Purification and characterization of an extracellular trypsin-like protease of Fusarium oxysporum var. lini

An alkaline serineprotease, capable of hydrolyzing Nalpha-benzoyl- dl arginine p-nitroanilide, was secreted by Fusarium oxysporum var. lini grown in the presence of gelatin as the sole nitrogen and carbon source. The protease was purified 65-fold to electrophoretic homogenity from the culture supernatant in a three-step procedure comprising QSepharose chromatography, affinity chromatography, and FPLC on a MonoQ column. SDS-PAGE analysis of the purified protein indicated an estimated molecular mass of 41 kDa. The protease had optimum activity at a reaction temperature of 45 degrees C and showed a rapid decrease of activity at 48 degrees C. The optimum pH was around 8.0. Characterization of the protease showed that Ca2+ and Mg2+ cations increased the activity, which was not inhibited by EDTA or 1,10-phenanthroline. The enzyme activity on Nalpha-benzoyl-DL arginine p-nitroanilide was inhibited by 4-(2-aminoethyl)-benzenesulfonyl fluoride hydrochloride, p-aminobenzamidine dihydrochloride, aprotinin, 3-4 dichloroisocoumarin, and N-tosyl-L-lysine chloromethyl ketone. The enzyme is also inhibited by substrate concentrations higher than 2.5 x 10(-4)M. The protease had a Michaelis-Menten constant of 0.16 mM and a V(max) of 0.60 mumol released product.min(-1).mg(-1) enzyme when assayed in a non-inhibiting substrate concentration. The activity on Nalpha-benzoyl- dl arginine p-nitroanilide was competitively inhibited by p-aminobenzamidine dihydrochoride. A K(i) value of 0.04 mM was obtained.     

Cellulose Nanowhiskers Extracted from Tempo-Oxidized Curaua Fibers

ABSTRACT

The objective of this study was to obtain nanowhiskers from bleached and milled curaua fibers. Nanowhiskers were obtained via a TEMPO system. TEM micrographies proved the fibers were obtained. FTIR analysis showed characteristic peaks of the oxidation products for the samples submitted to the treatment, suggesting that the it was effective for cellulose. The results show that the TEMPO method increased crystallinity and decreased the degree of polymerization. As for thermal stability, the nanowhiskers were more thermally stable than the other samples. Based on our findings, the use of nanowhiskers may be suitable for a variety of applications, such as the production of nanocomposites.     

Classification of Brazilian honeys by physical and chemical analytical methods and low field nuclear magnetic resonance (LF H NMR)

This study evaluated the possibility of differentiating the botanical origin of honeys using Low Field Nuclear Magnetic Resonance (LF ¹H NMR). Eighty samples of honey from 8 different botanical sources (eucalyptus, “assa-lipto”, oranges, Barbados cherry, cashew tree, “assa-peixe”, “cipó-uva” and polyfloral) were analyzed. A close correlation (p < 0.01) was established between the LF ¹H NMR analysis and physical and chemical measurements, including water content, water activity, pH and color. Bi-exponential fitting of the transverse relaxation (T₂) data revealed two water populations in all samples, T₂₁ and T₂₂, corresponding to relaxation times of 0.6–1.8 ms and 2.3–5.4 ms respectively. The observed differences in the relaxation times suggest that these were influenced by the differences in botanical origins. Good linear correlations were observed between the T₂ and T₂₁ parameters and the physical and chemical data. This study demonstrated that LF ¹H NMR can be a viable technique for use in classifying honeys by their botanical origin.     

Cytotoxic effects of thiamethoxam in the midgut and malpighian tubules of Africanized Apis mellifera (Hymenoptera: Apidae)

Due to its expansion, agriculture has become increasingly dependent on the use of pesticides. However, the indiscriminate use of insecticides has had additional effects on the environment. These products have a broad spectrum of action, and therefore the insecticide affects not only the pests but also non‐target insects such as bees, which are important pollinators of agricultural crops and natural environments. Among the most used pesticides, the neonicotinoids are particularly harmful. One of the neonicotinoids of specific concern is thiamethoxam, which is used on a wide variety of crops and is toxic to bees. Thus, this study aimed to analyze the effects of this insecticide in the midgut and Malpighian tubule cells of Africanized Apis mellifera. Newly emerged workers were exposed until 8 days to a diet containing a sublethal dose of thiamethoxam equal to 1/10 of LC₅₀ (0.0428 ng a.i./l L of diet). The bees were dissected and the organs were processed for transmission electron microscopy. The results showed that thiamethoxam is cytotoxic to midgut and Malpighian tubules. In the midgut, the damage was more evident in bees exposed to the insecticide on the first day. On the eighth day, the cells were ultrastructurally intact suggesting a recovery of this organ. The Malpighian tubules showed pronounced alterations on the eighth day of exposure of bees to the insecticide. This study demonstrates that the continuous exposure to a sublethal dose of thiamethoxam can impair organs that are used during the metabolism of the insecticide. Microsc. Res. Tech. 77:274–281, 2014. © 2014 Wiley Periodicals, Inc.     

Consumer food safety knowledge, practices, and demographic differences: findings from a meta-analysis

Risk communication and consumer education to promote safer handling of food can be the best way of managing the risk of foodborne illness at the consumer end of the food chain. Thus, an understanding of the overall status of food handling knowledge and practices is needed. Although traditional qualitative reviews can be used for combining information from several studies on specific food handling behaviors, a structured approach of meta-analysis can be more advantageous in a holistic assessment. We combined findings from 20 studies using meta-analysis methods to estimate percentages of consumers engaging in risky behaviors, such as consumption of raw food, poor hygiene, and cross-contamination, separated by various demographic categories. We estimated standard errors to reflect sampling error and between-study random variation. Then we evaluated the statistical significance of differences in behaviors across demographic categories and across behavioral measures. There were considerable differences in behaviors across demographic categories, possibly because of socioeconomic and cultural differences. For example, compared with women, men reported greater consumption of raw or undercooked foods, poorer hygiene, poorer practices to prevent cross-contamination, and less safe defrosting practices. Mid-age adults consumed more raw food (except milk) than did young adults and seniors. High-income individuals reported greater consumption of raw foods, less knowledge of hygiene, and poorer cross-contamination practices. The highest raw ground beef and egg consumption and the poorest hygiene and cross-contamination practices were found in the U.S. Mountain region. Meta-analysis was useful for identifying important data gaps and demographic groups with risky behaviors, and this information can be used to prioritize further research.     

Antimicrobial activity of Satureja montana L. essential oil against Clostridium perfringens type A inoculated in mortadella-type sausages formulated with different levels of sodium nitrite

This research evaluated the antimicrobial effect of the winter savory (Satureja montana L.) essential oil (EO) against Clostridium perfringens type A (ATCC 3624) inoculated in mortadella-type sausages formulated with different levels of sodium nitrite (NaNO₂: 0 ppm, 100 ppm and 200 ppm) in addition to EO at concentrations of 0.0%, 0.78%, 1.56% and 3.125% stored at 25 °C for 30 days. The EO extracted by hydrodistillation and analyzed by gas chromatography-mass spectrometry (CG-MS) was tested in vitro using an agar well diffusion method for determination of minimum inhibitory concentration (MIC) on C. perfringens. According to compositional analysis of the winter savory EO, 26 chemical compounds were identified, and the major constituents were thymol (28.99%), p-cymene (12.00%), linalool (11.00%) and carvacrol (10.71%). The results obtained showed that EO applied at a concentration of 1.56%, which was defined as the MIC, exhibited antimicrobial activity against C. perfringens in the in vitro assays, and the transmission electron microscopy (TEM) revealed structural damage and cell lysis of C. perfringens caused by EO treatment. A synergistic effect between NaNO₂ and EO was observed. In mortadella-type sausages formulated with 100 ppm of NaNO₂ and EO at all concentrations tested, the population of target microorganisms was reduced (p ≤ 0.05) compared to control samples during all storage period. This data suggests the potential combined use of savory EO and minimal amounts of the synthetic additive, NaNO₂ to control C. perfringens in mortadella, which goes according to current market trends, where consumers are requesting natural products.     

Determination of Inorganic Elements in Teas Using Inductively Coupled Plasma Optical Emission Spectrometry and Classification with Exploratory Analysis

Multivariate optimization was employed to obtain the best conditions of the inductively coupled optical emission spectrometer (ICP OES) (nebulization gas flow rate of 0.47 L min^?1 and applied power of 1.36 kW) for the determination of Al, Ba, Ca, Cu, Fe, K, Mg, Na, and Mn in 27 green tea samples. In the hierarchical cluster analysis, it was possible to observe the formation of five different groups (imported Japanese samples, samples without specifications, organically cultivated samples, samples in capsules, and ready-to-drink iced tea samples) besides the separation according to brand. In the principal component analysis we verified that the first four main components explained 99.98 % of the total variance. The ICP OES technique and the exploratory analysis were shown effective tools that can be used jointly in the quality control and classification of green tea samples.     

Cytosolic 5′-Nucleotidase II Silencing in Lung Tumor Cells Regulates Metabolism through Activation of the p53/AMPK Signaling Pathway

Cytosolic 5′-nucleotidase II (cN-II) is an allosteric catabolic enzyme that hydrolyzes IMP, GMP, and AMP. The enzyme can assume at least two different structures, being the more active conformation stabilized by ATP and the less active by inorganic phosphate. Therefore, the variation in ATP concentration can control both structure and activity of cN-II. In this paper, using a capillary electrophoresis technique, we demonstrated that a partial silencing of cN-II in a pulmonary carcinoma cell line (NCI-H292) is accompanied by a decrease in adenylate pool, without affecting the energy charge. We also found that cN-II silencing decreased proliferation and increased oxidative metabolism, as indicated by the decreased production of lactate. These effects, as demonstrated by Western blotting, appear to be mediated by both p53 and AMP-activated protein kinase, as most of them are prevented by pifithrin-α, a known p53 inhibitor. These results are in line with our previous observations of a shift towards a more oxidative and less proliferative phenotype of tumoral cells with a low expression of cN-II, thus supporting the search for specific inhibitors of this enzyme as a therapeutic tool for the treatment of tumors.     

Epigallocatechin-3-Gallate Modulates Postoperative Pain by Regulating Biochemical and Molecular Pathways

Treating postoperative (PO) pain is a clinical challenge. Inadequate PO pain management can lead to worse outcomes, for example chronic post-surgical pain. Therefore, acquiring new information on the PO pain mechanism would increase the therapeutic options available. In this paper, we evaluated the role of a natural substance, epigallocatechin-3-gallate (EGCG), on pain and neuroinflammation induced by a surgical procedure in an animal model of PO pain. We performed an incision of the hind paw and EGCG was administered for five days. Mechanical allodynia, thermal hyperalgesia, and motor dysfunction were assessed 24 h, and three and five days after surgery. At the same time points, animals were sacrificed, and sera and lumbar spinal cord tissues were harvested for molecular analysis. EGCG administration significantly alleviated hyperalgesia and allodynia, and reduced motor disfunction. From the molecular point of view, EGCG reduced the activation of the WNT pathway, reducing WNT3a, cysteine-rich domain frizzled (FZ)1 and FZ8 expressions, and both cytosolic and nuclear β-catenin expression, and the noncanonical β-catenin–independent signaling pathways, reducing the activation of the NMDA receptor subtype NR2B (pNR2B), pPKC and cAMP response element-binding protein (pCREB) expressions at all time points. Additionally, EGCG reduced spinal astrocytes and microglia activation, cytokines overexpression and nuclear factor kappa-light-chain-enhancer of activated B cells (NFkB) pathway, downregulating inducible nitric oxide synthase (iNOS) activation, cyclooxygenase 2 (COX-2) expression, and prostaglandin E2 (PGE2) levels. Thus, EGCG administration managing the WNT/β-catenin signaling pathways modulates PO pain related neurochemical and inflammatory alterations.