Isolation of a cotton NADP(H) oxidase homologue induced by drought stress

The aim of this study was to identify and isolate genes that are differentially expressed in four selected cotton (Gossypium hirsutum L.) genotypes contrasting according to their tolerance to water deficit. The genotypes studied were Siokra L-23, Stoneville 506, CS 50 and T-1521. Physiological, morphological and developmental changes that confer drought tolerance in plants must have a molecular genetic basis. To identify and isolate the genes, the mRNA Differential Display (DD) technique was used. Messenger RNAs differentially expressed during water deficit were identified, isolated, cloned and sequenced. The cloned transcript A12B15-5, a NADP(H) oxidase homologue, was up regulated only during the water deficit stress and only in Siokra L-23, a drought tolerant genotype. Ribonuclease protection assay confirmed that transcription.

Genetic distance estimated by RAPD markers and its relationship with hybrid performance in maize

The objective of this work was to evaluate the genetic diversity of 16 maize inbred lines, and to determine the correlation between genetic distance and hybrid performance, using random amplified polymorphic DNA (RAPD) molecular markers. Twenty-two different random primers were used, which resulted in the amplification of 265 fragments, 237 (84.44%) of them being polymorphic. A genetic similarity matrix was created from the RAPD data, using Jaccard coefficient, and a dendrogram was constructed. Hybrid analyses were carried out using random block design and Griffing method VI for diallel crossings. The genetic associations showed five distinct heterotic groups. Correlations between genetic divergences detected by RAPD, as well as the means observed in the diallel crossings were positive and significant for plant height, ear height, prolificacy, and grain weight. The correlation of genetic divergences, detected by RAPD, and the specific combining ability between heterotic group associations, showed significance in all characteristics under study, except prolificacy. A direct relationship between genetic divergence and productivity was found in 79.2% of the 120 hybrids confirming the hypothesis that genetic divergence is directly related to the performance of hybrids and is efficient in predicting it.

Extraction and isolation of dictamnine, obacunone and fraxinellone from Dictamnus dasycarpus Turcz. by supercritical fluid extraction and high-speed counter-current chromatography

Supercritical fluid extraction was used to extract active compounds from the Chinese traditional medicinal D. dasycarpus under the pressure of 30 MPa and temperature of 45 ºC. Further separation and purification was established by high-speed counter-current chromatography (HSCCC) with a two-phase solvent system composed of n-hexane-ethyl acetate-methanol-water (1:0.8:1.3:0.9, volume ratio). The separation yielded a total of 47 mg of dictamnine, 24 mg of obacunone and 83 mg of fraxinellone from 1.0 g of the crude extract in one step separation with the purity of 99.2, 98.4 and 99.0%, respectively, as determined by HPLC. The chemical structures of these compounds were identified by ESI-MS, IR, ¹H-NMR and 13C-NMR.

PREDICTING THE BOILING POINT OF PCDD/Fs BY THE QSPR METHOD BASED ON THE MOLECULAR DISTANCE-EDGE VECTOR INDEX

The quantitative structure property relationship (QSPR) for the boiling point (Tb) of polychlorinated dibenzo-p-dioxins and polychlorinated dibenzofurans (PCDD/Fs) was investigated. The molecular distance-edge vector (MDEV) index was used as the structural descriptor. The quantitative relationship between the MDEV index and Tb was modeled by using multivariate linear regression (MLR) and artificial neural network (ANN), respectively. Leave-one-out cross validation and external validation were carried out to assess the prediction performance of the models developed. For the MLR method, the prediction root mean square relative error (RMSRE) of leave-one-out cross validation and external validation was 1.77 and 1.23, respectively. For the ANN method, the prediction RMSRE of leave-one-out cross validation and external validation was 1.65 and 1.16, respectively. A quantitative relationship between the MDEV index and Tb of PCDD/Fs was demonstrated. Both MLR and ANN are practicable for modeling this relationship. The MLR model and ANN model developed can be used to predict the Tb of PCDD/Fs. Thus, the Tb of each PCDD/F was predicted by the developed models.

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.

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.

Anthocyanins standards (cyanidin-3-O-glucoside and cyanidin-3-O-rutinoside) isolation from freeze-dried açaí (Euterpe oleraceae Mart.) by HPLC

Availability of analytical standards is a critical aspect in developing methods for quantitative analysis of anthocyanins. The anthocyanins cyanidin-3-O-glucoside and cyanidin-3-O-rutinoside were isolated from samples of freeze-dried açaí (Euterpe oleraceae Mart.), which is a round and purple well-known palm fruit in Brazil, and then used as standards. The isolation of the anthocyanins was performed by High Performance Liquid Chromatography (HPLC), using an adapted six-channel selection valve. The identification of anthocyanin pigments in açaí was based on mass spectrometric data for molecular ions and MS-MS product ions and on previous published data. After the collection procedure, standards with a high purity grade were obtained and an external standard curve of each anthocyanin was plotted.

Isolation and recovery of glycomacropeptide from milk whey by means of thermal treatment

During enzymatic process of cheese manufacturing, rennin cleaves κ-casein releasing two fractions: para-κ-casein and glycomacropeptide (GMP), which remains soluble in milk whey. GMP is a peptide with structural particularities such as chain carbohydrates linked to specific threonine residues, to which a great variety of biological activities is attributed. Worldwide cheese production has increased generating high volumes of milk whey that could be efficiently used as an alternative source of high quality peptide or protein in foodstuff formulations. In order to evaluate isolation and recovery on whey GMP by means of thermal treatment (90 °C), 18 samples (2 L each) of sweet whey, resuspended commercial whey (positive control) and acid whey (negative control) were processed. Indirect presence of GMP was verified using chemical tests and PAGE-SDS 15%. At 90 °C treated sweet whey, 14, 20 and 41 kDa bands were observed. These bands may correspond to olygomers of GMP. Peptide recovery showed an average of 1.5 g/L (34.08%). The results indicate that industrial scale GMP production is feasible; however, further research must be carried out for the biological and nutritional evaluation of GMP's incorporation to foodstuff as a supplement.

Isolation and characterization of bacterial strains with a hydrolytic profile with potential use in bioconversion of agroindustial by-products and waste

There is a trend towards the use of novel technologies nowadays, mainly focused on biological processes, for recycling and the efficient utilization of organic residues that can be metabolized by different microorganisms as a source of energy. In the present study the isolation of bacterial strains from six different agro-industrial by-products and waste was performed with the objective of evaluating their hydrolytic capacities and suitability for use in bioconversion of specific substrates. The 34 isolated strains were screened in specific culture media for the production of various hydrolytic enzymes (lipase, protease, cellulase, and amylase). It was found that 28 strains exhibited proteolytic activity, 18 had lipolytic activity, 13 had caseinolytic activity, 15 had amylolytic activity, and 11 strains exhibited cellulolytic activity. The strains that showed the highest hydrolytic capacities with biotechnological potential were selected, characterized genotipically, and identified as Bacillus, Serratia, Enterococcus, Klebsiella, Stenotrophomonas, Lactococcus, and Escherichia genera. It was concluded that the strain isolates have a high potential for use in the bioconversion of agro-industrial waste, both as a pure culture and as a microbial consortium.

Isolation and serological identification of enteropathogenic Escherichia coli in pasteurized milk in Brazil

OBJECTIVE: To evaluate the microbiological quality of pasteurized milk commercialized in Rio de Janeiro, Brazil, and determine serologically enteropathogenic Escherichia coli (EPEC) strains in E. coli isolates obtained from milk samples. METHODS: Ninety samples of pasteurized milk -- types B and C -- of three different commercial brands, purchased in supermarkets and bakeries in Rio de Janeiro, were examined. The amount of total and fecal coliform bacteria was estimated using the Most Probable Number technique. Mesophilic, psychrotrophic, and thermoduric microorganism counts were determined by the Standard Plate Count technique. Isolation and identification of E. coli were carried out using conventional physiological tests. Commercial antisera were used for serological characterization of EPEC. RESULTS: The three milk brands analyzed revealed bacterial counts above the regulated values of the Brazilian government. It was found that among 208 strains of E. coli isolated, 46 (22.1%) were serologically classified as EPEC. The most common EPEC serogroup was O55 (15.2%). CONCLUSIONS: Though recent studies on virulence factors indicate that not all strains serologically classified as EPEC are able to attaching/effacing lesion, it is believed that the isolation of EPEC serogroups from pasteurized milk represent a potential risk for children, as well as an indicative of the presence of other enteropathogens.

Acute pulmonary histoplasmosis and first isolation of Histoplasma capsulatum from soil of Rio Grande do Sul, Brazil

A case of acute pulmonary histoplasmosis, where the clinical histoiy and epidemiological data led to the identification of H. capsulatum natural source, is described. Specimens of spleen and liver, obtained after intraperitonial inoculation in mice, grew H. capsulatum in culture from the soil of rural area of General Câmara, by the first time in Rio Grande do Sul.

Detection of circulating polysaccharide antigen of Schistosoma mansoni in hamster sera by crossed immunoelectrophoresis

Crossed immunoelectrophoresis (IEC) was used for detection of free and complexed circulating polisaccharide anodic antigen (AgCA) of Schistosoma mansoni in sera of infected hamsters. An attempt was also done to detect AgCA in human sera from patients infected with S. mansoni. The conditions for isolation and detection of complexed AgCA were established. The sensitivity of IEC was increased by incorporation of 2% polyethylene glicol (PEG) to the agarose and by maintaining the system at 4°C during the electrophoretic run. Free AgCA was detected in 12 and the complexed in 30 of the 37 hamsters sera analysed. Correlation between AgCA (free and complexed) and the parasite load was observed. AgCA was not detected, under the experimental conditions used, in human sera from 7 patients in the acute and 23 in the chronic phase of infection.

Comparative studies of Yersinia pestis outer membrane isolation techniques and their potential use in plague epidemiology

In the present study three techniques for obtaining outer membrane enriched fractions from Yersinia pestis were evaluated. The techniques analysed were: differential solubilization of the cytoplasmic membrane with Sarkosyl or Triton X-100, and centrifugation in sucrose density gradients. The sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of outer membrane isolated by the different methods resulted in similar protein patterns. The measurement of NADH-dehydrogenase and succinate dehydrogenase (inner membrane enzymes) indicated that the outer membrane preparations obtained by the three methods were pure enough for analytical studies. In addition, preliminary evidences on the potential use of outer membrane proteins for the identification of geographic variants of Y. pestis wild isolates are presented.

Isolation of Leishmania sp. from aqueous humor of a patient with cutaneous disseminated leishmaniasis and bilateral iridocyclitis (preliminary report)

The Authors report an uncommon case of leishmaniasis with disseminated cutaneous lesions, systemic manifestations and ocular involvement, the latter being characterized by bilateral nongranulomatous iridocyclitis. The severity of the oph-thalmologic lesions and its unresponsiveness to therapy (in spite of satisfactory regression of both systemic and cutaneous manifestations) lead to a needle aspiration of the anterior eye chamber, content. From this material Leishmania sp was isolated. To our knowledge this is the first time that Leishmania has been shown into the ocular globe.

Isolation and characterization of partially purified leptospiral antigens

The methanol extract of Leptospira interrogans serovar canicola was purified by precipitation with acetone or acetone and chloroform. The antigenicity of the antigen was not altered by heating or treatment with pepsin and pronase. However the antigenicity was lost when the antigen was treated with periodic acid. Chemical analysis revealed the presence of 40% carbohydrate (22% methylpentose, 28%; hexoses),4% protein, 20% lipid and 2,7% phosphate. The complement fixation test with sera from patients with leptospirosis agreed with the microscopic agglutination reaction.