The production and characterization of a new active lipase from Acremonium alcalophilum using a plant bioreactor

Background: Microorganisms are the most proficient decomposers in nature, using secreted enzymes in the hydrolysis of lignocellulose. As such, they present the most abundant source for discovery of new enzymes. Acremonium alcalophilum is the only known cellulolytic fungus that thrives in alkaline conditions and can be cultured readily in the laboratory. Its optimal conditions for growth are 30°C and pH 9.0-9.2. The genome sequence of Acremonium alcalophilum has revealed a large number of genes encoding biomass-degrading enzymes. Among these enzymes, lipases are interesting because of several industrial applications including biofuels, detergent, food processing and textile industries. Results: We identified a lipA gene in the genome sequence of Acremonium alcalophilum, encoding a protein with a predicted lipase domain with weak sequence identity to characterized enzymes. Unusually, the predicted lipase displays ≈ 30% amino acid sequence identity to both feruloyl esterase and lipase of Aspergillus niger. LipA, when transiently produced in Nicotiana benthamiana, accumulated to over 9% of total soluble protein. Plant-produced recombinant LipA is active towards p-nitrophenol esters of various carbon chain lengths with peak activity on medium-chain fatty acid (C8). The enzyme is also highly active on xylose tetra-acetate and oat spelt xylan. These results suggests that LipA is a novel lipolytic enzyme that possesses both lipase and acetylxylan esterase activity. We determined that LipA is a glycoprotein with pH and temperature optima at 8.0 and 40°C, respectively. Conclusion: Besides being the first heterologous expression and characterization of a gene coding for a lipase from A. alcalophilum, this report shows that LipA is very versatile exhibiting both acetylxylan esterase and lipase activities potentially useful for diverse industry sectors, and that tobacco is a suitable bioreactor for producing fungal proteins

Heterologous Production and Characterization of Cell Wall Degrading Enzymes Using Plants as a Bioreactor

Plants are wonderful living organisms. They are able to store solar energy into carbohydrates by fixing CO2 through photosynthesis which can be subsequently harvested and used for fuel production. However, one of the major limitations for transforming these carbohydrates into liquid fuels is the recalcitrance of the plant cell wall. Although microorganisms have evolved a series of cell wall degrading enzymes to harvest efficiently this energy and are considered the main source of these biocatalysts, harnessing these microorganisms for the production of enzymes is a costly process and a major factor limiting the commercialization of lignocellulosic biomass-to-ethanol processes. The production of cell wall degrading enzymes in plants has been proposed as an alternative platform to lower cost of production. However, due to the variability of biomass feedstock availability and plant cell wall complexity, specialized accessory enzymes are necessary for the complete plant cell wall deconstruction into fermentable sugars. The expression of three key accessory enzymes was investigated in Nicotiana benthamiana. A new esterase gene from Acremonium alcalophilum was identified, produced in plants and characterized. It accumulated to high levels, 9% of total soluble protein (TSP), and it was found to have both acetyl xylan esterase and lipase activity. A polygalacturonase I from Aspergillus niger (AnPGI) and a feruloyl esterase from Anaeromyces mucronatus (Fae1A), were also produced using a combination of subcellular targeting and protein fusions with elastin-like polypeptide (ELP) and hydrophobin I (HFBI). Accumulation levels of 3.6, and 3.9% of TSP were achieved for AnPGI and Fae1A respectively. Based on our observations, although the use of protein fusions lowered protein activity, the ELP fusion had the most significant impact on enzyme production, with a higher amount of total activity recovered from fresh leaf weight than from HFBI fusions or unfused proteins. The impact of ELP and HFBI fusions on protein accumulation was protein-specific and should be evaluated individually in the future. The production of these three enzymes can lead to a minimal enzymatic cocktail for degrading the pectin component of the plant cell wall and should have an impact on the use of pectin-rich biomass residues for biofuel production

Detection of serum and salivary IgE and IgG1 immunoglobulins specific for diagnosis of food allergy.

Given the growing incidence and prevalence of life-threatening food allergies, health concerns have raised new perspectives for in vivo and in vitro diagnostic methodologies, pointing to saliva as a promising material, already used to diagnose other pathologies. Based on the above considerations, this study aimed to verify the possible use of saliva for the detection of IgE and IgG1 in the diagnosis of food allergy. This was a randomized, cross-sectional clinical study with a quantitative approach, developed at a hospital referral center in allergy in the state of Ceará, from January to July 2015. The sample consisted of 36 children of both sexes, aged between 1 and 60 months, with a diagnosis of cow's milk protein allergy (CMPA) by the RAST test. Children hospitalized or under immunosuppressive drugs were excluded from the study. Serum and saliva samples of the participants were collected and subsequently subjected to the indirect immunoenzymatic assay (ELISA) for the detection of specific serum and salivary immunoglobulins for food: corn, papaya, cow's milk, egg white, wheat, soybeans, peanuts, nuts, kiwi, cacao, fish, shrimp, bananas and tomatoes. For comparison of serum and saliva results, the T-test of independent samples and Mann-Whitney were adopted, for samples with normal and non-normal distribution respectively. A confidence interval of 95% was adopted for significant results. It was observed that 100% (n = 36) of the participants presented cow's milk allergy through the indirect ELISA, detecting IgE or IgG1 in serum and saliva. When serum IgE and IgG1 concentrations were compared, there was no statistical difference (p > 0.05) in 12 of the 14 foods evaluated. The same amount (n = 12) of non-significant differences (p > 0.05) was observed in the comparison of the 14 foods under IgE and IgG1 contractions in saliva. In the verification of the average values of IgE present in the serum and saliva of the foods, only cow's milk, fish and papaya showed statistically significant differences (p < 0.05). Of the total food evaluated, only the average levels of IgG1 present in serum and saliva showed a significant value (p < 0.05) in banana and tomato. These findings indicate that the detection of IgE and IgG1 in saliva proves to be as efficient as in the serum. The use of the salivary technique for use in the diagnosis of food allergy is suggested

Antidiabetic effects of galactomannans from Adenanthera pavonina L. in streptozotocin-induced diabetic mice

Objective: To evaluate the antidiabetic effect of galactomannans extracted from Adenanthera pavonina's L. seeds (GAP) in streptozotocin (STZ) induced diabetic mice. Methods: The preliminary galactomannan yield from Adenanthera pavonina L. plant and extraction products composition were evaluated. Various chemical characterization methods like thin layer chromatography, Fourier transform infrared spectroscopy, 1H and 13C nuclear magnetic resonance, and molecular weight by gel permeation chromatography have been employed to characterize the extracted galactomannan. The mice were divided in four groups: Normal control, diabetic control, GAP (1% and 2%) treated and standard drug treated groups. Diabetic mice received treatment daily for 30 d. Diabetes was induced by STZ at a single dose of 120 mg/kg. Body weight, water and food intake, fasting blood glucose, total cholesterol and triglycerides were measured. Histopathological analysis of pancreas and liver were performed to evaluate STZ-induced tissue injuries. Results: The isolated and extracted galactomannan from Adenanthera pavonina was confirmed by various chemical characterization methods. GAP exhibited a 1.46:1 mannose: galactose ratio, and high molar weight. Both GAP enriched food decreased glycaemia, total cholesterol and triacylglycerol. GAP didn't interfere on food intakes or body weight, although it increased water intake. Furthermore, the relative liver weight indicated toxic galactomannan effects on the histopathological changes of the pancreas in STZ induced diabetes. Conclusions: It is concluded that GAP is a natural product that contains potent galactomannan and is useful in preventing and treating diabetes

Humoral and cellular immune responses in BALB/c and C57BL/6 mice immunized with cytoplasmic (CRA) and flagellar (FRA) recombinant repetitive antigens, in acute experimental Trypanosoma cruzi infection.

Submitted by Ana Maria Fiscina Sampaio ([email protected]) on 2015-04-14T17:12:13Z No. of bitstreams: 1 Pereira VRA Humoral and cellular immune.........pdf: 283731 bytes, checksum: d693fb5d4ffab78266f737a194ac7a59 (MD5)Approved for entry into archive by Ana Maria Fiscina Sampaio ([email protected]) on 2015-04-14T17:25:18Z (GMT) No. of bitstreams: 1 Pereira VRA Humoral and cellular immune.........pdf: 283731 bytes, checksum: d693fb5d4ffab78266f737a194ac7a59 (MD5)Made available in DSpace on 2015-04-14T17:25:18Z (GMT). No. of bitstreams: 1 Pereira VRA Humoral and cellular immune.........pdf: 283731 bytes, checksum: d693fb5d4ffab78266f737a194ac7a59 (MD5) Previous issue date: 2005Fundação Oswaldo Cruz. Centro de Pesquisas Aggeu Magalhães. Recife, PE, BrasilFundação Oswaldo Cruz. Centro de Pesquisas Aggeu Magalhães. Recife, PE, BrasilFundação Oswaldo Cruz. Centro de Pesquisas Aggeu Magalhães. Recife, PE, BrasilFundação Oswaldo Cruz. Centro de Pesquisas Aggeu Magalhães. Recife, PE, BrasilFundação Oswaldo Cruz. FarManguinhos. Rio de Janeiro, RJ, Brasil /Fundação Oswaldo Cruz. Centro de Pesquisas Gonçalo Moniz. Salvador, BA, BrasilFundação Oswaldo Cruz. FarManguinhos. Rio de Janeiro, RJ, Brasil /Fundação Oswaldo Cruz. Centro de Pesquisas Gonçalo Moniz. Salvador, BA, BrasilFundação Oswaldo Cruz. Instituto Oswaldo Cruz. Rio de Janeiro, RJ, BrasilFundação Oswaldo Cruz. Instituto Oswaldo Cruz. Rio de Janeiro, RJ, BrasilFundação Oswaldo Cruz. Centro de Pesquisas Gonçalo Moniz. Salvador, BA, BrasilFundação Oswaldo Cruz. Centro de Pesquisas Aggeu Magalhães. Recife, PE, BrasilFundação Oswaldo Cruz. Centro de Pesquisas René Rachou. Belo Horizonte, MG, BrasilFundação Oswaldo Cruz. Centro de Pesquisas Aggeu Magalhães. Recife, PE, BrasilIn previous studies, cytoplasmic repetitive antigen (CRA) and flagellar repetitive antigen (FRA) proteins induced specific humoral and cellular immune responses in susceptible and resistant mice in the absence of Trypanosoma cruzi infection with a significant induction of the Interferon-gamma (IFN-gamma) production in those animals. In this follow-up paper, the immunostimulatory and protective effects of these proteins were evaluated by immunizing with CRA or FRA antigens, BALB/c and C57BL/6 mice and challenging with a T. cruzi (Y strain). Both proteins induced humoral response with high levels of IgG isotypes as well as cellular immunity with high levels of IFN-gamma when compared to controls. However, the lymphocyte proliferative response was minimal. The survival rate at 30 days post-infection was significant in CRA (60%) or FRA (50%)--immunized BALB/c mice and CRA (83.3%)--immunized C57BL/6 mice. Taken as a whole these findings indicate that CRA and FRA are immunogenic and potentially important for protective immunity

Immunization with cytoplasmic repetitive antigen and flagellar repetitive antigen of Trypanosoma cruzi stimulates a cellular immune response in mice

Submitted by Ana Maria Fiscina Sampaio ([email protected]) on 2015-04-10T14:12:27Z No. of bitstreams: 1 Pereira V R A Immunization with....pdf: 111858 bytes, checksum: 4793453ce315eeed8d73c164a1323364 (MD5)Approved for entry into archive by Ana Maria Fiscina Sampaio ([email protected]) on 2015-04-10T14:27:34Z (GMT) No. of bitstreams: 1 Pereira V R A Immunization with....pdf: 111858 bytes, checksum: 4793453ce315eeed8d73c164a1323364 (MD5)Made available in DSpace on 2015-04-10T14:27:34Z (GMT). No. of bitstreams: 1 Pereira V R A Immunization with....pdf: 111858 bytes, checksum: 4793453ce315eeed8d73c164a1323364 (MD5) Previous issue date: 2004Fundação Oswaldo Cruz. Centro de Pesquisas Aggeu Magalhães. Recife, PE, Brasil.Fundação Oswaldo Cruz. Centro de Pesquisas Aggeu Magalhães. Recife, PE, Brasil.Universidade Federal de Pernambuco. Recife, PE, Brasil.Fundação Oswaldo Cruz. Centro de Pesquisas Aggeu Magalhães. Recife, PE, Brasil.Fundação Oswaldo Cruz. Bio-Manguinhos. Rio de Janeiro, RJ, Brasil / Fundação Oswaldo Cruz. Centro de Pesquisas Gonçalo Moniz. Salvador, BA, Brasil.Fundação Oswaldo Cruz. Bio-Manguinhos. Rio de Janeiro, RJ, Brasil / Fundação Oswaldo Cruz. Centro de Pesquisas Gonçalo Moniz. Salvador, BA, Brasil.Universidade Federal de Pernambuco. Recife, PE, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Rio de Janeiro, RJ, Brasil / Instituto de Biologia Molecular do Paraná. Curitiba, PR, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Rio de Janeiro, RJ, Brasil / Instituto de Biologia Molecular do Paraná. Curitiba, PR, Brasil.Fundação Oswaldo Cruz. Centro de Pesquisas Gonçalo Moniz. Salvador, BA, Brasil.Fundação Oswaldo Cruz. Centro de Pesquisas René Rachou. Belo Horizonte, MG, Brasil.Fundação Oswaldo Cruz. Centro de Pesquisas Aggeu Magalhães. Recife, PE, Brasil.In previous studies, we demonstrated that CRA and FRA recombinant proteins, used for diagnosis of Chagas' disease, elicited a humoral immune response in susceptible and resistant mice. To understand better the immune response to these proteins, we have evaluated, the cellular immune response in CRA- and in FRA-immunized BALB/c and C57BL/6 mice. A specific cellular lymphoproliferative response was observed in both strains of mice. Spleen cell cultures mainly from CRA-immunized C57BL/6 and FRA-immunized BALB/c mice produced high levels of IFN-y, indicating the induction of a Type 1 immune response. Regarding the T cell subsets, CD4+ T cells were the major source of IFN-y in CRA- and FRA-immunized mice. These results suggest that CRA and FRA are important immunogens in inducing a Type 1 immune response and that they may be considered as potential vaccine antigens

Avaliação hematológica e histopatológica de camundongos BALB/c e C57BL/6 expostos aos antígenos recombinantes Cytoplasmic Repetitive Antigen e Flagellar Repetitive Antigen de Trypanosoma cruzi Hematological and histopathological evaluation of BALB/c and C57BL/6 mice exposed to Cytoplasmic Repetitive Antigen and Flagellar Repetitive Antigen recombinant antigens of Trypanosoma cruzi

Os antígenos recombinantes Cytoplasmic Repetitive Antigen e Flagellar Repetitive Antigen de Trypanosoma cruzi foram inoculados em camundongos BALB/c e C57BL/6 e o seu efeito avaliado a nível hematológico e histopatológico. Os resultados mostraram que o padrão histológico normal dos órgãos e o perfil hematológico dos camundongos não foram modificados sugerindo que esses antígenos não parecem causar dano ao animal.<br>The Cytoplasmic Repetitive Antigen and Flagellar Repetitive Antigen recombinant antigens of Trypanosoma cruzi were inoculated into BALB/c and C57BL/6 mice and its effects evaluated at hematological and histopathological levels. The results showed that the histological pattern of the organs and the hematological profile of mice were not modified suggesting that these antigens are not harmful for the animal

Subcutaneous, Oral, and Intranasal Immunization of BALB/c Mice with <i>Leishmania infantum</i> K39 Antigen Induces Non-Protective Humoral Immune Response

Visceral leishmaniasis is a high-burden disease caused by parasites of the Leishmania genus. The K39 kinesin is a highly antigenic protein of Leishmania infantum, but little is known about the immune response elicited by this antigen. We evaluated the humoral immune response of female BALB/c mice (n = 6) immunized with the rK39-HFBI construct, formed by the fusion of the K39 antigen to a hydrophobin partner. The rK39-HFBI construct was administered through subcutaneous, oral, and intranasal routes using saponin as an adjuvant. We analyzed the kinetics of IgG, IgG1, and IgG2a production. The groups were then challenged by an intravenous infection with L. infantum promastigote cells. The rK39-HFBI antigen-induced high levels of total IgG (p p < 0.05), suggesting a potential secondary immune response following the booster dose. There was no reduction in the splenic parasite load; thus, the rK39-HFBI failed to protect the mice against infection under the tested conditions. The results presented here demonstrate that the high antigenicity of the K39 antigen does not contribute to a protective immune response against visceral leishmaniasis

Characterization of Cnidoscolus quercifolius Pohl bark root extract and evaluation of cytotoxic effect on human tumor cell lines

Objective: To evaluate the chemical components of active extract from Cnidoscolus quercifolius root bark and its cytotoxic potential against several tumor strains. Methods: The high-performance liquid chromatography with diode-array detection and 1H and 13C nuclear magnetic resonance spectroscopy of the extract were used to distinguish the existence of possible functional groups in the root bark extract. The in vitro cytotoxic activity of methanol extract on human colon cancer cell lines was evaluated using OVCAR-8, SF-295, HCT-116, HL-60 strains and the samples were assessed by 3-(4,5-dimethylthiazol2-yl)-2,5-diphenyltetrazolium bromide method. Results: The analysis of nuclear magnetic spectra of the active chloroform fraction revealed the presence of absorptions bands correspondent to a mixture of favelines such as neofavelanone, deoxofaveline or methyl-faveline, which structures were confirmed by ultraviolet spectra upon high-performance liquid chromatography with diode-array detection analysis. The active fraction showed cytotoxic effects in the tested strains, HCT-116, SF-295, OVCAR-8 and HL-60 cells with IC50 of 72 hours ranging from 4.95 to 15.23 μg/mL. Conclusions: The results suggest that the substances present in faveleira (Cnidoscolus quercifolius) root bark extract have a cytotoxic potential against several tumor lines, showing a broader antitumour potential, and in addition no adverse to healthy cells. Therefore, the root bark extract of Cnidoscolus quercifolius has a possibility of use for anticarcinogenic therapies