NAD in pathological cardiac remodeling: Metabolic regulation and beyond.

Nicotinamide adenine dinucleotide (NAD) coenzymes are carriers of high energy electrons in metabolism and also play critical roles in numerous signaling pathways. NAD metabolism is decreased in various cardiovascular diseases. Importantly, stimulation of NAD biosynthesis protects against heart disease under different pathological conditions. In this review, we describe pathways for both generation and catabolism of NAD coenzymes and the respective changes of these pathways in the heart under cardiac diseases, including pressure overload, myocardial infarction, cardiometabolic disease, cancer treatment cardiotoxicity, and heart failure. We next provide an update on the strategies and treatments to increase NAD levels, such as supplementation of NAD precursors, in the heart that prevent or reverse cardiomyopathy. We also introduce the approaches to manipulate NAD consumption enzymes to ameliorate cardiac disease. Finally, we discuss the mechanisms associated with improvements in cardiac function by NAD coenzymes, differentiating between mitochondria-dependent effects and those independent of mitochondrial metabolism.

Harnessing enzyme cofactors and plant metabolism: an essential partnership.

Cofactors are fundamental to the catalytic activity of enzymes. Additionally, because plants are a critical source of several cofactors (i.e., including their vitamin precursors) within the context of human nutrition, there have been several studies aiming to understand the metabolism of coenzymes and vitamins in plants in detail. For example, compelling evidence has been brought forth regarding the role of cofactors in plants; specifically, it is becoming increasingly clear that an adequate supply of cofactors in plants directly affects their development, metabolism, and stress responses. Here, we review the state-of-the-art knowledge on the significance of coenzymes and their precursors with regard to general plant physiology and discuss the emerging functions attributed to them. Furthermore, we discuss how our understanding of the complex relationship between cofactors and plant metabolism can be used for crop improvement.

Synthetic application of chalcogenonium salts: beyond sulfonium.

The application of chalcogenonium salts in organic synthesis has grown enormously in the past decades since the discovery of the methyltransferase enzyme cofactor S-adenosyl-L-methionine (SAM), featuring a sulfonium center as the reactive functional group. Chalcogenonium salts can be employed as alkylating agents, sources of ylides and carbon-centered radicals, partners for metal-catalyzed cross-coupling reactions and organocatalysts. Herein, we will focus the discussion on heavier chalcogenonium salts (selenonium and telluronium), presenting their utility in synthetic organic transformations and, whenever possible, drawing comparisons in terms of reactivity and selectivity with the respective sulfonium analogues.

Role of Micronutrients and Gut Microbiota-Derived Metabolites in COVID-19 Recovery.

A balanced and varied diet provides diverse beneficial effects on health, such as adequate micronutrient availability and a gut microbiome in homeostasis. Besides their participation in biochemical processes as cofactors and coenzymes, vitamins and minerals have an immunoregulatory function; meanwhile, gut microbiota and its metabolites coordinate directly and indirectly the cell response through the interaction with the host receptors. Malnourishment is a crucial risk factor for several pathologies, and its involvement during the Coronavirus Disease 2019 pandemic has been reported. This pandemic has caused a significant decline in the worldwide population, especially those with chronic diseases, reduced physical activity, and elder age. Diet and gut microbiota composition are probable causes for this susceptibility, and its supplementation can play a role in reestablishing microbial homeostasis and improving immunity response against Coronavirus Disease 2019 infection and recovery. This study reviews the role of micronutrients and microbiomes in the risk of infection, the severity of disease, and the Coronavirus Disease 2019 sequelae.

Improving the sensitivity for hydrogen peroxide determination with active V2O5 nanocubes incorporated on mesoporous TiO2.

The capacity to generate a constant signal response from an enzyme on an electrode surface has been a fascinating topic of research from the past three decades. To nourish the enzymatic activity during electrochemical reactions, the immobilization of dual enzymes on the electrode surface could prevent the enzymatic loss without denaturation and thus long-term stability can be achieved. For effective immobilization of dual enzymes, mesoporous materials are the ideal choice because of its numerous advantages such as 1. The presence of porous structure facilitates high loading of enzymes 2. The formation of protective environment can withstand the enzymatic activity even at acidic or basic pH values and even at elevated temperatures. Herein, we develop bienzymatic immobilization of horseradish peroxidase (HRP) and cholesterol oxidase (ChOx) on mesoporous V2O5-TiO2 based binary nanocomposite for effective sensing of hydrogen peroxide (H2O2) in presence of redox mediator hydroquinone (HQ). The utilization of redox mediator in second-generation biosensing of H2O2 can eliminate the interference species and reduces the operating potential with higher current density for electrochemical reduction reaction. Using this mediator transfer process approach at HRP/ChOx/V2O5-TiO2 modified GC, the H2O2 can be determined at operating potential (-0.2 V) with good linear range (0.05-3.5 mM) higher sensitivity (1040 µAµM-1 cm-2) and lower detection limit of about 20 µM can be attained, which is due to higher mediation of electrons were transferred to the enzyme cofactors. These interesting characteristics could be due to mesoporous structure of V2O5-TiO2 can induce large immobilization and facilitate higher interaction with enzymes for wide range of biosensing applications.

A complete biomimetic iron-sulfur cubane redox series.

Synthetic iron-sulfur cubanes are models for biological cofactors, which are essential to delineate oxidation states in the more complex enzymatic systems. However, a complete series of [Fe4S4]n complexes spanning all redox states accessible by 1-electron transformations of the individual iron atoms (n = 0-4+) has never been prepared, deterring the methodical comparison of structure and spectroscopic signature. Here, we demonstrate that the use of a bulky arylthiolate ligand promoting the encapsulation of alkali-metal cations in the vicinity of the cubane enables the synthesis of such a series. Characterization by EPR, 57Fe Mössbauer spectroscopy, UV-visible electronic absorption, variable-temperature X-ray diffraction analysis, and cyclic voltammetry reveals key trends for the geometry of the Fe4S4 core as well as for the Mössbauer isomer shift, which both correlate systematically with oxidation state. Furthermore, we confirm the S = 4 electronic ground state of the most reduced member of the series, [Fe4S4]0, and provide electrochemical evidence that it is accessible within 0.82 V from the [Fe4S4]2+ state, highlighting its relevance as a mimic of the nitrogenase iron protein cluster.

Mitochondrial dysfunction in anti-3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR) immune-mediated necrotising myopathy.

Mitochondrial dysfunction is a plausible cause of muscle fibre damage in a number of myopathies including immune-mediated necrotising myopathy. However, histopathological evidence of mitochondrial dysfunction is not often described in immune-mediated necrotising myopathy and, when present, it is often attributed to patient age. The purpose of this study was to describe features of mitochondrial dysfunction on muscle biopsy in anti-3­hydroxy-3-methylglutaryl-CoA reductase immune-mediated necrotising myopathy and explore whether these features are age-related. In this observational case control study, a statistically significant increase in the number of muscle fibres with increased lipid content (p = 0.004) and cytochrome c oxidase-negative/succinate dehydrogenase-positive fibres (p = 0.037) in anti-3­hydroxy-3-methylglutaryl-coenzyme immune-mediated necrotising myopathy was found compared to age-matched controls. Therefore, histopathological features of mitochondrial dysfunction are more frequent in anti-3­hydroxy-3-methylglutaryl-coenzyme immune-mediated necrotising myopathy than aged-matched controls and therefore, may be contributing to the pathogenesis.

Avaliação da influência da Coenzima Q10 sobre o processo de reparo periimplantar de implantes instalados em tíbias de ratos expostos ou não à nicotina / Evaluation of the influence of Coenzyme Q10 on the periimplant repair processo of implants installed in the tíbias of rats exposed or not to nicotine

O propósito do presente estudo foi avaliar a influência da Coenzima Q10 (CoQ10) no reparo periimplantar em implantes instalados em tíbias de ratos modificados sistemicamente ou não pela nicotina. Oitenta ratos machos (Wistar), foram divididos em quatro grupos (n=20). No dia 0 os animais receberam um implante (4 x 2,2mm ­ SLA) na metáfise proximal das tíbias direita e esquerda. Nos 30 dias que antecedem o procedimento cirúrgico e nos 28 que o sucedem, os animais receberam duas injeções subcutâneas diárias de 3mg/kg de hemissulfato de nicotina ou solução salina na região dorsal, com 12 horas de intervalo entre elas. Logo após à cirurgia, o protocolo se constituiu na administração via gavagem gástrica de 1 ml de glicerina vegetal, ou suplementação diária com 120 mg de CoQ10, ambos até o final do experimento. SS (SHAM): o protocolo de aplicação utilizado foi o de solução salina subcutânea, e os animais receberam gavagem gástrica diária de 1 ml de glicerina vegetal. SS-CoQ10: o protocolo de aplicação utilizado foi o de solução salina subcutânea e, como suplementação, receberam 120 mg de Coenzima Q10 via gavagem gástrica. NIC: o protocolo de aplicação utilizado foi o de nicotina, e os animais receberam gavagem gástrica diária de 1 ml de glicerina vegetal. NIC-CoQ10: o protocolo de aplicação utilizado foi o de nicotina e, como suplementação, os animais receberam 120 mg de CoQ10 via gavagem gástrica. As eutanásias foram aos 7 e 28 dias pós-operatórios. As peças coletadas foram processadas com desmineralização para as análises histológica, histométrica (PTON) e imunoistoquímica para detecção de BMP/2, OCN e TRAP; e sem desmineralização para análise da área do contato direto osso/implante (BIC). Após análise de normalidade e homocedasticidade, os dados foram submetidos aos testes mais adequados com significância de 5% (p≤0,05). Com relação ao contato osso implante, o grupo SS, SS-Q10 e NIC- Q10, apresentaram maior BIC em todos os períodos experimentais quando comparado com o grupo NIC. Os grupos SS, SS-Q10 e NIC-Q10 apresentaram também maior PTON em todos os períodos experimentais quando comparado com o grupo NIC. A análise histológica dos tecidos periimplantares mostrou que o grupo NIC-Q10 apresentou características histológicas que se mostraram similares ao grupo controle, no entanto, com maior quantidade de tecido ósseo periimplantar e menor quantidade de tecido conjuntivo. Nos padrões de marcação imunoistoquímica, quando comparado ao grupo SS, o grupo NIC-Q10 apresentou menor imunomarcação para e OCN, menor marcação para TRAP e não houve diferenças quanto a marcação de BMP2. Dentro dos limites do presente estudo, pode-se concluir que a Coenzima Q10 exerceu uma influência positiva na remodelação óssea periimplantar em implantes osseointegrados(AU)

The purpose of the presente study was to evaluate the influence of Coenzyme Q10 (CoQ10) on periimplant repair in implants installed in the tíbia of rats modificated sistemically or not by nicotine. Eighty male rats (Wistar) were divided into four groups(n=20). On day 0 the animals received na implant (4x2,2mm-SLA) in the proximal metaphasys of the right and left tíbias. In the 30 days preceding the surgical procedure and the 28 days following it, the animals received two daily subcutaneous injections of 3 mg/kg of nicotine hemissulfate or saline solution in the dorsal region, with a 12-hour interval between them. Soon after surgery, the protocol consisted of the administration via gastric gavage of 1ml of vegetable glycerin, or daily supplementation with 120 mg of CoQ10, both until the endo f of the experiment. SS (SHAM): the application protocol used was subcutaneous saline solution, and the animals received daily gastric gavage of 1 ml of vegetal glycerin. SS-CoQ10: the application protocol used was subcutaneous saline solution and, as a supplement, they received 120 mg of Coenzyme Q10 via gastric gavage. NIC: the application protocol used was nicotine, and the animals received daily gastric gavage of 1 ml of vegetable glycerin. NIC-CoQ10: the application protocol used was nicotine and, as a supplement, the animals received 120 mg of CoQ10 via gastric gavage. Euthanasias were performed at 7 and 28 days after surgery. The colleted pieces were processed with desmineralization for histological analysis, área of neofomad bone tissue, histomorfometric analysis (PTON) and immunohistochemistry for the detection of BMP2, OCN and TRAP; and without desmineralization for direct bone/implant contat (BIC) analysis. Regarding bonéimplant contact, the SS, SS-Q10 and NIC-Q10 groups showed higher BIC in all experimental periods When compared to the NIC group. The histological analysis of the periimplant tissues showed that the NIC-Q10 group presented histological characteristics that were similar to the control group, however, with a greater amount of periimplant bone tissue and less connective tissue. In immunohistochemical staining patterns, when compared to the SS group, the NICQ10 group showed lower immunostaining for and OCN, lower staining for TRAP and there were no diferences regarding BMP2 staining. Within the limits of the presente study, it can be concluded that Coenzyme Q10 exerted a positive influence on periimplant bone remodeling in osseointegrated implants(AU)

Spectroscopic analysis of coenzyme binding to betaine aldehyde dehydrogenase dependent on potassium.

Glycine betaine is the main osmolyte synthesized and accumulated in mammalian renal cells. Glycine betaine synthesis is catalyzed by the enzyme betaine aldehyde dehydrogenase (BADH) using NAD+ as the coenzyme. Previous studies have shown that porcine kidney betaine aldehyde dehydrogenase (pkBADH) binds NAD+ with different affinities at each active site and that the binding is K+ dependent. The objective of this work was to analyze the changes in the pkBADH secondary and tertiary structure resulting from variable concentrations of NAD+ and the role played by K+ . Intrinsic fluorescence studies were carried out at fixed-variable concentrations of K+ and titrating the enzyme with varying concentrations of NAD+ . Fluorescence analysis showed a shift of the maximum emission towards red as the concentration of K+ was increased. Changes in the exposure of tryptophan located near the NAD+ binding site were found when the enzyme was titrated with NAD+ in the presence of potassium. Fluorescence data analysis showed that the K+ presence promoted static quenching that facilitated the pkBADH-NAD+ complex formation. DC data analysis showed that binding of K+ to the enzyme caused changes in the α-helix content of 4% and 12% in the presence of 25 mM and 100 mM K+ , respectively. The presence of K+ during NAD+ binding to pkBADH increased the thermal stability of the complex. These results indicated that K+ facilitated the pkBADH-NAD+ complex formation and suggested that K+ caused small changes in secondary and tertiary structures that could influence the active site conformation.

Paraparesia espástica complicada como fenótipo neurológico em OPA1 / Complex spastic paraplegia as a neurological phenotype in an opa1 patient

RESUMO: A atrofia óptica autossômica dominante (ADOA) é uma das formas mais comuns de atrofias ópticas hereditárias, e causada por mutações no gene OPA1. Os pacientes afetados por essa doença geralmente apresentam perda visual na primeira década de vida, podendo apresentar manifestações extraoftalmológicas no decorrer dos anos, configurando uma síndrome chamada OPA1 plus ou ADOA-plus. Objetivos: Relatar caso de paciente portadora da síndrome ADOA-plus, estabelecendo correlações com casos descritos na literatura. Relato de caso: Paciente feminino, 30 anos, foi encaminhada para avaliação de quadro de atrofia óptica progressiva associada a sintomas de neuropatia periférica. Aos dois anos, foi diagnosticada com perda visual parcial em consulta de puericultura. Não relatou outros sintomas associados durante a infância e a adolescência. Aos 20 anos, apresentou dificuldades de deambular, fraqueza em membros inferiores e falta de equilíbrio. Aos 25 anos, após extensa investigação, foi identificada, através de sequenciamento de exoma, mutação patológica no gene OPA1 confirmando o diagnóstico ADOA-plus e iniciado tratamento com Coenzima Q10. Atualmente a paciente relata ataxia sensitiva, diminuição da acuidade visual progressiva, fasciculações e câimbras em MMII, disfagia e dispneia. Discussão: Muitos pacientes com ADOA-plus apresentam surdez neurossensorial como sintoma extraoftalmológico mais comum, além de quadros de parkinsonismo e demência, ataxia e ptose. Paciente relatada constitui um caso de atrofia óptica associado à neuropatia periférica, ataxia e miopatia. Devido à ampla variabilidade clínica dessa doença, deve-se investigar mutações no OPA1 em casos de paraparesia espástica progressiva associada à atrofia óptica, visto que possibilidade de tratamento com Coenzima Q10. (AU)

ABSTRACT: Introduction: Autosomal dominant optic atrophy (ADOA) is one of the most common forms of inherited optic atrophies and is caused by mutations in the OPA1 gene. Patients affected by this disease usually present visual loss in the first decade of life, and may present extra-ophthalmologic manifestations over the years, configuring a syndrome called OPA1 plus or ADOA-plus. Objectives: to report the case of a patient with ADOA-plus syndrome, establishing correlations with cases described in the literature, Case report: a 30-year-old female patient was referred for evaluation of progressive optic atrophy associated with symptoms of peripheral neuropathy. At two years of age, she was diagnosed with partial visual loss during a childcare visit. She reported no other associated symptoms during childhood and adolescence. At the age of 20, she presented with difficulty walking, lower limb weakness, and poor balance. At 25, after extensive investigation, a pathological mutation in the OPA1 gene was identified through exome sequencing, confirming the diagnosis of ADOA-plus, and treatment with Coenzyme Q10 was initiated. Currently the patient reports sensory ataxia, progressive decrease in visual acuity, fasciculations and cramps in the lower limbs, dysphagia and dyspnea. Discussion: Many patients with ADOA-plus present sensorineural deafness as the most common extra-ophthalmologic symptom, in addition to parkinsonism and dementia, ataxia and ptosis. The patient reported is a case of optic atrophy associated with peripheral neuropathy, ataxia and myopathy. Due to the wide clinical variability of this disease, OPA1 mutations should be investigated in cases of progressive spastic paraparesis associated with optic atrophy, since the possibility of treatment with Coenzyme Q10. (AU)

Effect of COQ9 and STAT5A polymorphisms on reproductive performance in a Holstein cow herd in Mexico

Coenzyme Q9 (COQ9), a coenzyme Q (CoQ) precursor, is an essential component of the mitochondrial electron transport chain that drives adenosine triphosphate production. COQ9 polymorphism 18:25527339 is characterized by substitution of guanine (allele G) for adenine (allele A), which modifies the function of the protein encoded by the gene. In Holsteins, allele A has been associated with better reproductive performance in terms of the conception rate, number of services per conception (SPC) and days open (DO). The signal transducer and activator of transcription (STAT) protein is a transcription factor activated in the presence of cytokines and growth factors. STAT5A polymorphism 19:42407732 in exon 8 has been associated with higher fertility and embryonic survival rates. The objective of this study was to determine the relationship of COQ9 and STAT5A polymorphisms with reproductive parameters [calving to first heat interval (CFHI), DO and SPC]. Blood samples were taken from 112 lactating Holstein from a herd in México for allele genotyping by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). To estimate the association between reproductive parameters and genotypes, a linear mixed-effect model was performed. The COQ9 AG genotype was associated significantly with lower SPC (P<0.05) but not with DO or CFHI. No significant association with any reproductive parameter was found for STAT5A. Our findings suggest that the COQ9 18:25527339 polymorphism is a useful molecular marker for improvement of reproductive performance in dairy herds.

Effect of COQ9 and STAT5A polymorphisms on reproductive performance in a Holstein cow herd in Mexico

Coenzyme Q9 (COQ9), a coenzyme Q (CoQ) precursor, is an essential component of the mitochondrial electron transport chain that drives adenosine triphosphate production. COQ9 polymorphism 18:25527339 is characterized by substitution of guanine (allele G) for adenine (allele A), which modifies the function of the protein encoded by the gene. In Holsteins, allele A has been associated with better reproductive performance in terms of the conception rate, number of services per conception (SPC) and days open (DO). The signal transducer and activator of transcription (STAT) protein is a transcription factor activated in the presence of cytokines and growth factors. STAT5A polymorphism 19:42407732 in exon 8 has been associated with higher fertility and embryonic survival rates. The objective of this study was to determine the relationship of COQ9 and STAT5A polymorphisms with reproductive parameters [calving to first heat interval (CFHI), DO and SPC]. Blood samples were taken from 112 lactating Holstein from a herd in México for allele genotyping by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). To estimate the association between reproductive parameters and genotypes, a linear mixed-effect model was performed. The COQ9 AG genotype was associated significantly with lower SPC (P<0.05) but not with DO or CFHI. No significant association with any reproductive parameter was found for STAT5A. Our findings suggest that the COQ9 18:25527339 polymorphism is a useful molecular marker for improvement of reproductive performance in dairy herds.(AU)

Biochemical Characterization of Heat-Tolerant Recombinant L-Arabinose Isomerase from Enterococcus faecium DBFIQ E36 Strain with Feasible Applications in D-Tagatose Production.

D-Tagatose is a ketohexose, which presents unique properties as a low-calorie functional sweetener possessing a sweet flavor profile similar to D-sucrose and having no aftertaste. Considered a generally recognized as safe (GRAS) substance by FAO/WHO, D-tagatose can be used as an intermediate for the synthesis of other optically active compounds as well as an additive in detergent, cosmetic, and pharmaceutical formulations. This study reports important features for L-arabinose isomerase (EC 5.3.1.4) (L-AI) use in industry. We describe arabinose (araA) gene virulence analysis, gene isolation, sequencing, cloning, and heterologous overexpression of L-AI from the food-grade GRAS bacterium Enterococcus faecium DBFIQ E36 in Escherichia coli and assess biochemical properties of this recombinant enzyme. Recombinant L-AI (rL-AI) was one-step purified to homogeneity by Ni2+-agarose resin affinity chromatography and biochemical characterization revealed low identity with both thermophilic and mesophilic L-AIs but high degree of conservation in residues involved in substrate recognition. Optimal conditions for rL-AI activity were 50 °C, pH 5.5, and 0.3 mM Mn2+, exhibiting a low cofactor concentration requirement and an acidic optimum pH. Half-life at 45 °C and 50 °C were 1427 h and 11 h, respectively, and 21.5 h and 39.5 h at pH 4.5 and 5.6, respectively, showing the high stability of the enzyme in the presence of a metallic cofactor. Bioconversion yield for D-tagatose biosynthesis was 45% at 50 °C after 48 h. These properties highlight the technological potential of E. faecium rL-AI as biocatalyst for D-tagatose production.

Cofactor Specificity Switch on Peach Glucitol Dehydrogenase.

Most oxidoreductases that use NAD+ or NADP+ to transfer electrons in redox reactions display a strong preference for the cofactor. The catalytic efficiency of peach glucitol dehydrogenase (GolDHase) for NAD+ is 1800-fold higher than that for NADP+. Herein, we combined structural and kinetic data to reverse the cofactor specificity of this enzyme. Using site-saturation mutagenesis, we obtained the D216A mutant, which uses both NAD+ and NADP+, although with different catalytic efficiencies (1000 ± 200 and 170 ± 30 M-1 s-1, respectively). This mutant was used as a template to introduce further mutations by site-directed mutagenesis, using information from the fruit fly NADP-dependent GolDHase. The D216A/V217R/D218S triple mutant displayed a 2-fold higher catalytic efficiency with NADP+ than with NAD+. Overall, our results indicate that the triple mutant has the potential to be used for metabolic and cellular engineering and for cofactor recycling in industrial processes.

Biochemistry of Peroxynitrite and Protein Tyrosine Nitration.

Peroxynitrite is a short-lived and reactive biological oxidant formed from the diffusion-controlled reaction of the free radicals superoxide (O2•-) and nitric oxide (•NO). In this review, we first analyze the biochemical evidence for the formation of peroxynitrite in vivo and the reactions that lead to it. Then, we describe the principal reactions that peroxynitrite undergoes with biological targets and provide kinetic and mechanistic details. In these reactions, peroxynitrite has roles as (1) peroxide, (2) Lewis base, and (3) free radical generator. Physiological levels of CO2 can change the outcome of peroxynitrite reactions. The second part of the review assesses the formation of protein 3-nitrotyrosine (NO2Tyr) by peroxynitrite-dependent and -independent mechanisms, as one of the hallmarks of the actions of •NO-derived oxidants in biological systems. Moreover, tyrosine nitration impacts protein structure and function, tyrosine kinase signal transduction cascades and protein turnover. Overall, the review is aimed to provide an integrated biochemical view on the formation and reactions of peroxynitrite under biologically relevant conditions and the impact of this stealthy oxidant and one of its major footprints, protein NO2Tyr, in the disruption of cellular homeostasis.

Molecular and functional characterization of two malic enzymes from Leishmania parasites.

Leishmania parasites cause a broad spectrum of clinical manifestations in humans and the available clinical treatments are far from satisfactory. Since these pathogens require large amounts of NADPH to maintain intracellular redox homeostasis, oxidoreductases that catalyze the production of NADPH are considered as potential drug targets against these diseases. In the sequenced genomes of most Leishmania spp. two putative malic enzymes (MEs) with an identity of about 55% have been identified. In this work, the ME from L. major (LmjF24.0770, Lmj_ME-70) and its less similar homolog from L. mexicana (LmxM.24.0761, Lmex_ME-61) were cloned and functionally characterized. Both MEs specifically catalyzed NADPH production, but only Lmex_ME-61 was activated by l-aspartate. Unlike the allosterically activated human ME, Lmex_ME-61 exhibited typical hyperbolic curves without any sign of cooperativity in the absence of l-aspartate. Moreover, Lmex_ME-61 and Lmj_ME-70 differ from higher eukaryotic homologs in that they display dimeric instead of tetrameric molecular organization. Homology modeling analysis showed that Lmex_ME-61 and Lmj_ME-70 notably differ in their surface charge distribution; this feature encompasses the coenzyme binding pockets as well. However, in both isozymes, the residues directly involved in the coenzyme binding exhibited a good degree of conservation. Besides, only Lmex_ME-61 and its closest homologs were immunodetected in cell-free extracts from L. mexicana, L. amazonensis and L. braziliensis promastigotes. Our findings provide a first glimpse into the biochemical properties of leishmanial MEs and suggest that MEs could be potentially related to the metabolic differences among the species of Leishmania parasites.

Human MMAA induces the release of inactive cofactor and restores methylmalonyl-CoA mutase activity through their complex formation.

Human mitochondrial methylmalonyl-CoA mutase (hMCM) is an isomerase that converts methylmalonyl-CoA to succinyl-CoA, a crucial step for the incorporation of some compounds derived from the diet into the central metabolism. hMCM employs highly reactive radicals from its cofactor (adenosylcobalamin, AdoCbl) to perform its reaction. Our previous work demonstrated that hMCM loses activity during catalysis and that the interaction with human MMAA (hMMAA), a GTPase protein, avoided this loss or restored hMCM activity. Even so, the mechanism by which hMMAA exerted these chaperone functions has not been described. In this work report that the formation and accumulation of OH2Cbl, the oxidized form of the AdoCbl cofactor formed during catalysis, is the cause of hMCM inactivation. Additionally, we demonstrate that the complex formation of hMCM/hMMAA decreases the rate of oxidized cofactor formation, protecting the hMCM enzyme. Moreover, an inactive model of hMCM was used to demonstrate that hMMAA is able to remove the damaged cofactor through GTP hydrolysis. Additionally, a modification in the kinetic parameters of hMCM in presence of hMMAA was observed, and for the first time, the in vivo localization of hMMAA and its colocalization with hMCM in human fibroblasts mitochondria were demonstrated.

Carcass traits and immune response of broiler chickens fed dietary l-carnitine, coenzyme Q10 and ractopamine

This study was conducted to evaluate the effects of coenzyme Q10, L-carnitine and ractopamine supplementation, alone and in combinations, on carcass traits and immune response of broiler chickens. Five hundred and twelve one-day-old Ross 308 male broiler chickens were randomly allocated into eight treatments with four replicates each. A 2×2×2 factorial arrangement was applied, with two levels of coenzyme Q10 (0 and 40 mg/kg), two levels of L-carnitine (0 and 200 mg/kg) and two levels of ractopamine (0 and 10 mg/kg). The birds were reared until day 42 of age under standard conditions. Blood samples were collected at the end of grower and finisher periods from the wing vein. Four birds per group were sacrificed at day 42 of age. Except for carcass yield, other carcass traits were not significantly affected (p>0.05) by different levels of coenzyme Q10, L-carnitine, or ractopamine. Immune response parameters were significantly (p 0.05) different between the treatments. The lowest antibody titers against Newcastle disease virus and relative spleen weight were observed in control group. The results of this study suggest that addition of coenzyme Q10 and L-carnitine to broiler diets has benefit effect on immune response of broiler chickens.

Carcass traits and immune response of broiler chickens fed dietary l-carnitine, coenzyme Q10 and ractopamine

This study was conducted to evaluate the effects of coenzyme Q10, L-carnitine and ractopamine supplementation, alone and in combinations, on carcass traits and immune response of broiler chickens. Five hundred and twelve one-day-old Ross 308 male broiler chickens were randomly allocated into eight treatments with four replicates each. A 2×2×2 factorial arrangement was applied, with two levels of coenzyme Q10 (0 and 40 mg/kg), two levels of L-carnitine (0 and 200 mg/kg) and two levels of ractopamine (0 and 10 mg/kg). The birds were reared until day 42 of age under standard conditions. Blood samples were collected at the end of grower and finisher periods from the wing vein. Four birds per group were sacrificed at day 42 of age. Except for carcass yield, other carcass traits were not significantly affected (p>0.05) by different levels of coenzyme Q10, L-carnitine, or ractopamine. Immune response parameters were significantly (p 0.05) different between the treatments. The lowest antibody titers against Newcastle disease virus and relative spleen weight were observed in control group. The results of this study suggest that addition of coenzyme Q10 and L-carnitine to broiler diets has benefit effect on immune response of broiler chickens.(AU)

The structure of a Trypanosoma cruzi glucose-6-phosphate dehydrogenase reveals differences from the mammalian enzyme.

The enzyme glucose-6-phosphate dehydrogenase from Trypanosoma cruzi (TcG6PDH) catalyses the first step of the pentose phosphate pathway (PPP) and is considered a promising target for the discovery of a new drug against Chagas diseases. In the present work, we describe the crystal structure of TcG6PDH obtained in a ternary complex with the substrate ß-d-glucose-6-phosphate (G6P) and the reduced 'catalytic' cofactor NADPH, which reveals the molecular basis of substrate and cofactor recognition. A comparison with the homologous human protein sheds light on differences in the cofactor-binding site that might be explored towards the design of new NADP(+) competitive inhibitors targeting the parasite enzyme.