Characterization of two Acanthoscelides obtectus alpha-amylases and their inactivation by wheat inhibitors

Wheat alpha-amylase inhibitors represent an important tool in engineering crop plants against bean bruchids. Because Acanthoscelides obtectus is a devastating storage bean insect-pest, we attempted to purify and characterize its gut alpha-amylases, to study their interaction with active proteinaceous inhibitors. Two digestives alpha-amylases (AoA1 and AoA2) were purified from gut larvae, showing molecular masses of 30 and 45 kDa for each one, respectively. The stoichiometry interaction between these alpha-amylases with two wheat inhibitors (0.19 and 0.53) showed a binding complex of 1:1 enzyme:inhibitor. In vivo activities of these inhibitors against A. obtectus were also evaluated using a rich ammonium sulfate inhibitor fraction (F(20)(-)(40)) and purified inhibitors after reversed phase high-performance liquid chromatography columns. Incorporation of three different inhibitor concentrations (0.25, 0.5, and 1.0% w/w) into artificial seeds showed that addition of the purified 0.19 inhibitor at the highest concentration (1.0%) reduced the larval weight by 80%. Similar data were observed when 0.53 inhibitor was incorporated at 0.5%. When the concentration of purified 0.53 was enhanced to 1.0%, no larvae or adult emergence were observed. Our data suggest that these alpha-amylase inhibitors present great potential for use in Phaseolus genetic improvement programs.     

Antioxidant activity of dietary fruits, vegetables, and commercial frozen fruit pulps

Fruits, vegetables, and commercial frozen pulps (FP) consumed in the Brazilian diet were analyzed for antioxidant activities using two different methods, one that determines the inhibition of copper-induced peroxidation of liposome and another based on the inhibition of the co-oxidation of linoleic acid and beta-carotene. The anthocyanin-rich samples showed the highest, concentration-dependent, antioxidant activities in both systems. In the liposome system, at both 10 and 50 microM gallic acid equivalent (GAE) addition levels, the neutral and acidic flavonoids of red cabbage, red lettuce, black bean, mulberry, Gala apple peel, jambolao, acai FP, mulberry FP, and the acidic flavonoids of acerola FP showed the highest antioxidant activities (>85% inhibition). In the beta-carotene bleaching system, the samples cited above plus red guava gave inhibition values >70%. On the other hand, some samples showed pro-oxidant activity in the liposome system coincident with a low antioxidant activity in the beta-carotene system. There was no relationship between total phenolics content, vitamin C, and antioxidant activity, suggesting that the antioxidant activity is a result of a combination of different compounds having synergic and antagonistic effects.     

Activity, cloning, and expression of an isoamylase-type starch-debranching enzyme from banana fruit

Unripe bananas have a high content of starch (almost 20%) that is metabolized during fruit ripening with a concomitant synthesis of soluble sugars. Since starch granules are composed of amylose and amylopectin, several enzymes have to be involved in its mobilization during banana ripening, with a necessary participation of one starch-debranching enzyme (DBE) to hydrolyze the alpha-1,6-branches of amylopectin. Banana DBE seems to be an isoamylase-type enzyme, as indicated by substrate specificity and the cloning of a 1575 bp cDNA, similar to the isoamylase sequences from potato, Arabdopsis, and maize. The assays for DBE indicated only minor changes in activity during ripening, and the results of the northern and western blots with antiserum against the recombinant banana isoamylase were in agreement with the steady-state level of activity, since no significant changes in gene expression were observed. The high activity on beta-limit dextrin and the similarity to the potato isoform 3 suggest that during banana ripening the hydrolysis of alpha-1,6-linkage of amylopectin results from the activity of a pre-existing isoamylase-type debranching enzyme in coordination with other amylolitic enzymes. To the best of our knowledge, this is the first evaluation of activity and expression of a DBE from a fruit.     

Effects of gamma-irradiation on the free radical and antioxidant contents in nine aromatic herbs and spices

Nine spice and aromatic herb samples (i.e., basil, bird pepper, black pepper, cinnamon, nutmeg, oregano, parsley, rosemary, and sage) were gamma-irradiated at a dose of 10 kGy according to commercial practices. The effects of the disinfection treatment on the content of organic radicals and some nutrients (namely, vitamin C and carotenoids) in the samples were investigated by chromatographic and spectroscopic techniques. Irradiation resulted in a general increase of quinone radical content in all of the investigated samples, as revealed by electron paramagnetic resonance spectroscopy. The fate of these radicals after storage for 3 months was also investigated. The cellulose radical was clearly observed in a few samples. Significant losses of total ascorbate were found for black pepper, cinnamon, nutmeg, oregano, and sage, whereas a significant decrease of carotenoids content was observed for cinnamon, oregano, parsley, rosemary, bird pepper, and sage.     

Morphometry of the heart of greater rhea (Rhea americana americana,Linnaeus, 1758)

Heart diseases in birds are frequent and generate significant production disorders. Morphometry is a valuable tool to provide fundamental information about heart conditions. Few studies have addressed morphological aspects of the heart of ratite birds, such as the Greater rhea. The present study aimed to analyse rhea heart morphometry, comparing young and adult subjects, in order to provide relevant information for the diagnosis of heart disease in this species. Hearts of young (n = 10) and adult (n = 10) female rheas were used in this research. Heart length and width and sternum length were measured using a caliper. Heart length and width and sternum length in adults were approximately three times greater than in young individuals. The left ventricular wall (LVW) was thicker than the right ventricular wall (RVW) at all ages, and the RVW was thicker in adults when compared to young subjects. The basal and middle RVW regions thicken with advancing age, and the thickness of the interventricular septum (ISW) occupies an intermediate position between the LVW and RVW. In general, an increase in rhea heart thickness and size relative to age is observed. The morphometric variations between young and adult rhea hearts observed in the present study may serve as a comparative subsidy for the diagnosis of cardiac abnormalities observed in this species.     

Along or across the visual axis: a comparison of two ultrasound screen,needle and transducer orientation techniques

ObjectiveTo evaluate two transducer and needle handling methods, along the visual axis (AL) and across the visual axis (AC), in non-skilled and skilled clinicians.Study designProspective randomized crossover study.MethodA total of 26 students with no ultrasound locoregional anaesthesia experience (non-skilled group) and six clinicians experienced and familiar with ultrasound locoregional anaesthesia (skilled group) were enrolled. The non-skilled group was asked to perform two tasks: the first on a phantom and the second on canine cadavers, whilst the skilled group performed only the second task. The tasks consisted of guiding the tip of the needle to a target point (simulated nerve on the jelly phantom and sciatic nerve on the cadavers) using two different methods of needle handling—AL or AC. All operators performed each task three times for each method. The time to drive the needle to the target for the two methods was analysed with a paired Student t test, and the number of times the needle was not visualized on the screen between the groups was compared using an unpaired Student t test. Data are presented as mean ± standard deviation. Value of p < 0.05 was considered significant.ResultsThe AL method, compared with the AC method, resulted in shorter performance time in both skilled (9 ± 5 versus 20 ± 8 seconds for the second task) and non-skilled groups (9 ± 8 versus 17 ± 15 seconds for the first task and 18 ± 11 versus 32 ± 26 seconds for the second task).Conclusion and clinical relevanceIn both groups, the AL method significantly reduced the time to complete the task. Results from this study indicate that the AL method should be the preferred method for learning/teaching ultrasound-guided regional anaesthesia.     

Competition and persistence of Rhizobium tropici and Rhizobium etli in tropical soil during successive bean (Phaseolus vulgaris L.) cultures

Strains of Rhizobium tropici IIB, CIAT899 and F98.5, both showing good N₂ fixation, and a R. etli strain W16.3SB were introduced into a field which had no history of bean culture. Plant dilution estimates showed that in the presence of its host (Phaseolus vulgaris cv. Carioca) during the cropping seasons and the subsequent fallow summer periods, the bean rhizobial populations increased from less than 30 to 10³ g^–1 dry soil after 1 year and to 10⁴ g^–1 dry soil after 2 years. In the 1st year crop, the inoculated strains occupied most of the nodules, which resulted in a higher nodulation and C₂H₂ reduction activity. Without reinoculation for the second and third crops, however, little R. tropici IIB was recovered from the nodules and the bean population consisted mainly of R. etli, R. leguminosarum bv. phaseoli, and R. tropici IIA. Reinoculation with our superior R. tropici IIB strains before the second crop resulted in R. tropici IIB occupying the main part of the nodules and a positive effect on nodulation and C₂H₂ reduction activity, but reintroduction of the inoculant strain in the third season did not have any effect.     

Gas chromatography-olfactometry (GC-O) and proton transfer reaction-mass spectrometry (PTR-MS) analysis of the flavor profile of grana padano,parmigiano reggiano,and grana trentino cheeses

Gas chromatography-olfactometry (GC-O) and proton transfer reaction-mass spectrometry (PTR-MS) techniques were used to deduce the profile of odor-active and volatile compounds of three grana cheeses: Grana Padano (GP), Parmigiano Reggiano (PR), and Grana Trentino (GT). Samples for GC-O analysis were prepared by dynamic headspace extraction, while a direct analysis of the headspace formed over cheese was performed by PTR-MS. The major contributors to the odor profile were ethyl butanoate, 2-heptanone, and ethyl hexanoate, with fruity notes. A high concentration of mass 45, tentatively identified as acetaldehyde, was found by PTR-MS analysis. Low odor threshold compounds, e.g., methional and 1-octen-3-one, which contributed to the odor profile but were not detected by FID, were detected by PTR-MS. Principal component analysis on both GC-O and PTR-MS data separated the three cheese samples well and showed specific compounds related to each sample.     

Dimethylmercury and dimethylmercury-sulfide of microbial origin in the biogeochemical cycle of HG

The transformation of MeHg under anaerobic conditions in axenic cultures ofDesulfovibrio desulfuricans strain LS is compared to that in anoxic marine sediments contaminated by Hg of industrial origin. MeHg was added to cultures ofD. desulfuricans strain LS and incubated at 28°C for two weeks. Significant amounts of dimethylmercury and metacinnabar were produced. These two Hg compounds were formed from the slow decomposition of the intermediate dimethylmercury-sulfide. Other collateral compounds, such as methane and ionic Hg, were also detected during the MeHg degradation process. On the other hand a sample of fresh sediment (1.5 g d.w.) was spiked with 10 μg of MeHg and 2 mmoles.ml^?1 of pyruvate, as carbon source for sulfate-reducing bacteria. After 9 days of incubation at 28°C, significant amounts of dimethylmercury were produced. A lower content of this volatile species was found in a subsample of sediment supplemented with sodium molybdate, which is a strong inhibitor of sulfatereducing activity. A kinetic study showed the disappearance of monomethylmercury from the sediment and the formation of dimethylmercury over the incubation period. The environmental significance of dimethylmercury and dimethylmercury-sulfide in the natural biogeochemical cycle of Hg is discussed.     

Induction of resistance to gray mold with benzothiadiazole modifies amino acid profile and increases proanthocyanidins in grape: primary versus secondary metabolism

Field treatments of grapevine (cv. Merlot) with the plant activator benzothiadiazole (BTH, 0.3 mM) induced resistance against gray mold caused by Botrytis cinerea. Both incidence and severity of the disease were reduced. The resistance was associated with an increase of total polyphenols in berry skins, in particular, the proanthocyanidin fraction, that increased up to 36%. The amino acid profile of leaves was also modified by treatments, particularly lysine, that augmented 4-fold. Other amino acids involved in resistance mechanisms to either biotic or abiotic stress increased as well. These results indicate that BTH treatments can be used to control gray mold, thereby limiting an excessive use of fungicides, and could be exploited to increase the content of micronutrients of high nutritional value, arising from both primary and secondary metabolisms.