An enzymatic alternative for the synthesis of nucleoside 5'-monophosphates.

A new procedure was carried out for the synthesis of nucleoside 5'-monophosphates, involving the use of two enzymes. The first step applied phospholipase D from Streptomyces netropsis and phosphatidylcholine as phosphatidyl donor, to give 5'-(3-sn-phosphatidyl) nucleosides (C, U, A, I). These were selectively hydrolysed in the second step by the action of phospholipase C from Bacillus cereus to produce the respective 5'-nucleotides. Application of this methodology on a preparative scale conducted to 5'-adenosine monophosphate in 63% overall yield from adenosine. The regioselectivity of these enzymes avoids protection steps, the overall synthesis is performed under mild reaction conditions and product isolation is easily achieved.

Genome sequence of Perigonia lusca single nucleopolyhedrovirus: insights into the evolution of a nucleotide metabolism enzyme in the family Baculoviridae.

The genome of a novel group II alphabaculovirus, Perigonia lusca single nucleopolyhedrovirus (PeluSNPV), was sequenced and shown to contain 132,831 bp with 145 putative ORFs (open reading frames) of at least 50 amino acids. An interesting feature of this novel genome was the presence of a putative nucleotide metabolism enzyme-encoding gene (pelu112). The pelu112 gene was predicted to encode a fusion of thymidylate kinase (tmk) and dUTP diphosphatase (dut). Phylogenetic analysis indicated that baculoviruses have independently acquired tmk and dut several times during their evolution. Two homologs of the tmk-dut fusion gene were separately introduced into the Autographa californica multiple nucleopolyhedrovirus (AcMNPV) genome, which lacks tmk and dut. The recombinant baculoviruses produced viral DNA, virus progeny, and some viral proteins earlier during in vitro infection and the yields of viral occlusion bodies were increased 2.5-fold when compared to the parental virus. Interestingly, both enzymes appear to retain their active sites, based on separate modeling using previously solved crystal structures. We suggest that the retention of these tmk-dut fusion genes by certain baculoviruses could be related to accelerating virus replication and to protecting the virus genome from deleterious mutation.

Equações estruturais aplicadas a modelos causais de câncer de pulmão / Structural equation models applied to lung cancer causal models

Introdução: O câncer de pulmão (CP) é o tipo de câncer que mais mata no mundo e o cigarro ainda é sua causa mais importante. Além disso, a alimentação tem sido associada ao CP, por ser fonte de vitaminas e aminoácidos que fazem parte do metabolismo do carbono (MC). O MC é considerado mecanismo chave na manutenção da integridade do DNA e na regulação da expressão gênica, que, dessa forma, deve estar relacionado à carcinogênese. A ativação da imunidade está associada ao envelhecimento em indivíduos saudáveis, assim como a uma série de patologias, incluindo o câncer. Objetivo: Estudar como o MC, a ativação da imunidade e o tabaco estão relacionados ao risco de CP em um estudo caso-controle aninhado à coorte do EPIC (European Prospective Investigation into Cancer and Nutrition). Métodos: Para avaliar se os níveis plasmáticos de cotinina são um bom biomarcador da exposição ao tabaco, foram utilizados modelos lineares generalizados. Para avaliar os efeitos do tabaco, do MC e da ativação da imunidade no risco de CP, foram aplicados modelos de equações estruturais (MEE) de duas maneiras diferentes (com e sem variáveis latentes).Resultados: Com base nas respostas aos questionários de qualidade de vida, com relação às questões sobre fumo ativo e passivo, a cotinina se mostrou um bom biomarcador de exposição recente ao tabaco (tanto o aumento da exposição passiva quanto ativa foram significativas, P<0,001 e P<0,001 respectivamente). Em um MEE com variáveis observadas, incluindo o MC e a via de ativação da imunidade, a metionina e o folato como causas proximais apresentaram uma forte e inversa associação com o risco de CP. O aumento em um desvio-padrão nos níveis séricos de metionina e de folato significou uma redução no risco de 19 por cento (P<0,01) e 12 por cento (P=0,03) respectivamente...

Background: Lung cancer (LC) continues to be the most common cancer death in the world. Tobacco exposure continues to be the most important cause. In addition, micronutrient intake has been linked to LC, because they are the main source of vitamins and amino acids involved in the one-carbon metabolism (OCM) which is considered key in maintaining DNA integrity, regulating gene expression, and may thus affect carcinogenesis. Immune activation is involved in the aging process in normal healthy individuals as well as in a number of pathologies, including cancer. Objectives: To investigate how OCM, immune activation and tobacco are related to LC incidence in a nested case-control study from the European Prospective Investigation into Cancer and Nutrition (EPIC) cohort. Methods: To validate plasma cotinine levels as a good biomarker for tobacco exposure, a generalized linear model was applied. To evaluate the effects of tobacco, OCM and immune activation in LC, structural equation models (SEM) were applied in two different ways. Results: Based on questions about smoking, passive smoking and number of cigarettes smoked, it was shown that cotinine is a good biomarker for tobacco exposure (passive and active exposure with significant relation, p<0.001 and P<0.001, respectively). In a SEM model with only observed variables, including OCM and immune activation, methionine and folate as proximal causes presented a strong and inverse relation with LC risk. An increase in one standard deviation of serum levels of methionine and folate meant a 19 per cent (P<0.01) and 12 per cent (P<0.01) reduction in LC risk, respectively...

UDP-galactose 4-epimerase: a key enzyme in exopolysaccharide formation by Lactobacillus casei CRL 87 in controlled pH batch cultures.

AIMS: To evaluate the relationship between exopolysaccharide (EPS) production and the sugar nucleotide biosynthetic enzymes in Lactobacillus casei CRL 87 under optimum growth conditions for polymer formation: controlled pH on galactose or glucose. Studies with an EPS mutant were carried out to determine the key enzymes in EPS synthesis under the above culture conditions. METHODS AND RESULTS: EPS concentration was estimated by the phenol/sulphuric acid method, while the activities of the biosynthetic enzymes were determined spectrophotometrically by measuring the formation or disappearance of NAD(P)H at 340 nm. An environmental pH of 5.0, using galactose as carbon source, markedly improved not only polymer production and yield but also, cell growth and lactic acid production. Analysis of the activities of the EPS precursor-forming enzymes revealed that polysaccharide synthesis was correlated with uridine-diphosphate (UDP)-glucose pyrophosphorylase and UDP-galactose 4-epimerase under these growth conditions. CONCLUSIONS: EPS synthesis by Lact. casei CRL 87 was considerably improved at a controlled pH of 5.0 with galactose as carbon source, and was correlated with the activity of UDP-glucose pyrophosphorylase and UDP-galactose 4-epimerase. The results obtained with the wild-type and EPS- strains suggest that UDP-galactose 4-epimerase plays an essential role in EPS formation. SIGNIFICANCE AND IMPACT OF THE STUDY: Unravelling the key enzymes involved in EPS biosynthesis under optimum culture conditions for polymer production provides important information for the design of strategies, via genetic engineering, to enhance polysaccharide formation.

Los nucleótidos como nutrimentos ¿Son semiesenciales para los neonatos? / The nucleotides. It is essentials for neonates?

La adición de nucleótidos a las fórmulas de iniciación se ha hecho en base al papel que juegan estos compuestos químicos en las diferentes facetas del proceso de maduración del niño, durante los primeros meses de la vida. En este informe se revisan los estudios en que fundamenta la decisión de incluirlos en las fómulas lácteas