Gastric anti-ulcer activity of leaf fractions obtained of polar extract from Wilbrandia ebracteata in mice

Leaf fractions of Wilbrandia ebracteata were investigated for anti-ulcerogenic effects in ethanol and indomethacin-induced gastric ulcer assays in mice. Protective anti-ulcer effects were detected only in the ethanol-induced ulcer assay effects after pre-treatment with MeOH extract, MeOH chlorophyll-free, chlorophyll residue, HEX, DCM, aqueous MeOH fraction, ethyl acetate (EtOAc) and aqueous fractions. A potent anti-ulcerogenic effect was determined after pre-treatment of animals with EtOAc fraction, which was fractionated for isolation of active constituents. Seven flavonoids, 3`,4`,5,6,7,8-hexahydroxyflavonol, orientin, isoorientin, vitexin, isovitexin, luteolin, 6-methoxi-luteolin were isolated from the leaves of W. ebracteata (Cucurbitaceae) by chromatographic methods and identified by their spectral data. The data suggest that flavonoids are active anti-ulcerogenic compounds from leaves of W. ebracteata. The ability of scavenging free radicals was evaluated by DPPH reduction assay by TLC of flavonoids isolated.

Detection of urease in the cell wall and membranes from leaf tissues of bromeliad species

Urea is an important nitrogen source for some bromeliad species, and in nature it is derived from the excretion of amphibians, which visit or live inside the tank water. Its assimilation is dependent on the hydrolysis by urease (EC: 3.5.1.5), and although this enzyme has been extensively studied to date, little information is available about its cellular location. In higher plants, this enzyme is considered to be present in the cytoplasm. However, there is evidence that urease is secreted by the bromeliad Vriesea gigantea, implying that this enzyme is at least temporarily located in the plasmatic membrane and cell wall. In this article, urease activity was measured in different cell fractions using leaf tissues of two bromeliad species: the tank bromeliad V. gigantea and the terrestrial bromeliad Ananas comosus (L.) Merr. In both species, urease was present in the cell wall and membrane fractions, besides the cytoplasm. Moreover, a considerable difference was observed between the species: while V. gigantea had 40% of the urease activity detected in the membranes and cell wall fractions, less than 20% were found in the same fractions in A. comosus. The high proportion of urease found in cell wall and membranes in V. gigantea was also investigated by cytochemical detection and immunoreaction assay. Both approaches confirmed the enzymatic assay. We suggest this physiological characteristic allows tank bromeliads to survive in a nitrogen-limited environment, utilizing urea rapidly and efficiently and competing successfully for this nitrogen source against microorganisms that live in the tank water.

Morphology and anatomy of leaf miners in two species of Commelinaceae (Commelina diffusa Burm. f. and Floscopa glabrata (Kunth) Hassk)

In specialized literature, reports on anatomy of miners in host plants are few in number. These agents trigger excavations, or paths, by consumption of plant inner tissues by larvae of several insects. The aim of this work was to investigate leaf miner occurrence in Commelina diffusa (a cosmopolitan plant) and Floscopa glabrata (an amphibious plant) using anatomical techniques. The place where the plants were collected is subjected to seasonal floods, consequently both the species were exposed to the same weather conditions and seasonal floods. This study showed that members of Agromyzidae and Chironomidae families, which are Diptera endophytophagous larvae types, were responsible for the tunnels. Moreover, in Commelina diffusa Agromyzidae larvae were found, while in Floscopa glabrata three Chironomidae cephalic exuviae were found. The miners, as can be seen from anatomical studies, used only mesophyll parenchyma tissues for feeding, causing the formation of linear mines. In addition, in both the species, the epidermis and the medium-sized vascular units were kept intact, showing no structural modification, such as neoformation of tissues.

A comparison of the leaf-litter ant fauna in a secondary atlantic forest with an adjacent pine plantation in southeastern Brazil

We investigated the influence of Pinus afforestation on the structure of leaf-litter ant communities in the southeastern Brazilian Atlantic Forest, studying an old secondary forest and a nearly 30 year-old never managed Pinus elliottii reforested area. A total of 12,826 individual ants distributed among 95 species and 32 genera were obtained from 50 1 m² samples/ habitat. Of these, 60 species were recorded in the pine plantation and 82 in the area of Atlantic forest; almost 50% of the species found in the secondary forest area were also present in the pine plantation. The number of species per sample was significantly higher in the secondary forest than in the pine plantation. Forest-adapted taxa are the most responsible for ant species richness differences between areas, and the pine plantation is richer in species classified as soil or litter omnivorous-dominants. The specialized ant predators registered in the pine plantation, as seven Dacetini, two Basiceros, two Attini and two Discothyrea, belong to widely distributed species. The NMDS (non-metric multidimensional scaling) ordination also suggested strong differences in similarity among samples of the two areas. Furthermore, this analysis indicated higher sample heterogeneity in the secondary forest, with two clusters of species, while in the pine plantation the species belong to a single cluster. We applied the ant mosaic hypothesis to explain the distribution of the leaf-litter fauna and spatial autocorrelation tests among samples. We argue that the results are likely related to differences in quality and distribution of the leaf-litter between the pine plantation and the secondary area.

Determinação eletroquímica da capacidade antioxidante de sucos de frutas industrializados usando o CRAC assay

Fruits juices are natural sources of several compounds that present antioxidant action. Together with the fruits, they contribute with almost 40% of the antioxidant capacity in a healthy diet avoiding and preventing diseases deriving from oxidative stress. The present study determined the antioxidant capacity of seven samples of industrialized fruits juices applying CRAC (Ceric Reducing/Antioxidant Capacity) assay, a new electrochemistry assay that evaluates, by means of chronoamperometric measurements, the ability of a sample in reducing species Ce4+ in acid media. At the end of the assay was obtained the following classification: cashew > guava > grape > mango > apple > orange > passion fruit.

Biotin/avidin sandwich enzyme-linked immunosorbent assay for Culicidae mosquito blood meal identification

The knowledge of mosquitoes Culicidae host feeding patterns is basic to understand the roles of different species and to indicate their importance in the epidemiology of arthropod-borne diseases. A laboratory assay was developed aiming at standardizing the biotin-avidin sandwich enzyme-linked immunosorbent assay, which was unprecedented for mosquito blood meal identification. The enzyme-linked immunosorbent assay (ELISA) activity was evaluated by the detection of titers on each sample of the 28 blood-fed Culex quinquefasciatus. In light of the high sensitivity that the technique permits, by means of small quantities of specific antibodies commercially provided and phosphatase substrate which reinforces additional dilutions, human and rat blood meals were readily identified in all laboratory-raised Culex quinquefasciatus tested. The assay was effective to detect human blood meal dilutions up to 1:4,096, which enables the technique to be applied in field studies. Additionally, the present results indicate a significant difference between the detection patterns recorded from human blood meal which corroborate the results of host feeding patterns.

Simplified flow cytometric assay to detect minimal residual disease in childhood with acute lymphoblastic leukemia

The detection of minimal residual disease (MRD) is an important prognostic factor in childhood acute lymphoblastic leukemia (ALL) providing crucial information on the response to treatment and risk of relapse. However, the high cost of these techniques restricts their use in countries with limited resources. Thus, we prospectively studied the use of flow cytometry (FC) with a simplified 3-color assay and a limited antibody panel to detect MRD in the bone marrow (BM) and peripheral blood (PB) of children with ALL. BM and PB samples from 40 children with ALL were analyzed on days (d) 14 and 28 during induction and in weeks 24-30 of maintenance therapy. Detectable MRD was defined as > 0.01% cells expressing the aberrant immunophenotype as characterized at diagnosis among total events in the sample. A total of 87% of the patients had an aberrant immunophenotype at diagnosis. On d14, 56% of the BM and 43% of the PB samples had detectable MRD. On d28, this decreased to 45% and 31%, respectively. The percentage of cells with the aberrant phenotype was similar in both BM and PB in T-ALL but about 10 times higher in the BM of patients with B-cell-precursor ALL. Moreover, MRD was detected in the BM of patients in complete morphological remission (44% on d14 and 39% on d28). MRD was not significantly associated to gender, age, initial white blood cell count or cell lineage. This FC assay is feasible, affordable and readily applicable to detect MRD in centers with limited resources.

Detection of anti-Toxoplasma gondii antibodies in experimentally and naturally infected non-human primates by Indirect Fluorescence Assay (IFA) and indirect ELISA

The Indirect Fluorescence Assay (IFA) and the indirect ELISA were comparatively used to detect IgG and IgM antibodies for Toxoplasma gondii in experimentally and naturally infected primates. In the experimentally infected group, antibodies of diagnostic value were detected at day 9 post-infection (PI) with the IFA (IgG and IgM) and with IgG-ELISA. IgM-ELISA detected antibodies for T. gondii starting at day 3 PI until the end of the experiment (102 days PI). Of the 209 naturally infected sera tested, from many zoos of State of Sao Paulo, 64.59 and 67.94% were positive in the IgG-IFA test and IgG-ELISA respectively. IgM-ELISA test detected seropositivity in 52.63% of the sera although IgM-IFA test detected it in only in 0.96% of the samples. The differential toxoplasmosis diagnosis was accomplished with Neospora caninum by IFA, observing 61 (29.2%) seropositive animals for this parasite and 149 (70.8%) negative. Sixty animals were positive for both T. gondii and N. caninum. Pneumonia, splenomegaly, and intestinal ulcers were macroscopically observed. Unremarkable interstitial pneumonia, enteritis, colitis, splenitis, and glomerulitis were microscopically observed. The immunohistochemical stain could not detect the presence of T. gondii in the tissues of the animals infected experimentally.

Sensitivity evaluation of a single-step PCR assay using Ehrlichia canis p28 gene as a target and its application in diagnosis of canine ehrlichiosis

The aim of this study was to optimize a PCR assay that amplifies an 843 pb fragment from the p28 gene of Ehrlichia canis and compare it with two other PCR methods used to amplify portions of the 16S rRNA and dsb genes of Ehrlichia. Blood samples were collected from dogs suspected of having a positive diagnosis for canine ehrlichiosis. Amplification of the p28 gene by PCR produced an 843-bp fragment and this assay could detect DNA from one gene copy among 1 billion cells. All positive samples detected by the p28-based PCR were also positive by the 16S rRNA nested-PCR and also by the dsb-based PCR. Among the p28-based PCR negative samples, 55.3% were co-negatives, but 27.6% were positive in 16S rRNA and dsb based PCR assays. The p28-based PCR seems to be a useful test for the molecular detection of E. canis, however improvements in this PCR sensitivity are desired, so that it can become an important alternative in the diagnosis of canine ehrlichiosis.

Differentiation of Bovine coronavirus (BCoV) genotypes by a restriction enzyme assay

This article reports the use of the GsuI restriction enzyme to differentiate genotypes of Bovine Coronavirus (BCoV), based on an 18-nucleotide deletion of S1-coding region found in one of the two genotypes. It was concluded that this assay can be used as a rapid tool for BCoV genotypes differentiation.

Biotin/Avidin sandwich enzyme-linked immunosorbent assay for Culicidae mosquito blood meal identification

The knowledge of mosquitoes Culicidae host feeding patterns is basic to understand the roles of different species and to indicate their importance in the epidemiology of arthropod-borne diseases. A laboratory assay was developed aiming at standardizing the biotin-avidin sandwich enzyme-linked immunosorbent assay, which was unprecedented for mosquito blood meal identification. The enzyme-linked immunosorbent assay (ELISA) activity was evaluated by the detection of titers on each sample of the 28 blood-fed Culex quinquefasciatus. In light of the high sensitivity that the technique permits, by means of small quantities of specific antibodies commercially provided and phosphatase substrate which reinforces additional dilutions, human and rat blood meals were readily identified in all laboratory-raised Culex quinquefasciatus tested. The assay was effective to detect human blood meal dilutions up to 1:4,096, which enables the technique to be applied in field studies. Additionally, the present results indicate a significant difference between the detection patterns recorded from human blood meal which corroborate the results of host feeding patterns

Modified Steels for Cold-Forming U-Bolts Used In Leaf Springs Systems

In this work, a low alloy steel and a fabrication process were developed to produce U-Bolts for commercial vehicles. Thus, initially five types of no-heat treated steel were developed with different additions of chrome, nickel, and silicon to produce strain hardening effect during cold-forming processing of the U-Bolts, assuring the required mechanical properties. The new materials exhibited a fine perlite and ferrite microstructure due to aluminum and vanadium additions, well known as grain size refiners. The mechanical properties were evaluated in a servo-hydraulic test machine system-MTS 810 according to ASTM A370-03; E739 and E08m-00 standards. The microstructure and fractography analyses of the cold-formed steels were performed by using optical and scanning electronic microscope techniques. To evaluate the performance of the steels and the production process, fatigue tests were carried out under load control (tensile-tensile), R = 0.1 and f = 30 Hz. The Weibull statistic methodology was used for the analysis of the fatigue results. At the end of this work the 0.21% chrome content steel, Alloy 2, presented the best fatigue performance.

Importance of considering a material micro-failure criterion in the numerical modelling of the shot peening process applied to parabolic leaf springs

Shot peening is a cold-working mechanical process in which a shot stream is propelled against a component surface. Its purpose is to introduce compressive residual stresses on component surfaces for increasing the fatigue resistance. This process is widely applied in springs due to the cyclical loads requirements. This paper presents a numerical modelling of shot peening process using the finite element method. The results are compared with experimental measurements of the residual stresses, obtained by the X-rays diffraction technique, in leaf springs submitted to this process. Furthermore, the results are compared with empirical and numerical correlations developed by other authors.

High-throughput SNP genotyping in the highly heterozygous genome of Eucalyptus: assay success, polymorphism and transferability across species

Background: High-throughput SNP genotyping has become an essential requirement for molecular breeding and population genomics studies in plant species. Large scale SNP developments have been reported for several mainstream crops. A growing interest now exists to expand the speed and resolution of genetic analysis to outbred species with highly heterozygous genomes. When nucleotide diversity is high, a refined diagnosis of the target SNP sequence context is needed to convert queried SNPs into high-quality genotypes using the Golden Gate Genotyping Technology (GGGT). This issue becomes exacerbated when attempting to transfer SNPs across species, a scarcely explored topic in plants, and likely to become significant for population genomics and inter specific breeding applications in less domesticated and less funded plant genera. Results: We have successfully developed the first set of 768 SNPs assayed by the GGGT for the highly heterozygous genome of Eucalyptus from a mixed Sanger/454 database with 1,164,695 ESTs and the preliminary 4.5X draft genome sequence for E. grandis. A systematic assessment of in silico SNP filtering requirements showed that stringent constraints on the SNP surrounding sequences have a significant impact on SNP genotyping performance and polymorphism. SNP assay success was high for the 288 SNPs selected with more rigorous in silico constraints; 93% of them provided high quality genotype calls and 71% of them were polymorphic in a diverse panel of 96 individuals of five different species. SNP reliability was high across nine Eucalyptus species belonging to three sections within subgenus Symphomyrtus and still satisfactory across species of two additional subgenera, although polymorphism declined as phylogenetic distance increased. Conclusions: This study indicates that the GGGT performs well both within and across species of Eucalyptus notwithstanding its nucleotide diversity >= 2%. The development of a much larger array of informative SNPs across multiple Eucalyptus species is feasible, although strongly dependent on having a representative and sufficiently deep collection of sequences from many individuals of each target species. A higher density SNP platform will be instrumental to undertake genome-wide phylogenetic and population genomics studies and to implement molecular breeding by Genomic Selection in Eucalyptus.

Evaluation of the spouted bed dried leaf extract of Bauhinia forficata for the treatment of experimental diabetes in rats

Previously, we have demonstrated that treatment of experimental diabetes with a decoction of Bauhinia forficata leaves is beneficial. In this study, we prepared a two-fold concentrate of this extract and tested its effects on physiological, biochemical and toxicity markers in streptozotocin-diabetic rats. Dried and ground leaves were extracted with warm 70% hydroethanol and the filtrate concentrated by evaporation at 50 degrees C. This solution was mixed with colloidal silicon dioxide (Tixosil-333 (R)) and dried in a spouted bed (BfT). Rats were treated with water, insulin and Tixosil particles at low or high doses, alone or coated with dried BfT. Animals were periodically weighed and monitored for water and food intake; urinary volume, glucose, urea and protein; blood glucose, serum lipids, liver toxicity markers transaminase and phosphatase and masses of adipose tissue and skeletal muscle. Insulin treatment gave best rat growth and lowest values for all other markers. No other treatment affected any diabetic marker, but the enzyme activities were changed by diabetes and BfT. Thus, BfT toxicity could arise from secondary products of plant constituents or Tixosil interaction. Therefore, BfT prepared in the spouted bed as described, is unsuitable for treatment of diabetes, which implies that the method of preparation of any medicine is critical for its efficacy and toxicity.