5' and 3' UTR thymidylate synthase polymorphisms modulate the risk of colorectal cancer independently of the intake of methyl group donors

Thymidylate synthase, as a rate-limiting step in DNA synthesis, catalyses the conversion of dUMP into dTMP using 5,10-methylenotetrahydrofolate as the methyl donor. Two polymorphisms have been described in this gene: a repeat polymorphism in the 5' promoter enhancer region (3R versus 2R) and a 6 bp deletion in the 3' unstranslated region. Both of these may affect protein levels. The present case control study was aimed at investigating the influence of these two polymorphisms on the development of colorectal cancer (CRC), as well as their potential interaction with folate, vitamin B6 and vitamin B12 intake. A total of 196 cases and 200 controls, matched for age and sex distribution, were included in the study. No association was found between CRC and the 28 bp repeat polymorphism, but it was observed that individuals with the 6 bp/del and del/del genotypes had a significantly lower risk of developing the disease (OR=0.47; 95% CI 0.30-0.72). A combined genotype (2R/2R; 6 bp/del+del/del) was also found, which was associated with an even lower risk of developing of the disease (OR=0.42; 95% CI 0.26-0.69). No significant interaction between these polymorphisms and vitamin intake was observed. These results indicate for the first time that the 6 bp/del allele might be a protective factor in the development of CRC, independent of the intake of methyl group donors.

Determination of chitin content in fungal cell wall: an alternative flow cytometric method

The conventional methods used to evaluate chitin content in fungi, such as biochemical assessment of glucosamine release after acid hydrolysis or epifluorescence microscopy, are low throughput, laborious, time-consuming, and cannot evaluate a large number of cells. We developed a flow cytometric assay, efficient, and fast, based on Calcofluor White staining to measure chitin content in yeast cells. A staining index was defined, its value was directly related to chitin amount and taking into consideration the different levels of autofluorecence. Twenty-two Candida spp. and four Cryptococcus neoformans clinical isolates with distinct susceptibility profiles to caspofungin were evaluated. Candida albicans clinical isolate SC5314, and isogenic strains with deletions in chitin synthase 3 (chs3Δ/chs3Δ) and genes encoding predicted Glycosyl Phosphatidyl Inositol (GPI)-anchored proteins (pga31Δ/Δ and pga62Δ/Δ), were used as controls. As expected, the wild-type strain displayed a significant higher chitin content (P < 0.001) than chs3Δ/chs3Δ and pga31Δ/Δ especially in the presence of caspofungin. Ca. parapsilosis, Ca. tropicalis, and Ca. albicans showed higher cell wall chitin content. Although no relationship between chitin content and antifungal drug susceptibility phenotype was found, an association was established between the paradoxical growth effect in the presence of high caspofungin concentrations and the chitin content. This novel flow cytometry protocol revealed to be a simple and reliable assay to estimate cell wall chitin content of fungi.

Risk of colorectal cancer associated with the C677T polymorphism in 5,10-methylenetetrahydrofolate reductase in Portuguese patients depends on the intake of methyl-donor nutrients

Background: Polymorphisms located in genes involved in the metabolism of folate and some methyl-related nutrients are implicated in colorectal cancer (CRC). Objective: We evaluated the association of 3 genetic polymorphisms [C677T MTHFR (methylene tetrahydrofolate reductase), A2756G MTR (methionine synthase), and C1420T SHMT (serine hydroxymethyltransferase)] with the intake of methyl-donor nutrients in CRC risk. Design: Patients withCRC(n 196) and healthy controls (n 200) matched for age and sex were evaluated for intake of methyl-donor nutrients and the 3 polymorphisms. Results: Except for folate intake, which was significantly lower in patients (P 0.02), no differences were observed in the dietary intake of other methyl-donor nutrients between groups. High intake of folate ( 406.7 g/d) was associated with a significantly lower risk of CRC (odds ratio: 0.67; 95% CI: 0.45, 0.99). The A2756G MTR polymorphism was not associated with the risk of developing CRC. In contrast, homozygosity for the C677TMTHFRvariant (TT) presented a 3.0-fold increased risk of CRC (95% CI: 1.3, 6.7). Similarly, homozygosity for the C1420T SHMT polymorphism also had a 2.6-fold increased risk (95% CI: 1.1, 5.9) of developing CRC. When interactions between variables were studied, low intake of all methyl-donor nutrients was associated with an increased risk ofCRC in homozygous participants for the C677T MTHFR polymorphism, but a statistically significant interaction was only observed for folate (odds ratio: 14.0; 95% CI: 1.8, 108.5). No significant associations were seen for MTR or SHMT polymorphisms. Conclusion: These results show an association between the C677T MTHFR variant and different folate intakes on risk of CRC.

A high throughput colorimetric assay of beta-1,3-D-glucans by Congo red dye

Mushroom strains contain complex nutritional biomolecules with a wide spectrum of therapeutic and prophylactic properties. Among these compounds, β-d-glucans play an important role in immuno-modulating and anti-tumor activities. The present work involves a novel colorimetric assay method for β-1,3-d-glucans with a triple helix tertiary structure by using Congo red. The specific interaction that occurs between Congo red and β-1,3-d-glucan was detected by bathochromic shift from 488 to 516 nm (> 20 nm) in UV–Vis spectrophotometer. A micro- and high throughput method based on a 96-well microtiter plate was devised which presents several advantages over the published methods since it requires only 1.51 μg of polysaccharides in samples, greater sensitivity, speed, assay of many samples and very cheap. β-d-Glucans of several mushrooms (i.e., Coriolus versicolor, Ganoderma lucidum, Pleurotus ostreatus, Ganoderma carnosum, Hericium erinaceus, Lentinula edodes, Inonotus obliquus, Auricularia auricular, Polyporus umbellatus, Cordyseps sinensis, Agaricus blazei, Poria cocos) were isolated by using a sequence of several extractions with cold and boiling water, acidic and alkaline conditions and quantified by this microtiter plate method. FTIR spectroscopy was used to study the structural features of β-1,3-d-glucans in these mushroom samples as well as the specific interaction of these polysaccharides with Congo red. The effect of NaOH on triple helix conformation of β-1,3-d-glucans was investigated in several mushroom species.

A novel colorimetric assay of beta-D-glucans in basidiomycete strains by alcian blue dye in a 96-well microtiter plate

Basidiomycete strains synthesize several types of beta-D-glucans, which play a major role in the medicinal properties of mushrooms. Therefore, the specific quantification of these beta-D-glucans in mushroom strains is of great biochemical importance. Because published assay methods for these beta-D-glucans present some disadvantages, a novel colorimetric assay method for beta-D-glucan with alcian blue dye was developed. The complex formation was detected by following the decrease in absorbance in the range of 620 nm and by hypsochromic shift from 620 to 606 nm (similar to 14 nm) in UV-Vis spectrophotometer. Analysis of variance was used for optimization of the slope of the calibration curve by using the assay mixture containing 0.017% (w/v) alcian blue in 2% (v/v) acetic acid at pH 3.0. The high-throughput colorimetric assay method on microtiter plates was used for quantification of beta-D-glucans in the range of 0-0.8 mu g, with a slope of 44.15 x 10(-2) and a limit of detection of 0.017 mu g/well. Recovery experiments were carried out by using a sample of Hericium erinaceus, which exhibited a recovery of 95.8% for beta-1,3-D-glucan. The present assay method exhibited a 10-fold higher sensitivity and a 59-fold lower limit of detection compared with the published method with congo red beta-D-glucans of several mushrooms strains were isolated from fruiting bodies and mycelia, and they were quantified by this assay method. This assay method is fast, specific, simple, and it can be used to quantify beta-D-glucans from other biological sources. (C) 2015 American Institute of Chemical Engineers

Generation of high-affinity monoclonal antibodies of IgG class against native β-d-glucans from basidiomycete mushrooms

β-d-glucans from basidiomycete strains are powerful immunomodulatory agents in several clinical conditions. Therefore, their assay, purification and characterization are of great interest to understand their structure-function relationship. Hybridoma cell fusion was used to raise monoclonal antibodies (Mabs) against extracellular β-d-glucans (EBGs) from Pleurotus ostreatus. Two of the hybridoma clones (1E6-1E8-B5 and 3E8-3B4) secreting Mabs against EBGs were selected. This hybridoma cell line secreted Mabs of the IgG class which were then purified by hydroxyapatite chromatography to apparent homogeneity on native and SDS-PAGE. Mabs secreted by 1E6-1E8-B5 clone were found to recognize a common epitope on several β-d-glucans from different basidiomycete strains. This Mab exhibited high affinity constant (KA) for β-d-glucans from several mushroom strains in the range of 3.20 × 109 ± 3.32 × 103-1.51 × 1013 ± 3.58 × 107 L/mol. Moreover, they reacted to some heat-treated β-d-glucans in a different mode when compared with the native forms; these data suggest that this Mab binds to a conformational epitope on the β-d-glucan molecule. The epitope-binding studies of Mabs obtained from 1E6-1E8-B5 and 3E8-3B4 revealed that the Mabs bind to the same epitope on some β-d-glucans and to different epitopes in other antigen molecules. Therefore, these Mabs can be used to assay for β-d-glucan from basidiomycete mushrooms. © 2015 Elsevier Ltd. All rights reserved.