Inhibition of Chara vulgaris oospore germination by sulfidic sediments

The germinability of Chara vulgaris oospores collected from the sediments of four Ontario lakes varies considerably, ranging in germination percentage from 7% to 54%. Chemical analysis of the interstitial water of the sediments indicated that oospores with low germination occurred in lakes which have high acid volatile sulfides (H₂S, FeS, HS⁻) and high soluble Fe²⁺. The inhibitory effects of sediment on oospore germination were demonstrated by transplant experiments, and suggested that sulfide was the toxic agent. Exposure of high-germinating sedimentary oospores to free sulfide concentrations greater than 2.0 mM caused a greater than 30% reduction in oospore germination. The presence of sulfide in sediments was shown to result from sulfate reduction by bacteria in sediment pore water of those lakes where oospore viability was lowest. Differences in oospore germination percentage appear, therefore, to be due to the toxicity of H₂S produced in the sediment, either by a direct effect on the oospore, or on the parent plants.     

Structural characterization of the cell wall D-glucans isolated from the mycelium of Botryosphaeria rhodina MAMB-05

Three D-glucans were isolated from the mycelium of the fungus Botryosphaeria rhodina MAMB-05 by sequential extraction with hot-water and hot aqueous KOH (2% w/v) followed by ethanol precipitation. Following their purification by gel permeation chromatography on Sepharose CL-4B, the structural characteristics of the D-glucans were determined by FT-IR and 13C NMR spectroscopy and, after methylation, by GC-MS. The hot-water extract produced a fraction designated Q1A that was a beta-(1-->6)-D-glucan with the following structure: [Formula: see text] The alkaline extract, when subjected to repeated freeze-thawing, yielded two fractions: K1P (insoluble) that comprised a beta-(1-->3)-D-glucan with beta-D-glucose branches at C-6 with the structure: [Formula: see text] and K1SA (soluble) consisting of a backbone chain of alpha-(1-->4)-linked D-glucopyranosyl residues substituted at O-6 with alpha-D-glucopyranosyl residues: [Formula: see text]     

Physicochemical and mechanical characterization of galactomannan from Mimosa scabrella: Effect of drying method

The galactomannan from Mimosa scabrella Bentham was extracted on a pilot plant scale and dried either by vacuum oven (GVO) or by spray-drier (GSD) to evaluate the effect of the drying technique on the powder quality and its applicability as excipient in solid dosage forms. The analysis by high performance size exclusion chromatography (HPSEC) coupled to multiangle laser light scattering (MALLS) suggests that both products behave as semi-flexible polymers, although the GSD showed more aggregation at molecular level (～10%) and higher chain stiffness (Lp 9.1 nm). TG and DSC analysis showed weight loss event with peak at 299.7 and 311.9 °C to GVO and GSD, respectively, as well and higher ash content for GSD sample, in both inert and oxidant atmosphere. The X-ray diffraction confirmed the amorphous nature of both galactomannans although GVO showed high crystallinity. The GSD showed lower density (1.009 g/cm³), higher cohesiveness (repose angle 35.5°, compressibility 32.2% and absence of flow), smaller and more spherical particles than GVO sample, both with high polydispersion. As vacuum oven-drying resulted in a like fibrous material, spray-drying appears as an alternative method easy to extrapolate in industry, requiring a glidant incorporation to improve the powder flowability.     

Anti-Influenza Neuraminidase Inhibitor Oseltamivir Phosphate Induces Canine Mammary Cancer Cell Aggressiveness

Oseltamivir phosphate is a widely used anti-influenza sialidase inhibitor. Sialylation, governed by sialyltransferases and sialidases, is strongly implicated in the oncogenesis and progression of breast cancer. In this study we evaluated the biological behavior of canine mammary tumor cells upon oseltamivir phosphate treatment (a sialidase inhibitor) in vitro and in vivo. Our in vitro results showed that oseltamivir phosphate impairs sialidase activity leading to increased sialylation in CMA07 and CMT-U27 canine mammary cancer cells. Surprisingly, oseltamivir phosphate stimulated, CMT-U27 cell migration and invasion capacity in vitro, in a dose-dependent manner. CMT-U27 tumors xenograft of oseltamivir phosphate-treated nude mice showed increased sialylation, namely α2,6 terminal structures and SLe(x) expression. Remarkably, a trend towards increased lung metastases was observed in oseltamivir phosphate-treated nude mice. Taken together, our findings revealed that oseltamivir impairs canine mammary cancer cell sialidase activity, altering the sialylation pattern of canine mammary tumors, and leading, surprisingly, to in vitro and in vivo increased mammary tumor aggressiveness.     

Ribose 5-Phosphate Isomerase B Knockdown Compromises Trypanosoma brucei Bloodstream Form Infectivity

Ribose 5-phosphate isomerase is an enzyme involved in the non-oxidative branch of the pentose phosphate pathway, and catalyzes the inter-conversion of D-ribose 5-phosphate and D-ribulose 5-phosphate. Trypanosomatids, including the agent of African sleeping sickness namely Trypanosoma brucei, have a type B ribose-5-phosphate isomerase. This enzyme is absent from humans, which have a structurally unrelated ribose 5-phosphate isomerase type A, and therefore has been proposed as an attractive drug target waiting further characterization. In this study, Trypanosoma brucei ribose 5-phosphate isomerase B showed in vitro isomerase activity. RNAi against this enzyme reduced parasites'' in vitro growth, and more importantly, bloodstream forms infectivity. Mice infected with induced RNAi clones exhibited lower parasitaemia and a prolonged survival compared to control mice. Phenotypic reversion was achieved by complementing induced RNAi clones with an ectopic copy of Trypanosoma cruzi gene. Our results present the first functional characterization of Trypanosoma brucei ribose 5-phosphate isomerase B, and show the relevance of an enzyme belonging to the non-oxidative branch of the pentose phosphate pathway in the context of Trypanosoma brucei infection.     

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.     

Expression of exogenous proteins and short hairpin RNAs in human primary thyrocytes

Recently, it has been shown that commercial human thyroid lines were in fact derived from colon, mammary carcinoma, or melanoma. Others have demonstrated the absence of a common pattern of gene expression between available thyroid cancer cell lines and tumors from patients. Thus, it is important to use several primary cells with a common pathological origin to achieve reproducible results, and it is necessary to find common methods for manipulation of protein expression in such various cultures. We have standardized a transfection method for efficient expression of exogenous proteins in human primary thyroid cultures. We compared lipid-based techniques with three electroporation systems (Electroporator PulseAgile [PA]-4000, Microporator MP-100, and Nucleofector II). Nucleofection was unquestionably the most efficient even for promoter regulation studies, and it was effective in cultures from different origins as normal thyroid, papillary carcinoma, or lymphoid node metastasis. We also standardized, through lentiviral infection, the short hairpin RNA downregulation of protein expression generating human thyrocytes with low levels of p27KIP1 as a model system.     

The defensive functions of plant inhibitors are not restricted to insect enzyme inhibition

Three plant proteinase inhibitors BbKI (kallikrein inhibitor) and BbCI (cruzipain inhibitor) from Bauhinia bauhinioides, and a BrTI (trypsin inhibitor) from B. rufa, were examined for other effects in Callosobruchus maculatus development; of these only BrTI affected bruchid emergence. BrTI and BbKI share 81% identities in their primary sequences and the major differences between them are the regions comprising the RGD and RGE motifs in BrTI. These sequences were shown to be essential for BrTI insecticidal activity, since a modified BbKI [that is a recombinant form (BbKIm) with some amino acid residues replaced by those found in BrTI sequence] also strongly inhibited insect development. By using synthetic peptides related to the BrTI sequence, YLEAPVARGDGGLA-NH₂ (RGE) and IVYYPDRGETGL-NH₂ (RGE), it was found that the peptide with an RGE sequence was able to block normal development of C. maculatus larvae (ED₅₀ 0.16% and LD₅₀ 0.09%), this being even more effective than the native protein.     

<Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> glycerol/H<Superscript>+</Superscript> symporter Stl1p is essential for cold/near-freeze and freeze stress adaptation. A simple recipe with high biotechnological potential is given

Background  

Freezing is an increasingly important means of preservation and storage of microbial strains used for many types of industrial applications including food processing. However, the yeast mechanisms of tolerance and sensitivity to freeze or near-freeze stress are still poorly understood. More knowledge on this regard would improve their biotechnological potential. Glycerol, in particular intracellular glycerol, has been assigned as a cryoprotectant, also important for cold/near-freeze stress adaptation. The S. cerevisiae glycerol active transporter Stl1p plays an important role on the fast accumulation of glycerol. This gene is expressed under gluconeogenic conditions, under osmotic shock and stress, as well as under high temperatures.