Glucose-6-Phosphate dehydrogenase deficiency in children from 0 to 14 years hospitalized at the Pediatric Hospital David Bernardino, Luanda, Angola

The Glucose-6-phosphate dehydrogenase (G6PD) deficiency is the most common enzymatic defect in the world. The most common clinical manifestations are acute hemolytic anemia associated with drugs, infections, neonatal jaundice and hemolytic non-spherocytic chronic anemia. The main aim of this study was to determine the frequency of major genetic variants of G6PD leading to enzyme deficiency in children from 0 to 14 years at a Pediatric Hospital in Luanda, Angola. A cross-sectional and descriptive analytical study covered a total of 194 children aged from 0 to 14 years, of both genders and hospitalized at the Pediatric Hospital David Bernardino, Luanda between November and December, 2011. The G202A, A376G and C563T mutations of the G6PD gene were determined by real-time PCR with Taqman probes. The disabled A-/A- genotype was detected in 10 girls (10.9%). Among the boys, 21 (20.6%) presented the genotype A-. Considering all the samples, the A- variant was observed in 22.4% of cases. The Mediterranean mutation was not detected in the Angolan sample. Furthermore, no association was found between genotype and anemia, nutritional state and mucosa color. A significant association, however, was observed with jaundice. Based on the results obtained, there is a clear need to identify those with the disabled genotype in the Angolan population in order to avoid cases of drug-induced anemia, particularly in the treatment of malaria, so prevalent in Angola.

Viscerocutaneous form of loxoscelism and erythrocyte glucose-6-phosphate deficiency

In a period of time of five years, all patients who exhibited viscerocutaneous form of loxoscelism were investigated for erythrocyte glucose-6-phosphate deficiency, and in two patients out of seven it was found this deficiency. This finding suggests that this genetical enzyme deficiency could account for the hemolysis after Loxosceles bite, at least in some of the cases.

The expression in saccharomyces cerevisiae of a glucose/xylose symporter from Candida intermedia is affected by the presence of a glucose/xylose facilitator

Microbiology, 154

Glucose-6-phosphate dehydrogenase and glutathione reductase activity in methemoglobin reduction by methylene blue and cyst amine: study on glucose-6-phosphate dehydrogenase-deficient individuals, on normal subjects and on riboflavin-treated subjects

The authors have standardized methods for evaluation of the activity of the glucose-6-phosphate dehydrogenase and of glutathione reductase. The general principle of the first method was based on methemoglobin formation by sodium nitrite followed by stimulation of the glucose-6-phosphate dehydrogenase with methylene blue. Forty six adults (23 males and 23 females) were studied. Subjects were not G6PD deficient and were aged 20 to 30 years. The results showed that methemoglobin reduction by methylene blue was 154.40 and 139.90 mg/min (p<0.05) for males and females, respectively, in whole blood, and 221.10 and 207.85 mg/min (n.s.), respectively, in washed red cells. These data showed that using washed red cells and 0.7g% sodium nitrite concentration produced no differences between sexes and also shortened reading time for the residual amount of methemoglobin to 90 minutes. Glutathione reductase activity was evaluated on the basis of the fact that cystamine (a thiol agent) binds to the SH groups of hemoglobin, forming complexes. These complexes are reversed by the action of glutathione reductase, with methemoglobin reduction occurring simultaneously with this reaction. Thirty two adults (16 males and 16 females) were studied. Subjects were not G6PD deficient and were aged 20 to 30 years. Methemoglobin reduction by cystamine was 81.27 and 91.13 mg/min (p<0.01) for males and females, respectively. These data showed that using washed red cells and 0.1 M cystamine concentration permits a reading of the residual amount of methemoglobin at 180 minutes of incubation. Glutathione reductase activity was evaluated by methemoglobin reduction by cystamine in 14 females before and after treatment with 10 mg riboflavin per day for 8 days. The results were 73.69 and 94.26 jug/min (p<0.01) before and after treatment, showing that riboflavin treatment increase glutathione reductase activity even in normal individuals. Three Black G6PD-deficient individuals (2 males and 1 female) were also studied. The G6PD and glutathione reductase were partially activated, the change being more intense in males. On the basis of race and of the laboratory characteristics observed, it is possible to suggest that the G6PD deficiency of these individuals is of the African type and that the female is heterozygous for this deficiency. Analysis of the results as a whole permitted us to conclude that the methods proposed here were efficient for evaluating the activity of the glucose-6-phosphate dehydrogenase and of glutathione reductase. The latter is dependent on the pentose pathway, which generates NADPH, and on riboflavin, a FAD precursor vitamin.

Structure to function studies in UDP-glucose dehydrogenases and nitroreductases

The thesis is divided into two parts corresponding to structural studies on two different proteins. The first part concerns the study of two UDP-glucose dehydrogenases (UGDs) from Sphingomonas elodea ATCC 31461 and Burkholderia cepacia IST 408, both involved in exopolysaccharide production. Their relevance arises because some of these bacterial exopolysaccharides are valuable as established biotechnological products, the former case, whilst others are highly problematic, when used by pathogens in biofilm formation over biological surfaces, as the latter case, namely in the human lungs. The goal of these studies is to increase our knowledge regarding UGDs structural properties, which can potentiate either the design of activity enhancers to respond to the increased demand of useful biofilms, or the design of inhibitors of biofilm production, in order to fight invading pathogens present in several infections. The thesis reports the production and crystallisation of both proteins, the determination of initial phases by single-wavelength anomalous dispersion (SAD) in S. elodea crystals using a seleno-methionine isoform, and phasing of B. cepacia crystals by molecular replacement (MR) using the S. elodea model, as well as the refinement, structural analysis and comparison between the several UGDs structures available during this work.(...)

trans-Acting factors and cis elements involved in glucose repression of arabinan degradation in Bacillus subtilis.

Journal of Bacteriology (Nov 2007) 8371-8376

Non-enzymatic assay for glucose by using immobilized whole-cells of E. coli containing glucose binding protein fused to fluorescent proteins

Glucose monitoring in vivo is a crucial issue for gaining new understanding of diabetes. Glucose binding protein (GBP) fused to two fluorescent indicator proteins (FLIP) was used in the present study such as FLIP-glu- 3.2 mM. Recombinant Escherichia coli whole-cells containing genetically encoded nanosensors as well as cell-free extracts were immobilized either on inner epidermis of onion bulb scale or on 96-well microtiter plates in the presence of glutaraldehyde. Glucose monitoring was carried out by Förster Resonance Energy Transfer (FRET) analysis due the cyano and yellow fluorescent proteins (ECFP and EYFP) immobilized in both these supports. The recovery of these immobilized FLIP nanosensors compared with the free whole-cells and cell-free extract was in the range of 50–90%. Moreover, the data revealed that these FLIP nanosensors can be immobilized in such solid supports with retention of their biological activity. Glucose assay was devised by FRET analysis by using these nanosensors in real samples which detected glucose in the linear range of 0–24 mM with a limit of detection of 0.11 mM glucose. On the other hand, storage and operational stability studies revealed that they are very stable and can be re-used several times (i.e. at least 20 times) without any significant loss of FRET signal. To author's knowledge, this is the first report on the use of such immobilization supports for whole-cells and cell-free extract containing FLIP nanosensor for glucose assay. On the other hand, this is a novel and cheap high throughput method for glucose assay.

Fast-to-fed shift in glucose homeostasis: clues to an earlier detection of human prediabetic states

FCM: UC Bioquímica I - PhD Thesis

Explicit series solution for a glucose-induced electrical activity model of pancreatic beta-cells

In this work, we present the explicit series solution of a specific mathematical model from the literature, the Deng bursting model, that mimics the glucose-induced electrical activity of pancreatic beta-cells (Deng, 1993). To serve to this purpose, we use a technique developed to find analytic approximate solutions for strongly nonlinear problems. This analytical algorithm involves an auxiliary parameter which provides us with an efficient way to ensure the rapid and accurate convergence to the exact solution of the bursting model. By using the homotopy solution, we investigate the dynamical effect of a biologically meaningful bifurcation parameter rho, which increases with the glucose concentration. Our analytical results are found to be in excellent agreement with the numerical ones. This work provides an illustration of how our understanding of biophysically motivated models can be directly enhanced by the application of a newly analytic method.

Alterações de pH após adição de pulsos de glucose a Saccharomyces cerevisiae: estimativa de parâmetros a partir da modelação matemática do comportamento cinéticometabólico

Dissertação para a obtenção do grau de mestre em Bioquímica Estrutural e Funcional

Glucose consume and growth of E. coli under electromagnetic field

E. coli was submitted to a 5G electromagnetic field generated by a alternate 60 Hz voltage source. The differences on growth and glucose consume in control and exposed groups were evaluated using the non-parametric Mann-Whitney U-test. There was a significant difference in glucose consume and growth in E. coli after 8 hours of exposition to electromagnetic field. It can be concluded that electromagnetic field had a positive effect in consume of glucose and growth of E. coli. The cause of these results can be explained by an increasing of glucose entrance through membrane due to the stimulated transport system via Facility Diffusion or cyclotron resonance. The growth can be caused by shortening of lag phase and excitement of log phase.

SPR based Studies for Pentagalloyl Glucose Binding to α-Amylase

Astringency is an organoleptic property resulting mostly from the interaction of salivary proteins with dietary polyphenols. It is of great importance to consumers but being typically measured by sensorial panels it turns out subjective and expensive. The main goal of the present work is to develop a sensory system to estimate astringency relying on protein/polyphenol interactions. For this purpose, a model protein was immobilized on a sensory gold surface and its subsequent interaction with polyphenols was measured by Surface Plasma Resonance (SPR). α-amylase and pentagalloyl glucose (PGG) were selected as model protein and polyphenol, respectively. To ensure specific binding between these, various surface chemistries were tested. Carboxylic terminated thiol decreased the binding ability of PGG and allowed covalent attachment of α-amylase to the surface. The pH 5 was the optimal condition for α-amylase immobilization on the surface. Further studies focus on Localized SPR sensor and application to wine samples, providing objectivity when compared to a trained panel.

Variante Bética da Glucose-6-Fosfato-Desidrogenase: a Propósito de um Caso Clínico

O défice de Glucose-6-fosfato Desidrogenase (G6PD) é, provavelmente, a mutação clinicamente significativa mais frequente a nível mundial, sendo Portugal um país de baixa prevalência (cerca de 0,51%). A G6PD é o enzima que catalisa o primeiro passo na via das pentoses fosfato transformando a glicose- 6- fosfato em 6- fosfogluconato com redução do NADP a NADPH. Apesar de ser expressa em todos os tecidos, a sua deficiência apenas se faz sentir nos eritrocitos, levando a hemólise dos mesmos em situações de stress oxidativo. Já foram descritas mais de 400 variantes da G6PD. Os autores apresentam um caso de uma mulher portadora da variante Bética da G6PD, sendo a doença manifestada por favismo.

Optical measurements of rat muscle samples under treatment with ethylene glycol and glucose

With the objective to study the variation of optical properties of rat muscle during optical clearing, we have performed a set of optical measurements from that kind of tissue. The measurements performed were total transmittance, collimated transmittance, specular reflectance and total reflectance. This set of measurements is sufficient to determine diffuse reflectance and absorbance of the sample, also necessary to estimate the optical properties. All the performed measurements and calculated quantities will be used later in inverse Monte Carlo (IMC) simulations to determine the evolution of the optical properties of muscle during treatments with ethylene glycol and glucose. The results obtained with the measurements already provide some information about the optical clearing treatments applied to the muscle and translate the mechanisms of turning the tissue more transparent and sequence of regimes of optical clearing.

The characteristic time of glucose diffusion measured for muscle tissue at optical clearing

The study of agent diffusion in biological tissues is very important to understand and characterize the optical clearing effects and mechanisms involved: tissue dehydration and refractive index matching. From measurements made to study the optical clearing, it is obvious that light scattering is reduced and that the optical properties of the tissue are controlled in the process. On the other hand, optical measurements do not allow direct determination of the diffusion properties of the agent in the tissue and some calculations are necessary to estimate those properties. This fact is imposed by the occurrence of two fluxes at optical clearing: water typically directed out of and agent directed into the tissue. When the water content in the immersion solution is approximately the same as the free water content of the tissue, a balance is established for water and the agent flux dominates. To prove this concept experimentally, we have measured the collimated transmittance of skeletal muscle samples under treatment with aqueous solutions containing different concentrations of glucose. After estimating the mean diffusion time values for each of the treatments we have represented those values as a function of glucose concentration in solution. Such a representation presents a maximum diffusion time for a water content in solution equal to the tissue free water content. Such a maximum represents the real diffusion time of glucose in the muscle and with this value we could calculate the corresponding diffusion coefficient.