RESUMO
The partitioning and purification of lectins from the crude extract of Cratylia mollis seeds (Cramoll 1,4) was investigated in aqueous two-phase systems (ATPS). A factorial design model (24) was used to evaluate the influence of polyethylene glycol (PEG) molar mass (1500-8000 g/mol), PEG concentration (12.5-17.5% w/w), phosphate (10-15% w/w) concentration, and pH (6-8) on the differential partitioning, purification factor, and yield of the lectin. Polymer and salt concentration were the most important variables affecting partition of lectin and used to find optimum purification factor by experimental Box-Behnken design together with the response surface methodology (RSM). ATPS showed best conditions composed by 13.9% PEG1500, 15.3% phosphate buffer at pH 6, which ensured purification factor of 4.70. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed a single band of protein with 26.1 kDa. Furthermore, results demonstrated a thermostable lectin presenting activity until 60 °C and lost hemagglutinating activity at 80 °C. According to the obtained data it can be inferred that the ATPS optimization using RSM approach can be applied for recovery and purification of lectins.
Assuntos
Lectinas/química , Lectinas/isolamento & purificação , Phaseolus/química , Extratos Vegetais/química , Eletroforese em Gel de Poliacrilamida , Hemaglutininas/química , Concentração de Íons de Hidrogênio , Fosfatos/química , Polietilenoglicóis/química , Proteínas/química , Sementes/química , Espectrofotometria , Propriedades de Superfície , TemperaturaRESUMO
Glycoconjugates found on cell walls of Candida species are fundamental for their pathogenicity. Laborious techniques have been employed to investigate the sugar composition of these microorganisms. Herein, we prepared a nanotool, based on the fluorescence of quantum dots (QDs) combined with the specificity of Cramoll lectin, to evaluate glucose/mannose profiles on three Candida species. The QDs-Cramoll conjugates presented specificity and bright fluorescence emission. The lectin preserved its biological activity after the conjugation process mediated by adsorption interactions. The labeling of Candida species was analyzed by fluorescence microscopy and quantified by flow cytometry. Morphological analyses of yeasts labeled with QDs-Cramoll conjugates indicated that C. glabrata (2.7 µm) was smaller when compared to C. albicans (4.0 µm) and C. parapsilosis sensu stricto (3.8 µm). Also, C. parapsilosis population was heterogeneous, presenting rod-shaped blastoconidia. More than 90% of cells of the three species were labeled by conjugates. Inhibition and saturation assays indicated that C. parapsilosis had a higher content of exposed glucose/mannose than the other two species. Therefore, QDs-Cramoll conjugates demonstrated to be effective fluorescent nanoprobes for evaluation of glucose/mannose constitution on the cell walls of fungal species frequently involved in candidiasis.
Assuntos
Candida/química , Corantes Fluorescentes/química , Glucose/análise , Lectinas/química , Manose/análise , Microscopia de Fluorescência/métodos , Candida/crescimento & desenvolvimento , Candida/isolamento & purificação , Candida/metabolismo , Candidíase/diagnóstico , Candidíase/microbiologia , Parede Celular/química , Parede Celular/metabolismo , Glucose/metabolismo , Humanos , Manose/metabolismo , Microscopia de Fluorescência/instrumentação , Nanopartículas/química , Pontos Quânticos/químicaRESUMO
Gastrointestinal nematodes are considered one of the most important sanitary problems affectingruminants globally; whether parasitizing alone or acting with other pathogens, they are responsiblefor severe economic losses related to animal husbandry. Endoparasites in small ruminants areusually controlled with anti-helminthic drugs. However, certain strains of endoparasites are resistantto such drugs. Supplementation of the diet with protein-rich foods is one strategy for reducing thenegative impact of gastrointestinal parasites. Cratylia argentea has anthelmintic properties and othercharacteristics that favor its use as an animal feed supplement. In the present study, we subdividedcrossbred lambs into homogeneous groups and fed them C. argentea (G1), C. argentea plus Brachiariaspp. (G2), or Brachiaria spp. (G3) only. The animals in group G1 had a lower average number of eggsper gram of feces (EPG) at Day 42 (p 0.05), indicating the absenceof anemia and normal globular volume. This reinforces the importance of the measurement of EPG; thisis a quantitative method and does not depend on the subjective interpretation of the results.
Os nematódeos gastrointestinais são considerados um dos problemas sanitários mais importantes dosruminantes no mundo, seja em infecções isoladas ou atuando com outros patógenos, são responsáveis porseveras perdas econômicas relacionadas à produção animal. O controle dos endoparasitas em pequenosruminantes normalmente é feito por meio de drogas anti-helmínticas. No entanto, determinadas cepasde endoparasitos são resistentes a esses fármacos. A suplementação da dieta com alimentos ricos emproteína é uma estratégia para reduzir o impacto negativo das parasitoses gastrointestinais. Cratylia argentea tem efeito anti-helmíntico, além de outras características que favorecem seu uso comosuplemento para alimentação animal. No presente estudo, borregas cruzadas foram subdivididas emgrupos homogêneos e alimentadas com C. argentea (G1), C. argentea mais Brachiaria spp. (G2) ouapenas Brachiaria spp. (G3). Os animais do grupo G1 apresentaram menor valor médio de ovos porgrama de fezes (OPG) aos 42 dias (p0,05), indicando ausência de anemia e volume globularnormal. Isso reforça a importância da avaliação por OPG, que é um método quantitativo e não dependede interpretação subjetiva de resultados.
Assuntos
Animais , Infecções por Nematoides/dietoterapia , Ovinos/parasitologia , Ração Animal , Suplementos Nutricionais , Dietoterapia/veterinária , Helmintíase Animal/dietoterapiaRESUMO
Gastrointestinal nematodes are considered one of the most important sanitary problems affectingruminants globally; whether parasitizing alone or acting with other pathogens, they are responsiblefor severe economic losses related to animal husbandry. Endoparasites in small ruminants areusually controlled with anti-helminthic drugs. However, certain strains of endoparasites are resistantto such drugs. Supplementation of the diet with protein-rich foods is one strategy for reducing thenegative impact of gastrointestinal parasites. Cratylia argentea has anthelmintic properties and othercharacteristics that favor its use as an animal feed supplement. In the present study, we subdividedcrossbred lambs into homogeneous groups and fed them C. argentea (G1), C. argentea plus Brachiariaspp. (G2), or Brachiaria spp. (G3) only. The animals in group G1 had a lower average number of eggsper gram of feces (EPG) at Day 42 (p < 0.05), and the average count was ten times lower than that inthe control at the last evaluated time-point (Day 56). The animals exhibited linear weight gain, andthose in group G1 gained the most weight. The combined use of drugs and plants containing bioactivecompounds can minimize the evolution of multidrug resistance, prolonging the efficacy of the drugs.The FAMACHA® score showed little variation during the experiment (p > 0.05), indicating the absenceof anemia and normal globular volume. This reinforces the importance of the measurement of EPG; thisis a quantitative method and does not depend on the subjective interpretation of the results.(AU)
Os nematódeos gastrointestinais são considerados um dos problemas sanitários mais importantes dosruminantes no mundo, seja em infecções isoladas ou atuando com outros patógenos, são responsáveis porseveras perdas econômicas relacionadas à produção animal. O controle dos endoparasitas em pequenosruminantes normalmente é feito por meio de drogas anti-helmínticas. No entanto, determinadas cepasde endoparasitos são resistentes a esses fármacos. A suplementação da dieta com alimentos ricos emproteína é uma estratégia para reduzir o impacto negativo das parasitoses gastrointestinais. Cratylia argentea tem efeito anti-helmíntico, além de outras características que favorecem seu uso comosuplemento para alimentação animal. No presente estudo, borregas cruzadas foram subdivididas emgrupos homogêneos e alimentadas com C. argentea (G1), C. argentea mais Brachiaria spp. (G2) ouapenas Brachiaria spp. (G3). Os animais do grupo G1 apresentaram menor valor médio de ovos porgrama de fezes (OPG) aos 42 dias (p<0,05) e sua contagem média foi dez vezes mais baixa que a docontrole no último período de tempo avaliado (56 dias). Os animais apresentaram aumento linear doganho de peso e aqueles do grupo G1 apresentaram o maior ganho. O escore FAMACHA® mostroupouca variação durante o experimento (p>0,05), indicando ausência de anemia e volume globularnormal. Isso reforça a importância da avaliação por OPG, que é um método quantitativo e não dependede interpretação subjetiva de resultados.(AU)
Assuntos
Animais , Ovinos/parasitologia , Ração Animal , Suplementos Nutricionais , Infecções por Nematoides/dietoterapia , Dietoterapia/veterinária , Helmintíase Animal/dietoterapiaRESUMO
The use of natural products having therapeutic properties can be considered an alternative approach for the control of helminths in ruminants, reducing the indiscriminate use of chemicals as well as production costs. The anthelmintic effectiveness of Cratylia argentea on sheep endoparasites was evaluated both in vitro by assessing the number of third-stage larvae recovered per gram of feces (LPG) from coprocultures treated with pods/ground seeds, macerated leaves or vermiculite (control), and in vivo using the Fecal Egg Count Reduction Test (FECRT) in two groups of lambs (n = 8/group) that received 1 mL kg-1 body weight of C. argentea solution (treated group) or distilled water (control group) orally. When compared to control group, there was a significant reduction in the percentage of LPG in the leaves group (55.88%; p ? 0.05) and pods/ground seeds group (82.48%; p ? 0.01). There was no significant reduction (p > 0.05) in the average count of EPG upon treatment with the aqueous solution of C. argentea. These findings suggest that C. argentea extract mainly obtained from pods/ground seeds has a promising potential for the control of young stages of gastrointestinal nematodes. However, further studies are needed to validate the use of C. argentea as an alternative method for the control of parasitic diseases of ruminants.(AU)
O uso de produtos naturais com propriedades terapêuticas pode ser considerado uma abordagem alternativa no controle de helmintos em ruminantes, reduzindo o uso indiscriminado de produtos químicos, bem como os custos de produção. A eficácia anti-helmíntica de Cratylia argentea sobre endoparasitos de ovinos foi avaliada tanto in vitro, mediante teste de recuperação de larvas infectantes por grama de fezes (LPG) a partir de coproculturas tratadas com vagens/sementes trituradas, folhas maceradas e vermiculita (controle), como in vivo, por meio de Teste de Redução da Contagem de Ovos por Grama de Fezes (TRCOF) em dois grupos de borregas (n = 8/grupo) que receberem por via oral 1 mL kg-1 PV de solução de C. argentea (grupo tratado) ou água destilada (grupo controle). Quando comparado ao grupo controle, houve redução estatisticamente significativa na porcentagem de LPG a partir do grupo tratado com folhas (55,88%; p ? 0,05) e do grupo tratado com vagens/sementes (82,48%; p ? 0,01). Não houve redução significativa (p > 0,05) na contagem média de OPG após o tratamento com a solução aquosa de C. argentea. Essas descobertas sugerem que o extrato de C. argentea obtido principalmente a partir de vagens/sementes tem potencial promissor para o controle de estágios jovens de nematódeos gastrointestinais. Contudo, são necessários novos estudos, a fim de validar o uso desta planta como um método alternativo no controle das parasitoses de ruminantes.(AU)
Assuntos
Animais , Ovinos/parasitologia , Anti-Helmínticos/análise , Fabaceae/parasitologia , Gastroenteropatias/parasitologia , Gastroenteropatias/veterináriaRESUMO
Cryptococcus gattii is one of the main causative agents of cryptococcosis in immunocompetent individuals. Treatment of the infection is based on the use of antimycotics, however, the toxicity of these drugs and the increase of drug-resistant strains have driven the search for more effective and less toxic therapies for cryptococcosis. pCramoll are isolectins purified from seeds of Cratylia mollis, a native forage plant from Brazil, which has become a versatile tool for biomedical application. We evaluated the effect of pCramoll alone and in combination with fluconazole for the treatment of mice infected with C. gatti. pCramoll alone or in combination with fluconazole increased the survival, reduced the morbidity and improved mice behavior i.e., neuropsychiatric state, motor behavior, autonomic function, muscle tone and strength and reflex/sensory function. These results were associated with (i) decreased pulmonary and cerebral fungal burden and (ii) increased inflammatory infiltrate and modulatory of IFNγ, IL-6, IL-10, and IL-17A cytokines in mice treated with pCramoll. Indeed, bone marrow-derived macrophages pulsed with pCramoll had increased ability to engulf C. gattii, with an enhanced production of reactive oxygen species and decrease of intracellular fungal proliferation. These findings point toward the use of pCramoll in combination with fluconazole as a viable, alternative therapy for cryptococcosis management.
Assuntos
Criptococose/tratamento farmacológico , Cryptococcus gattii/efeitos dos fármacos , Cryptococcus gattii/patogenicidade , Combinação de Medicamentos , Fabaceae/química , Fluconazol/uso terapêutico , Lectinas/uso terapêutico , Extratos Vegetais/uso terapêutico , Acetilglucosaminidase/metabolismo , Animais , Encéfalo/microbiologia , Encéfalo/patologia , Brasil , Proliferação de Células , Criptococose/fisiopatologia , Citocinas/metabolismo , Modelos Animais de Doenças , Fluconazol/farmacologia , Imunomodulação , Interferon gama/metabolismo , Interleucina-10/metabolismo , Interleucina-17/metabolismo , Interleucina-6/metabolismo , Lectinas/farmacologia , Pulmão/microbiologia , Pulmão/patologia , Macrófagos/microbiologia , Camundongos , Camundongos Endogâmicos C57BL , Peroxidase/metabolismo , Fagocitose , Extratos Vegetais/farmacologia , Espécies Reativas de Oxigênio , Sementes/química , Taxa de SobrevidaRESUMO
The use of natural products having therapeutic properties can be considered an alternative approach for the control of helminths in ruminants, reducing the indiscriminate use of chemicals as well as production costs. The anthelmintic effectiveness of Cratylia argentea on sheep endoparasites was evaluated both in vitro by assessing the number of third-stage larvae recovered per gram of feces (LPG) from coprocultures treated with pods/ground seeds, macerated leaves or vermiculite (control), and in vivo using the Fecal Egg Count Reduction Test (FECRT) in two groups of lambs (n = 8/group) that received 1 mL kg-1 body weight of C. argentea solution (treated group) or distilled water (control group) orally. When compared to control group, there was a significant reduction in the percentage of LPG in the leaves group (55.88%; p ? 0.05) and pods/ground seeds group (82.48%; p ? 0.01). There was no significant reduction (p > 0.05) in the average count of EPG upon treatment with the aqueous solution of C. argentea. These findings suggest that C. argentea extract mainly obtained from pods/ground seeds has a promising potential for the control of young stages of gastrointestinal nematodes. However, further studies are needed to validate the use of C. argentea as an alternative method for the control of parasitic diseases of ruminants.
O uso de produtos naturais com propriedades terapêuticas pode ser considerado uma abordagem alternativa no controle de helmintos em ruminantes, reduzindo o uso indiscriminado de produtos químicos, bem como os custos de produção. A eficácia anti-helmíntica de Cratylia argentea sobre endoparasitos de ovinos foi avaliada tanto in vitro, mediante teste de recuperação de larvas infectantes por grama de fezes (LPG) a partir de coproculturas tratadas com vagens/sementes trituradas, folhas maceradas e vermiculita (controle), como in vivo, por meio de Teste de Redução da Contagem de Ovos por Grama de Fezes (TRCOF) em dois grupos de borregas (n = 8/grupo) que receberem por via oral 1 mL kg-1 PV de solução de C. argentea (grupo tratado) ou água destilada (grupo controle). Quando comparado ao grupo controle, houve redução estatisticamente significativa na porcentagem de LPG a partir do grupo tratado com folhas (55,88%; p ? 0,05) e do grupo tratado com vagens/sementes (82,48%; p ? 0,01). Não houve redução significativa (p > 0,05) na contagem média de OPG após o tratamento com a solução aquosa de C. argentea. Essas descobertas sugerem que o extrato de C. argentea obtido principalmente a partir de vagens/sementes tem potencial promissor para o controle de estágios jovens de nematódeos gastrointestinais. Contudo, são necessários novos estudos, a fim de validar o uso desta planta como um método alternativo no controle das parasitoses de ruminantes.
Assuntos
Animais , Anti-Helmínticos/análise , Fabaceae/parasitologia , Gastroenteropatias/parasitologia , Gastroenteropatias/veterinária , Ovinos/parasitologiaRESUMO
The research for new biomarkers of cancer has studied the role of fetuin glycoprotein on the metastatic disease diagnosis. Cratylia mollis is a lectin with high finity to fetuin, and used here to differentiate prostate cancer and benign prostatic hyperplasia. A label-free electrochemical nanosensor based on assembled carboxylated carbon nanotubes (COOH-CNTs) and poly-L-lysine (PLL) film was developed and applied to serum samples of prostate cancer positive for Gleason score. The electrode analytical response to fetuin in PBS samples, obtained by square wave voltammetry, exhibited a linear range from 0.5 to 25µgmL(-1), with a high correlation coefficient (r=0.994, p<0.001) and low limit of detection (0.017µgmL(-1)). The lectin nanoelectrode showed a good repeatability (1.24% RSD) and reproducibility (4.24% RSD). A pool of serum samples from prostate cancer patients with known the Gleason score were tested showing a significant statistically correlation. Thus, the lectin nanoelectrode was able to distinguish the degree of staging prostate cancer, providing the diagnostic differentiation of benign and malign hyperplasia. To the best of our knowledge, it is the first biosensor for this application using a lectin.
Assuntos
Técnicas Eletroquímicas/métodos , Fabaceae/química , Fetuínas/análise , Nanotubos de Carbono/química , Lectinas de Plantas/química , Hiperplasia Prostática/diagnóstico , Neoplasias da Próstata/diagnóstico , Técnicas Biossensoriais/métodos , Diagnóstico Diferencial , Eletrodos , Humanos , Proteínas Imobilizadas/química , Limite de Detecção , Masculino , Polilisina/química , Próstata/patologia , Hiperplasia Prostática/sangue , Neoplasias da Próstata/sangue , Reprodutibilidade dos TestesRESUMO
Lectins have been described as glycoproteins that reversibly and specifically bind to carbohydrates. Legume lectins isolated from the subtribe Diocleinae (Canavalia, Dioclea andCratylia) are structurally homologous with respect to their primary structures. The Diocleinae lectins of Canavalia brasiliensis, Dioclea guianensis andCanavalia ensiformis have been shown to distinctly alter physiological parameters in isolated rat kidneys. Thus, the aim of this study was to investigate the effect of Cratylia floribunda lectin (CFL) on renal hemodynamics and ion transport in rats. In isolated perfused kidneys, CFL (10 mg/mL, n=5) increased RPP, RVR and decreased %TK+, but did not change urinary flow, glomerular filtration rate, sodium or chloride tubular transport. In isolated perfused mesenteric bed, CFL (3 and 10 mg/mL/min; n=4) did not alter tissue basal tonus or tissue contraction by phenylephrine (1 mM/mL/min). In conclusion, the seed lectin of Cratylia floribunda increased renal hemodynamic parameters showing a kaliuretic effect. This effect could be of tubular origin, rather than a result from haemodynamic alterations.
As lectinas são descritas como (glico)proteínas que se ligam, especificamente e reversivelmente, a carboidratos. Lectinas de leguminosas isoladas da subtribo Diocleinae (Canavalia, Dioclea eCratylia) são estruturalmente homólogas em relação às suas estruturas primárias. Demonstrou-se que as lectinas de DiocleinaeCanavalia brasiliensis, Dioclea guianensis eCanavalia ensiformis alteram diferentemente parâmetros fisiológicos em rins isolados de ratos. Dessa maneira, o objetivo deste estudo foi investigar o papel da lectina de Cratylia floribunda (CFL) na hemodinâmica renal e no transporte de íons em ratos. Em rins isolados perfundidos, CFL (10 mg/mL, n=5) aumentou a pressão de perfusão renal, a resistência vascular renal e reduziu o percentual do transporte tubular de K+, mas não alterou o fluxo urinário, a taxa de filtração glomerular e o percentual de transporte tubular dos íons sódio e cloreto. No leito mesentérico isolado perfundido, CFL (3 e 10 mg/mL/min, n=4) não alterou o tônus basal ou a contração do tecido induzida por fenilefrina (1 mM/mL/min). Em conclusão, a lectina de sementes de Cratylia floribunda altera parâmetros hemodinâmicos renais, provavelmente de origem tubular, e não por alterações hemodinâmicas.
Assuntos
Ratos , Transporte de Íons , Lectinas de Plantas/análise , Dioclea , Hemodinâmica , Amilorida/análiseRESUMO
PREMISE OF THE STUDY: This work aimed to develop microsatellite markers for Cratylia mollis as tools to assess its genetic diversity and structure and to evaluate their potential cross-amplification in related species. ⢠METHODS AND RESULTS: Microsatellite markers were developed using a microsatellite-enriched library and an intersimple sequence repeat library. From a set of 19 markers, 12 microsatellite loci were polymorphic and presented considerable variation in allele number (2-11), expected heterozygosity (0.226-0.883), and polymorphism information content per locus (0.212-0.870). Cross-amplification in C. argentea was successful in 16 loci, 12 of which were polymorphic (2-10 alleles). ⢠CONCLUSIONS: The polymorphism of this set of microsatellite markers for C. mollis, as well as their successful cross-amplification in C. intermedia and C. bahiensis and their transferability to C. argentea, supports their use in future comparative studies to understand the mechanism involved in population divergence and speciation in the genus.
RESUMO
The aim of this study was to characterize rhizobial isolates from Cratylia mollis Mart. ex Benth, Calliandra depauperata Benth. and Mimosa tenuiflora (Willd.) Poir. by means of rhizobial colonies morphology and restriction analysis of the 16S ribosomal gene (16S rDNA-ARDRA). Nodules were collected in the field and from plants cultivated in a greenhouse experiment using Caatinga soil samples. Sixty seven isolates were described by morphological analysis. Forty seven representative isolates were used for ARDRA analysis using seven restriction enzymes. We observed high diversity of both slow and fast-growing rhizobia that formed three morpho-physiological clusters. A few fast-growing isolates formed a group of strains of the Bradyrhizobium type; however, most of them diverged from the B. japonicum and B. elkanii species. Cratylia mollis nodule isolates were the most diverse, while all Mimosa tenuiflora isolates displayed fast growth with no pH change and were clustered into groups bearing 100 percent similarity, according to ARDRA results.
Assuntos
Ativação Enzimática/genética , Técnicas In Vitro , Rhizobiaceae/citologia , Rhizobiaceae/genética , Enzimas Reparadoras do DNA/genética , Enzimas Reparadoras do DNA/isolamento & purificação , Genes de Plantas , Variação Genética , Reação em Cadeia da Polimerase , Rhizobiaceae/crescimento & desenvolvimentoRESUMO
The aim of this study was to characterize rhizobial isolates from Cratylia mollis Mart. ex Benth, Calliandra depauperata Benth. and Mimosa tenuiflora (Willd.) Poir. by means of rhizobial colonies morphology and restriction analysis of the 16S ribosomal gene (16S rDNA-ARDRA). Nodules were collected in the field and from plants cultivated in a greenhouse experiment using Caatinga soil samples. Sixty seven isolates were described by morphological analysis. Forty seven representative isolates were used for ARDRA analysis using seven restriction enzymes. We observed high diversity of both slow and fast-growing rhizobia that formed three morpho-physiological clusters. A few fast-growing isolates formed a group of strains of the Bradyrhizobium type; however, most of them diverged from the B. japonicum and B. elkanii species. Cratylia mollis nodule isolates were the most diverse, while all Mimosa tenuiflora isolates displayed fast growth with no pH change and were clustered into groups bearing 100% similarity, according to ARDRA results.
RESUMO
The aim of this study was to characterize rhizobial isolates from Cratylia mollis Mart. ex Benth, Calliandra depauperata Benth. and Mimosa tenuiflora (Willd.) Poir. by means of rhizobial colonies morphology and restriction analysis of the 16S ribosomal gene (16S rDNA-ARDRA). Nodules were collected in the field and from plants cultivated in a greenhouse experiment using Caatinga soil samples. Sixty seven isolates were described by morphological analysis. Forty seven representative isolates were used for ARDRA analysis using seven restriction enzymes. We observed high diversity of both slow and fast-growing rhizobia that formed three morpho-physiological clusters. A few fast-growing isolates formed a group of strains of the Bradyrhizobium type; however, most of them diverged from the B. japonicum and B. elkanii species. Cratylia mollis nodule isolates were the most diverse, while all Mimosa tenuiflora isolates displayed fast growth with no pH change and were clustered into groups bearing 100% similarity, according to ARDRA results.