RESUMO
Current antifungal drugs present poor effectiveness and there is no available vaccine for fungal infections. Thus, novel strategies to treat or prevent invasive mycosis, such as cryptococcosis, are highly desirable. One strategy is the use of immunomodulators of polysaccharide nature isolated from mushrooms. The purpose of the present work was to evaluate the immunostimulatory activity of ß-(1,3)-glucan-containing exopolysaccharides (EPS) from the edible mushrooms Auricularia auricula in phagocytes and mice infected with Cryptococcus neoformans. EPS triggered macrophages and dendritic cell activation upon binding to Dectin-1, a pattern recognition receptor of the C-type lectin receptor family. Engagement of Dectin-1 culminated in pro-inflammatory cytokine production and cell maturation via its canonical Syk-dependent pathway signaling. Furthermore, upon EPS treatment, M2-like phenotype macrophages, known to support intracellular survival and replication of C. neoformans, repolarize to M1 macrophage pattern associated with enhanced production of the microbicidal molecule nitric oxide that results in efficient killing of C. neoformans. Treatment with EPS also upregulated transcript levels of genes encoding products associated with host protection against C. neoformans and Dectin-1 mediated signaling in macrophages. Finally, orally administrated ß-glucan-containing EPS from A. auricular enhanced the survival of mice infected with C. neoformans. In conclusion, the results demonstrate that EPS from A. auricula exert immunostimulatory activity in phagocytes and induce host protection against C. neoformans, suggesting that polysaccharides from this mushroom may be promising as an adjuvant for vaccines or antifungal therapy.
Assuntos
Agaricales/química , Criptococose/prevenção & controle , Polissacarídeos Fúngicos/imunologia , Fagócitos/efeitos dos fármacos , Fagócitos/imunologia , beta-Glucanas/imunologia , Animais , Criptococose/imunologia , Cryptococcus neoformans/imunologia , Citocinas/imunologia , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/imunologia , Células Dendríticas/microbiologia , Fatores Imunológicos/farmacologia , Lectinas Tipo C/imunologia , Pneumopatias Fúngicas , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Macrófagos/microbiologia , Camundongos , Camundongos Endogâmicos C57BL , Fagócitos/microbiologia , Transdução de Sinais , beta-Glucanas/farmacologiaRESUMO
Drought and rice blast disease caused by Magnaporthe oryzae are two of the most serious threats to global rice production. To explore the mechanisms underlying gene expression induced in rice by stresses, studies involving transcriptome analyses have been conducted over the past few years. Thus, it is crucial to have a reliable set of reference genes to normalize the expression levels of rice genes affected by different stresses. To identify potential reference genes for studies of the differential expression of target genes in rice under M. oryzae infection and drought conditions, the present study evaluated five housekeeping genes for the normalization of gene expression. The stability of the expression of these genes was assessed using the analytical software packages geNorm and NormFinder. For all samples analyzed, the stability rank was UBQ5 > GAPDH > eIF-4α> ß-TUB > 18S rRNA. The data showed that the UBQ5, GAPDH, and eIF-4αgenes are appropriate, high-performing reference genes and will be highly useful in future expression studies of fungal infections and drought in rice.
Assuntos
Regulação da Expressão Gênica de Plantas , Genes Essenciais , Genes de Plantas , Oryza/genética , Reação em Cadeia da Polimerase em Tempo Real/normas , Secas , Fator de Iniciação 4A em Eucariotos/genética , Gliceraldeído-3-Fosfato Desidrogenase (Fosforiladora)/genética , Magnaporthe/patogenicidade , Magnaporthe/fisiologia , Oryza/microbiologia , Doenças das Plantas/genética , Doenças das Plantas/microbiologia , Padrões de Referência , Estresse Fisiológico , Transcriptoma , Ubiquitina/genéticaRESUMO
α-Amylases are common enzymes responsible for hydrolyzing starch. Insect-pests, whose larvae develop in seeds, rely obligatorily on α-amylase activity to digest starch, as their major food source. Considering the relevance of insect α-amylases and the natural α-amylase inhibitors present in seeds to protect from insect damage, we report here the molecular cloning and nucleotide sequence of the full-length AmyHha cDNA of the coffee berry borer, Hypothenemus hampei, a major insect-pest of coffee crops. The AmyHha sequence has 1879 bp, containing a 1458 bp open reading frame, which encodes a predicted protein with 485 amino acid residues, with a predicted molecular mass of 51.2 kDa. The deduced protein showed 55-79% identity to other insect α-amylases, including Anthonomus grandis, Ips typographus and Sitophilus oryzae α-amylases. In depth analysis revealed that the highly conserved three amino acid residues (Asp184, Glu220, and Asp285), which compose the catalytic site are also presented in AmyHha amylase. The AmyHha gene seems to be a single copy in the haploid genome and AmyHha transcription levels were found higher in L2 larvae and adult insects, both corresponding to major feeding phases. Modeling of the AmyHha predicted protein uncovered striking structural similarities to the Tenebrio molitor α-amylase also displaying the same amino acid residues involved in enzyme catalysis (Asp184, Glu220 and Asp285). Since AmyHha gene was mostly transcribed in the intestinal tract of H. hampei larvae, the cognate α-amylase could be considered a high valuable target to coffee bean insect control by biotechnological strategies.
Assuntos
Besouros/fisiologia , DNA Complementar/genética , Comportamento Alimentar , alfa-Amilases/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , Besouros/classificação , Besouros/enzimologia , Modelos Moleculares , Dados de Sequência Molecular , Fases de Leitura Aberta , Filogenia , Reação em Cadeia da Polimerase em Tempo Real , Homologia de Sequência de Aminoácidos , alfa-Amilases/químicaRESUMO
Eugenia dysenterica is a plant typically found in the Cerrado biome and commonly used in popular medicine due to its pharmacological properties, which include antidiarrheal, skin healing, and antimicrobial activities. The effects of ethanolic extract, aqueous extract and infusion of E. dysenterica leaves on intestinal motility and antidiarrheal activity were evaluated using ricin oil-induced diarrhea in rats. At doses of 400 and 800 mg·Kg(-1), the ethanolic extract decreased intestinal motility while the other extracts showed no significant effects. Moreover, serum levels of chloride, magnesium, and phosphorus were also measured in rats. Histopathologic and enzymatic analyses were also performed to investigate any toxic effect. Animals treated with infusion, ethanolic extract, ricin oil, and loperamide presented morphological alterations in the small intestine, such as mucosa lesion, epithelial layer damage, and partial loss and/or morphological change of villi. Furthermore, the liver showed congestion and hydropic degeneration. Serum levels of alanine aminotransferase increased significantly in all treatments, but none rose above reference values. In summary, our results suggest that compounds present in leaves of E. dysenterica may have therapeutic benefits on recovery from diarrhea despite their toxic effects.
RESUMO
Plants have contributed over the years to the discovery of various pharmacological products. Amongst the enormous diversity of herbs with remarkable medicinal use and further pharmacological potential, here in this report we evaluated pulp extracts from Eugenia dysenterica fruits and further identified the active principle involved in such laxative activity in rats. For protein isolation, fruits were macerated with an extraction solution following precipitation with (NH(4))(2)SO(4) (100%). After dialysis, the peptide was applied onto a reversed-phase semi-preparative HPLC column, and the major fraction was eluted with 26% and 66% acetonitrile. The evaluation of molecular masses by MALDI-TOF and Tris/Tricine SDS-PAGE of HPLC fractions showed the presence of a major peptide with approximately 7 kDa. The N-terminal amino acid peptide sequence was determined and showed no similarity to other proteins deposited in the Data Bank. Peptide from E. dysenterica was able to enhance rats' intestinal motility by approximately 20.8%, probably being responsible for laxative activity. Moreover, these proteins were non-toxic to mammals, as observed in histopathology and hemolytic analyses. In conclusion, results here reported indicate that, in the near future, proteins synthesized by E. dysenterica fruits could be utilized in the development of novel biotechnological pharmaceutics with laxative properties for use in chronic constipation and irritable bowel syndrome treatment.
Assuntos
Constipação Intestinal/tratamento farmacológico , Frutas/metabolismo , Síndrome do Intestino Irritável/tratamento farmacológico , Laxantes/farmacologia , Peptídeos/farmacologia , Proteínas de Plantas/farmacologia , Syzygium/metabolismo , Sequência de Aminoácidos , Animais , Brasil , Doença Crônica/tratamento farmacológico , Frutas/efeitos adversos , Motilidade Gastrointestinal/efeitos dos fármacos , Hemólise/efeitos dos fármacos , Intestino Delgado/efeitos dos fármacos , Intestino Delgado/patologia , Laxantes/efeitos adversos , Laxantes/química , Laxantes/isolamento & purificação , Fígado/efeitos dos fármacos , Fígado/patologia , Masculino , Medicina Tradicional , Dados de Sequência Molecular , Peso Molecular , Peptídeos/efeitos adversos , Peptídeos/isolamento & purificação , Proteínas de Plantas/efeitos adversos , Proteínas de Plantas/química , Proteínas de Plantas/isolamento & purificação , Proteínas de Plantas/metabolismo , Ratos , Ratos Wistar , Homologia de Sequência de AminoácidosRESUMO
A beta-N-Acetylhexosaminidase (EC 3.2.1.52) was purified from hepatic extracts of Sotalia fluviatilis, order Cetacea. The protein was purified by using ammonium sulfate fractionation and four subsequent chromatographies (Biogel A 1.5 m, Chitin, Deae-Biogel and hydroxyapatite resins). After these purification steps, the enzyme was purified 380.5-fold with an 8.4% yield. The molecular mass (10 kDa) was estimated by SDS-PAGE and MALDI-TOF analysis. A Km of 2.72 mM and Vmax 9.5 x 10(-6) micromol/(min x mg) were found for this enzyme, determined by p-nitrophenyl-beta-D: -hexosaminide substrate digestion. Optimal pH and temperature for beta-N-Acetylhexosaminidase activity were 5.0 and 60 degrees C, respectively. Enzyme activity was inhibited by sodium selenate (Na(2)SeO(4)), mercuric chloride (HgCl(2)) and sodium dodecyl sulfate (C(12)H(25)SO(4)Na), and activated by zinc, calcium, barium and lithium ions. Characterization of the beta-N-Acetylhexosaminidase in Sotalia fluviatilis can be a basis for physiological studies in this species.
Assuntos
Golfinhos/metabolismo , Fígado/enzimologia , beta-N-Acetil-Hexosaminidases/isolamento & purificação , Animais , Carboidratos , Cromatografia em Gel , Eletroforese em Gel de Poliacrilamida , Concentração de Íons de Hidrogênio , Cinética , Fígado/química , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Temperatura , beta-N-Acetil-Hexosaminidases/química , beta-N-Acetil-Hexosaminidases/metabolismoRESUMO
The biochemical properties of the digestive alpha-amylase from Tecia solanivora larvae, an important and invasive insect pest of potato (Solanum tuberosum), were studied. This insect has three major digestive alpha-amylases with isoelectric points 5.30, 5.70 and 5.98, respectively, which were separated using native and isoelectric focusing gels. The alpha-amylase activity has an optimum pH between 7.0 and 10.0 with a peak at pH 9.0. The enzymes are stable when heated to 50 degrees C and were inhibited by proteinaceous inhibitors from Phaseolus coccineus (70% inhibition) and P. vulgaris cv. Radical (87% inhibition) at pH 6.0. The inhibitors present in an amaranth hybrid inhibited 80% of the activity at pH 9.0. The results show that the alpha-amylase inhibitor from amaranth seeds may be a better candidate to make genetically-modified potatoes resistant to this insect than inhibitors from common bean seeds.
Assuntos
Inibidores Enzimáticos/química , Mariposas/enzimologia , Proteínas de Plantas/química , alfa-Amilases/química , Animais , Estabilidade Enzimática , Concentração de Íons de Hidrogênio , Larva/enzimologia , Mariposas/crescimento & desenvolvimento , Temperatura , alfa-Amilases/antagonistas & inibidores , alfa-Amilases/fisiologiaRESUMO
Canatoxin, a urease isoform from Canavalia ensiformis seeds, shows insecticidal activity against different insect species. Its toxicity relies on an internal 10 kDa peptide (pepcanatox), released by hydrolysis of Canatoxin by cathepsins in the digestive system of susceptible insects. In the present work, based on the N-terminal sequence of pepcanatox, we have designed primers to amplify by PCR a 270-bp fragment corresponding to pepcanatox using JBURE-II cDNA (one of the urease isoforms cloned from C. ensiformis, with high identity to JBURE-I, the classical urease) as a template. This amplicon named jaburetox-2 was cloned into pET 101 vector to obtain heterologous expression in Escherichia coli of the recombinant protein in C-terminal fusion with V-5 epitope and 6-His tag. Jaburetox-2Ec was purified on Nickel-NTA resin and bioassayed in insect models. Dysdercus peruvianus larvae were fed on cotton seed meal diets containing 0.01% (w/w) Jaburetox-2Ec and, after 11 days, all individuals were dead. Jaburetox-2Ec was also tested against Spodoptera frugiperda larvae and caused 100% mortality. In contrast, high doses of Jaburetox-2Ec were innocuous when injected or ingested by mice and neonate rats. Modeling of Jaburetox-2Ec, in comparison with other peptide structures, revealed a prominent beta-hairpin motif consistent with an insecticidal activity based on either neurotoxicity or cell permeation.
Assuntos
Canavalia/enzimologia , Inseticidas/isolamento & purificação , Urease/química , Sequência de Aminoácidos , Sequência de Bases , Clonagem Molecular , Primers do DNA , Eletroforese em Gel de Poliacrilamida , Inseticidas/química , Inseticidas/toxicidade , Dados de Sequência Molecular , Proteínas de Plantas , Reação em Cadeia da Polimerase , Homologia de Sequência de Aminoácidos , Urease/genética , Urease/isolamento & purificação , Urease/toxicidadeRESUMO
Acanthoscelides obtectus is a devastating storage insect pest capable of causing severe bean crop losses. In order to maintain their own development, insect pest larvae feed continuously, synthesizing efficient digestive enzymes. Among them, cysteine proteinases (CPs) are commonly produced as inactive precursors (procysteines), requiring a cleavage of the peptide proregion to become active. The proregion fits tightly into the active site of procysteines, efficiently preventing their activity. In this report, a CP cDNA (cpao) was isolated from A. obtectus midgut larvae. In silico studies indicated that the complete CP sequence contains a hydrophobic signal peptide, a prodomain and a conserved catalytic region. Moreover, the encoding cDNA contains 963bp translating into a 321 residue protein, CPAo, which was expressed in E. coli, fused with thioredoxin. Enzymatic assays using the recombinant protein revealed that the enzyme was catalytically active, being able to cleave the synthetic substrate Z-Phe-Arg-7-AMC. Additionally, this report also focuses the cpao propeptide (PCPAo) subcloning and expression. The expressed propeptide efficiently inhibited CPAo, as well as digestive CP of other bean bruchids. Little or no activity was found against proteolytic enzymes of two other coleopterans: Rhyzopertha dominica and Anthonomus grandis. The data reported here indicate the possibility of endogenous propeptides as a novel strategy on bruchids control, which could be applicable to bean improvement programs.
Assuntos
Besouros/enzimologia , Cisteína Endopeptidases/metabolismo , Inibidores de Cisteína Proteinase/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Sítios de Ligação , Clonagem Molecular , Sequência Conservada , Cisteína Endopeptidases/química , Cisteína Endopeptidases/genética , Cisteína Endopeptidases/isolamento & purificação , Inibidores de Cisteína Proteinase/química , Inibidores de Cisteína Proteinase/genética , DNA Complementar/genética , Escherichia coli/genética , Dados de Sequência Molecular , Ligação Proteica , Sinais Direcionadores de Proteínas , Estrutura Terciária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Homologia de Sequência de Aminoácidos , Especificidade por Substrato , Tiorredoxinas/metabolismoRESUMO
A 6.5 kDa serine protease inhibitor was purified by anion-exchange chromatography from the crude extract of the Inga umbratica seeds, containing inhibitor isoforms ranging from 6.3 to 6.7 kDa and protease inhibitors of approximately 19 kDa. The purified protein was characterized as a potent inhibitor against trypsin and chymotrypsin and it was named I. umbratica trypsin and chymotrypsin inhibitor (IUTCI). MALDI-TOF spectra of the IUTCI, in the presence of DTT, showed six disulfide bonds content, suggesting that this inhibitor belongs to Bowman-Birk family. The circular dichroism spectroscopy indicates that IUTCI is predominantly formed by unordered and beta-sheet secondary structure. It was also characterized, by fluorescence spectroscopy, as a stable protein at range of pH from 5.0 to 7.0. Moreover, this inhibitor at concentration of 75 microM presented a remarkable inhibitory activity (60%) against digestive serine proteases from boll weevil Anthonomus grandis, an important economical cotton pest.