A limonoid from Trichilia estipulata

The limonoid 21,24,25,26,27-pentanor-15,22-oxo-7alpha,23-dihydroxy-apotirucalla(eupha)-1-en-3-one was isolated from the dichloromethane extract of the stem bark of Trichilia estipulata. Its structure was established by spectroscopic methods (UV, EIMS, 1H and 13C NMR, HMQC and HMBC).     

SELECTIVE PERMEABILITY OF THE EXTRACELLULAR ENVELOPE OF THE MICROALGA SPONDYLOSIUM PANDURIFORME (CHLOROPHYCEAE) AS REVEALED BY ELECTRON PARAMAGNETIC RESONANCE

The aim of this work was to investigate the role of the polysaccharide sheath of the microalga Spondylosium panduriforme (Chlorophyceae, Desmidiaceae) in the selective permeability and transport of molecules into the interior of the cell. We have used the electron paramagnetic resonance (EPR) technique applied to a variety of spin labels of a hydrophobic nature with different substitutents on the ring (−OH, =O, −N=C=S, −NH₃⁺, and others). The spin label EPR signals were destroyed as a consequence of metabolic processes once the spin probes had entered the cells. The decay time of the EPR signal was regulated by the diffusion mechanism across the polysaccharide sheath, cell wall, and membrane. To discriminate the effect of the polysaccharide sheath from that of the cell wall and membrane, the polysaccharide sheath was removed by ultrasonic treatment. The decay times for the cells without capsule were faster than those for intact cells, and a possible mechanism of interaction involving hydrogen bonds between the spin labels and the −OH groups of the polysaccharide sheath is presented. These were expressed by their diffusion and friction coefficients as derived from Ficks' Second Law and the Einstein-Stokes equation and were summarized in terms of diffusion coefficients ( D ₁) for the capsule medium in the order: =O < −OH < −phe < −H < −N=C=S; and for cell wall and membrane ( D ₂): −OH < −H < =O < −NH₃⁺≅−phe < −N=C=S. For the friction coefficients ( f ₁ and f ₂), the order was inverted. These results suggest the capsule plays a role in selectivity as a result of polar interactions with the spin labels.     

l-3,4-Dihydroxyphenylalanine (l-DOPA) secreted by oyster (Crassostrea gigas) mantle cells: functional aspects

l-DOPA is present in many molluscan periostraca and is thought to play an important role in the shell protein sclerotization mechanism. We analyzed l-DOPA content in organic membranes produced by the oyster (Crassostrea gigas) mantle as a reaction against shell damage, such as induced by Polydora sp. infestation, an artificial perforation of the shell, and in normal oysters mantle. When oysters are secreting the protective organic membrane against a shell perforation, the area of mantle close to the repairing region had a greater l-DOPA content than the remaining mantle. Mantle border as a whole had the same l-DOPA content as the central mantle. Nevertheless there is a variation pattern in mantle border l-DOPA content, the area near the umbo having a higher content and decreasing towards the frontal region. l-DOPA content in younger oysters was greater than that in older oysters. The results of perforation experiment suggest a good correlation between organic membrane synthesis and l-DOPA mantle border content. Concerning normal oysters, a higher l-DOPA content in mantle border than in central mantle would be expected but we couldn’t detect any differences. From all the results there is some evidence that mantle l-DOPA is related to other functions in addition to participating in the sclerotization mechanism.     

Interchain disulfide bonds in crotamine self-association

Crotamine, a basic neurotoxic protein, was isolated from the venom of the Southern Brazilian rattlesnake (Crotalus durissus terrificus) by gel filtration. The isolated protein showed a single band on PAGE at pH 4.5 and 7% (w/v) gel concentration, but two or more bands at 14% gel concentration, even in the presence of 4 M urea. After reduction and carboxymethylation, however, a single band was again detected. SDS-PAGE as well as ultracentrifugal analysis of the native (NC) and of the reduced and carboxymethylated (RCC) crotamine revealed a molecular weight of 4,500-5,000 for RCC and 9,000-10,000 for NC. Both components of a two-band crotamine preparation were isolated by preparative PAGE and characterized. Their particular electrophoretic mobility was retained. Their amino acid composition. N-terminal residue, and apparent toxicity were the same as those of the original sample. It was concluded that crotamine is able to form a dimer of 9,760 Da with two identical polypeptide chains crosslinked by interchain disulfide bonds and a shape not very far from spherical, which covalently binds extra subunits of 4,880 Da each.     

Detection of resistance genes and susceptibility patterns in Bacteroides and Parabacteroides strains

Susceptibility to five antimicrobials was determined for Bacteroides spp. (n = 52) and Parabacteroides distasonis (n = 8). All isolates were susceptible to metronidazole. The resistance rates to ampicillin, cefoxitin, tetracycline and clindamycin were 98%, 9.6%, 65.3% and 19.2% of the Bacteroides strains, respectively. The genes cepA, cfiA, cfxA, tetQ, ermF and nim were found in 69.2%, 17.3% 9.6%, 50%, 7.7% and 3.8% for these strains respectively. All P. distasonis strains were resistant to ampicilin. Cefoxitin, tetracycline and clindamycin resistance rates were 75%, 87.5% and 50%, respectively. The ermF and nim genes were absent and 37.5%, 12.5%, 12.5% and 87.5% of this strains possessed cepA, cfiA, cfxA and tetQ genes, respectively. Ten cfiA gene positive strains of Bacteroides and Parabacteroides were submitted to E-test with imipenem and amoxicillin–clavulanate. The resistance rate to imipenem was 4.1% and 8.3% to amoxicillin–clavulanate. This feature is for the first time described in Brazil.     

New strategy to apply perfluorodecalin as an oxygen carrier in lipase production: minimisation and reuse

A novel strategy for the production of lipase by Bacillus sp. ITP-001 in a stirred tank fermenter using perfluorodecalin (PFD) was studied. Firstly, a response surface methodology 2² with three central points was employed to optimise the effect of agitation speed and aeration rate in lipase production. According to the response from the experimental designs, 300 rpm (revolutions per minute) and 0.5 vvm (air volume/liquid volume per minute) were found to provide the best condition (lipolytic activity: LA = 3,140.76 U mL^?1). Then, the influence of PFD concentration on the fermentation process was evaluated. Incorporation of PFD at all concentrations above 1 % had no statistically significant influence on lipase production, that is, the previous optimisation allowed the reduction of the amount of PFD added besides increasing lipase production. Furthermore, PFD could be used in three sequential fermentations without altering the statistical production of lipase, reducing by 67 % the cost of PFD addition.     

Evaluation of a High Throughput Method for the Detection of Mutations Associated with Thrombosis and Hereditary Hemochromatosis in Brazilian Blood Donors

BackgroundThe aim of this study was to evaluate the OpenArray platform for genetic testing of blood donors and to assess the genotype frequencies of nucleotide-polymorphisms (SNPs) associated with venous thrombosis (G1691A and G20210A), hyperhomocysteinemia (C677T, A1298C), and hereditary hemochromatosis (C282Y, H63D and S65C) in blood donors from Sao Paulo, Brazil.MethodsWe examined 400 blood donor samples collected from October to November 2011. The SNPs were detected using OpenArray technology. The blood samples were also examined using a real-time PCR–FRET system to compare the results and determine the accuracy of the OpenArray method.ResultsWe observed 100% agreement in all assays tested, except HFE C282Y, which showed 99.75% agreement. The HFE C282Y assay was further confirmed through direct sequencing, and the results showed that OpenArray analysis was accurate. The calculated frequencies of each SNP were FV G1691A 98.8% (G/G), 1.2% (G/A); FII G2021A 99.5% (G/G), 0.5% (G/A); MTHFR C677T 45.5% (C/C), 44.8% (C/T), 9.8% (T/T); MTHFR A1298C 60.3% (A/A), 33.6% (A/C), 6.1% (C/C); HFE C282Y 96%(G/G), 4%(G/A), HFE H63D 78.1%(C/C), 20.3% (C/G), 1.6% (G/G); and HFE S65C 98.1% (A/A), 1.9% (A/T).ConclusionTaken together, these results describe the frequencies of SNPs associated with diseases and are important to enhance our current knowledge of the genetic profiles of Brazilian blood donors, although a larger study is needed for a more accurate determination of the frequency of the alleles. Furthermore, the OpenArray platform showed a high concordance rate with standard FRET RT-PCR.     

Optimization and Technological Development Strategies of an Antimicrobial Extract from Achyrocline alata Assisted by Statistical Design

Achyrocline alata, known as Jateí-ka-há, is traditionally used to treat several health problems, including inflammations and infections. This study aimed to optimize an active extract against Streptococcus mutans, the main bacteria that causes caries. The extract was developed using an accelerated solvent extraction and chemometric calculations. Factorial design and response surface methodologies were used to determine the most important variables, such as active compound selectivity. The standardized extraction recovered 99% of the four main compounds, gnaphaliin, helipyrone, obtusifolin and lepidissipyrone, which represent 44% of the extract. The optimized extract of A. alata has a MIC of 62.5 μg/mL against S. mutans and could be used in mouth care products.     

Developmental Block and Programmed Cell Death in Bos indicus Embryos: Effects of Protein Supplementation Source and Developmental Kinetics

The aims of this study were to determine if the protein source of the medium influences zebu embryo development and if developmental kinetics, developmental block and programmed cell death are related. The culture medium was supplemented with either fetal calf serum or bovine serum albumin. The embryos were classified as Fast (n = 1,235) or Slow (n = 485) based on the time required to reach the fourth cell cycle (48 h and 90 h post insemination - hpi -, respectively). The Slow group was further separated into two groups: those presenting exactly 4 cells at 48 hpi (Slow/4 cells) and those that reached the fourth cell cycle at 90 hpi (Slow). Blastocyst quality, DNA fragmentation, mitochondrial membrane potential and signs of apoptosis or necrosis were evaluated. The Slow group had higher incidence of developmental block than the Fast group. The embryos supplemented with fetal calf serum had lower quality. DNA fragmentation and mitochondrial membrane potential were absent in embryos at 48 hpi but present at 90 hpi. Early signs of apoptosis were more frequent in the Slow and Slow/4 cell groups than in the Fast group. We concluded that fetal calf serum reduces blastocyst development and quality, but the mechanism appears to be independent of DNA fragmentation. The apoptotic cells detected at 48 hpi reveal a possible mechanism of programmed cell death activation prior to genome activation. The apoptotic cells observed in the slow-developing embryos suggested a relationship between programmed cell death and embryonic developmental kinetics in zebu in vitro-produced embryos.