Scielo RSS <![CDATA[Brazilian Archives of Biology and Technology]]> vol. 57 num. 4 lang. es <![CDATA[SciELO Logo]]> <![CDATA[cDNA cloning, Phylogenic Analysis and Gene Expression Pattern of Phenylalanine ammonia-lyase in Sugarcane (<em>Saccharum officinarum</em> L.)]]> The aim of the present study was to clone and characterize a full length cDNA of sugarcane (Saccharum officinarum) phenylalanine ammonia-lyase (SoPAL). Differential tissue expression pattern of the SoPAL transcript and its enzyme activity was also analyzed during the tillering stage of growth. The full-length of SoPAL cDNA was 2118 bp long and contained a protein with 706 amino acids, determined by encoding technique. The amino acid sequence and phylogenic analysis of the cloned SoPAL showed high similarity to PAL from other monocotyledonous such as sorghum (96%), maize (93%) and Bamboos (87.12%). The highest levels of SoPAL transcript were observed in the root and stem, while its minimal gene expression levels were in the leaves and sheath, respectively. The highest level of SoPAL enzyme activity was in the leaves. These results helped to understanding the characteristics of PAL biosynthesis and its regulation at the molecular level in sugarcane. This information could be critical for the manipulation of phenylpropanoid biosynthesis in the plant using biotechnological processes. <![CDATA[Effects of Chitosan on the Secretion of Cytokines and Expression of Inducible Nitric Oxide Synthase mRNA in Peritoneal Macrophages of Broiler Chicken]]> An in vitro experiment was conducted to study the effects of chitosan on the secretion of cytokines and expression of inducible nitric oxide synthase mRNA in peritoneal macrophages of broiler chicken. In the experiment, peritoneal macrophages were incubated for 24 h in culture medium supplemented with 0 (control), 40, 80, 160 and 320 µg/mL chitosan. The results showed that chitosan tended to increase quadratically the levels of interleukin-1 (P = 0.093) and interleukin-2 (P = 0.106) in the culture fluid of peritoneal macrophage. Chitosan also significantly enhanced inducible nitric oxide synthase mRNA expression of peritoneal macrophage in a quadratic dose-dependent manner (P &lt; 0.05) and tended to promote quadratically the secretion of nitric oxide (P = 0.053) and inducible nitric oxide synthase (P = 0.157) in peritoneal macrophages. This result implied that one of the mechanisms by which chitosan modulated immune functions in chickens might be chitosan activating expression of inducible nitric oxide synthase and then improving the secretion of nitric oxide. <![CDATA[First Report of Citrus <em>Aleurocanthus woglumi</em> Ashby (Hemiptera: Aleyrodidae) in the State of Paraná, Brazil]]> The citrus blackfly Aleroucanthus woglumi Ashby, (Hemiptera Aleyrodidae) is an important pest that occurs in citrus groves, native to south-east Asia. In Brazil, according to the Ministry of Agriculture, this is a quarantine pest (A2) under official control IN 52, 2007 (MAPA) and is not widespread in the country. The insect can infest more than 300 host plants, including cultivated plants, ornamentals and weeds, but mostly occurs in the plants of the genus citrus. This paper is the first report of citrus blackfly in the State of Paraná. <![CDATA[Leaf Morphological Plasticity of Tree Species from Two Developmental Stages in Araucaria Forest]]> This study compared the morphological and anatomical variations of the leaves of four shade-tolerant tree species Allophylus edulis (St.-Hil.) Radlk (Sapindaceae), Casearia sylvestris Sw. (Salicaceae), Cupania vernalis Cambess. (Sapindaceae) and Luehea divaricata Mart. (Malvaceae) from a fragment of Araucaria forest in two developmental stages. Morphological and anatomical traits, such as leaf and tissue thickness, leaf area, leaf dry mass, specific leaf area, leaf density and stomata density were measured from 30 leaves of each developmental stage. The phenotypic plasticity index was also calculated for each quantitative trait. The results showed that the four species presented higher mean values ​​for specific leaf area and spongy/palisade parenchyma ratio at young stage, and higher mean values ​​for stomata density, total and palisade parenchyma thickness in the adult stage. The plasticity index demonstrated that L. divricata presented highest plasticity for both the morphological and anatomical traits while A. edulis displayed the lowest plasticity index. The results of this study indicated that the leaves of these species exhibited distinct morphological traits at each stage of development to cope with acting environmental factors. <![CDATA[Management of Root-Knot Disease on Tomato with Bioformulated <em>Paecilomyces lilacinus</em> and Leaf Extract of <em>Lantana camara</em>]]> Glasshouse experiments were conducted to evaluate the efficacy of application frequency of a bioformulated Paecilomyces lilacinus in combination with five concentration of Lantana camara crude aqueous leaf extract against Meloidogyne incognita race I on tomato. The experiment was a 3x5 factorial laid out in a completely randomized design (CRD) with four replications. Each seedling was inoculated with 5000 eggs of M. incognita. Application of the bionematicide and L. camara leaf extract alone significantly (P≤ 0.05) inhibited root galling and egg production compared with their respective control. However, the severity of root galling and egg mass production was more significantly (P&lt; 0.05) suppressed with the application of P. lilacinus than L. camara leaf extract. Double inoculation with P. lilacinus in combination with 0.80 g mL-1 of the L. camara leaf extract changed the susceptibility of the tomato cultivar with gall index (GI=4.00) to GI=1.50. Application of P. lilacinus twice (at transplanting and two weeks after transplanting) in combination with 0.80g mL-1 of L. camara leaf extract was the most effective treatment in gall and egg mass inhibition, growth enhancement and dry matter accumulation. This environment-friendly approach could be incorporated into integrated root-knot disease management in tomato. <![CDATA[Production, Composition, Fatty Acids Profile and Stability of Milk and Blood Composition of Dairy Cows Fed High Polyunsaturated Fatty Acids Diets and Sticky Coffee Hull]]> Four lactating Holstein cows were assigned to a 4 × 4 Latin square design to determine the effects of feeding sticky coffee hull (SCH) as a source of antioxidants on dairy cows fed with high PUFA diets. The treatments (on DM basis) were control diet, diet with 30 g/kg of soybean oil, diet with 30 g/kg of soybean oil and 100 g/kg of SCH, and diet with 30 g/kg of soybean oil and 150 g/kg of SCH. Inclusion of 150 g/kg of SCH decreased the crude protein digestibility. Lower values of NDF digestibility were also observed when cows were fed with 100 g/kg and 150g/kg of SCH. The digestibility of NDT was lower in the control and 150 g/kg of SCH diets. Milk production and composition did not differ among the treatments. Inclusion of SCH increased the total polyphenols and flavonoids in the milk and reducing power as well. Soybean oil and SCH supplementation increased the LDL and total cholesterol concentration in the plasma. Milk fatty acid profile was barely altered by the treatments. In conclusion, the results confirmed that SCH added up to 15% in the diet did not alter milk production, improved its stability, and incorporated antioxidants substances in the milk, improving its quality for human health. <![CDATA[Bark Extract of <em>Bathysa cuspidata</em> in the Treatment of Liver Injury Induced by Carbon Tetrachloride in Rats]]> The aim of this study was to investigate the effect of bark extract Bathysa cuspidata (BCE) in the reconstitution of hepatic parenchyma and stroma after dysfunction induced by the CCl4. Liver lesions were induced by intraperitoneal administration of CCl4 (1 mL/kg) every 48 h for 12 days. The animals were treated with B. cuspidata extract (BCE) administered by gavage for another 12 days. Forty-nine rats were randomized into seven treatment groups with seven animals receiving CCl4, BCE (200, or 400 mg/kg) and the vehicle DMSO alone, or in different combinations. The extract alone showed no evidence of hepatic toxicity. In general, rats acutely exposed to CCl4 without the treatment with BCE presented high ALT and AST serum levels and high tissue content of lipid hydroperoxides, malondialdehyde, and lipid droplets. BCE administration, especially at 400 mg/kg attenuated significantly all these parameters. Furthermore, the antioxidant activities of the enzymes superoxide dismutase and catalase were significantly increased in the groups receiving this extract. These results showed that the extract of B. cuspidata stem bark stimulated the antioxidant defense system and reduced the morphological and functional liver damage in Wistar rats previously exposed to CCl4. <![CDATA[Peptidase with Keratinolytic Activity Secreted by <em>Aspergillus terreus</em> During Solid-State Fermentation]]> The aim of this study was to evaluate peptidase production by Aspergillus terreus in solid-state bioprocess and evaluate its parameters. The best conditions were 5.0 g of wheat bran as substrate, incubation temperature 30°C, inoculum 2.0x105 spores/g and 75% saline volume, with production reaching 677 U/mL (5400 U/g culture medium) after 72 h of fermentation. Biochemical characterization of the crude enzymatic extract showed the optimum pH and temperature of 6.5 and 55°C, respectively. The stability at different temperatures and pH values showed that the extract could endure different pH. The evaluation of the ions influence and inhibitors proved that the enzyme required an ion for better activity, which was corroborated with the inhibition of EDTA and PMSF, characterizing serine and/or metallo peptidase. The extract was also tested for specific activities and showed promising results for keratinolytic and collagenolytic activities (0.252 and 0.165 OD/mL, respectively). <![CDATA[Production and Characterization of IgY against Canine IgG: Prospect of a New Tool for the Immunodiagnostic of Canine Diseases]]> This study describes the production of a new avian polyclonal antibody (IgY) against canine IgG, as another tool for the immunodiagnostic using IgY technology. The immunization protocol caused neither deaths nor pathologies, and no decline in egg laying capacity was detected. The total concentration of isolated IgY was constant, without significant difference (p&gt; 0.05), with average of 97.55 mg of IgY/yolk. The IgY revealed a strong sensitivity and specificity in recognition against canine IgG by ELISA. After the immunization, there was a significant increase in the production of IgY specific from the first to the second month (p &lt;0.05), reaching a stable peak without decrease in the production by the end of the analysis period (p &gt; 0.05). The IgY demonstrated a suitable specificity in Western blot against the purified and serum canine IgG, not enabling recognition of canine IgM or IgG of other animal species. The specific IgY in the egg yolks of immunized hens proved to be a molecule with an appropriate purity and desired specificity against the immunizing antigen. Moreover, its constant production in large quantities during the four months analyzed indicated that IgY antibody production technology could be considered as an excellent alternative to the standard methods. <![CDATA[The Risk Evaluation of Tungsten Oxide Nanoparticles in Cultured Rat Liver Cells for Its Safe Applications in Nanotechnology]]> Tungsten (VI) oxide (WO3) nanoparticles (NPs) are used for many industrial purposes in everyday life. However, their effects on human health have not been sufficiently evaluated. Therefore, the present study was designed to investigate the toxicity potentials of various concentrations (0 to 1000 ppm) of WO3 NPs (&lt;100 nm particle size) in cultured primary rat hepatocytes. The results of cell viability assay showed that the higher concentrations of dispersed WO3 NPs (300, 500 and 1000 ppm) caused significant (p&lt;0.05) decreases of cell viability. Also, dose dependent negative alterations were observed in oxidative status and antioxidant capacity levels after the application of WO3 in cultured rat primary hepatocytes. The results of genotoxicity tests revealed that these NPs did not cause significant increases of micronucleated hepatocytes (MNHEPs) but increased 8-oxo-2-deoxyguanosine (8-OH-dG) levels as compared to the control culture. <![CDATA[An Alternative and Rapid Method for the Extraction of Nucleic Acids from Ixodid Ticks by Potassium Acetate Procedure]]> Four variants of the potassium acetate procedure for DNA extraction from ixodid ticks at different stage of their life cycles were evaluated and compared with phenol-chloroform and ammonium hydroxide methods. The most rapid and most efficient variant was validated in the DNA extraction procedure from the engorged ticks collected from bovine, canine as well as from house ticks for the screening of Borrelia burgdorferi sensu lato, Anaplasma spp. and Babesia spp. The ammonium hydroxide procedure was used for non-engorged ticks. All the variants were efficient and allowed obtaining PCR-quality material according to the specific amplification of 16S rRNA gene fragment of the original tick. DNA extracted from the ticks under the study was tested by multiplex PCR for the screening of tick-borne pathogens. Anaplasma spp. and Babesia spp. amplification products were obtained from 29/48 extracts. Ammonium hydroxide protocol was not efficient for two extracts. Detection of amplification products from the PCR indicated that DNA had been successfully extracted. The potassium acetate procedure could be an alternative, rapid, and reliable method for DNA extraction from the ixodid ticks, mainly for poorly-resourced laboratories. <![CDATA[Application of RAPD-PCR for Determining the Clonality of Methicillin Resistant <em>Staphylococcus aureus</em> Isolated from Different Hospitals]]> Randomly amplified polymorphic DNA (RAPD)-PCR was applied with ten random 10-mer primers to examine the molecular diversity among methicillin resistant Staphylococcus aureus (MRSA) strains in the hospitals and to investigate the epidemiological spread of these strains between different hospitals. The main objective of the study was to identify appropriate primers, which successfully established the clonality of MRSA. Three of the primers yielded particularly discriminatory patterns and they were used to perform the RAPD analysis which revealed different bands ranging from 200 to 1500 bp. Dendogram was created by the un-weighted pair group method using arithmetic (UPGMA) average clustering and it was constructed based on the combination results of the new primers (S224, S232 and S395) which represented a novel approach for rapid screening of the strains and also provided the opportunity for monitoring the emergence and determining clonal dissemination of MRSA strains between the hospitals. Dendogram generated two main groups (Group I and II) with three clusters (A, B and C) and indicated that the strains isolated from the same hospital were closely related and they placed together in the same group. This technique could be of attractive use in controlling the sources and routes of transmission, tracking the spread of strains within hospital and between the hospitals, and especially preventing the nosocomial infections caused by the MRSA. <![CDATA[Characterization and Metal Detoxification Potential of Moderately Thermophilic <em>Bacillus cereus</em> from Geothermal Springs of Himalaya]]> Two thermophilic Bacillus cereus strains (B. cereus-TA2 and B. cereus-TA4) used in the present study were isolated from the geothermal spring of Hunza valley, Gilgit, Pakistan. They showed the ability to withstand and grow at high temperature (85°C). Both these strains could resist multiple metals (copper, cadmium, mercury, manganese, zinc, arsenic, chromium and selenium). Strain B. cereus-TA4 reduced Cr (VI) at pH 5.0 to 9.0 but maximum reduction (83%) was observed at pH 7.0 after 48 h when initially supplied with 200 µg mL-1 of K2CrO4. Lower initial concentrations such as 100 µg mL-1 supported higher reduction (90 to 95%) than that of high concentration such as 500 µg mL-1 (20 to 30%). Both the strains reduced nearly 70% of Se (IV) after 48 h of growth at pH 7.0 when initially supplied with 200 µg mL-1 of Na2SeO3. The optimum temperature for maximum Se (IV) reduction was 45°C for both the strains. <![CDATA[Physiological Characterization of Fungal Inoculum for Biotechnological Remediation of Soils]]> The aim of this work was to study the bioremediating potential of Lentinus crinitus CCIBt2611 according to the physiological condition of the inoculum. Inoculum was prepared using sugarcane ground husk (C:N 90), at several physiological ages and applied in soil contaminated with pentachlorophenol. The inoculum's potential was assessed by evaluating the mycelium's vigor at soil's colonization, determination of peroxidase and phenoloxidase activities, in vitro degradation of Remazol Brilliant Blue R and in vivo degradation of pentachlorophenol. The results showed that the assessed parameters were relevant to identify the quality of the inoculum. For L. crinitus, 10 day old inoculum showed good soil-colonization speed with significant enzymatic activities, indicating the role of Manganese-dependent peroxidase and laccase in degradation, and efficient degradation of pentachlorophenol. <![CDATA[Production of <em>Beauveria bassiana</em> Air Conidia by Means of Optimization of Biphasic System Technology]]> Air conidia production of Beauveria bassiana, strain CA-603 was studied based on modified diphasic system. The biomass yield obtained in the first phase based on submerged cultivation of fungus was processed using methodology providing different contact with air space. Our study indicated that productivity of the second stage of diphasic system is had inversely proportional dependence on depth of liquid fungal biomass. Increase of biomass depth is significantly decreased production of air conidia. Two methodology of biomass processing extending contact biomass with air space including distribution of fungal material on surface of hygroscopic paper and starch packaging peanuts were investigated. The novel substrates provided optimal contact between the submerged fungal biomass and the air, and overall, conidial production was directly proportional to the total area of air-to-fungal surface. Technologies based on the starch peanuts and hygroscopic paper were the most productive in comparison to the common technology where the submerged culture was transferred to flat containers. The advantages and disadvantages of these different production methods are discussed. <![CDATA[Immobilization of Lipases Produced by the Endophytic Fungus <em>Cercospora kikuchii</em> on Chitosan Microparticles]]> This work studied the immobilization of Cercospora kikuchii lipases on chitosan microparticles by chemical attachment on chitosan acetate microparticles activated by glutaraldehyde (CAM) added before or after the enzyme and physical adsorption on highly deacetylated chitosan hydrochloride microparticles (CHM). Lipases covalently immobilized on pre-activated CAM showed better performance retaining 88.4% of the enzymatic activity, with 68.2% of immobilization efficiency (IE). The immobilized enzyme retained an activity of about 53.5 % after five reuses, using p-NPP as substrate. Physical adsorption of lipase onto highly deacetylated CHM showed 46.2 % of enzymatic activity and 28.6% of IE. This immobilized derivative did not lose activity up to 80 days of storage at 4°C, while lipases immobilized on pre-activated CAM maintained its activity up to 180 days at same conditions. Taken together the results indicate that chitosan microparticles provide an optimal microenvironment for the immobilized enzyme to maintain good activity and stability. <![CDATA[Purification and Characterization of a Polygalacturonase Produced by <em>Wickerhamomyces anomalus</em>]]> The aim of this work was to study the purification and physicochemical properties of an endo-polygalacturonase (PG) produced by Wickerhamomyces anomalus isolated from the citrus fruit peels. The enzyme was purified to homogeneity from the culture filtrate of W. anomalus grown on the yeast nitrogen base medium with glucose as carbon and energy source and citrus pectin as inductor. After anion-exchange chromatography and gel filtration chromatography, PG activity was eluted as a single peak, yielding 21% of the original activity. After dialysis and cation-exchange chromatography, only one fraction with PG activity was obtained, recovering 56% of initial enzyme activity and 1.3-fold increase in specific activity. The molecular weight of the enzyme was estimated as 43 kDa by the SDS-PAGE. The enzyme exhibited maximal activity at pH 4.2 and was stable over a pH range from 3.5 to 6.0 and up to 49ºC for 10 h. The Vmax and Km values with polygalacturonic acid as substrate were 0.26 mmol/L.min and 0.173 mg/mL, respectively. Cations such as Cu+2, Fe+3, Mg+2, Mn+2 and Zn+2 did not show any significant effect on PG activity but K+ and Ca+2 reduced it. The purified PG was able to macerate cassava tissues. <![CDATA[Effects of Allelochemicals from <em>Ficus microcarpa </em>on <em>Chlorella pyrenoidosa</em>]]> This study was performed in order to isolate and identify unknown allelochemicals from Ficus microcarpa, and to investigate the inhibitory to bloom-forming of green alga Chlorella pyrenoidosa. Through gradient elution, fraction C2, whose inhibition of alga growth in diverse extracts was the strongest was shown to cause significant reductions of maximum quantum yield, as well as electron transport rates of C. pyrenoidosa. The study data also showed that the increase of fraction C2 concentration decreased the activity of total superoxide dismutase (SOD), but increased the activities of catalase (CAT) and malondialdehyde (MDA) content. These results demonstrate that the active fraction C2 not only induced the photoinhibition or photodamage of PSII reaction centers, but also triggered the synthesis of reactive oxygen species which may change cell membrane penetrability, thereby leading to the eventual death of C. pyrenoidosa. Furthermore, the gas chromatography/mass spectrometry (GC/MS) analyses showed that the most potential allelochemical in active fraction C2 was 2-Propyl phenol, which may exhibit potent allelopathy. <![CDATA[Enhanced Production of Vitamin K<sub>2</sub> from <em>Bacillus subtilis (natto) </em>by Mutation and Optimization of the Fermentation Medium]]> The aim of this study was to enhance the production of vitamin K2 by using N-methyl-N-nitro-N-nitroso-guanidine (NTG) and low energy ion beam implantation and optimizing the fermentation medium. Mutation resulted in 1.66-fold higher production of vitamin K2 than that of the parentl strain. The production by the mutant BN-P15-11-1was increased 55% and reached 3.593±0.107 mg/L by using the Plackett-Burman and Box-Behnken designs to optimize the fermentation medium. The optimal fermentation culture medium was composed of (g/L) glycerol 69.6, sucrose 34.5, K2HPO4 4.0, peptone 20, yeast extract 25 and fermented at 37 °C and 150 rpm for 72 h. The results showed that the NTG and low energy ion beam implantation mutations and optimizing fermentation medium were effective methods to enhance vitamin K2 production. <![CDATA[Enzyme-based Colorimetric and Potentiometric Biosensor for Detecting Pb (II) Ions in Milk]]> The aim of the present work was to study a simple colorimetric and potentiometric biosensor based on urease inhibition by Pb (II) ions for its estimation in milk samples. Urease immobilized on nylon membrane by hydrosol gel method was used as the biocomponent to demonstrate the metal effect on the enzyme activity using phenol red as the pH indicator. A lower limit detection of 38.6µm was achieved in the milk and the enzyme membranes were stable for more than two months at 4ºC. In potentiometric approach, response of an ion selective electrode (ISE) to changing ammonium ion concentration as a consequence of urease inhibition by Pb (II) ions was explored to achieve a detection limit of 9.66 µm. Lead specificity was attained by means of masking agents 1,10 - phenanthroline and sodium potassium tartarate. Validation of the developed biosensors was carried out with spiked milk samples.