Multiple forms of cotyledonary b-galactosidases from Vigna unguiculata quiescent seeds

Cotyledonary b-galactosidases were isolated and partially purified from Pitiúba cowpea (Vigna unguiculata (L.) Walp.) quiescent seeds. The purification steps consisted of precipitation of the crude extract with ammonium sulphate in the range of 20-60% saturation, acid precipitation, DEAE-Sephadex ion-exchange chromatography and Lactosyl-Sepharose affinity chromatography. This purification process gave rise to three b-galactosidases-rich fractions: b-gal I, b-gal II and b-gal III, which were purified about 5, 509, and 62 fold, respectively. They reached maximal enzyme activity at different pH ranges: 3.5-4.5 for b-gal I, 3.0-3.5 for b-gal II, and 3.0-4.0 for b-gal III. Their maximal activities were reached when the temperature of the assay medium was 60° C, and preincubation of the enzymes at different temperatures has shown that they were heat-stable up to 50° C. There were no significant differences among the partially purified enzymes as far as their response to the different effectors tested, except for Mn2+ and EDTA, which affected differently b-gal I, b-gal II, and b-gal III. They were slightly affected by Mg2+, Ca2+, Zn2+, Co2+, tartarate, molybdate, glucose, and lactose, strongly inhibited by Cu2+ and galactose, and inactivated by Hg2+. These chemical and physical properties are similar to the ones found for other plant b-galactosidases. Although through this process of purification three isoforms of this enzyme were obtained, isoelectric focusing in polyacrylamide slab gel of these enzyme-proteins suggest that cotyledons of Pitiúba cowpea quiescent seeds possess four isoforms of b-galactosidases.

Chemical composition, acetylcholinesterase inhibitory and antifungal activities of Pera glabrata (Schott) Baill. (Euphorbiaceae)

Pera glabrata (Schott) Baill. was selected for this study after showing a preliminary positive result in a screening of Atlantic Forest plant species in the search for acetylcholinesterase inhibitors and antifungal compounds. The bioassays were conducted with crude ethanol extract of the leaves using direct bioautography method for acetylcholinesterase and antifungal activities. This extract was partitioned with hexane, chloroform and ethyl acetate solvents. The active chloroform fraction was submitted to silica gel chromatography column affording 12 groups. Caffeine, an alkaloid, which showed detection limits of 0.1 and 1.0 µg for anticholinesterasic and antifungal activities, respectively, was isolated from group nine. After microplate analyses, only groups four, nine, 10, 11 and 12 showed acetylcholinesterase inhibitory activity of 40% or higher. The group 12 was purified by preparative layer chromatography affording four sub-fractions. Two sub-fractions from this group were analyzed by gas chromatography-mass spectrometry and gas chromatography-flame ionization detector. The first sub-fraction showed anticholinesterasic activity and contained two major compounds: 9-hydroxy-4-megastigmen-3-one (84%) and caffeine (6%). The second sub-fraction presented five major compounds identified as 9-hydroxy-4-megastigmen-3-one, isololiolide, (-) loliolide, palmitic acid and lupeol and did not show activity.

Test of hypotheses about herbivory and chemical defences of Qualea parviflora (Vochysiaceae) in Brazilian Cerrado

(Test of hypotheses about herbivory and chemical defences of Qualea parviflora (Vochysiaceae) in Brazilian Cerrado). Qualea parviflora Mart. (Vochysiaceae), a widely distributed tree found in different habitats in Brazilian Cerrado (savanna), provides resources for a great variety of insects. In this study, we tested two hypotheses about plant investment in anti-herbivore defences along a fertility gradient in Cerrado: the carbon/nitrogen balance (CNBH) and resource availability (RAH). We also investigated how the pattern of herbivory varies through the year and among three types of vegetation in Brazilian Cerrado - campo sujo, cerrado sensu strictu and cerradão. Sampling was conducted in three types of vegetation and in rainy (January and November) and dry months (April and July). Damage on 20 completely expanded leaves, leaf nutrients, sclerophlylly, total phenols and tannins were recorded for each plant (n = 30). When leaves were young, less sclerophyllous, and with higher concentration of nutrients and tannins, damage by herbivores was about 7% in cerrado sensu stricto and 3% in campo sujo. Mature leaves did not show any significant difference on herbivory among habitats, that varied from 6 to 9%. Nutrient availability to plants is an important factor determining production of secondary metabolites in Q. parviflora, corroborating the CNBH. The absence of correlation between damaged leaf area and tannin concentration did not corroborate the RAH, suggesting that tannin production is not strongly influenced by herbivores on Q. parviflora.

Connexin domains relevant to the chemical gating of gap junction channels

Most cells exchange ions and small metabolites via gap junction channels. These channels are made of two hemichannels (connexons), each formed by the radial arrangement of six connexin (Cx) proteins. Connexins span the bilayer four times (M1-M4) and have both amino- and carboxy-termini (NT, CT) at the cytoplasmic side of the membrane, forming two extracellular loops (E1, E2) and one inner (IL) loop. The channels are regulated by gates that close with cytosolic acidification (e.g., CO2 treatment) or increased calcium concentration, possibly via calmodulin activation. Although gap junction regulation is still unclear, connexin domains involved in gating are being defined. We have recently focused on the CO2 gating sensitivity of Cx32, Cx38 and various mutants and chimeras expressed in Xenopus oocytes and studied by double voltage clamp. Cx32 is weakly sensitive to CO2, whereas Cx38 is highly sensitive. A Cx32 chimera containing the second half of the inner loop (IL2) of Cx38 was as sensitive to CO2 as Cx38, indicating that this domain plays an important role. Deletion of CT by 84% did not affect CO2 sensitivity, but replacement of 5 arginines (R) with sparagines (N) at the beginning of CT (C1) greatly enhanced the CO2 sensitivity of Cx32. This suggests that whereas most of CT is irrelevant, positive charges of C1 maintain the CO2 sensitivity of Cx32 low. As a hypothesis we have proposed a model that involves charge interaction between negative residues of the beginning of IL1 and positive residues of either C1 or IL2. Open and closed channels would result from IL1-C1 and IL1-IL2 interactions, respectively

Plasma cortisol levels in captive wild felines after chemical restraint

Eight Panthera onca (Po), 13 Felis concolor (Fc), 7 Felis yagouaroundi (Fy), 7 Felis tigrina (Ft) and 5 Felis pardalis (Fp) specimens from São Paulo State zoos were used. All animals were restrained with darts containing 10 mg/kg ketamine and 1 mg/kg xylazine. Venous blood samples were collected as soon as possible (within 15-20 min) and serum was frozen until the time for cortisol quantification. Cortisol was determined using a solid phase radioimmunoassay with an intra-assay coefficient of 8.51%. Data were analyzed statistically by the Kruskal-Wallis test, followed by Dunn's multiple comparisons test, and the one-sample t-test, with the level of significance set at P<0.05. Data are reported as means ± SEM. Cortisol levels differed among the captive felines: Po = 166 ± 33a, Fc = 670 ± 118b, Fy = 480 ± 83b, Ft = 237 ± 42ab, Fp = 97 ± 12a nmol/l (values followed by different superscript letters were significantly different (P<0.001)). Since most of the veterinary procedures on these species involve chemical restraint, these results show the necessity of preventive measures in order to minimize the effect of restraint stress on more susceptible species

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.

Heterofucans from Dictyota menstrualis have anticoagulant activity

Fucan is a term used to denote a family of sulfated L-fucose-rich polysaccharides which are present in the extracellular matrix of brown seaweed and in the egg jelly coat of sea urchins. Plant fucans have several biological activities, including anticoagulant and antithrombotic, related to the structural and chemical composition of polysaccharides. We have extracted sulfated polysaccharides from the brown seaweed Dictyota menstrualis by proteolytic digestion, followed by separation into 5 fractions by sequential acetone precipitation. Gel electrophoresis using 0.05 M 1,3-diaminopropane-acetate buffer, pH 9.0, stained with 0.1% toluidine blue, showed the presence of sulfated polysaccharides in all fractions. The chemical analyses demonstrated that all fractions are composed mainly of fucose, xylose, galactose, uronic acid, and sulfate. The anticoagulant activity of these heterofucans was determined by activated partial thromboplastin time (APTT) using citrate normal human plasma. Only the fucans F1.0v and F1.5v showed anticoagulant activity. To prolong the coagulation time to double the baseline value in the APTT, the required concentration of fucan F1.0v (20 µg/ml) was only 4.88-fold higher than that of the low molecular weight heparin Clexane® (4.1 µg/ml), whereas 80 µg/ml fucan 1.5 was needed to obtain the same effect. For both fucans this effect was abolished by desulfation. These polymers are composed of fucose, xylose, uronic acid, galactose, and sulfate at molar ratios of 1.0:0.8:0.7:0.8:0.4 and 1.0:0.3:0.4:1.5:1.3, respectively. This is the fist report indicating the presence of a heterofucan with higher anticoagulant activity from brown seaweed.

Some physico-chemical parameters that influence proteinase K resistance and the infectivity of PrP Sc after high pressure treatment

Crude brain homogenates of terminally diseased hamsters infected with the 263 K strain of scrapie (PrP Sc) were heated and/or pressurized at 800 MPa at 60ºC for different times (a few seconds or 5, 30, 120 min) in phosphate-buffered saline (PBS) of different pH and concentration. Prion proteins were analyzed on immunoblots for their proteinase K (PK) resistance, and in hamster bioassays for their infectivity. Samples pressurized under initially neutral conditions and containing native PrP Sc were negative on immunoblots after PK treatment, and a 6-7 log reduction of infectious units per gram was found when the samples were pressurized in PBS of pH 7.4 for 2 h. A pressure-induced change in the protein conformation of native PrP Sc may lead to less PK resistant and less infectious prions. However, opposite results were obtained after pressurizing native infectious prions at slightly acidic pH and in PBS of higher concentration. In this case an extensive fraction of native PrP Sc remained PK resistant after pressure treatment, indicating a protective effect possibly due to induced aggregation of prion proteins in such buffers.

Inducing mutations in the mouse genome with the chemical mutagen ethylnitrosourea

When compared to other model organisms whose genome is sequenced, the number of mutations identified in the mouse appears extremely reduced and this situation seriously hampers our understanding of mammalian gene function(s). Another important consequence of this shortage is that a majority of human genetic diseases still await an animal model. To improve the situation, two strategies are currently used: the first makes use of embryonic stem cells, in which one can induce knockout mutations almost at will; the second consists of a genome-wide random chemical mutagenesis, followed by screening for mutant phenotypes and subsequent identification of the genetic alteration(s). Several projects are now in progress making use of one or the other of these strategies. Here, we report an original effort where we mutagenized BALB/c males, with the mutagen ethylnitrosourea. Offspring of these males were screened for dominant mutations and a three-generation breeding protocol was set to recover recessive mutations. Eleven mutations were identified (one dominant and ten recessives). Three of these mutations are new alleles (Otop1mlh, Foxn1sepe and probably rodador) at loci where mutations have already been reported, while 4 are new and original alleles (carc, eqlb, frqz, and Sacc). This result indicates that the mouse genome, as expected, is far from being saturated with mutations. More mutations would certainly be discovered using more sophisticated phenotyping protocols. Seven of the 11 new mutant alleles induced in our experiment have been localized on the genetic map as a first step towards positional cloning.

Intrinsic denervation of the colon is associated with a decrease of some colonic preneoplastic markers in rats treated with a chemical carcinogen

Denervation of the colon is protective against the colon cancer; however, the mechanisms involved are unknown. We tested the hypothesis that the denervated colonic mucosa could be less responsive to the action of the chemical carcinogen dimethylhydrazine (DMH). Three groups of 32 male Wistar rats were treated as follows: group 1 (G1) had the colon denervated with 0.3 mL 1.5 mM benzyldimethyltetradecylammonium (benzalkonium chloride, BAC); G2 received a single ip injection of 125 mg/kg DMH; G3 was treated with BAC + the same dose and route of DMH. A control group (Sham, N = 32) did not receive any treatment. Each group was subdivided into four groups according to the sacrifice time (1, 2, 6, and 12 weeks after DMH). Crypt fission index, ß-catenin accumulated crypts, aberrant crypt foci, and cell proliferation were evaluated and analyzed by ANOVA and the Student t-test. G3 animals presented a small number of aberrant crypt foci and low crypt fission index compared to G2 animals after 2 and 12 weeks, respectively. From the second week on, the index of ß-catenin crypt in G3 animals increased slower than in G2 animals. From the 12th week on, G2 animals presented a significant increase in cell proliferation when compared to the other groups. Colonic denervation plays an anticarcinogenic role from early stages of colon cancer development. This finding can be of importance for the study of the role of the enteric nervous system in the carcinogenic process.

Provitamin a activity of Brazilian carrots: leaves and roots, raw and cooked and their chemical composition

The purpose of this study was to determine through carotenoid analysis, the provitamin A value of two carrots (Daucus carota L.) cultivars (Brasilia and Beta3), leaves and roots, raw and submitted to two ways of cooking: boiling and microwave. Proximate analysis results are also presented for a better characterization of these vegetables (moisture, ash, lipids, proteins, fiber and total carbohydrates). The main carotenoids sources of provitamin A in this vegetable, both in leaves or roots was β-carotene and α-carotene. α-carotene shows half of the provitamin A value of β-carotene. Samples of the Brasilia cultivar presented the better provitamin A value both for leaves and roots. The results for raw samples of the Brasilia cultivar were 464.48 RE/100g for leaves and 606.42 RE/100g for roots. There were no significant losses of provitamin A with the boiling or microwave cooking methods used.

Chemical and nutritional evaluation of kernels of bocaiuva, Acrocomia aculeata (Jacq.) Lodd.

Protein characterization and results of proximate composition and mineral analyses of fruit kernels of bocaiuva, Acrocomia aculeata (Jacq.) Lodd., are reported. The kernels presented high contents of oil (51.7%), protein (17.6%) and fiber (15.8%). The seeds´ soluble proteins were isolated according to their solubility. The main separated proteins were globulins (53.5%) and glutelins (40.0%). Moreover, the presence of low molecular mass proteases in these two fractions was shown by the SDS-PAGE method. The assays of protease-inhibitory and hemagglutinating activities showed that bocaiuva´s protein fractions were not resistant to trypsin or chymotrypsin activities and that both had low lectin content. The globulin in vitro digestibility assay resembled a casein standard. Neither globulin nor glutelin enzymatic hydrolyses increased significantly (p < 0.05) after heat treatment. Threonine and lysine are the most limiting amino acids, respectively from two major protein fractions of the bocaiuva kernel, globulin (47.1% amino acid score) and glutelin (49.5% amino acid score), in terms of the theoretical profiles for children in the age range of 2 to 5 years recommended by the FAO/WHO. Bocaiuva kernels are found to be rich in calcium, phosphorus and manganese compared to some fruit nuts such as cashew and coconut.

Postharvest respiratory activity and changes in some chemical constituents during maturation of yellow mombin (Spondias mombin) fruit

Mature fruit from the yellow mombin (Spondias mombin) was monitored for its respiration activity. Mature green fruit from the yellow mombin was stored in closed glass chambers and the concentration of oxygen and carbon dioxide at the end of a six hour respiration period was determined. At the same interval of time, the lid of the chamber was opened for air renewal. The increase in carbon dioxide and decrease in oxygen concentration demonstrated that the fruit was climacteric. The maximum liberation of CO2 54.2 mL Kg-1 h-1 and maximum absorption of O2 49.0 mL Kg-1 h-1 occurred 186 hours after the harvest which, obviously, represented the optimum fruit quality after the senescence process started. The respiratory quotient of fruit at a climacteric maximum was 1.11 representing the oxidation of carbohydrates. Total soluble solids increased from 9.1 °Brix (initial) to 13.7 °Brix (climacteric maximum) during maturation, while the total number of acids in the fruit decreased during maturation i.e. from 1.55% initially to 1.40% at pre-climacteric, 1.0% at climacteric maximum and 0.8% in the post-climacteric stage. A similar behaviour was observed in the case of ascorbic acid. There was a continuous decrease in chlorophyll and a continuous increase in the carotenoid content of fruit during maturation.

Interference of heating on the antimicrobial activity and chemical composition of Origanum vulgare L. (Lamiaceae) essential oil

Origanum vulgare L. (oregano), Lamiaceae, essential oil has a variety of biological properties and its antimicrobial activity has received a renewed interest for use in food conservation. The aim of this study was to evaluate the interference of heating on the antimicrobial activity and chemical composition of O. vulgare essential oil. The antimicrobial activity of the essential oil kept at room temperature and exposed to different heating temperatures (60, 80, 100 and 120 °C during 1 hour) was evaluated by observing antimicrobial effectiveness at absolute concentration and determining MIC values by the solid medium diffusion procedure. The essential oil chemical composition analysis was performed by GC-MS. O. vulgare essential oil showed interesting antimicrobial activity on all assayed microbial strains (Candida albicans, C.krusei, C. tropicalis, Bacillus cereus, Escherichia coli, Staphylococcus aureus, Yersinia enterocolitica, Salmonella enterica, Serratia marcencens), noted by large growth inhibition zones (30-42 mm). Heating treatment showed no significant interference (p < 0.05) on the essential oil antimicrobial activity, noted by the development of microbial growth inhibition zones with similar or close diameters when evaluating the essential oil kept at room temperature and after exposure to different thermal treatments. MIC values oscillated between 10and 40 µL.mL-1 (20µL.mL-1 for most strains). However, no significant difference (p < 0.05) was noted among the MIC values found for the essential oil aliquots exposed to different temperatures. Moreover, heating did not significantly (p < 0.05) affect the chemical composition of O. vulgare essential oil. Monoterpenes, terpenic compounds and sesquiterpenes were found in the essential oil, with carvacrol (68.06-70.27%) and p-cymene (12.85-15.81%) being the compounds found in the highest amounts. These results showed the thermal stability and intense antimicrobial properties of O. vulgare essential oil and support its possible concomitant use with heating temperatures in order to reach microbial safety in foods.

Physico-chemical characterization and bioactive compounds of blackberry fruits (Rubus sp.) grown in Brazil

Five blackberry cultivars (Rubus sp.) were evaluated for antioxidant capacity, bioactive compounds and composition. Ascorbic acid levels, consisting of dehydro-ascorbic acid, ranged from 9.8 to 21.4 mg.100 g-1 fresh weight. Cyanidin (66 to 80% of total flavonoids), epicatechin, quercetin and traces of kaempferol were the main flavonoids found in all cultivars. The five cultivars presented high antioxidant capacity in the β-carotene/linoleic acid system, with inhibition similar to the synthetic antioxidant BHT, at a 50 µM concentration. Caingangue cultivar presented high vitamin C and total phenolics content, while Guarani had the highest cyanidin, total anthocyanin and total flavonoids levels and also the highest antioxidant capacity. These cultivars also presented good TSS/TA ratios. From the data, at a quantitative level, blackberry can be considered a good source of bioactive compounds, as well as potentially beneficial to human health.