Examination of Potential Virulence Factors of <Emphasis Type="Italic">Candida tropicalis</Emphasis> Clinical Isolates From Hospitalized Patients

Candida tropicalis has been reported to be one of the Candida species which is most likely to cause bloodstream and urinary tract infections in hospitalized patients. Accordingly, the aim of this study was to characterize the virulence of C. tropicalis by assessing antifungal susceptibility and comparing the expression of several virulence factors. This study was conducted with seven isolates of C. tropicalis from urine and blood cultures and from central venous catheter. C. tropicalis ATCC 750 was used as reference strain. Yeasts adhered (2 h) to epithelial cells and silicone and 24 h biofilm biomass were determined by crystal violet staining. Pseudohyphae formation ability was determined after growth in fetal bovine serum. Enzymes production (hemolysins, proteases, phospholipases) was assessed by halo formation on agar plates. Susceptibility to antifungal agents was determined by E-test. Regarding adhesion, it can be highlighted that C. tropicalis strains adhered significantly more to epithelium than to silicone. Furthermore, all C. tropicalis strains were able to form biofilms and to express total hemolytic activity. However, protease was only produced by two isolates from urine and by the isolates from catheter and blood. Moreover, only one C. tropicalis (from catheter) was phospholipase positive. All isolates were susceptible to voriconazole, fluconazole and amphotericin B. Four strains were susceptible-dose dependent to itraconazole and one clinical isolate was found to be resistant.     

A two-dimensional proteome reference map of Herbaspirillum seropedicae proteins

Herbaspirillum seropedicae is an endophytic diazotroph associated with economically important crops such as rice, sugarcane, and wheat. Here, we present a 2-D reference map for H. seropedicae. Using MALDI-TOF-MS we identified 205 spots representing 173 different proteins with a calculated average of 1.18 proteins/gene. Seventeen hypothetical or conserved hypothetical ORFs were shown to code for true gene products. These data will support the genome annotation process and provide a basis on which to undertake comparative proteomic studies.     

Effect of trehalose on a phospholipid membrane under mechanical stress

Explicit solvent molecular dynamics simulations were used to investigate at atomic resolution the effect of trehalose on a hydrated phospholipid bilayer under mechanical stress (stretching forces imposed in the form of negative lateral pressure). Simulations were performed in the absence or presence of trehalose at 325 K, and with different values for negative lateral pressure. In the concentration regime (2 molal) and range of lateral pressures (1 to −250 bar) investigated, trehalose was found to interact directly with the membrane, partially replacing water molecules in the formation of hydrogen bonds with the lipid headgroups. Similar to previous findings in the context of thermal stress, the number, degree of bridging, and reaching depth of these hydrogen bonds increased with the magnitude of perturbation. However, at the concentration considered, trehalose was not sufficient to preserve the integrity of the membrane structure and to prevent its extreme elongation (and possible disruption) under the effect of stretching forces.     

Antiproliferative activity of Pterodon pubescens Benth. seed oil and its active principle on human melanoma cells

On a preliminary screening, relevant in vitro antiproliferative activity was observed to the crude ethanolic extract of Pterodon pubescens seed oil against the human melanoma cell line SK MEL 37. The diethyl ether fraction from crude ethanolic extract which exhibited stronger activity was submitted to fractionation by gradient elution with hexane/ethyl acetate. Subfraction A, eluted by hexane/ethyl acetate (80:20), was essentially the most active between all the assayed subfractions with an IC₅₀ of 37 μg/ml calculated by the MTT colorimetric method. At this concentration, subfraction A caused morphological features and internucleosomal DNA fragmentation pattern of apoptosis. Through chromatographic separation, the furane diterpene 1 was isolated from this active subfraction and identified by spectral techniques. Compound 1 showed an IC₅₀ value of 32 μM and fluorescence staining with DAPI revealed some typical nuclear changes which are characteristic of apoptosis. These findings support a role for diterpenoids vouacapan-type skeleton as a model to develop new anticancer agents.     

High dietary salt decreases antioxidant defenses in the liver of fructose-fed insulin-resistant rats

In this study we investigated the hypothesis that a high-salt diet to hyperinsulinemic rats might impair antioxidant defense owing to its involvement in the activation of sodium reabsorption to lead to higher oxidative stress. Rats were fed a standard (CON), a high-salt (HS), or a high-fructose (HF) diet for 10 weeks after which, 50% of the animals belonging to the HF group were switched to a regimen of high-fructose and high-salt diet (HFS) for 10 more weeks, while the other groups were fed with their respective diets. Animals were then euthanized and their blood and liver were examined. Fasting plasma glucose was found to be significantly higher (approximately 50%) in fructose-fed rats than in the control and HS rats, whereas fat liver also differed in these animals, producing steatosis. Feeding fructose-fed rats with the high-salt diet triggered hyperinsulinemia and lowered insulin sensitivity, which led to increased levels of serum sodium compared to the HS group. This resulted in membrane perturbation, which in the presence of steatosis potentially enhanced hepatic lipid peroxidation, thereby decreasing the level of antioxidant defenses, as shown by GSH/GSSG ratio (HFS rats, 7.098±2.1 versus CON rats, 13.2±6.1) and superoxide dismutase (HFS rats, 2.1±0.05 versus CON rats, 2.3±0.1%), and catalase (HFS rats, 526.6±88.6 versus CON rats, 745.8±228.7 U/mg ptn) activities. Our results indicate that consumption of a salt-rich diet by insulin-resistant rats may lead to regulation of sodium reabsorption, worsening hepatic lipid peroxidation associated with impaired antioxidant defenses.     

Real-time quantification of Pseudomonas fluorescens cell removal from glass surfaces due to bacteriophage varphiS1 application

Aims:  To study the efficacy of the lytic phage φS1 in eliminating Pseudomonas fluorescens in the early stage of biofilm formation, using an in situ and real time methodology for cell quantification.
Methods and Results:  Cell adhesion and phage infection studies were carried out in a parallel plate flow chamber under laminar conditions. Cells were allowed to adhere until reaching 1·7–1·8 × 10⁶ cells cm⁻² and phage infection was performed with two different phage concentrations (2 × 10⁹ PFU ml⁻¹ and 1 × 10¹⁰ PFU ml⁻¹). Phage concentration clearly affects the speed of infection. The less concentrated phage solution promoted a three times slower rate of cell removal but did not affect the overall percentage of cell removal. In fact, after a longer infection period the less concentrated phage solution reached the same 93% cell removal value.
Conclusions:  Phages are efficient in the eradication of bacterial cells at the early stage of biofilm formation and their presence at the surface did not allow bacterial recolonization of the surface.
Significance and Impact of the Study:  To date, no published studies have been made concerning in situ and real time quantification of cell removal from surfaces due to phage action.     

Mixture design of starchy substrates hydrolysis by an immobilized glucoamylase from Aspergillus brasiliensis

Starch has great importance in human diet, since it is a heteropolymer of plants, mainly found in roots, as potato, cassava and arrowroots. This carbohydrate is composed by a highly-branched chain: amylopectin; and a linear chain: amylose. The proportion between the chains varies according to the botanical source. Starch hydrolysis is catalyzed by enzymes of the amilolytic system, named amylases. Among the various enzymes of this system, the glucoamylases (EC 3.2.1.3 glucan 1,4-alpha-glucosidases) are the majority because they hydrolyze the glycosidic linkages at the end of starch chains releasing glucose monomers. In this work, a glucoamylase secreted in the culture medium, by the ascomycete Aspergillus brasiliensis, was immobilized in Dietilaminoetil Sepharose-Polyethylene Glycol (DEAE-PEG), since immobilized biocatalysts are more stable in long periods of hydrolysis, and can be recovered from the final product and reused for several cycles. Glucoamylase immobilization has shown great thermal stability improvement over the soluble enzyme, reaching 66% more activity after 6?h at 60?°C, and 68% of the activity after 10 hydrolysis cycles. A simplex centroid experimental mixture design was applied as a tool to characterize the affinity of the immobilized enzyme for different starchy substrates. In assays containing several proportions of amylose, amylopectin and starch, the glucoamylase from A. brasiliensis mainly hydrolyzed the amylopectin chains, showing to have preference by branched substrates.     

Phytochemical Characterization of Cannabis sativa L. Roots from Northeastern Brazil

This article describes the phytochemical study of Cannabis sativa roots from northeastern Brazil. The dried plant material was pulverized and subjected to exhaustive maceration with ethanol at room temperature, obtaining the crude ethanolic extract (Cs-EEBR). The volatile compounds were analyzed by gas chromatography coupled with mass spectrometry (GC/MS), which allowed to identify 22 compounds by comparing the linear retention index (LRI), the similarity index (SI) and the fragmentation pattern of the constituents with the literature. By this technique the major compounds identified were: friedelan-3-one and β-sitosterol. In addition, two fractions were obtained from Cs-EEBR by classical column chromatography and preparative thin layer chromatography. These fractions were analyzed by NMR and IR and together with the mass spectrometry data allowed to identify the compounds: epifriedelanol, friedelan-3-one, β-sitosterol and stigmasterol. The study contributed to the phytochemical knowledge of Cannabis sativa, specifically the roots, as there are few reports on the chemical constituents of this part of the plant.