CTAB, Triton X-100 and freezing-thawing treatments of Candida guilliermondii: Effects on permeability and accessibility of the glucose-6-phosphate dehydrogenase, xylose reductase and xylitol dehydrogenase enzymes

Cells of Candida guilliermondii (ATCC 201935) were permeabilised with surfactant treatment (CTAB or Triton X-100) or a freezing-thawing procedure. Treatments were monitored by in situ activities of the key enzymes involved in xylose metabolism, that is, glucose-6-phosphate dehydrogenase (G6PD), xylose reductase (XR) and xylitol dehydrogenase (XD). The permeabilising ability of the surfactants was dependent on its concentration and incubation time. The optimum operation conditions for the permeabilisation of C. guilliermondii with surfactants were 0.41 mM (CTAB) or 2.78 mM (Triton X-100), 30 degrees C, and pH 7 at 200 rpm for 50 min. The maximum permeabilisation measured in terms of the in situ G6PD activity observed was, in order, as follows: CTAB (122.4 +/- 15.7 U/g(cells)) > freezing-thawing, , (54.3 +/- 1.9 U/g(cells)) > Triton X-100 (23.5 +/- 0.0 U/g(cells)). These results suggest that CTAB surfactant is more effective in the permeabilisation of C. guilliermondii cells in comparison to the freezing-thawing and Triton X-100 treatments. Nevertheless, freezing-thawing was the only treatment that allowed measurable in situ XR activity. Therefore, freezing-thawing permeabilised yeast cells could be used as a source of xylose reductase for analytical purposes or for use in biotransformation process such as xylitol preparation from xylose. The level of in situ xylose reductase was found to be 13.2 +/- 0.1 U/g(cells).

Patient with toxoplasmosis and glucose-6-phosphate dehydrogenase deficiency: a case report

Abstract Introduction Toxoplasmosis, a zoonotic protozoal disease caused by toxoplasma gondii, is prevalent throughout the world, affecting a large proportion of persons who usually have no symptoms. Glucose 6 phosphate dehydrogenase deficiency, an X-linked inherited disorder, is present in over 400 million people world wide. It is more common in tropical and subtropical countries and is one of the important causes of hemolytic anemia. Case presentation This case report relates the occurrence of the two diseases simultaneously in a child of five years old. Conclusion Patients with glucose-6-phosphate dehydrogenase deficiency are more susceptible to toxoplasmosis and this case report, reinforce the findings of this propensity and alert us for such possibility, what it is important, therefore, the treatment of toxoplasmosis can cause serious hemolysis in these patients.

Underlying Factors Associated with Anemia in Amazonian Children: A Population-Based, Cross-Sectional Study

Background: Although iron deficiency is considered to be the main cause of anemia in children worldwide, other contributors to childhood anemia remain little studied in developing countries. We estimated the relative contributions of different factors to anemia in a population-based, cross-sectional survey. Methodology: We obtained venous blood samples from 1111 children aged 6 months to 10 years living in the frontier town of Acrelandia, northwest Brazil, to estimate the prevalence of anemia and iron deficiency by measuring hemoglobin, erythrocyte indices, ferritin, soluble transferrin receptor, and C-reactive protein concentrations. Children were simultaneously screened for vitamin A, vitamin B-12, and folate deficiencies; intestinal parasite infections; glucose-6-phosphate dehydrogenase deficiency; and sickle cell trait carriage. Multiple Poisson regression and adjusted prevalence ratios (aPR) were used to describe associations between anemia and the independent variables. Principal Findings: The prevalence of anemia, iron deficiency, and iron-deficiency anemia were 13.6%, 45.4%, and 10.3%, respectively. Children whose families were in the highest income quartile, compared with the lowest, had a lower risk of anemia (aPR, 0.60; 95% CI, 0.37-0.98). Child age (<24 months, 2.90; 2.01-4.20) and maternal parity (>2 pregnancies, 2.01; 1.40-2.87) were positively associated with anemia. Other associated correlates were iron deficiency (2.1; 1.4-3.0), vitamin B-12 (1.4; 1.0-2.2), and folate (2.0; 1.3-3.1) deficiencies, and C-reactive protein concentrations (>5 mg/L, 1.5; 1.1-2.2). Conclusions: Addressing morbidities and multiple nutritional deficiencies in children and mothers and improving the purchasing power of poorer families are potentially important interventions to reduce the burden of anemia.

Assessment of the sublethal toxicity of organochlorine pesticide endosulfan in juvenile common carp (Cyprinus carpio)

This study is aimed at evaluating the sublethal effects of endosulfan (EDS) in juvenile common carp (Cyprinus carpio). For this purpose, fish were exposed for 15 days to the technical EDS (95% pure) diluted in dimethyl sulfoxide (DMSO) 0.1% of the total volume in water solution in a semi-static system at sublethal concentration (1 mu g/L). Subsequently, the liver somatic index (LSI) and factor condition (K) were determined. The total cytocrome P450 (CYP), CYP1A isoform, and the ethoxyresorufin-O-deethylase (EROD) activity were determined from the hepatic microsomal fraction as well as the activity of the oxidative stress enzyme system such as superoxide dismutase (SOD), catalase (CAT), glutathione-S-transferase (GST), glutathione peroxidase (GP(X)), glutathione reductase (GR), and glucose-6-phosphate dehydrogenase (G6PDH). Among the parameters assessed, EDS at the sublethal concentration in subchronic exposure caused significant changes in liver somatic indices as well as induction of the phase I biotransformation system and oxidative stress in juvenile common carp (Cyprinus carpio). Thus, it is seen that the use of biochemical biomarkers of environmental contamination in this study proved to be an extremely important tool for detecting the adverse effects of xenobiotics in the aquatic environment, even at low concentration.

RNA extraction from ten year old formalin-fixed paraffin-embedded breast cancer samples: a comparison of column purification and magnetic bead-based technologies

Abstract Background The development of protocols for RNA extraction from paraffin-embedded samples facilitates gene expression studies on archival samples with known clinical outcome. Older samples are particularly valuable because they are associated with longer clinical follow up. RNA extracted from formalin-fixed paraffin-embedded (FFPE) tissue is problematic due to chemical modifications and continued degradation over time. We compared quantity and quality of RNA extracted by four different protocols from 14 ten year old and 14 recently archived (three to ten months old) FFPE breast cancer tissues. Using three spin column purification-based protocols and one magnetic bead-based protocol, total RNA was extracted in triplicate, generating 336 RNA extraction experiments. RNA fragment size was assayed by reverse transcription-polymerase chain reaction (RT-PCR) for the housekeeping gene glucose-6-phosphate dehydrogenase (G6PD), testing primer sets designed to target RNA fragment sizes of 67 bp, 151 bp, and 242 bp. Results Biologically useful RNA (minimum RNA integrity number, RIN, 1.4) was extracted in at least one of three attempts of each protocol in 86–100% of older and 100% of recently archived ("months old") samples. Short RNA fragments up to 151 bp were assayable by RT-PCR for G6PD in all ten year old and months old tissues tested, but none of the ten year old and only 43% of months old samples showed amplification if the targeted fragment was 242 bp. Conclusion All protocols extracted RNA from ten year old FFPE samples with a minimum RIN of 1.4. Gene expression of G6PD could be measured in all samples, old and recent, using RT-PCR primers designed for RNA fragments up to 151 bp. RNA quality from ten year old FFPE samples was similar to that extracted from months old samples, but quantity and success rate were generally higher for the months old group. We preferred the magnetic bead-based protocol because of its speed and higher quantity of extracted RNA, although it produced similar quality RNA to other protocols. If a chosen protocol fails to extract biologically useful RNA from a given sample in a first attempt, another attempt and then another protocol should be tried before excluding the case from molecular analysis.

Chronic cold stress in mice induces a regulatory phenotype in macrophages: Correlation with increased 11 beta-hydroxysteroid dehydrogenase expression

Susceptibility to infections, autoimmune disorders and tumor progression is strongly influenced by the activity of the endocrine and nervous systems in response to a stressful stimulus. When the adaptive system is switched on and off efficiently, the body is able to recover from the stress imposed. However, when the system is activated repeatedly or the activity is sustained, as during chronic or excessive stress, an allostatic load is generated, which can lead to disease over long periods of time. We investigated the effects of chronic cold stress in BALB/c mice (4 degrees C/4 h daily for 7 days) on functions of macrophages. We found that chronic cold stress induced a regulatory phenotype in macrophages, characterized by diminished phagocytic ability, decreased TNF-alpha and IL-6 and increased IL-10 production. In addition, resting macrophages from mice exposed to cold stress stimulated spleen cells to produce regulatory cytokines, and an immunosuppressive state that impaired stressed mice to control Trypanosoma cruzi proliferation. These regulatory effects correlated with an increase in macrophage expression of 11 beta-hydroxysteroid dehydrogenase, an enzyme that converts inactive glucocorticoid into its active form. As stress is a common aspect of modern life and plays a role in the etiology of many diseases, the results of this study are important for improving knowledge regarding the neuro-immune-endocrine interactions that occur during stress and to highlight the role of macrophages in the immunosuppression induced by chronic stress. (C) 2011 Elsevier Inc. All rights reserved.

Hyperinsulinism/hyperammonemia (HI/HA) syndrome due to a mutation in the glutamate dehydrogenase gene

The hyperinsulinism/hyperammonemia (HI/HA) syndrome is a rare autosomal dominant disease manifested by hypoglycemic symptoms triggered by fasting or high-protein meals, and by elevated serum ammonia. HI/HA is the second most common cause of hyperinsulinemic hypoglycemia of infancy, and it is caused by activating mutations in GLUD1, the gene that encodes mitochondrial enzyme glutamate dehydrogenase (GDH). Biochemical evaluation, as well as direct sequencing of exons and exon-intron boundary regions of the GLUD1 gene, were performed in a 6-year old female patient presenting fasting hypoglycemia and hyperammonemia. The patient was found to be heterozygous for one de novo missense mutation (c.1491A>G; p.Il497Met) previously reported in a Japanese patient. Treatment with diazoxide 100 mg/day promoted complete resolution of the hypoglycemic episodes. Arq Bras Endocrinol Metab. 2012;56(8):485-9

Crystallization and preliminary X-ray diffraction of malate dehydrogenase from Plasmodium falciparum

The expression, purification, crystallization and preliminary X-ray diffraction characterization of malate dehydrogenase (MDH) from the malarial parasite Plasmodium falciparum (PfMDH) are reported. In order to gain a deeper understanding of the function and role of PfMDH, the protein was purified to homogeneity. The purified protein crystallized in space group P1, with unit-cell parameters a = 72, b = 157, c = 159 angstrom, a = 105, beta = 101, ? = 95 degrees. The resulting crystals diffracted to a maximal resolution of 2.24 angstrom and the structure has been solved by molecular replacement, with 16 monomers in the asymmetric unit. The 16 monomers are arranged into four independent tetramers, in agreement with previous reports demonstrating the tetrameric solution state of PfMDH. The X-ray structure of PfMDH is expected to clarify the differences in catalysis by PfMDH compared with other MDH family members and to provide a basis for the structure-based design of specific PfMDH inhibitors as well as general MDH inhibitors.

Crystal structure of dihydroorotate dehydrogenase from Leishmania major

Dihydroorotate dehydrogenase (DHODH) is the fourth enzyme in the de novo pyrimidine biosynthetic pathway and has been exploited as the target for therapy against proliferative and parasitic diseases. In this study, we report the crystal structures of DHODH from Leishmania major, the species of Leishmania associated with zoonotic cutaneous leishmaniasis, in its apo form and in complex with orotate and fumarate molecules. Both orotate and fumarate were found to bind to the same active site and exploit similar interactions, consistent with a ping-pong mechanism described for class 1A DHODHs. Analysis of LmDHODH structures reveals that rearrangements in the conformation of the catalytic loop have direct influence on the dimeric interface. This is the first structural evidence of a relationship between the dimeric form and the catalytic mechanism. According to our analysis, the high sequence and structural similarity observed among trypanosomatid DHODH suggest that a single strategy of structure-based inhibitor design can be used to validate DHODH as a druggable target against multiple neglected tropical diseases such as Leishmaniasis, Sleeping sickness and Chagas' diseases. (C) 2012 Elsevier Masson SAS. All rights reserved.