Construction of multi-enzyme cascade reactions and its application in the synthesis of bifunctional chemicals / 生物工程学报

The synthesis of fine chemicals using multi-enzyme cascade reactions is a recent hot research topic in the field of biocatalysis. The traditional chemical synthesis methods were replaced by constructing in vitro multi-enzyme cascades, then the green synthesis of a variety of bifunctional chemicals can be achieved. This article summarizes the construction strategies of different types of multi-enzyme cascade reactions and their characteristics. In addition, the general methods for recruiting enzymes used in cascade reactions, as well as the regeneration of coenzyme such as NAD(P)H or ATP and their application in multi-enzyme cascade reactions are summarized. Finally, we illustrate the application of multi-enzyme cascades in the synthesis of six bifunctional chemicals, including ω-amino fatty acids, alkyl lactams, α, ω-dicarboxylic acids, α, ω-diamines, α, ω-diols, and ω-amino alcohols.

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)

Formate dehydrogenase and its application in biomanufacturing of chiral chemicals / 生物工程学报

The redox biosynthesis system has important applications in green biomanufacturing of chiral compounds. Formate dehydrogenase (FDH) catalyzes the oxidation of formate into carbon dioxide, which is associated with the reduction of NAD(P)+ into NAD(P)H. Due to this property, FDH is used as a crucial enzyme in the redox biosynthesis system for cofactor regeneration. Nevertheless, the application of natural FDH in industrial production is hampered by low catalytic efficiency, poor stability, and inefficient coenzyme utilization. This review summarized the structural characteristics and catalytic mechanism of FDH, as well as the advances in protein engineering of FDHs toward improved enzyme activity, catalytic efficiency, stability and coenzyme preference. The applications of using FDH as a coenzyme regeneration system for green biomanufacturing of chiral compounds were summarized.

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)

Design and assembly of bio-manufacturing "cell factory" / 生物工程学报

The chemical manufacturing industry that uses fossil resources as raw materials, consumes non-renewable resources and also causes damage to the ecological environment, stimulating the development of bio-manufacturing with renewable resources as raw materials. Unlike traditional chemical manufacturing, bio-manufacturing uses cells as a "production workshop", and each process in the "workshop" is catalyzed by enzymes. In addition to mild reaction conditions, the "cell factory" has strong plasticity, and can be used to synthesize various target chemicals according to demand adjustment or reconstitution of metabolic pathways. The design process of the "cell factory" follows the following guidelines: 1) Construct an optimal synthetic route from raw materials to products; 2) Balance the metabolic flux of each reaction in the metabolic pathway, so that the metabolic flux of this pathway is much higher than the primary metabolism of the cells; 3) Precursor supply in the pathway should be sufficient, and adjust multiple precursors supply ratio as needed; 4) enzymatic reactions often involve the participation of various cofactors, smooth metabolic pathways need to balance or regenerate various cofactors; 5) Through genetic modification or process improvement to remove metabolic intermediates and products feedback inhibition to achieve higher yields.

A Case of Neonatal Cerebral Infarction associated with Mutation in homozygous MTHFR C677T Gene / 대한주산의학회잡지

C677T mutation in the gene encoding 5,10-methylenetetrahydrofolate reductase (MTHFR) predisposes to hyperhomocysteinemia in vivo and is known to be one of the causes of perinatal ischemic stroke. As MTHFR plays a role in the metabolism of homocysteine, C677T mutation may account for reduced enzymatic activity resulting in hyperhomocysteinemia. This may be prevented by introducing activity-enhancing coenzymes such as folic acid, vitamin B6, and B12. Though C677T mutation is known as a significant risk factor for cerebral infarction, reported cases of cerebral infarction among affected neonates are scarce. This report describes a case of a neonate homozygous for C677T mutation who had a perinatal ischemic stroke, born in a mother whose folic acid and nutritional consumption had been reduced during pregnancy.

Práticas alimentares no primeiro ano de vida: representações sociais de mães adolescentes / Eating habits in the first year of life: social representations of young mothers / Los hábitos de alimentación en el primer año de vida: las representaciones sociales de las madres adolescentes

Trata-se de pesquisa qualitativa exploratória, que buscou analisar as representações sociais de mães adolescentes sobre as práticas alimentares do filho no primeiro ano de vida. Teve como sujeitos 10 mães adolescentes, cujos filhos encontravam-se na faixa etária de sete a doze meses de vida. Os dados foram coletados por meio de entrevista semi-estruturada, além da utilização de material visual. A análise seguiu a técnica de análise de conteúdo, apoiando-se no referencial da Teoria das Representações Sociais. Dessa análise, emergiram quatro temas: conflito do amamentar versus consagração do mingau; estabelecendo a alimentação complementar do filho; discurso cristalizado: "danoninho vale mais que um bifinho"; a (in)definição dos hábitos alimentares maternos: implicações para a alimentação infantil. As representações que conduzem as práticas maternas na escolha, preparo e oferta dos alimentos seguem uma lógica particular, onde as adolescentes reinterpretam os discursos técnicos nos termos da sua cultura.

This is qualitative research that investigates the social representations of adolescent mothers on child eating habits in the first year of life. Its subjects were 10 adolescent mothers, whose children were aged seven to twelve months. Data were collected through semi-structured interview, besides the use of visual material. The analysis followed the technique of content analysis, relying on the framework of Social Representations Theory. That analysis revealed four themes: the conflict of breastfeeding versus consecration of porridge; establishing complementary feeding of the child; crystallized speech: "yogurt is better than a little steak"; the (un)definition of maternal eating habits: implications for infant feeding. The representations that drive maternal practices in selecting, preparing and offering food follow a particular logic, where adolescents reinterpret technical speeches in terms of their culture.

Esta es una investigación cualitativa que investiga las representaciones sociales de madres adolescentes en las prácticas de alimentación infantil en el primer año de vida. El estudio incluyó a 10 madres adolescentes cuyos hijos tenían entre siete a doce meses. Los datos fueron obtenidos mediante entrevista semiestructurada, además de la utilización de material visual. El análisis se realizó por la técnica de análisis de contenido, basándose en el marco de la teoría de las representaciones sociales. Ese análisis revelo cuatro temas: el conflicto de la lactancia materna frente a la consagración de la papilla; el establecimiento de alimentación complementaria del niño; discurso cristalizado "el yogur es mejor que un filete"; la dieta materna sin definición: implicaciones para la alimentación infantil. Las representaciones que impulsan prácticas maternas en la selección, preparación y oferta de alimentos siguen una lógica particular, donde las adolescentes reinterpretan intervenciones técnicas en términos de su cultura.

Representações sociais de educação em saúde em tempos de AIDS / Social representations of health education in a time of AIDS / Representaciones sociales de educación para la salud en la era del SIDA

Estudo qualitativo e descritivo, cujo objetivo foi identificar e analisar as representações sociais de educação em saúde à pessoa vivendo com HIV entre profissionais de saúde. Os cenários foram três serviços de atenção à DST/HIV/AIDS, em Belém-PA, Brasil, e 37 profissionais de saúde participaram da pesquisa. A coleta de dados deu-se em 2012-2013 por meio de entrevista em profundidade; a análise utilizou o software Alceste 4.10. Com base no conjunto dos resultados foi possível vislumbrar que a educação em saúde pode ser compreendida a partir de categorias: a configuração do agir educativo; as condições sine qua non: educação no trabalho e estrutura da unidade; o processo pedagógico. Conclui-se que as representações sociais configuram-se como orientação-informação para precaução-prevenção e revelam-se no movimento do agir persistente ao emergente, o que suscita uma educação em saúde permanente para se chegar à integralidade nos serviços.

This is a qualitative and descriptive study, which aimed at identifying and analyzing social representations of health education to HIV patients among health professionals. The setting included three healthcare DST/HIV/AIDS services in Belém-PA, Brazil, and 37 health professionals participated in the study. Data collection was conducted in 2012-2013 on the basis of in-depth interviews and analysis was made on Alceste 4.0 software. Final results indicated that health education can be comprehended in light of categories: educational action; sine qua non: education and training at work, and unit structure; teaching-learning process. Conclusions show that social representations are set as guidance-information for precaution-prevention and that they come forth along continuous and emerging action flow, bringing about permanent health education to ensure healthcare services in full.

Estudio cualitativo y descriptivo, que objetivó identificar y analizar las representaciones sociales de educación en salud a la persona viviendo con HIV entre profesionales de salud. Los escenarios fueron tres servicios de atendimiento al DST/HIV/ SIDA, en Belém-PA, Brasil, y 37 profesionales de salud participaran del estudio. La colecta de datos se dio en 2012-2013, por medio de entrevista en profundidad y el análisis utilizo el software Alceste 4.10. Con base en el conjunto de los resultados fue posible vislumbrar que la educación en salud puede ser comprendida a partir de categorías: la configuración del acto educativo; las condiciones sine qua non: educación en el trabajo y estructura de la unidad; el proceso pedagógico. Se concluye que las representaciones sociales se configuran como orientación-información para precaución-prevención y se revelan en el movimiento del acto persistente al emergente, lo que suscita una educación en salud permanente para llegarse a la integralidad en los servicios.

Determination of a method for extraction of coenzyme Q10 in human plasma: optimization of the use of surfactants and other variables / Determinação de um método de extração de coenzima Q10 em plasma humano: otimização do uso de surfactantes e outras variáveis

Objective: To establish a routine for the extraction of the total levels of CoQ10 in human plasma through the Ultra High Performance Liquid Chromatography (UHPLC). Methods: Two extraction protocols were tested: a) methanol: hexane and b) 1-propanol. The following parameters were analyzed: extraction temperature (19ºC and 4ºC), extraction tubes (glass and polypropylene), and surfactants (SDS, Triton X-100, Tween-20) at different concentrations, i.e., 1%, 3%, 5% and 10%. Results: The results showed that the method of extraction of CoQ10 in a sample of human plasma at 4ºC, using solvents methanol: hexane (85:15, v/v) in the presence of surfactant Tween-20 at 3% and polypropylene tubes showed better efficiency and reproducibility when compared to the method with 1-propanol. Conclusion: By the analyses performed, it was possible to observe that the addition of the surfactant Tween-20 promoted an increase in the recovery of CoQ10 by the methanol:hexane extraction method. This method showed good reproducibility, with a low coefficient of variation and high sensitivity, since CoQ10 was detected in samples of plasma of a control individual using a UV-type detector. The use of UHPLC equipment allowed a total analysis with total run time of 3.5 minutes, enabling the rapid achievement of results, considered mandatory for laboratory routines.

Objetivo: Estabelecer uma rotina de extração dos níveis totais de CoQ10 em plasma humano por meio da análise por Cromatografia Líquida de Ultra Alta Eficiência (UHPLC). Métodos: foram testados dois protocolos de extração: a) metanol:hexano e b) 1-propanol. Os seguintes parâmetros foram analisados: temperatura de extração (19ºC e 4ºC), tubos de extração (vidro e polipropileno), surfactantes (SDS, Triton X-100, Tween-20) em diferentes concentrações 1%, 3%, 5% e 10%. Resultados: Os resultados mostraram que o método de extração de CoQ10 em amostra de plasma humano, a 4ºC, utilizando-se os solventes metanol:hexano (85:15, v/v) na presença do surfactante Tween-20 a 3% e tubos de polipropileno apresentou melhor eficiência e reprodutibilidade quando comparado ao método com 1-propanol. Conclusão: A adição do surfactante Tween-20 no processo de preparação de amostra promoveu um aumento na recuperação da CoQ10 pelo método de extração metanol:hexano observada pela boa reprodutibilidade das prelicatas, pelo baixo coeficiente de variação e alta sensibilidade uma vez que a CoQ10 foi detectada em amostras de plasma de um indivíduo controle utilizando-se um detector do tipo UV. Além disso, a utilização de um equipamento de UHPLC proporcionou a obtenção de uma análise com tempo total de corrida de 3,5 minutos, o que viabiliza a obtenção rápida de resultados, considerado mandatório para rotinas laboratoriais.

Metallo beta lactamase producing Pseudomonas aeruginosa and Acinetobacter baumannii.

This study aims in identifying MBLs particularly Zn requiring Molecular Class B enzymes produced by Pseudomonas aeruginosa and Acinetobacter baumannii .The resistance by these organisms are in a rise against all antibiotics including carbapenems and no prescribed CLSI guidelines is available for detecting them. Clinical isolates antibiotic susceptibility was determined by number of phenotypic tests by addition of 50mM of 10 μl zinc as cofactor for metallo beta lactamase production along with 0.5M ETDA of 5μl (930 μg per disk) plain disks. Increase in zone size of the meropenem -EDTA disk compared to the meropenem disk without EDTA was recorded positive. For Zn requiring MBLs zone towards both disks of EDTA and Zn along with meropenem is detected by DDST.

Zinc-dependent carbapenemases in clinical isolates of family Enterobacteriaceae.

Purpose: The emergence and spread of zinc-dependent carbapenem resistance has become a diagnostic challenge for clinical microbiologists. The objective of the present study was to screen zinc-dependent carbapenemase activity in clinical isolates of family Enterobacteriaceae. Materials and Methods: A total of 102 multidrug-resistant organisms (MDROs), non-repeat clinical isolates of family Enterobacteriaceae from two tertiary care centres in Delhi, were screened for carbapenemase production by a modified Hodge test (MHT) and additionally by a re-modified Hodge test, EDTA double disc synergy test, and combined disc test (or disc enhancement test) to determine zinc dependence of carbapenemases harbouring bacteria. Results: Of the total 102 clinical isolates (June through November 2010), 91 were from urine and 11 were from blood specimens. The isolates were obtained from patients visiting the outpatient department (18 isolates), admitted in non-ICU inpatient care units (74 isolates) and patients admitted in ICUs (4 isolates). MHT identified 92 (90.2%) isolates as carbapenemases producers. Among those found negative for MHT (n=10), metallo-beta-lactamases (MBLs) activity was demonstrated through the EDTA disc diffusion synergy test and the combined disc test in 8 and 9 isolates respectively. A total of 63 (61.7%) isolates demonstrated MBL activity despite in vitro sensitivity to Imipenem. Conclusions: The study demonstrated that supplementing the MHT with at least one of the screening methods increases the likelihood of picking up such isolates that may be missed by the MHT. The study also demonstrates the wide-spread presence of MBLs in Enterobacteriaceae members from patients visiting hospitals in east Delhi.

Zinc-dependent carbapenemases in clinical isolates of family Enterobacteriaceae.

Purpose: The emergence and spread of zinc-dependent carbapenem resistance has become a diagnostic challenge for clinical microbiologists. The objective of the present study was to screen zinc-dependent carbapenemase activity in clinical isolates of family Enterobacteriaceae. Materials and Methods: A total of 102 multidrug-resistant organisms (MDROs), non-repeat clinical isolates of family Enterobacteriaceae from two tertiary care centres in Delhi, were screened for carbapenemase production by a modified Hodge test (MHT) and additionally by a re-modified Hodge test, EDTA double disc synergy test, and combined disc test (or disc enhancement test) to determine zinc dependence of carbapenemases harbouring bacteria. Results: Of the total 102 clinical isolates (June through November 2010), 91 were from urine and 11 were from blood specimens. The isolates were obtained from patients visiting the outpatient department (18 isolates), admitted in non-ICU inpatient care units (74 isolates) and patients admitted in ICUs (4 isolates). MHT identified 92 (90.2%) isolates as carbapenemases producers. Among those found negative for MHT (n=10), metallo-beta-lactamases (MBLs) activity was demonstrated through the EDTA disc diffusion synergy test and the combined disc test in 8 and 9 isolates respectively. A total of 63 (61.7%) isolates demonstrated MBL activity despite in vitro sensitivity to Imipenem. Conclusions: The study demonstrated that supplementing the MHT with at least one of the screening methods increases the likelihood of picking up such isolates that may be missed by the MHT. The study also demonstrates the wide-spread presence of MBLs in Enterobacteriaceae members from patients visiting hospitals in east Delhi.

Effects of carnitine and coenzyme Q10 on lipid profile and serum levels of lipoprotein[a] in maintenance hemodialysis patients on statin therapy

Dyslipidemia and high serum lipoprotein[a] are among the risk factors for cardiovascular diseases in hemodialysis patients. Statins as a first line of therapy in hyperlipidemia does not always reduce the serum lipoprotein[a] level. Several studies have reported the lipid-lowering effects of carnitine and coenzyme Q10 in hemodialysis patients. This study was designed to investigate the effects of carnitine and coenzyme Q10 on serum lipid profile and lipoprotein[a] level in maintenance hemodialysis patients. This was a randomized placebo-controlled trial. We studied on hemodialysis patients who were on treatment with atorvastatin or lovastatin to assess the efficacy of supplement therapy. They were divided into 4 groups to receive carnitine, coenzyme Q10, both carnitine and coenzyme Q10, and placebo. After a 3-month experiment, blood samples were collected to measure serum levels of lipoprotein[a], triglyceride, total cholesterol, high-density lipoprotein cholesterol and low-density lipoprotein cholesterol. Fifty-two hemodialysis patients, 27 men and 25 women, completed the course of the study. Three months after supplement therapy, serum levels of lipoprotein[a] reduced significantly in the carnitine, coenzyme Q10, and combination groups compared to the baseline values and the 3-month value of lipoprotein[a] in the placebo group [P = .01]. Serum levels of triglyceride and other lipoproteins did not significantly alter. Our study showed that supplementation with carnitine and coenzyme Q10 could reduce serum levels of lipoprotein[a] in maintenance hemodialysis patients treated with statins

Biotin biosynthesis in Mycobacterium tuberculosis: physiology, biochemistry and molecular intervention

Biotin is an important micronutrient that serves as an essential enzyme cofactor. Bacteria obtain biotin either through de novo synthesis or by active uptake from exogenous sources. Mycobacteria are unusual amongst bacteria in that their primary source of biotin is through de novo synthesis. Here we review the importance of biotin biosynthesis in the lifecycle of Mycobacteria. Genetic screens designed to identify key metabolic processes have highlighted a role for the biotin biosynthesis in bacilli growth, infection and survival during the latency phase. These studies help to establish the biotin biosynthetic pathway as a potential drug target for new anti-tuberculosis agents.

Effect of concomitant lycopene biosynthesis on CoQ[10] accumulation in transformed Escherichia coli strains

CoQ[10] and lycopene are isoprenoid compounds with nutraceutical and pharmaceutical benefits. In this study, the effect of concomitant lycopene biosynthesis on CoQ[10] accumulation in transformed Escherichia coli DH5 alpha was studied. A lycopene production pathway including geranyl diphosphate synthase [crtE], phytoene synthase [crtB], and phytoene desaturase [crtI] from Erwinia herbicola was constructed in two CoQ[10]-producing E. coli strains. E. coli Ba and E. coli Br containing dds orthologs encoding for decaprenyl diphosphate synthase [Dds], respectively from Agrobacterium tumefaciens and Rhodobacter sphaeroides were transformed by the lycopene pathway resulting in E. coli Ba-lyc and E. coli Br-lyc. The lycopene pathway in E. coli Br-lyc interestingly resulted in a significant increase in CoQ[10] production from 564 +/- 28 to 989 +/- 22 micro g /g DCW. To confirm that the improvement of CoQ[10] production in E. coli Br-lyc was due to lycopene biosynthesis and not just geranylgeranyl diphosphate formation in the lycopene pathway, crtE was only introduced into E. coli Ba and E. coli Br strains. Surprisingly, crtE expression had adverse effects on CoQ[10] production in both strains. The results shed light on the Dds-catalyzed reaction as a bottleneck controlled by precursors; and the efficiency of a parallel lycopene pathway to streamline the flow of metabolites

Mutational research on the role of lysine 21 in the Pichia stipitis xylose reductase / 生物工程学报

The xylose reductase of Pichia stipitis is one of the most important enzymes. It can be used to build up recombinant Saccharomyces cerevisiae strain for utilizing xylose and producing ethanol. Intercellular redox imbalance caused by NADPH preference over NADH for Pichia stipitis xylose reductase (PsXR) has been considered to be one of the main factors for poor ethanol productivity. Some key amino acids of PsXR, which affect the activity or coenzyme preference, were investigated in our previous study. In this study, Lys21 were rational designed for site-directed mutagenesis to alter coenzyme specificity of PsXR from NADPH and NADH into single NADH. The wild gene and mutagenesis genes were ligated into pET28b, and were transferred into E.coli BL21(DE3). After induced by IPTG, the xylose reductases were purified. Purified mutants K21A (Lys21-->Ala), K21R(Lys21-->Arg) were characterized by steady-state kinetic analysis. The results showed that the coenzyme dependence of K21A was completely reversed to NADH.

Predicting the cofactors of oxidoreductases by the modified pseudo-amino acid composition / 生物工程学报

Types of cofactor independency for newly found oxidoreductases sequences are usually determined by experimental analysis. These experimental methods are both time-consuming and costly. With the explosion of oxidoreductases sequences entering into the databanks, it is highly desirable to explore the feasibility of selectively classifying newly found oxidoreductases into their respective cofactor independency classes by means of an automated method. In this study, we proposed a modified Chou's pseudo-amino acid composition method to extract features from sequences and the k-nearest neighbor was used as the classifier, and the results were very encouraging. When lambda = 48, w = 0.1, the areas under the ROC curve of k-nearest neighbor in 10-fold cross-validation was 0.9536; and the success rate was 92.0%, which was 3.5% higher than that of pseudo-amino acid composition. It was also better than all the other 7 feature extraction methods. Our results showed that predicting the cofactors of oxidoreductases was feasible and the modified pseudo-amino acid composition method may be a useful method for extracting features from protein sequences.

Serum and synovial fluid concentrations of soluble granzyme B in early rheumatoid arthritis

Cytotoxic cells possess specialized granules which contain perform and a group of serine proteinases termed granzymes. Granzyme B is a serine protease that have been identified in synovial fluid and tissue of rheumatoid arthritis [RA] patients, where they may play an important role as mediators of granule-mediated apoptosis, extracellular proteolysis, and cytokine induction. To assess plasma and synovial fluid soluble granzyme B concentrations in early RA patients and its association to seropositivity, ESR, C-reactive protein [CRP], disease activity and the rate of radiological progression. This was in an attempt to throw light on its possible role as predictive factor for disease severity and joint outcome. This study included 80 early RA patients presenting with knee effusion and 30 matched osteoarthritis [OA] patients presenting with traumatic non-hemorrhagic knee synovial effusion as a control group. Serum and synovial fluid granzyme B concentrations were determined with ELISA. Assessment of disease activity. Rheumatoid factor [RF], CRP and ESR were measured. Radiographs of the hands, wrists and forefeet were taken for all RA patients at baseline and after one year. Radiographical damage and scoring were evaluated. There was a highly significant increase in serum and synovial fluid concentrations of soluble granzyme B in RA patients as compared to OA control group. Also, there was a highly significant increase in synovial fluid concentrations of granzyme B as compared to serum concentrations in RA patients. There was a highly significant increase in serum and synovial fluid concentrations of granzyme B in seropositive RA patients as compared to seronegative patients. There were highly significant elevations in the mean serum and SF granzyme B concentrations at the entry of the study in patients showing radiological progression than non-progressive group. There was a statistically significant positive correlation between serum granzyme B concentration and CRP and a highly significant positive correlation as compared with disease duration, number of swollen joints, Ritchie articular index [RAI], general health status, ESR, disease activity score, RF, radiological progression or granzyme B synovial fluid concentration. High soluble granzyme B concentrations are found in early rheumatoid arthritis patients. Increase in serum concentrations of granzyme B in seropositive RA patients as compared to seronegative patients and increased concentrations associated with the development of radiographic erosions in those patients raise the attention to the importance of granzyme R as a marker for prediction of radiographic joint damage. So granzyme B concentration can be considered as a useful prognostic marker in early rheumatoid arthritis patients

Suppression of azoxymethane-induced colonic premalignant lesion formation by coenzyme Q10 in rats.

Reactive oxygen species cause damage to proteins, lipids and DNA. Coenzyme Q10 (CoQ10) is a compound with mitochondrial bioenergetic functions. The reduced form of CoQ10 shows antioxidant activity. In the present study, effects of CoQ10 on development of azoxymethane (AOM)-induced aberrant crypt foci (ACF) and mucin-depleted foci (MDF) in F344 male rats were investigated. To induce ACF and MDF, 6-week old rats were given two weekly subcutaneous injections of AOM (15 mg/kg body weight) and also received a control diet or experimental diets containing CoQ10 (200 or 500 ppm) for 4 weeks, starting one day before the first dose of AOM. At 10 weeks of age, all animals were sacrificed and their colons were evaluated for numbers and sizes of ACF and MDF. Administration of 200 and 500 ppm CoQ10 resulted in reduction of ACF numbers, to 77% and 68% of the carcinogen control value, respectively. The percentages of ACF consisting of more than 4 crypts in these groups were also significantly lower than in the controls. Treatment with 500 ppm CoQ10 furthermore decreased the number of sialomucin-producing ACF and MDF per colon to 42% and 38% of the carcinogen control value without CoQ10, respectively. These results suggest that CoQ10 may be an effective chemopreventive agent against colon carcinogenesis.

Modification of biochemical parameters of gentamicin nephrotoxicity by coenzyme Q10 and green tea in rats.

The present study was designed to investigate the possible potential protective role of coenzymeQ10 (CoQ10; 10 mg/kg/day, ip) and/or green tea (GT; 25mg/kg/day, po) against gentamicin (GM) nephrotoxicity. Marked increase in the level of serum urea. creatinine and lipid peroxidation (LPO) content was found after administration of gentamicin (80 mg/kg/day, ip) for eight days along with significant decrease in the antioxidant enzymes, superoxide dismutase (SOD), reduced glutathione (GSH), catalase (CAT) as well as brush border enzymes (Na+/K+ ATPase, Mg(+2)ATPase and Ca2+ ATPase).Treatment with CoQ10 or green tea alone with GM showed significant decrease in serum urea, creatinine and tissue LPO content and significant increase in antioxidant and membrane bound enzymes. Combined treatment with CoQ10 and green tea was more effective in mitigating adverse effect of GM nephrotoxicity. The present work indicated that CoQ10 and green tea due to their antioxidant activity modified the biochemical changes occurred during gentamicin nephrotoxicity and thus had a potential protective effect.