Scielo RSS <![CDATA[Brazilian Archives of Biology and Technology]]> vol. 58 num. 3 lang. es <![CDATA[SciELO Logo]]> <![CDATA[Design of signal peptide bombyxin and its effect on secretory expression efficiency and levels of <em>Helicobacter pylori</em> urease subunit B in silkworm cells and larvae]]> This study employed a Bac-to-Bac/Bombyx mori bioreactor to mass-produce immunogenic urease subunit B (UreB) from Helicobacter pylori. The signal peptide bombyxin from B. mori was used to promote secretory expression to improve expression levels and was designed and integrated into the UreB gene to generate the Bacmid/BmNPV/(signal peptide)-UreB baculovirus expression system. To determine whether the bombyxin signal peptide resulted in secretory expression of recombinant UreB (rUreB) and to determine the secretory efficiency, we tested the secretory expression level of rUreB in Bm5 cells using ELISA. To further investigate whether secretory expression affected cell viability, cells were evaluated using 0.4% trypan blue staining, and Bacmid/BmNPV/UreB without the signal peptide served as a control. The above recombinant bacmid constructs were injected to silkworm larvae, and the secretory expression level of rUreB was detected using SDS-PAGE and semi-quantitative western blot analysis. The results indicated that the bombyxin signal peptide directed the secretory expression of rUreB and that this expression improved the viability of Bm5 cells. Moreover, the results showed that the expression level of rUreB was 1.5 times higher with the Bacmid/BmNPV constructs containing the bombyxin signal sequence than those without the signal sequence. These results demonstrate that secretory expression can enhance rUreB expression levels and is likely to aid in the large-scale expression and yield of rUreB in silkworm larvae. <![CDATA[Evaluation of the microbial diversity in sequencing batch reactor treating linear alkylbenzene sulfonate under denitrifying and mesophilic conditions using swine sludge as inoculum]]> The objective of this study was to evaluate the degradation of Linear Alkylbenzene Sulfonate (LAS) in anaerobic sequencing batch reactor (ASBR) under denitrifying conditions using swine sludge as inoculum. The reactor was operated for 104 days with synthetic substrate containing nitrate, and LAS was added later (22 mg/L). Considering the added mass of the LAS, the adsorbed mass in the sludge and discarded along with the effluent, degradation of the surfactant at the end of operation was 87%, removal of chemical oxygen demand was 86% and nitrate was 98%. The bacterial community was evaluated by cutting the bands and sequencing of polymerase chain reaction (PCR) fragments and denaturing gradient gel electrophoresis (DGGE). The sequences obtained were related to the phylum Proteobacteria and the alpha-and beta-proteobacteria classes, these bacteria were probably involved in the degradation of LAS. The efficiently degraded LAS in the reactor was operated in batch sequences in denitrifying conditions. <![CDATA[Lentinan promotes the root of <em>Brassica Campestris</em> L.]]> The aim of this work was to study the effect of lentinan on Brassica campestris L (rape). Spraying on the leaves of lentinan B. campestris L. at 0.05×10-6 g ml-1 concentration significantly promoted the root elongation (P<0.05). The results for the first time showed that lentinan could prolongate roots as a new plant hormone. <![CDATA[Protease gene shuffling and expression in <em>Pichia pastoris</em>]]> Four kinds of neutral and alkaline protease genes from Aspergillus oryzae and Bacillus subtilis were isolated and shuffled. The shuffled genes were selected, inserted into pGAPZαA plasmid and transformed into Escherichia coli. The gene which could express high-activity protease was selected by screening the sizes of transparent zones around the colonies on casein plates. After an ideal protease gene was selected, it was sequenced and then transformed into Pichia pastoris X33. The result showed that the base in 1022th position of shuffled protease gene was changed from thymine to cytosine, inferring that cysteine was changed to arginine in the mutant protease. After 48 h incubation for the transformed P. pastoris with the mutant or native protease genes, the mutant protease activity was 36.4% higher than the native protease (P&lt;0.05). The optimal pH and temperature of the mutant protease were 6.5-8.0 and 30-70°C, respectively, which indicated better stability than the native protease (P&lt;0.05). <![CDATA[Solid-state fermentation of <em>Acanthogobius hasta</em> processing by-products for the production of antioxidant protein hydrolysates with <em>Aspergillus oryzae</em>]]> Functional properties and antioxidative activity of a protein hydrolysate prepared from Acanthogobius hasta processing by-product protein during solid-state fermentation with Aspergillus oryzae were investigated. Overall, protease activity increased with the degree of hydrolysis (DH) decreased during solid-state fermentation. All the protein hydrolysate had excellent solubility, possessed interfacial properties, and varying degrees of antioxidant activity which were governed by their concentrations and DH, molecular weight distribution and amino acid composition. After 5 days fermentation, the DH of the protein hydrolysate was 31.23%. The protein hydrolysate had the highest total hydrophobic amino acid content, the highest DPPH scavenging activity, reducing power, and the chelating activity. The radical-scavenging activity of the hydrolysates at 6 mg/mL was 78.6%. The reducing power of protein hydrolysate at the range of 0-6 mg/mL was lower than that of BHA at the range of 0-60 µg/mL, while the chelating activity of APs was similar to that of BHA at the range of 0-60 µg/mL. Moreover, the protein hydrolysate showed good emulsifying and foaming properties over a wide pH range from 2 to 12. Therefore, solid state fermentation provided a suitable and low-cost method for converting Acanthogobius hasta processing by-product protein into antioxidant protein hydrolysates. <![CDATA[Aqueous extract of <em>Huang-lian</em> induces apoptosis in lung cancer cells <em>via</em> P53- mediated mitochondrial apoptosis]]> The current study was designed to evaluate the activity of the aqueous extract of Huang-lian, and the main apoptosis pathway induced by the extracts of Huang-lian was detected on lung cancer. Antiproliferactive activity was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and TUNEL methods against human lung cancer cells (A549). Huang-lian regulated the Bcl-2 family protein-mediated mitochondrial pathway via p53 and was detected by western blot. It increased the activation of caspase-3 and caspase-3 cleavage increased as the time increased. These results suggested that Huang-lian regulated the Bcl-2 family protein-mediated mitochondrial via p53 pathway, suggesting that Huang-lian should be further investigation as a natural agent for treating and preventing cancer. <![CDATA[Bioefficacy of pectolinaringenin from <em>Clerodendrum phlomidis</em> Linn. F. against <em>Anopheles stephensi</em> and bhendi fruit borer, <em>Earias vittella</em> fab.]]> Larvicidal activity of pectolinaringenin from Clerodendrum phlomidis was evaluated against Anopheles stephensi and antifeedant, larvicidal and growth inhibitory activities were evaluated against Earias vittella. Pectolinaringenin exhibited larvicidal activity of 100 and 98.24% against 2nd and 4th instar larvae of Anopheles stephensi at 5ppm concentration. It exhibited LC50 values of 0.35 and 0.55 ppm for 2nd and 4th instar larvae, respectively. At 100 ppm concentration, pectolinaringenin exhibited maximum antifeedant activity of 74.00% and larvicidal activity of 89.98%. The LC50 values were 36.2 and 10.23 ppm for antifeedant and larvicidal, respectively. The compound completely prevented the adult emergence at 50 and 100 ppm concentrations. This is the first report of pectolinaringenin from C. phlomidis evaluated against An. stephensi and E. vittella. The results suggested that the pectolinaringenin from C. philomidis could be used to develop a new botanical formulation to manage vector mosquitoes and agricultural pests. <![CDATA[Characterization of an alcoholic hepatic steatosis model induced by ethanol and high-fat diet in rats]]> Alcoholic liver disease is characterized by a wide spectrum of liver damage, which increases when ethanol is associated with high-fat diets (HFD). This work aimed to establish a model of alcoholic hepatic steatosis (AHS) by using a combination of 10% ethanol and sunflower seeds as the source of HFD. Male rats received water or 10% ethanol and regular chow diet and/or HFD, which consisted of sunflower seeds. The food consumption, liquid intake and body weight of the rats were monitored for 30 days. After this period, blood was collected for biochemical evaluation, and liver samples were collected for histological, mitochondrial enzyme activity and oxidative stress analyses. Our results indicated that the combination of 10% ethanol and HFD induced micro- and macrosteatosis and hepatocyte tumefaction, decreased the levels of reduced glutathione and glutathione S-transferase activity and increased the level of lipoperoxidation and superoxide dismutase activity. The mitochondrial oxidation of NADH and succinate were partially inhibited. Complexes I and II were the main inhibition sites. Hepatic steatosis was successfully induced after 4 weeks of the diet, and the liver function was modified. The combination of 10% ethanol and sunflower seeds as an HFD produced an inexpensive model to study AHS in rats. <![CDATA[Comparative evaluation of anthelmintic and antibacterial activities in leaves and fruits of <em>Garcinia cambogia</em> (Gaertn.) desr. and <em>Garcinia indica</em> (Dupetit-Thouars) choisy]]> This study aimed to evaluate the in vitro anthelmintic activity and antibacterial activity of the extracts from the leaves and fruits of Garcinia indica (Dupetit-Thouars) Choisy and Garcinia cambogia (Gaertn.) Desr. using the Indian earthworm Pheretima posthuma. Two concentrations (25 and 50 mg/mL) of various extracts such as petroleum ether, chloroform, ethyl acetate, methanol and water were tested. Albendazole at the concentrations of 25 and 50 mg/mL was used as the standard reference. Significant anthelmintic effects of the fruits and leaves of G. cambogia and G. indica (P&lt;0.05) were observed and the results were expressed in terms of paralysis and death time. All the extracts showed the dose dependent paralysis and death of earthworms. Among all the extracts used, methanol extract exhibited the highest activity. G. cambogia leaf extract (50 mg/mL) had 30% faster paralysis effect on earthworms than the standard reference. Furthermore, the antimicrobial activity of the methanol extracts of the fruits and leaves showed significant (P&lt;0.05) activity against Gram-positive and Gram-negative bacteria. At a concentration of 500 µg/mL, G. indica fruit extract presented higher zones of inhibition against Pseudomonas aeruginosa and Staphylococcus aureus. Hence, it could be concluded that the leaves and fruits of G. indica and G. cambogia contained active anthelmintic and antibacterial phytochemicals, which could find their applications in pharmaceuticals. <![CDATA[Human FcRn can mediate the transport across intestinal mucosal barrier and prolong the half-life of rabbit IgG <em>in vivo</em>]]> FcRn (neonatal Fc receptor) plays an important role in IgG transportation, antigen presentation and signal transmission. In this study, the complement fixation test and flow cytometry test were performed to verify whether the heterologous antibody could be transmitted to the serum or leukocyte with FcγR (Fc gamma receptor) across the intestinal mucosa. The results showed that rabbit anti-bovine IgG could be detected in both the serum and the leukocytes, which indicated that the heterologous antibody could transport across the intestinal mucosa to enter the blood and be effectively delivered to the leukocytes with FcγR. In addition, the results also showed that the rabbit anti-bovine IgG still could be detected in the leukocyte group (P=0.044&lt;0.05) after 21 days. It indicated that the rabbit IgG could exist in the body for a long term (up to 21 days) after being transported to the cells containing FcγR. <![CDATA[Intrauterine sexual differentiation: biosyntesis and action of sexual steroid hormones]]> The objective of this review was to describe sexual differentiation events in mammals, relating them to biosynthesis of sexual steroid hormones and their mechanisms of action. Cholesterol is the precursor of sexual steroid hormone biosynthesis via action of several enzymes converting these hormones. Progestagens hormones serve as substrate for the production of androgens, which in turn serve as substrate for estrogen hormones. These hormones are responsible for sexual differentiation and reproductive cycles of mammals. Sexual differentiation process comprises determining the sexual chromosomes XX or XY + SRY and other genes linked to them, differentiation of gonads in testis or ovary, differentiation of internal and external male or female genital organs from undifferentiated anatomical structures present in the embryo, which is dependent on the presence or absence of testes and the production of anti-Müllerian hormone and testosterone; and secondary sexual differentiation, which is the response of various tissues to hormones produced by the gonads, interacting with genes linked to sexual chromosomes to increase or decrease the differences in sexual phenotype. However, some differences between the sexes and some anomalies of sexual differentiation are not explained only by these sexual hormonal effects, but also by the effect of genes encoded in sexual chromosomes. <![CDATA[Metabolic and behavioral effects of ractopamine at continuous low levels in rats under stress]]> This study aimed at evaluating the effect of ractopamine (RAC) on metabolism, zootechnical performance, body composition, and behavior in Wistar rats submitted to acute and chronic restrain stress. The oral dose of 5 mg/kg of RAC was administered in periods of 0, 7, 14, 21, and 28 days. The elevated plus-maze test (EPMT) was used for behavioral assessment. Blood, carcass and viscera characteristics were evaluated. Insulin-dependent glucose transporters (GLUT-4) were semi-quantified by Western Blot in epididymal adipocytes. RAC periods associated with chronic stress increased the GLUT-4 protein expression in adipose tissue in a time-dependent manner (P=0.01), i.e., the longer the RAC addition period, the higher the GLUT-4 concentration in chronically stressed animals (0=1.42; 7=1.19; 14=2.03; 21=1.59; 28=2.35). The stress periods combined with RAC increased the time spent in the opened arms of the maze (Chronic stress: 0=10.6; 7=8.7; 14=5.9; 21=12.3; 28=4.0; Acute stress 0=3.1; 7= 4.7; 14=7.5; 21=0.0; 28=2.8) (P=0.04). Chronic (entries on the closed arms [ECA]=3.60) and acute (ECA=3.80) stress reduced locomotive activity in the maze (P=0.03). The results suggested that stress could negatively affect the possible benefits offered by the RAC, mainly impairing the adipose tissue metabolism and behavior in the animals. <![CDATA[Dideoxy single allele-specific PCR - DSASP new method to discrimination allelic]]> Gastric cancer (GC) is a multifactorial disease with a high mortality rate in Brazil and worldwide. This work aimed to evaluate single nucleotide polymorphisms (SNP) rs1695, in the Glutathione S-Transferase Pi (GSTP1) gene in GC samples by comparative analysis Specific PCR - ASP and Dideoxy Single Allele-Specific PCR - DSASP methods. The DSASP is the proposed new method for allelic discrimination. This work analyzed 60 GC samples, 26 diffuse and 34 intestinal types. The SNP rs1695 of the GSTP1 gene was significantly associated with GC analyzed by DSASP method (χ2 = 9.7, P &lt; 0.05). A comparative analysis of the data obtained from both methods did not differ significantly (χ2 = 0.08, P &gt; 0.05). These results suggest that the SNP rs1695 of the GSTP1 gene was a risk factor associated with gastric carcinogens is and the DSASP method was a new successfully low-cost strategy to study allelic discrimination. <![CDATA[Multiple gene sequence analysis using genes of the bacterial DNA repair pathway]]> The ability to recognize and repair abnormal DNA structures is common to all forms of life. Physiological studies and genomic sequencing of a variety of bacterial species have identified an incredible diversity of DNA repair pathways. Despite the amount of available genes in public database, the usual method to place genomes in a taxonomic context is based mainly on the 16S rRNA or housekeeping genes. Thus, the relationships among genomes remain poorly understood. In this work, an approach of multiple gene sequence analysis based on genes of DNA repair pathway was used to compare bacterial genomes. Housekeeping and DNA repair genes were searched in 872 completely sequenced bacterial genomes. Seven DNA repair and housekeeping genes from distinct metabolic pathways were selected, aligned, edited and concatenated head-to-tail to form a super-gene. Results showed that the multiple gene sequence analysis using DNA repair genes had better resolution at class level than the housekeeping genes. As housekeeping genes, the DNA repair genes were advantageous to separate bacterial groups at low taxonomic levels and also sensitive to genes derived from horizontal transfer. <![CDATA[New aminoporphyrins bearing urea derivative substituents: synthesis, characterization, antibacterial and antifungal activity]]> This work studied the synthesis of 5,10,15-tris(4-aminophenyl)-20-(N,N-dialkyl/diaryl-N-phenylurea) porphyrins (P1-P4 with alkyl or aryl groups of Ph, iPr, Et and Me, respectively) and also the preparation of their manganese (III) and cobalt (II) complexes (MnP and CoP). The P1-P4 ligands were characterized by different spectroscopic techniques (1H NMR, FTIR, UV-Vis) and elemental analysis, and metalated with Mn and Co acetate salts. The antibacterial and antifungal activities of these compounds in vitro were investigated by agar-disc diffusion method against Escherichia coli (-), Pseudomonas aeruginosa (-), Staphylococcus aureus (+), Bacillus subtilis (+) and Aspergillus oryzae and Candida albicans. Results showed that antibacterial and antifungal activity of the test samples increased with increase of their concentrations and the highest activity was obtained when the concentration of porphyrin compounds was 100 µg/mL. The activity for the porphyrin ligands depended on the nature of the urea derivative substituents and increased in the order P1 &gt; P2 &gt; P3 &gt;P4, which was consistent with the order of their liposolubility. MnP and CoP complexes exhibited much higher antibacterial and antifungal activity than P1-P4 ligands. Further, the growth inhibitory effects of these compounds was generally in the order CoP complexes &gt; MnP complexes &gt; P1-P4 ligands. Among these porphyrin compounds, CoP1 displayed the highest antibacterial and antifungal activity, especially with a concentration of 100 µg/mL, against all the four tested bacteria and two fungi, and therefore it could be potential to be used as drug. <![CDATA[Biomarkers of oxidative stress and acetylcholinesterase activity in the blood of grass snake (<em>Natrix natrix</em> L.) during prehibernation and posthibernation periods]]> This work examined the enzymatic (superoxide dismutase-CuZn SOD, catalase-CAT, glutathione peroxidase-GSHPx, glutathione reductase-GR, and the biotransformation phase II enzyme glutathione-S-transferase-GST) and nonenzymatic (total glutathione-GSH and lipid peroxides-TBARS concentrations) biomarkers of oxidative stress and acetylcholinesterase (AChE) activity in the blood of the grass snake (Natrix natrix L.) during prehibernation and posthibernation. The animals were collected in October (prehibernation) and April (posthibernation) at the nature reserve Obedska Bara (OB) and industrial region Pancevacki Rit (PR) in Serbia. In posthibernation, decreased CAT activity and TBARS concentration in specimens from PR, and decreased GR and AChE activities, and TBARS concentration in specimens from OB were observed, whereas GR and GST activities and GSH concentration were significantly elevated in the specimens from PR. In prehibernation, CAT activity and GSH concentration were increased, while GSH-Px, GR, GST and AChE activities and TBARS concentration were decreased in the specimens from PR when compared to animals from OB. During the posthibernation, the activity of CuZn SOD was decreased, while GST and AChE activities were increased in the specimens from PR when compared to the specimens from OB. These differences represented an adaptive mechanism to oxidative stress induced by tissue reoxygenation during arousal from hibernation and could be modulated by environmental pollution. <![CDATA[Dynamics of ethanol production from deproteinized whey by <em>Kluyveromyces marxianus</em>: An analysis about buffering capacity,thermal and nitrogen tolerance]]> The production of value-added products could be a valuable option for cheese wastewater management. However, this kind of study cannot just focus alone on getting the final product. This also necessitates studies on the dynamics of bioprocesses. With these as background, the present investigation aimed at evaluating the buffering capacity of deproteinized whey and effect of temperature and nitrogen source on ethanol yields from it. The batch fermentation conditions used to evaluate ethanol production were temperatures 30, 35, 40°C and pH 4.5, 5.0, 5.5, 6.0. To study the influence of nitrogen source on ethanol yield, a design matrix was applied using yeast extract and (NH4)2SO4.The final pH was analyzed to evaluate the buffering capacity. The results showed that the Kluyveromyces marxianus was thermotolerance to produce ethanol at 35 and 40°C, which was not observed at 30°C. Results also showed that the deproteinization procedure did not affect the buffering capacity of cheese whey. Finally, higher ethanol production was obtained using yeast extract (3% v/v). These results could be important for developing low-cost method for industrial production of ethanol from deproteinized whey. <![CDATA[Effect of nitrogen limitation on cell growth, lipid accumulation and gene expression in <em>Chlorella sorokiniana</em>]]> The influence of nitrogen (N) limitation on the cell growth, chlorophyll content, intracellular lipid production and expression levels of two pathway genes in Chlorella sorokiniana was investigated in mixotrophic culture in this study. The maximum biomass concentration of C. sorokiniana cultured in modified BG11 medium containing 3, 6, 9, 12 and 15 mM N were 1.60, 2.21, 2.74, 3.18 and 3.21 g/L of dry cell weight, respectively with maximum specific growth rate of 0.180, 0.198, 0.201, 0.203, 0.206 day−1 during culture with an initial N feed of 3, 6, 9, 12 and 15 mM in the first eight days, respectively. The maximum lipid content was 51 % with 3 mM N. However, the maximum lipid productivity of 0.00884 g L−1 day−1 was achieved with 6 mM N. Expression levels of accD (heteromeric acetyl-CoA carboxylase beta subunit) and accI (homomeric acetyl-CoA carboxylase) genes in C. sorokiniana were studied by real-time PCR. Increased expression levels of accD reflected the increased lipid content in stationary phase. In contrast, expression of the acc1 gene always remained low, showing that the gene might not be critical for lipid accumulation. <![CDATA[Microencapsulation of a Colombian <em>Spodoptera frugiperda</em> Nucleopolyhedrovirus with Eudragit® S100 by spray drying]]> A Colombian Spodoptera frugiperda nucleopolyhedrovirus NPV003 with high potential for the development of an efficient biopesticide was microencapsulated by spray drying with a pH dependent polymer (Eudragit® S100). Conditions for microparticles production were standardized and microencapsulation process was validated. Physical properties, insecticide activity and photo-stability of microencapsulated virus were determined. The microparticles were spherical and irregular shaped, with sizes between 17.64 and 19.47 µm. Moisture content was 10.38 ± 0.87%; encapsulation efficiency 84.61± 13.09% and process yield was 91.20 ± 6.40%. Microencapsulation process did not affect viral insecticidal activity and provided efficient protection against UVB radiation. Results demonstrated technological feasibility of spray drying process to be used in formulating a biopesticide based on NPV003. <![CDATA[Thermodynamic study of asparagine and glycyl-asparagine using computational methods]]> This work aimed to develop an ab initio procedure for accurately calculating pKa values and applied it to study the acidity of asparagine and glycyl-asparagine. DFT methods with B3LYP composed by 6-31+G(d) basis set were applied for calculating the acidic dissociation constant of asparagine and glycyl-asparagine. The formation of intermolecular hydrogen bonds between the available species and water was analyzed using Tomasi,s method. Results showed that in alkaline solutions, the cation, anion and neutral species of asparagine and glycyl-asparagine were solvated with one, two, three and four molecules of water, respectively. There was an excellent similarity between the experimentally attained pKa values and the theoretically ones in this work.