Isolation of lignans glycosides from Alibertia sessilis (Vell.) K. Schum. (Rubiaceae) by preparative high-performance liquid chromatography

Enantiomeric aglycone lignans contained in a mixture were separated from a fraction of the extract of the stems of Alibertia sessilis (Vell.) K. Schum. (Rubiaceae) by preparative high-performance liquid chromatography. An efficient and fast separation can be achieved with methanol-water (30:70, v/v). Their structures were identified as (+)-lyoniresinol 3alpha-O-beta-glucopyranoside and (-)-lyoniresinol 3alpha-O-beta-glucopyranoside, being reported for the first time in Rubiaceae.

Humic substances from sediments of Lobos Pond (Argentina). Isolation, characterization and limnological implications

Humic substances (HS) were isolated from the sediments of Lobos Pond (Argentina) using mild conditions to preserve their native structure. The HS (humic and fulvic acids) were characterized by means of elemental analysis and FTIR spectroscopy. Also a by-product obtained during fulvic acids (FA) fractionation (an amorphous white solid residue) was analyzed. Results revealed possible interactions between FA and inorganic-organic substances that may have implications referring to bioavailability. Other limnological implications, such as autochtonous origin of HS linked with the hydrology, and change of pH during stormy weather that affects HS interactions, are discussed.

Semi-selective culture medium for Exserohilum turcicum isolation from corn seeds

Northern corn leaf blight, caused by Exserohilum turcicum (Et), is a disease of widespread occurrence in regions where corn, sweetcorn and popcorn are grown. This disease has great potential to cause damage and has been studied for years, but the association of its causal agent with seeds remains unconfirmed. Thus, the availability of a sensitive method to detect and quantify the inoculum in seeds, even at low incidence, is essential. The aim of this study was to develop a method to detect and quantify the presence of the fungus infecting and infesting corn and popcorn seeds. Artificially and naturally infected seeds were employed to develop the medium. The semi-selective medium was composed of carbendazim (active ingredient) (60 mg/L), captan (30 mg/L), streptomycin sulfate (500 mg/L) and neomycin sulfate (600 mg/L) aggregated to the medium lactose casein hydrolysate agar medium. By using this, Et was detected in naturally infected corn seeds, showing 0.124% incidence, in four out of ten analyzed samples. In addition, 1.04 conidia were detected per infested seed. By means of isolation, pathogenicity test, morphological characterization and comparison with descriptions of the species in the literature, the fungus isolated from the seeds was confirmed to be Et. Both infection and infestation were considered low; thus, for studies of Et detection in corn seeds, the use of semi-selective medium and more than 1,200 seeds/sample is suggested.

PRESERVATION OF PRIMARY FOREST CHARACTERISTICS DESPITE FRAGMENTATION AND ISOLATION IN A FOREST REMNANT FROM VIÇOSA, MG, BRAZIL1

According to its owners, the Forest of Seu Nico (FSN) from the Viçosa municipality, Minas Gerais, Brazil, never has been logged and is therefore considered a primary forest. Nevertheless, the forest patch suffered impacts due to selective wood and non-timber extraction, fragmentation and isolation. Aim of this study was to test if the FSN, despite impacts, preserved characteristics of primary forests, which are elevated percentages of non-pioneer (>90%), animal-dispersed (>80 %), understory (>50%) and endemic species (~40%). For that, all trees with diameter at breast height equal or major than 3.2 cm within a plot of 100 x 100 m were identified. With 218 tree species found within this hectare, the FSN's species richness is outstanding for the region. The percentages of non-pioneer (92 %), animal-dispersed (85 %), understory (55 %) and endemic species (39.2 %) from the FSN fulfill the criteria proposed for primary forest. Therefore, we conclude that the FSN maintained its characteristics as a primary forest which highlights its importance for the conservation of biotic resources in the region, where similar fragments are lacking or not described yet.

Benefits of automated acclimatization during the pre-milking phase of lactating girolando cows

The aim of this paper was to evaluate the automated acclimatization effects during pre-milking of cows on thermal conditioning, physiology, milk production and cost-benefit of the automated adiabatic evaporative cooling system (AECS). The treatments 20; 30; 40 min and control consisted of exposure time of pre-milking cows to the automated AECS. Sixteen cows were used with an average daily milk yield of 19 kg, distributed in a 4 x 4 Latin square design. The Tukey's test (P<0.05) was used to compare the means. The environmental variables, dry bulb temperature (DBT, ºC) and relative humidity (RH, %), were recorded every minute, which allowed the determination of the system efficiency through the Temperature and Humidity Index (THI). The respiratory rate (RR), rectal temperature (RT) and temperature of the coat (TC) were measured before and after the acclimatization. The 40 min treatment kept the environmental variables and the comfort indexes within recommended limits. The physiological variables (RR, RT and TC) were lower in the 40 min treatment and reflected positively on milk production, which increased 3.66% compared to the control treatment. The system was profitable, having a 43 days return on investment and a monthly revenue increase of R$ 1,992.67.

Isolation of microorganisms of cheese whey with lipolytic activity for removal of COD

The aim of this study was to isolate microorganisms that produce lipase and to assess the efficiency of COD removal intreatment of cheese whey under different operating conditions. The microorganisms were isolated from cheese whey and a commercial product; it was selectedthreemicroorganisms that obtained the best response to the lipolytic activity test through the enzyme index. Then, the microorganisms were inoculated in sterilized cheese whey samples, for two pH values (6.2 and 7.0), incubated at 35 °C and 150 rpm in shaker and the lipolityc activity and the efficiency of COD removal were measured in two time periods (24 and 48h). After incubation, it was observed that the treatments showed a good removal efficiency of COD for the pre-treatment and the isolated microorganism (S1) from the cheese whey showed the highest lipase production. Regarding the pH and time variables, there was not significant effect between the two evaluated factors. Among all treatments, T2 (S1, pH 7.0 and 24h) obtained more enzyme production (4.87 U mL-1).

Development of an automated bench top electronic penetrometer

The aim of this study was to develop a an automated bench top electronic penetrometer (ABEP) that allows performing tests with high rate of data acquisition (up to 19,600 Hz) and with variation of the displacement velocity and of the base area of cone penetration. The mechanical components of the ABEP are: a supporting structure, stepper motor, velocity reducer, double nut ball screw and six penetration probes. The electronic components of ABEP are: a "driver" to control rotation and displacement, power supply, three load cells, two software programs for running and storing data, and a data acquisition module. This penetrometer presented in compact size, portable and in 32 validation tests it proved easy to operate, and showed high resolution, high velocity in reliability in data collection. During the validation tests the equipment met the objectives, because the test results showed that the ABEP could use different sizes of cones, allowed work at different velocities, showed for velocity and displacement, were only 1.3% and 0.7%, respectively, at the highest velocity (30 mm s-1) and 1% and 0.9%, respectively for the lowest velocity (0.1 mm s-1).

Screening of antigenemia and isolation of Cryptococcus neoformans and C. gattii from cloaca and crop of birds in the state of Paraná, Brazil

Cryptococcus neoformans and C. gattii are associated with dry bird excreta but rarely recovered from birds' digestive tract. The objective of the present study was (1) to verify the existence of C. neoformans and C. gattii in crop and cloaca of wildlife and captivity birds hypothesizing about a possible primary source of this yeast in the excreta, and (2) to determine the fungi's invasive capability in avian species through latex agglutination. For that purpose, 172 cloacal and 77 crop samples of domestic pigeon, Passerine, and Psittacine birds were collected. None of these samples was positive, suggesting that the yeast is not saprobiotic in the digestive tract of these birds. Only one out of 82 serum samples collected from pigeons and Psittacine birds was positive (title 1:2) showing that Cryptococcus sp. probably has a low invasive capability in birds, and is thus considered only a dry excreta colonizer.

Isolation and characterization of equine peripheral blood-derived multipotent mesenchymal stromal cells

The objective of the study was to isolate, cultivate and characterize equine peripheral blood-derived multipotent mesenchymal stromal cells (PbMSCs). Peripheral blood was collected, followed by the isolation of mononuclear cells using density gradient reagents, and the cultivation of adherent cells. Monoclonal mouse anti-horse CD13, mouse anti-horse CD44, and mouse anti-rat CD90 antibodies were used for the immunophenotypic characterization of the surface of the PbMSCs. These cells were also cultured in specific media for adipogenic and chondrogenic differentiation. There was no expression of the CD13 marker, but CD44 and CD90 were expressed in all of the passages tested. After 14 days of cell differentiation into adipocytes, lipid droplets were observed upon Oil Red O (ORO) staining. Twenty-one days after chondrogenic differentiation, the cells were stained with Alcian Blue. Although the technique for the isolation of these cells requires improvement, the present study demonstrates the partial characterization of PbMSCs, classifying them as a promising type of progenitor cells for use in equine cell therapy.

First isolation and characterization of Leptospira interrogans serogroup Australis from swine in Brazil

The purpose of this study was to report the first recovery and characterization of Leptospira interrogans (serogroup Australis) from urine of swine in Brazil. The isolate was studied by serogrouping, MLVA, PGFE, and partial sequencing of rrs and secY. It was serogrouped as serogroup Australis, probably serovar Bratislava (titre 1,600), and sequenced as Leptospira interrogans. The MLVA and PGFE profiles also suggested the isolate as serovar Bratislava, since they were indistinguishable from reference strains Balico and Jez Bratislava. This is the first Leptospira interrogans serogroup Australis isolate, probably serovar Bratislava, obtained in Brazil.

Isolation, expansion and differentiation of cellular progenitors obtained from dental pulp of agouti (Dasyprocta prymnolopha Wagler, 1831)

Abstract: The study aimed to isolate, expand, differentiate and characterize progenitor cells existent in the dental pulp of agouti. The material was washed with PBS solution and dissociated mechanically with the aid of a scalpel blade on plates containing culture medium D-MEM/F-12, and incubated at 5% CO2-37⁰C. The growth curve, CFU assay, osteogenic/adipogenic differentiation and characterization were obtained from the isolation. The cells began to be released from the explant tissue around the 7th day of culture. By day 22 of culture, cells reached 80% confluence. At the UFC test, 81 colonies were counted with 12 days of cultivation. The growth curves before and after freezing showed a regular growth with intense proliferation and clonogenic potential. The cell differentiation showed formation of osteoblasts and fat in culture, starting at 15 days of culture in a specific medium. Flow cytometry (FACs) was as follows: CD34 (positive), CD14 (negative), CD45 (negative), CD73 (positive), CD79 (negative), CD90 (positive), CD105 (positive), demonstrating high specificity and commitment of isolated cells with mesenchymal stem cells strains. These results suggest the existence of a cell population of stem cells with mesenchymal features from the isolated tissue in the explants of agouti dental pulp, a potential model for study of stem cell strains obtained from the pulp tissue.

Reproductive isolation between Anastrepha bistrigata and A. striata (Diptera, Tephritidae)

The reproductive isolation between two closely related species, Anastrepha bistrigata and A. striata, was studied in the laboratory. Interespecific copulation attempts were observed, but examination of the spermathecae showed that sperm transference did not occur, even after a prolonged period of contact between the mating pairs. These results indicate prezygotic isolation. The analysis of the hourly distribution of mating activities under laboratory conditions, here described for the first time for A. bistrigata, clearly showed differences for the two species, the activities being concentrated in the afternoon period for A. striata and in the morning for A. bistrigata

Isolation and characterization of a reserve protein from the seeds of Opuntia ficus-indica (Cactaceae)

We describe here the isolation and characterization of a major albumin from the seeds of Opuntia ficus-indica (Cactaceae). This protein has a molecular mass of 6.5 kDa and was isolated by a combination of gel filtration chromatography and reverse-phase HPLC. The amino acid composition of this protein was determined and it was shown to have similarities with the amino acid composition of several proteins from the 2S albumin storage protein family. The N-terminal amino acid sequence of this protein is Asp-Pro-Tyr-Trp-Glu-Gln-Arg.

An optically coupled power stimulus isolation unit with high voltage and fast rise time output

Recent technological developments have created new devices that could improve and simplify the construction of stimulus isolators. HEXFET transistors can switch large currents and hundreds of volts in nanoseconds. The newer opto-isolators can give a pulse rise time of a few nanoseconds, with output compatible with MOSFET devices, in which delays are reduced to nanoseconds. Integrated DC/DC converters are now available. Using these new resources we developed a new electrical stimulus isolator circuit with selectable constant-current and constant-voltage modes, which are precise and easy to construct. The circuit works like a regulated power supply in both modes with output switched to zero or to free mode through an opto-isolator device. The isolator analyses showed good practical performance. The output to ground resistance was 1011 ohms and capacitance 35 picofarads. The rise time and fall time were identical (5 µs) and constant. The selectable voltage or current output mode made it very convenient to use. The current mode, with higher output resistance values in low current ranges, permits intracellular stimulation even with tip resistances close to 100 megaohms. The high compliance of 200 V guarantees the value of the current stimulus. The very low output resistance in the voltage mode made the device highly suitable for extracellular stimulation with low impedance electrodes. Most importantly, these characteristics were achieved with a circuit that was easy to build and modify and assembled with components available in Brazil.

Isolation of bovine plasma albumin by liquid chromatography and its polymerization for use in immunohematology

The aim of the method described here is to remove hemoglobin, the major contaminant in the bovine plasma obtained from slaughterhouses, by adding a mixture of 19% cold ethanol and 0.6% chloroform, followed by fibrinogen and globulin precipitation by the Cohn method and nonspecific hemagglutinin by thermocoagulation. The experimental volume of bovine plasma was 2,000 ml per batch. Final purification was performed by liquid chromatography using the ion-exchange gel DEAE-Sepharose FF. The bovine albumin thus obtained presented > or = 99% purity, a yield of 25.0 ± 1.2 g/l plasma and >71.5% recovery. N-acetyl-DL-tryptophan (0.04 mmol/g protein) and sodium caprylate (0.04 mmol/g protein) were used as stabilizers and the final concentration of albumin was adjusted to 22.0% (w/v), pH 7.2 to 7.3. Viral inactivation was performed by pasteurization for 10 h at 60°C. The bovine albumin for the hemagglutination tests used in immunohematology was submitted to chemical treatment with 0.06% (w/v) glutaraldehyde and 0.1% (w/v) formaldehyde at 37°C for 12 h to obtain polymerization. A change in molecular distribution was observed after this treatment, with average contents of 56.0% monomers, 23.6% dimers, 12.2% trimers and 8.2% polymers. The tests performed demonstrated that this polymerized albumin enhances the agglutination of Rho(D)-positive red cells by anti-Rho(D) serum, permitting and improving visualization of the results.