method. The time of ischemia was 180 min, and 120 min after reperfusion, the amount of glutamate (Glu), and γ-aminobutyric acid (GABA) in the brain were measured, and the ultrastructure-level changes in the cerebral cortex were examined using electron microscopy. Results: Compared to group C, Glu contents in group D, and I/R significantly increased. Compared to group I/R, Glu contents in group D significantly decreased. Compared to group C, GABA contents in group D, and I/R significantly increased, and those in group D significantly increased, as compared to group I/R. The cerebral ultrastructure was normal in group C. Vacuolar degeneration in the plastiosome and nervous processes, was more critical than in group D. Vascular endothelial cells (VEC) were damaged. On the contrary, these changes in group D significantly improved. Conclusion: Dexmedetomidine is capable of decreasing glutamergic content, and increasing GABAergic content, in order to decrease the injury of the cerebral ultrastructure, following cerebral ischemia-reperfusion injury.

Protein metabolism and physical training: any need for amino acid supplementation?

Muscle mass is the major deposit of protein molecules with dynamic turnover between net protein synthesis and degradation. In human subjects, invasive and non-invasive techniques have been applied to determine their skeletal muscle catabolism of amino acids at rest, during and after different forms of physical exercise and training. The aim of this review is to analyse the turnover flux and the relative oxidation rate of different types of muscle proteins after one bout of exercise as well as after resistance and endurance condition of training. Protein feeding in athletes appears to be a crucial nutrition necessity to promote the maintenance of muscle mass and its adaptation to the need imposed by the imposed technical requirements. In resting human individuals, there commended protein daily allowance is about 0.8 g (dry weight) kg−body weight per 24 h knowing that humans are unable to accumulate protein stores in muscle tissues. Nevertheless, practical feeding recommendations related to regular exercise practice are proposed to athletes by different bodies in order to foster their skills and performance. This review will examine the results obtained under endurance and resistance type of exercise while consuming single or repeated doses of various ingestions of protein products (full meat, essential amino acids, specific amino acids and derivatives, vegetarian food). From the scientific literature, it appears that healthy athletes(and heavy workers) should have a common diet of 1.25 g kg−24 h to compensate the exercise training muscle protein degradation and their resyn thesis within the following hours. A nitrogen-balance assay would berecommended to avoid any excessive intake of protein. Eventually, a daily equilibrated food intake would beof primer importance versus inadequate absorption of some specific by-products.

Influence of environmental hypertonicity on the induction of ureogenesis and amino acid metabolism in air-breathing walking catfish (Clarias batrachus, Bloch).

Effect of environmental hypertonicity, due to exposure to 300 mM mannitol solution for 7 days, on the induction of ureogenesis and also on amino acid metabolism was studied in the air-breathing walking catfish, C. batrachus, which is already known to have the capacity to face the problem of osmolarity stress in addition to other environmental stresses in its natural habitats. Exposure to hypertonic mannitol solution led to reduction of ammonia excretion rate by about 2-fold with a concomitant increase of urea-N excretion rate by about 2-fold. This was accompanied by significant increase in the levels of both ammonia and urea in different tissues and also in plasma. Further, the environmental hypertonicity also led to significant accumulation of different non-essential free amino acids (FAAs) and to some extent the essential FAAs, thereby causing a total increase of non-essential FAA pool by 2-3-fold and essential FAA pool by 1.5-2.0-fold in most of the tissues studied including the plasma. The activities of three ornithine-urea cycle (OUC) enzymes such as carbamoyl phosphate synthetase, argininosuccinate synthetase and argininosuccinate lyase in liver and kidney tissues, and four key amino acid metabolism-related enzymes such as glutamine synthetase, glutamate dehydrogenase (reductive amination), alanine aminotransaminase and aspartate aminotransaminase were also significantly up-regulated in different tissues of the fish while exposing to hypertonic environment. Thus, more accumulation and excretion of urea-N observed during hypertonic exposure were probably associated with the induction of ureogenesis through the induced OUC, and the increase of amino acid pool was probably mainly associated with the up-regulation of amino acid synthesizing machineries in this catfish in hypertonic environment. These might have helped the walking catfish in defending the osmotic stress and to acclimatize better under hypertonic environment, which is very much uncommon among freshwater teleosts.

Metabolic engineering: use of system-level approaches and application to fuel production in Escherichia coli

Metabolic engineering was formally defined more than two decades ago (Bailey, 1991) and it is now an established discipline. Metabolic engineering is generally defined as the directed improvement of product formation or cellular properties through the modification of specific biochemical reactions or the introduction of new ones with the use of recombinant DNA technology (Bailey, 1991; Stephanopoulos et al. 1998). Therefore, the analysis and engineering/synthesis of metabolic pathways is of central importance to metabolic engineering. The analytical part uses a number of experimental and modeling techniques for the systematic study of cellular responses (in terms of RNA, protein and metabolite levels, metabolic fluxes, etc.) to genetic and environmental perturbations. This facilitates a rational design of metabolic modifications, which are implemented using recombinant DNA technology. Both, the analysis and the synthesis of metabolic pathways will be covered in this review. Recent efforts on the engineering of fermentative and biosynthetic pathways for biofuel production in Escherichia coli, as well as those enabling the utilization of novel feedstocks, will be highlighted.

Protective effect of eicosapentaenoic acid-docosahexaenoic acid and α–linolenic acid rich phytosterol ester on brain antioxidant status and brain lipid composition in hypercholesterolemic rats.

With a view to investigate the ameliorative effects of sitosterol esters against degenerative effects of hypercholesterolemia brain antioxidant enzyme assays, brain lipid profile, brain phospholipid compositional change and brain neurotransmitter concentrates (glutamic acid, asparctic acid, glycine) were measured in hypercholesterolemic rats. The results indicated that phytosterol esters have a role in countering hypercholesterolemia-related changes in the brain by decreasing the cholesterol levels, increasing the phospholipid levels and increasing the level of antioxidant enzymes. The results suggest that phytosterol esters may be of therapeutic significance and may offer new and effective options for the treatment of hypercholesterolemia-induced changes in the brain.

Screening of Dihydropyrimidine Dehydrogenase Genetic Variants by Direct Sequencing in Different Ethnic Groups

Dihydropyrimidine dehydrogenase (DPYD) is an enzyme that regulates the rate-limiting step in pyrimidine metabolism, especially catabolism of fluorouracil, a chemotherapeutic agent for cancer. In order to determine the genetic distribution of DPYD, we directly sequenced 288 subjects from five ethnic groups (96 Koreans, 48 Japanese, 48 Han Chinese, 48 African Americans, and 48 European Americans). As a result, 56 polymorphisms were observed, including 6 core polymorphisms and 18 novel polymorphisms. Allele frequencies were nearly the same across the Asian populations, Korean, Han Chinese and Japanese, whereas several SNPs showed different genetic distributions between Asians and other ethnic populations (African American and European American). Additional in silico analysis was performed to predict the function of novel SNPs. One nonsynonymous SNP (+199381A > G, Asn151Asp) was predicted to change its polarity of amino acid (Asn, neutral to Asp, negative). These findings would be valuable for further research, including pharmacogenetic and drug responses studies.

Screening of Dihydropyrimidine Dehydrogenase Genetic Variants by Direct Sequencing in Different Ethnic Groups

Dihydropyrimidine dehydrogenase (DPYD) is an enzyme that regulates the rate-limiting step in pyrimidine metabolism, especially catabolism of fluorouracil, a chemotherapeutic agent for cancer. In order to determine the genetic distribution of DPYD, we directly sequenced 288 subjects from five ethnic groups (96 Koreans, 48 Japanese, 48 Han Chinese, 48 African Americans, and 48 European Americans). As a result, 56 polymorphisms were observed, including 6 core polymorphisms and 18 novel polymorphisms. Allele frequencies were nearly the same across the Asian populations, Korean, Han Chinese and Japanese, whereas several SNPs showed different genetic distributions between Asians and other ethnic populations (African American and European American). Additional in silico analysis was performed to predict the function of novel SNPs. One nonsynonymous SNP (+199381A > G, Asn151Asp) was predicted to change its polarity of amino acid (Asn, neutral to Asp, negative). These findings would be valuable for further research, including pharmacogenetic and drug responses studies.

In Vitro Maintenance of Clonorchis sinensis Adult Worms

Clonorchis sinensis is a biological carcinogen inducing human cholangiocarcinoma, and clonorchiasis is one of the important endemic infectious diseases in East Asia. The present study investigated survival longevity of C. sinensis adult worms in various in vitro conditions to find the best way of keeping the worms longer. The worms were maintained in 0.85% NaCl, 1xPBS, 1xLocke's solution, RPMI-1640, DMEM, and IMDM media, and in 1xLocke's solution with different supplements. All of the worms died within 3 and 7 days in 0.85% NaCl and 1xPBS, respectively, but survived up to 57 days in 1xLocke's solution. The worms lived for 106 days in DMEM, and 114 days in both RPMI-1640 and IMDM media. The survival rate in RPMI-1640 medium was the highest (50%) compared to that in DMEM (20+/-10%) and in IMDM (33.3+/-25.2%) after 3 months. The 1xLocke's solution with 0.005% bovine bile supplement showed increased duration of maximum survival from 42 days to 70 days. Higher concentration of bile supplements than 0.005% or addition of glucose were disadvantageous for the worm survival. The worms died rapidly in solutions containing L-aspartic acid, L-glutamic acid, and adenine compared to L-arginine, L-serine, and L-tryptophan. In conclusion, the 1xLocke's solution best supports the worms alive among inorganic solutions for 57 days, and the RPMI-1640 medium maintains living C. sinensis adults better and longer up to 114 days in vitro than other media.

Molecular battles between plant and pathogenic bacteria in the phyllosphere

The phyllosphere, i.e., the aerial parts of the plant, provides one of the most important niches for microbial colonization. This niche supports the survival and, often, proliferation of microbes such as fungi and bacteria with diverse lifestyles including epiphytes, saprophytes, and pathogens. Although most microbes may complete the life cycle on the leaf surface, pathogens must enter the leaf and multiply aggressively in the leaf interior. Natural surface openings, such as stomata, are important entry sites for bacteria. Stomata are known for their vital role in water transpiration and gas exchange between the plant and the environment that is essential for plant growth. Recent studies have shown that stomata can also play an active role in limiting bacterial invasion of both human and plant pathogenic bacteria as part of the plant innate immune system. As counter-defense, plant pathogens such as Pseudomonas syringae pv tomato (Pst) DC3000 use the virulence factor coronatine to suppress stomate-based defense. A novel and crucial early battleground in host-pathogen interaction in the phyllosphere has been discovered with broad implications in the study of bacterial pathogenesis, host immunity, and molecular ecology of bacterial diseases.

Restrição calórica e mitocôndrias: papel no envelhecimento de Saccharomyces cerevisiae / Caloric Restriction and Mitochondria: Role in Saccharomyces cerevisiae aging / Restriction calorique et mitochondrie: rôle dans le vieillissement de Saccharomyces cerevisiae

A restrição calórica (RC) é uma intervenção dietética capaz de estender a longevidade de vários organismos. O modelo para RC em Saccharomyces cerevisiae consiste da diminuição da concentração de glicose no meio de cultura e mostra um aumentado tanto do tempo de vida cronológico quanto replicativo. Nosso objetivo foi investigar experimentalmente a ação da RC, focando principalmente nas causas e consequências das modificações de geração de EROs mitocondriais e como estas estão associadas ao processo de envelhecimento. Em um primeiro período de estudos, verificamos quais as fontes mitocondriais de EROs, e comprovamos que uma quantidade significativa se origina de proteínas da matriz mitocondrial, e não da cadeia de transporte de elétrons. Nós estudamos a participação de glicose e de outras fontes de carbono sobre o tempo de vida cronológico em leveduras e mostramos que o aumento da longevidade promovida pela RC está associado à uma mudança de metabolismo fermentativo para respiratório, com participação da via de sinalização de glicose. No estágio realizado no laboratório do Professor Francis Sluse na Université de Liegè, Bélgica, estudamos a ação da RC em leveduras focando nas consequências das modificações no proteoma mitocondrial. Em nosso estudo proteômico, encontramos grandes modificações em proteínas envolvidas com o metabolismo de aminoácidos. Monitoramos a atividade de enzimas relacionadas ao metabolismo de aminoácidos e o tempo de vida cronológico de S. cerevisiae e as mutantes nulas bat2Δ, gdh1Δ, gdh2Δ e gdh3Δ, que codificam a aminotransferase de aminoácidos de cadeia ramificada citosólica, NADP glutamato desidrogenase citosólica, a NAD glutamato desidrogenase mitocondrial, e a NADP glutamato desidrogenase mitocondrial, respectivamente. A atividade da NAD glutamato desidrogenase é aumentada em RC, mas a de NADP glutamato desidrogenase decresce em células controle. Aumentos do tempo de vida cronológico foram observados nas mutantes...

Calorie restriction (CR) is a dietary intervention capable of extending lifespans in a wide range of organisms. A yeast model of CR has been developed in which limiting the concentration of glucose in growth media of Saccharomyces cerevisiae leads to enhanced chronological and replicative life spans. Our aim was to experimentally investigate the effects of CR, focusing mainly on the causes and consequences of changes in mitochondrial reactive oxygen species (ROS) generation and how these are associated with the aging process. Initially, we looked for sources of mitochondrial ROS, and found that a significant amount of ROS comes from mitochondrial matrix enzymes and not from the electron transport chain. We studied the participation of glucose and other carbon sources in chronological lifespan and show that increased longevity promoted by CR is associated with a metabolism change from fermentation to respiration, with participation of glucose repression pathway. During studies performed in the laboratory of Professor Francis Sluse at the Université de Liège, Belgium, we studied the effect of CR in yeast with focus on the consequences of changes in the mitochondrial proteome. We found large proteomic changes in proteins involved in amino acid metabolism. We monitored the activity of enzymes related to amino acid metabolism and chronological life span of S. cerevisiae null mutants bat2Δ, gdh1Δ, gdh2Δ, and gdh3Δ, which encode for the cytosolic branched-chain amino acid aminotransferase, cytosolic NADP glutamate dehydrogenase, mitochondrial NAD glutamate dehydrogenase and mitochondrial NADP glutamate dehydrogenase, respectively. The activity of NAD glutamate dehydrogenase is increased in CR, but NADP glutamate dehydrogenase decreases in control cells. Increases in chronological life span due to RC were observed in bat2Δ and gdh1Δ mutants, but no significant difference was found in Gdh2p and Gdh3p null mutants in the stationary phase…

La restriction calorique (RC) est une intervention diététique capable de prolonger la durée de vie dans divers organismes. Un modèle de RC pour la levure a été developpé dans lequel limiter la concentration de glucose dans le milieu de culture de Saccharomyces cerevisiae a montré une augmentation de vieillessement chronologique et réplicative. Notre objectif était d’étudierexpérimentalement l’effet de la RC, en se concentrant principalement sur les causes et les conséquences des changements dans la production des espèces d’oxygène réactive (ROS) mitochondriales et de la façon dont elles sont associée au processus de vieillessement. Dans une première période d’études, qui a trouvé la source de ROS mitochondriale, nous montrons qu’une quantité importante vient d’enzyme de la matrice et pas de la chaîne de transport d’électrons. Nous avons étudié la participation de glucose et d’autres sources de carbone sur le vieillissement chronologique dans la levure et nous avons montré que l’augmentation de la longévité promure par RC est associée à un changement du métabolisme fermentaire à respiratoire, avec la participation de la voie de signalisation du glucose. Dans le laboratoire du professeur Francis Sluse à l’Université de Liège, en Belgique, nous avons étudié l’effet du RC dans la levure en se concentrant sur les conséquences des changements du protéome mitochondrial. Dans notre étude, nous avons constaté de grands changements des protéines impliquées dans le métabolisme des acides amines. Nous avons surveillé l’activité des enzymes liées au métabolisme des acides aminés et le vieillissement chronologique de S. cerevisiae et des mutants nul bat2Δ, gdh1Δ, gdh2Δ et gdh3Δ, qui codent aminotransférase pour les acides aminés à chaîne ramifiée cytosolique, NADP glutamate déshydrogénase cytologique, NAD glutamate déshydrogénase mitochondriale et NADP glutamate déshydrogénase mitochondriale, respectivement. L’activité de la NAD glutamate...

Cuantificación de aminoácidos en plasma empleando Cromatografía Líquida de Alta Eficiencia / Plasma amino acid quantification using High Performance Liquid Chromatography

Las aminoacidopatías son errores innatos del metabolismo intermediario de los aminoácidos. Su confirmación diagnóstica y seguimiento se realiza con la cuantificación de aminoácidos libres en fluidos biológicos por técnicas como la cromatografía líquida de alta eficiencia (HPLC), para lo que es necesario comparar con valores de referencia normales. La población colombiana no cuenta con estos valores disponibles y el diagnóstico es realizado por comparación con los de otras poblaciones. En el presente trabajo se obtuvieron valores de referencia de aminoácidos en plasma en una población de niños (n=36) y adultos no afectados (n=17), mediante HPLC por derivatización postcolumna con ninhidrina. Los valores de referencia obtenidos fueron ligeramente más elevados que los informados para otras poblaciones y permitieron la identificación de doce casos de aminoacidopatías, incluyendo fenilcetonuria clásica, hiperfenilalaninemia, hiperglicinemia no cetósica, desórdenes del ciclo de la urea, tirosinemia. La implementación de la cuantificación de aminoácidos por HPLC y la obtención de los valores de referencia de aminoácidos en plasma permitirán aumentar el conocimiento sobre la incidencia de las aminoacidopatías en el país para garantizar, junto con otros factores, su diagnóstico preciso y oportuno y la implementación de un adecuado seguimiento nutricional.

Aminoacidopathies are inborn errors of the amino acid intermediary metabolism. The benchmark method used for their diagnosis and monitoring is the quanti!cation of free amino acids in biological fluids using High Performance Liquid Chromatography (HPLC), which needs to be compared against normal reference values. However, those amino acid reference values are not available for the Colombian population and the diagnosis is usually made using values from American or European populations. In this work, plasma amino acid reference values in non-affected children (n=36) and adults (n=17) were established, using an HPLC method with a postcolumn derivatization with ninhidrine. Plasma amino acid reference values in a Colombian population were slightly higher compared with those reported for other populations, and enabled the identification of twelve aminoacidopathies including urea cycle disorders, phenylketonuria, hyperphenylalaninemia, nonketotichyperglycinemia, hepatorrenaltyrosinemia and maple syrup urine disease. The implementation of amino acid cuantification by HPLC and the construction of plasma amino acid reference values is very useful for a suitable and precise diagnosis of amino acid disorders, the implementation of proper nutritional treatments, and an increased knowledge of aminoacidopathy incidence in Colombia.