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1.
Molecules ; 26(4)2021 Feb 09.
Artigo em Inglês | MEDLINE | ID: mdl-33572445

RESUMO

Anomalocalyx uleanus (Pax & K. Hoffm.) Ducke (Euphorbiaceae) is a singular species in the genus and is restricted and exclusive to the Brazilian Amazon. A phytochemical study of A. uleanus leaves was performed, yielding the isolation of five major compounds: catechin/epicatechin, afzelin, quercetin 3-O-α-L-rhamnopyranoside, and astilbin. The phytochemical compositions of the methanolic extracts of leaves, roots, bark, and stem bark were determined using a dereplication approach. Forty-six compounds were annotated from the liquid chromatography-mass spectrometry (LC-MS/MS) data, while four lipids were identified using gas chromatography-mass spectrometry (GC-MS). In total, fifty compounds were detected, and they belonged to the primary metabolism and several classes of natural products such as flavonoids, flavonoids O-glycosides, flavonoids C-glycosides, biflavonoids, procyanidin, triterpene, triterpenes esterified with phenylpropanoids, phenylpropanoid derivatives, flavonolignans, coumarins, quinic acid derivatives, and benzoic acid derivatives. This is the first report on the phytochemical data of the genus Anomalocalyx, and the results of this study will contribute to the chemosystematic knowledge of the Euphorbiaceae family and justify the need for investigation of the pharmacological potential of the species A. uleanus.


Assuntos
Euphorbiaceae/química , Euphorbiaceae/metabolismo , Compostos Fitoquímicos/análise , Compostos Fitoquímicos/metabolismo , Extratos Vegetais/análise , Extratos Vegetais/metabolismo , Cromatografia Líquida de Alta Pressão/métodos , Flavonoides/análise , Flavonoides/isolamento & purificação , Flavonoides/metabolismo , Frutas/química , Frutas/metabolismo , Cromatografia Gasosa-Espectrometria de Massas/métodos , Compostos Fitoquímicos/isolamento & purificação , Extratos Vegetais/isolamento & purificação , Folhas de Planta/química , Folhas de Planta/metabolismo , Raízes de Plantas/química , Raízes de Plantas/metabolismo , Espectrometria de Massas em Tandem/métodos
2.
Sci Rep ; 9(1): 7276, 2019 05 13.
Artigo em Inglês | MEDLINE | ID: mdl-31086208

RESUMO

Alzheimer's disease (AD) is a multifactorial pathology characterized by amyloid deposits, neurofibrillary formation, oxidative stress and cholinergic system dysfunction. In this sense, here we report the rational design of a multi-target directed ligand (MTDL) for AD based on virtual screening and bioinformatic analyses, exploring the molecular targets ß-secretase (BACE-1), glycogen synthase kinase-3ß (GSK-3ß) and acetylcholinesterase (AChE). After this screening, the compound with higher molecular docking affinity was selected, the 1-(7-chloroquinolin-4-yl)-N-(4-methoxybenzyl)-5-methyl-1H-1,2,3-triazole-4 carboxamide(QTC-4-MeOBnE). To further our studies, the protective effect of QTC-4-MeOBnE (0.1 and 1 mg/kg for 20 days) on STZ-induced sporadic AD mice was determined. QTC-4-MeOBnE pretreatment attenuated cognitive and memory deficit induced by STZ in an object recognition test, Y-maze, social recognition test and step-down passive avoidance. The mechanisms underlying this action might be attributed to the reduction of lipid peroxidation and reactive species formation in the prefrontal cortex and hippocampus of mice submitted to STZ. In addition, QTC-4-MeOBnE pretreatment abolished the up-regulation of AChE activity and the overexpression of GSK 3ß and genes involved in amyloid cascade such as BACE-1, protein precursor amyloid, у-secretase, induced by STZ. Moreover, toxicological parameters were not modified by QTC-4-MeOBnE chronic treatment. This evidence suggests that QTC-4-MeOBnE exerts its therapeutic effect through multiple pathways involved in AD.


Assuntos
Doença de Alzheimer/tratamento farmacológico , Cognição/efeitos dos fármacos , Fármacos Neuroprotetores/uso terapêutico , Quinolinas/uso terapêutico , Triazóis/uso terapêutico , Doença de Alzheimer/induzido quimicamente , Doença de Alzheimer/patologia , Animais , Modelos Animais de Doenças , Masculino , Aprendizagem em Labirinto/efeitos dos fármacos , Transtornos da Memória/induzido quimicamente , Transtornos da Memória/tratamento farmacológico , Camundongos , Simulação de Acoplamento Molecular , Estreptozocina
3.
Int J Biol Macromol ; 119: 811-820, 2018 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-30071232

RESUMO

Lectins are proteins of non-immune origin present throughout all kingdoms of life. They are capable of binding to specific carbohydrates reversibly, thus performing several biological roles. Plant lectins are the most studied ones, with hundreds of isolated and characterized hemagglutinins. Most of the known lectins have been isolated from plants belonging to family Leguminosae, which includes genus Bauhinia. This genus comprises over 300 species located in the tropical zones of the planet, where these are utilized in folk medicine because of their numerous medicinal effects, such as anti-inflammatory and antidiabetic actions. Despite being studied for over fifty years, the literature regarding Bauhinia hemagglutinins is scarce, describing just ten proteins isolated from seven different species. Structurally as well as biophysically, there is great similarity among all the known Bauhinia lectins, which may classify them as chemotaxonomic markers; however, the carbohydrate-binding sites and further specificities are unique for each of these proteins. The activities identified for these lectins include growth inhibition in cancer cell lines, cell marking, anti-inflammatory and insecticidal effects, which are just a few among their various other activities of high economic importance. Besides their versatility, four recombinant Bauhinia lectins have already been successfully expressed in heterologous microbial systems, further suggesting that these proteins could serve as promising biotechnological products in future.


Assuntos
Bauhinia/química , Lectinas de Plantas/química , Lectinas de Plantas/farmacologia , Biotecnologia , Fenômenos Químicos , Lectinas de Plantas/isolamento & purificação , Proteínas Recombinantes , Relação Estrutura-Atividade
4.
Mem. Inst. Oswaldo Cruz ; 113(2): 80-86, Feb. 2018. tab, graf
Artigo em Inglês | LILACS | ID: biblio-894891

RESUMO

BACKGROUND Leptospirosis is the most widespread zoonotic disease. It is caused by infection with pathogenic Leptospira species, of which over 300 serovars have been described. The accurate identification of the causative Leptospira spp. is required to ascertain the pathogenic status of the local isolates. OBJECTIVES This study aimed to obtain the complete genome sequence of a virulent Leptospira interrogans strain isolated from southern Brazil and to describe its genetic features. METHODS The whole genome was sequenced by next-generation sequencing (Ion Torrent). The genome was assembled, scaffolded, annotated, and manually reviewed. Mutations were identified based on a variant calling analysis using the genome of L. interrogans strain Fiocruz L1-130 as a reference. FINDINGS The entire genome had an average GC content of 35%. The variant calling analysis identified 119 single nucleotide polymorphisms (SNPs), from which 30 led to a missense mutation. The structural analyses identified potential evidence of genomic inversions, translocations, and deletions in both the chromosomes. MAIN CONCLUSIONS The genome properties provide comprehensive information about the local isolates of Leptospira spp., and thereby, could facilitate the identification of new targets for the development of diagnostic kits and vaccines.


Assuntos
Filogenia , Microbiologia da Água , Leptospira interrogans/isolamento & purificação , Leptospira interrogans/genética , Virulência , Dados de Sequência Molecular , Genoma Bacteriano
5.
Mem. Inst. Oswaldo Cruz ; 113(2): 137-141, Feb. 2018. tab, graf
Artigo em Inglês | LILACS | ID: biblio-894894

RESUMO

A previous study by our group reported the isolation and characterisation of Leptospira borgpetersenii serogroup Ballum strain 4E. This strain is of particular interest because it is highly virulent in the hamster model. In this study, we performed whole-genome shotgun genome sequencing of the strain using the SOLiD sequencing platform. By assembling and analysing the new genome, we were able to identify novel features that have been previously overlooked in genome annotations of other strains belonging to the same species.


Assuntos
Animais , Cobaias , Camundongos , Leptospira/classificação , Leptospira/genética , Leptospira/patogenicidade , Virulência
6.
Mem Inst Oswaldo Cruz ; 113(2): 80-86, 2018 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-29236923

RESUMO

BACKGROUND Leptospirosis is the most widespread zoonotic disease. It is caused by infection with pathogenic Leptospira species, of which over 300 serovars have been described. The accurate identification of the causative Leptospira spp. is required to ascertain the pathogenic status of the local isolates. OBJECTIVES This study aimed to obtain the complete genome sequence of a virulent Leptospira interrogans strain isolated from southern Brazil and to describe its genetic features. METHODS The whole genome was sequenced by next-generation sequencing (Ion Torrent). The genome was assembled, scaffolded, annotated, and manually reviewed. Mutations were identified based on a variant calling analysis using the genome of L. interrogans strain Fiocruz L1-130 as a reference. FINDINGS The entire genome had an average GC content of 35%. The variant calling analysis identified 119 single nucleotide polymorphisms (SNPs), from which 30 led to a missense mutation. The structural analyses identified potential evidence of genomic inversions, translocations, and deletions in both the chromosomes. MAIN CONCLUSIONS The genome properties provide comprehensive information about the local isolates of Leptospira spp., and thereby, could facilitate the identification of new targets for the development of diagnostic kits and vaccines.


Assuntos
Genoma Bacteriano , Leptospira interrogans/genética , Virulência/genética , Microbiologia da Água , Brasil , Leptospira interrogans/isolamento & purificação , Dados de Sequência Molecular , Filogenia , Polimorfismo Genético
7.
Mem Inst Oswaldo Cruz ; 113(2): 137-141, 2018 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-29236926

RESUMO

A previous study by our group reported the isolation and characterisation of Leptospira borgpetersenii serogroup Ballum strain 4E. This strain is of particular interest because it is highly virulent in the hamster model. In this study, we performed whole-genome shotgun genome sequencing of the strain using the SOLiD sequencing platform. By assembling and analysing the new genome, we were able to identify novel features that have been previously overlooked in genome annotations of other strains belonging to the same species.


Assuntos
Leptospira/genética , Leptospira/patogenicidade , Virulência/genética , Animais , Leptospira/classificação , Camundongos
8.
Genet Mol Biol ; 40(3): 553-576, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28898352

RESUMO

The introduction of next-generation sequencing (NGS) had a significant effect on the availability of genomic information, leading to an increase in the number of sequenced genomes from a large spectrum of organisms. Unfortunately, due to the limitations implied by the short-read sequencing platforms, most of these newly sequenced genomes remained as "drafts", incomplete representations of the whole genetic content. The previous genome sequencing studies indicated that finishing a genome sequenced by NGS, even bacteria, may require additional sequencing to fill the gaps, making the entire process very expensive. As such, several in silico approaches have been developed to optimize the genome assemblies and facilitate the finishing process. The present review aims to explore some free (open source, in many cases) tools that are available to facilitate genome finishing.

9.
Genet. mol. biol ; 40(3): 553-576, July-Sept. 2017. tab, graf
Artigo em Inglês | LILACS | ID: biblio-892419

RESUMO

Abstract The introduction of next-generation sequencing (NGS) had a significant effect on the availability of genomic information, leading to an increase in the number of sequenced genomes from a large spectrum of organisms. Unfortunately, due to the limitations implied by the short-read sequencing platforms, most of these newly sequenced genomes remained as "drafts", incomplete representations of the whole genetic content. The previous genome sequencing studies indicated that finishing a genome sequenced by NGS, even bacteria, may require additional sequencing to fill the gaps, making the entire process very expensive. As such, several in silico approaches have been developed to optimize the genome assemblies and facilitate the finishing process. The present review aims to explore some free (open source, in many cases) tools that are available to facilitate genome finishing.

10.
Front Immunol ; 8: 463, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28496441

RESUMO

Leptospira spp. are diderm (two membranes) bacteria that infect mammals causing leptospirosis, a public health problem with global implications. Thousands of people die every year due to leptospirosis, especially in developing countries with tropical climates. Prophylaxis is difficult due to multiple factors, including the large number of asymptomatic hosts that transmit the bacteria, poor sanitation, increasing numbers of slum dwellers, and the lack of an effective vaccine. Several leptospiral recombinant antigens were evaluated as a replacement for the inactivated (bacterin) vaccine; however, success has been limited. A prospective vaccine candidate is likely to be a surface-related protein that can stimulate the host immune response to clear leptospires from blood and organs. In this study, a comprehensive bioinformatics approach based on reverse and structural vaccinology was applied toward the discovery of novel leptospiral vaccine candidates. The Leptospira interrogans serovar Copenhageni strain L1-130 genome was mined in silico for the enhanced identification of conserved ß-barrel (ßb) transmembrane proteins and outer membrane (OM) lipoproteins. Orthologs of the prospective vaccine candidates were screened in the genomes of 20 additional Leptospira spp. Three-dimensional structural models, with a high degree of confidence, were created for each of the surface-exposed proteins. Major histocompatibility complex II (MHC-II) epitopes were identified, and their locations were mapped on the structural models. A total of 18 ßb transmembrane proteins and 8 OM lipoproteins were identified. These proteins were conserved among the pathogenic Leptospira spp. and were predicted to have epitopes for several variants of MHC-II receptors. A structural and functional analysis of the sequence of these surface proteins demonstrated that most ßb transmembrane proteins seem to be TonB-dependent receptors associated with transportation. Other proteins identified included, e.g., TolC efflux pump proteins, a BamA-like OM component of the ßb transmembrane protein assembly machinery, and the LptD-like LPS assembly protein. The structural mapping of the immunodominant epitopes identified the location of conserved, surface-exposed, immunogenic regions for each vaccine candidate. The proteins identified in this study are currently being evaluated for experimental evidence for their involvement in virulence, disease pathogenesis, and physiology, in addition to vaccine development.

11.
Int J Biol Macromol ; 95: 1072-1081, 2017 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-27984144

RESUMO

Lectins are proteins able to interact specifically and reversibly with carbohydrates. They are present in all living beings, particularly in legume seeds, which have many biological functions. The aim of this study was to isolate, characterize and verify antioxidant, anti-hemolytic, antitumor and gastroprotective activities in a lectin present in seeds of Phaseolus lunatus L. var. cascavel (PLUN). The isolation of lectin was performed by size exclusion chromatography on Sephadex G-100, which was isolated from a protein capable of agglutinating only human erythrocytes type A, being this the only inhibited haemagglutination n-acetyl-d-galactosamine. Its weight was estimated by PAGE is 128kDa. The lectin is thermostable up to 80°C and is active between pH 2-11. As 8M urea was able to denature the lectin. PLUN is a glycoprotein consisting of 2% carbohydrate and has antioxidant action with ascorbic acid equivalent antioxidant capacity (µMAA/g) of 418.20, 326 and 82.9 for total antioxidant activity, ABTS radical capture and capture of DPPH radical, respectively. The lectin has antitumor activity against melanoma derived cells at doses of 100 and 50mg/ml, reducing up to 83% tumor cells, and gastroprotective action, reducing up to 63% damaged area of ​​the stomach induced by ethanol.


Assuntos
Antineoplásicos Fitogênicos/farmacologia , Antioxidantes/farmacologia , Fármacos Gastrointestinais/farmacologia , Phaseolus/química , Lectinas de Plantas/farmacologia , Úlcera Gástrica/tratamento farmacológico , Acetilgalactosamina/química , Animais , Antineoplásicos Fitogênicos/química , Antineoplásicos Fitogênicos/isolamento & purificação , Antioxidantes/química , Antioxidantes/isolamento & purificação , Benzotiazóis/antagonistas & inibidores , Benzotiazóis/química , Compostos de Bifenilo/antagonistas & inibidores , Compostos de Bifenilo/química , Linhagem Celular Tumoral , Eritrócitos/efeitos dos fármacos , Etanol , Fármacos Gastrointestinais/química , Fármacos Gastrointestinais/isolamento & purificação , Testes de Hemaglutinação , Humanos , Masculino , Camundongos , Peso Molecular , Picratos/antagonistas & inibidores , Picratos/química , Lectinas de Plantas/química , Lectinas de Plantas/isolamento & purificação , Desnaturação Proteica , Sementes/química , Extração em Fase Sólida/métodos , Estômago/efeitos dos fármacos , Estômago/patologia , Úlcera Gástrica/induzido quimicamente , Úlcera Gástrica/patologia , Ácidos Sulfônicos/antagonistas & inibidores , Ácidos Sulfônicos/química , Ureia/química
12.
FEMS Microbiol Lett ; 363(23)2016 12.
Artigo em Inglês | MEDLINE | ID: mdl-27856568

RESUMO

Next-generation sequencing has significantly reduced the cost of genome-sequencing projects, resulting in an expressive increase in the availability of genomic data in public databases. The cheaper and easier is to sequence new genomes, the more accurate the annotation steps have to be to avoid both the loss of information and the accumulation of erroneous features that may affect the accuracy of further analysis. In the case of bacteria genomes, a range of web annotation software has been developed; however, many applications have yet to incorporate the steps required to improve their result, including the removal of false-positive/spurious and a more complete identification of non-coding features. We present Genix, a new web-based bacterial genome annotation pipeline. A comparison of the results generated by Genix for four reference genomes against those generated by other annotation tools indicated that our pipeline is able to provide results that are closer to the reference genome annotation, with a smaller amount of false-positive proteins and missing functional annotated proteins. Additionally, the metrics obtained by Genix were slightly better than those obtained by Prokka, a state-of-art standalone annotation system. Our results indicate that Genix is a useful tool that is able to provide a more refined result, and may be a user-friendly way to obtain high-quality results.


Assuntos
Escherichia coli K12/genética , Genoma Bacteriano/genética , Leptospira interrogans/genética , Listeria monocytogenes/genética , Anotação de Sequência Molecular/métodos , Mycobacterium tuberculosis/genética , Proteínas de Bactérias/genética , Bases de Dados Genéticas , Processamento Eletrônico de Dados/métodos , Perfilação da Expressão Gênica/métodos , Sequenciamento de Nucleotídeos em Larga Escala , Internet
13.
Rev Bras Parasitol Vet ; 24(2): 148-54, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26083692

RESUMO

Neosporosis is a disease caused by the protozoon Neospora caninum that leads to significant economic losses in many countries. In the present study, we report on use of the recombinant protein NcSRS2 of N. caninum expressed in Pichia pastoris in an indirect immunoenzymatic assay (ELISA) for diagnosing neosporosis infection in sheep and dogs. We observed that the ELISA test yielded specificity of 94.5% and sensitivity of 100% for sheep and specificity of 93.3% and sensitivity of 100% for dogs. We observed that the sensitivity was higher than shown by the indirect fluorescent antibody test, and this was confirmed by means of Western blot. The results from this study suggest that the recombinant protein expressed in P. pastoris is a suitable antigen for use in immunodiagnosis to detect N. caninum in two important species exposed to this parasitosis.


Assuntos
Antígenos de Protozoários/sangue , Antígenos de Superfície/sangue , Doenças do Cão/sangue , Doenças do Cão/parasitologia , Ensaio de Imunoadsorção Enzimática , Neospora , Infecções Protozoárias em Animais/sangue , Proteínas de Protozoários/sangue , Doenças dos Ovinos/sangue , Doenças dos Ovinos/parasitologia , Animais , Antígenos de Protozoários/biossíntese , Antígenos de Protozoários/imunologia , Antígenos de Superfície/biossíntese , Antígenos de Superfície/imunologia , Doenças do Cão/diagnóstico , Cães , Neospora/imunologia , Pichia/metabolismo , Infecções Protozoárias em Animais/diagnóstico , Proteínas de Protozoários/biossíntese , Proteínas de Protozoários/imunologia , Ovinos , Doenças dos Ovinos/diagnóstico
14.
Rev. bras. parasitol. vet ; 24(2): 148-154, Apr-Jun/2015. graf
Artigo em Inglês | LILACS | ID: lil-750757

RESUMO

Neosporosis is a disease caused by the protozoon Neospora caninum that leads to significant economic losses in many countries. In the present study, we report on use of the recombinant protein NcSRS2 of N. caninum expressed in Pichia pastoris in an indirect immunoenzymatic assay (ELISA) for diagnosing neosporosis infection in sheep and dogs. We observed that the ELISA test yielded specificity of 94.5% and sensitivity of 100% for sheep and specificity of 93.3% and sensitivity of 100% for dogs. We observed that the sensitivity was higher than shown by the indirect fluorescent antibody test, and this was confirmed by means of Western blot. The results from this study suggest that the recombinant protein expressed in P. pastoris is a suitable antigen for use in immunodiagnosis to detect N. caninum in two important species exposed to this parasitosis.


A neosporose é uma doença causada pelo protozoário Neospora caninum que leva a perdas econômicas importantes em muitos países. No presente estudo, é descrita a utilização da proteína recombinante NcSRS2 de N. caninum expressa em Pichia pastoris em um ensaio imunoenzimático indireto (ELISA) para o diagnóstico de infecção por Neospora em ovelhas e cães. Observou-se, que utilizando-se um ELISA, o teste produziu uma especificidade de 94,5% e uma sensibilidade de 100% para ovinos; e uma especificidade de 93,3% e sensibilidade de 100% para cães. Uma maior sensibilidade foi observada em relação à IFI que foi confirmada por Western blot. Os resultados deste estudo sugerem que a proteína recombinante expressa em P. pastoris é bom antígeno para ser utilizado no diagnóstico imunológico para detectar N. caninum em duas espécies importantes expostas a esta parasitose.


Assuntos
Animais , Cães , Infecções Protozoárias em Animais/sangue , Doenças dos Ovinos/sangue , Ensaio de Imunoadsorção Enzimática , Proteínas de Protozoários/sangue , Neospora/imunologia , Doenças do Cão/parasitologia , Doenças do Cão/sangue , Antígenos de Protozoários/sangue , Antígenos de Superfície/sangue , Pichia/metabolismo , Infecções Protozoárias em Animais/diagnóstico , Doenças dos Ovinos/diagnóstico , Doenças dos Ovinos/parasitologia , Ovinos , Proteínas de Protozoários/biossíntese , Proteínas de Protozoários/imunologia , Doenças do Cão/diagnóstico , Antígenos de Protozoários/biossíntese , Antígenos de Protozoários/imunologia , Antígenos de Superfície/biossíntese , Antígenos de Superfície/imunologia
15.
Ciênc. rural ; 44(10): 1830-1836, 10/2014. graf
Artigo em Português | LILACS | ID: lil-726288

RESUMO

A piroplasmose equina causada por Theileria equi acomete os equinos de forma endêmica no Brasil e em diversos outros países tropicais e subtropicais. Considerada uma das mais importantes doenças de equinos, causa danos à saúde animal e perdas econômicas. A proteína equi merozoite antigen (EMA-2) é uma das principais proteínas de superfície, expressa nos diversos estágios do ciclo do parasita, estimula resposta imune em animais infectados, tornando-se um possível candidato para utilização em diagnóstico. O gene EMA-2 foi clonado e expresso na levedura Pichia pastoris. A proteína EMA-2 recombinante (rEMA-2) foi caracterizada antigenicamente por Western Blot e por ELISA indireto, utilizando-se soro de equino positivo para theileriose. O resultado do ELISA demonstrou uma especificidade de 90,9% e sensibilidade de 83,3%, quando comparado ao padrão, sendo superior à imunofluorescência (80,6% de especificidade e 75,0% de sensibilidade), o que sugere que a rEMA-2 expressa em P. pastoris é um promissor antígeno para ser utilizado como ferramenta no imunodiagnóstico de theileriose equina.


Theileria equi, the causative agent of equine piroplasmosis, is endemic in Brazil and many other tropical and subtropical countries. It is considered one of the most important diseases of horses causing animal health problems and significant economic loss. The Protein equi merozoite antigen-2 (EMA-2) is a major surface protein that is expressed in different parasite cycle stages and induces immune response in infected animals, being a possible candidate to be used in diagnose. EMA-2 gene was cloned and expressed in the yeast Pichia pastoris, and the recombinant protein EMA-2 (rEMA-2) was characterized by Western Blot and indirect ELISA using equine positive sera. The ELISA results demonstrated a specificity of 90.9% and a sensitivity of 83.3% compared to the standard ELISA and being superior to immunofluorescence (80.6% of specificity and 75.0% of sensitivity) suggesting that the rEMA-2 expressed in P. pastoris is a promising antigen to be used as a tool in immunodiagnostic of theileriasis.

16.
Pathog Glob Health ; 107(3): 116-21, 2013 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-23683365

RESUMO

Neospora caninum is considerd a major cause of abortion in cattle worldwide. The antigenic domain of NcSRS2 in N. caninum is an important surface antigen present in the membrane of this parasite. In the present study, the Pichia pastoris expression system proved to be a useful tool for the production of recombinant protein. The truncated NcSRS2 gene (by removal of the N-terminal hydrophobic sequence), was cloned in the vector pPICZalphaB, and integrated on the genome of the methylotrophic yeast P. pastoris. Subsequently, the NcSRS2 protein was expressed, purified, and characterized using naturally infected cattle sera and Mab 6xhistag. The recombinant protein NcSRS2 was present in the supernatant of the culture, where later it was concentrated and purified using ammonium sulfate (∼100 mg/ml). An indirect immunoenzymatic assay (ELISA) was performed using cattle sera from endemic N. caninum area.


Assuntos
Antígenos de Protozoários/isolamento & purificação , Antígenos de Superfície/isolamento & purificação , Doenças dos Bovinos/diagnóstico , Coccidiose/veterinária , Proteínas de Protozoários/isolamento & purificação , Animais , Antígenos de Protozoários/genética , Antígenos de Superfície/genética , Biotecnologia/métodos , Bovinos , Fracionamento Químico , Clonagem Molecular , Coccidiose/diagnóstico , Expressão Gênica , Vetores Genéticos , Pichia/genética , Proteínas de Protozoários/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Testes Sorológicos/métodos , Medicina Veterinária/métodos
17.
Mycopathologia ; 175(1-2): 147-51, 2013 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-23161017

RESUMO

The search for new compounds with antifungal activity is accelerating due to rising yeast and fungal resistance to commonly prescribed drugs. Among the molecules being investigated, plant lectins can be highlighted. The present work shows the potential of six plant lectins which were tested in vitro against yeasts of medical importance, Candida albicans, Candida tropicalis, Candida parapsilosis, Cryptococcus gattii, Cryptococcus neoformans, Malassezia pachydermatis, Rhodotorula sp. and Trichosporon sp. Broth microdilution susceptibility testing was performed in accordance with standard protocols to evaluate antifungal activity. Minimum inhibitory concentration (MIC) was determined at 80% yeast growth inhibition, whereas the minimum fungicidal concentration (MFC) was evaluated after making the subcultures of each dilution. Only C. parapsilosis growth was inhibited by the lectins tested. Abelmoschus esculentus lectin showed the highest MIC (0.97 µg ml(-1)). Lectins from Canavalia brasiliensis, Mucuna pruriens and Clitoria fairchildiana presented the highest MFC at (3.90 µg ml(-1)). These results encourage further studies with wider yeast strain selections, and open new perspectives for the development of pharmacological molecules.


Assuntos
Antifúngicos/farmacologia , Fungos/efeitos dos fármacos , Lectinas/farmacologia , Plantas/química , Antifúngicos/isolamento & purificação , Lectinas/isolamento & purificação , Testes de Sensibilidade Microbiana , Viabilidade Microbiana/efeitos dos fármacos
18.
Rev Bras Parasitol Vet ; 21(3): 254-62, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-23070436

RESUMO

The southern cattle fever tick, Rhipicephalus (Boophilus) microplus, is no doubt the most economically important ectoparasite of cattle globally. The inappropriate use of chemical acaricides has driven the evolution of resistance in populations of R. (B.) microplus. Anti-tick vaccines represent a technology that can be combined with acaricides in integrated control programs to mitigate the impact of R. (B.) microplus. The recombinant form of Bm86 antigen from the Campo Grande (rBm86-CG) strain of R. (B.) microplus was produced using the Pichiapastoris expression system to test its ability to immunoprotect cattle against tick infestation. Secretion of rBm86-CG by P. pastoris through the bioprocess reported here simplified purification of the antigen. A specific humoral immune response was detected by ELISA in vaccinated cattle. Immunoblot results revealed that polyclonal antibodies from vaccinated cattle recognized a protein in larval extracts with a molecular weight corresponding to Bm86. The rBm86-CG antigen showed 31% efficacy against the Campo Grande strain of R. (B.) microplus infesting vaccinated cattle. The rBm86-CG is an antigen that could be used in a polyvalent vaccine as part of an integrated program for the control of R. (B.) microplus in the region that includes Mato Grosso do Sul.


Assuntos
Doenças dos Bovinos/prevenção & controle , Doenças dos Bovinos/parasitologia , Glicoproteínas de Membrana/imunologia , Glicoproteínas de Membrana/uso terapêutico , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/uso terapêutico , Rhipicephalus/imunologia , Infestações por Carrapato/veterinária , Vacinas/imunologia , Animais , Bovinos , Feminino , Masculino , Infestações por Carrapato/prevenção & controle , Vacinas/uso terapêutico
19.
Rev. bras. parasitol. vet ; 21(3): 254-262, July-Sept. 2012. ilus, tab
Artigo em Inglês | LILACS | ID: lil-653714

RESUMO

The southern cattle fever tick, Rhipicephalus (Boophilus) microplus, is no doubt the most economically important ectoparasite of cattle globally. The inappropriate use of chemical acaricides has driven the evolution of resistance in populations of R. (B.) microplus. Anti-tick vaccines represent a technology that can be combined with acaricides in integrated control programs to mitigate the impact of R. (B.) microplus. The recombinant form of Bm86 antigen from the Campo Grande (rBm86-CG) strain of R. (B.) microplus was produced using the Pichiapastoris expression system to test its ability to immunoprotect cattle against tick infestation. Secretion of rBm86-CG by P. pastoris through the bioprocess reported here simplified purification of the antigen. A specific humoral immune response was detected by ELISA in vaccinated cattle. Immunoblot results revealed that polyclonal antibodies from vaccinated cattle recognized a protein in larval extracts with a molecular weight corresponding to Bm86. The rBm86-CG antigen showed 31% efficacy against the Campo Grande strain of R. (B.) microplus infesting vaccinated cattle. The rBm86-CG is an antigen that could be used in a polyvalent vaccine as part of an integrated program for the control of R. (B.) microplus in the region that includes Mato Grosso do Sul.


O carrapato Rhipicephalus (Boophilus) microplus é, sem dúvidas, o ectoparasito economicamente mais importante para o gado a nível mundial. A utilização inadequada de acaricidas tem impulsionado a evolução da resistência em populações de R. (B.) microplus. Vacinas contra o carrapato representam uma tecnologia que pode ser combinada com acaricidas em programas de controle integrado para diminuir o impacto de R. (B.) microplus. A forma recombinante da Bm86 da cepa Campo Grande (rBm86-CG) de R. (B.) microplus foi produzido utilizando o sistema de expressão em Pichia pastoris para testar sua capacidade de imunoproteção ao gado contra a infestação de carrapatos. A secreção de rBm86-CG em P. pastoris pelo bioprocesso, simplificou a purificação do antígeno. A resposta imune humoral específica foi detectada por ELISA em soros de bovinos vacinados. Resultados de "imunoblot" revelaram que anticorpos policlonais de bovinos vacinados reconheceram uma proteína em extratos de larvas com um peso molecular correspondente à Bm86. O antígeno rBm86-CG mostrou eficácia de 31% contra a amostra CG de R. (B.) microplus utilizada para infestar os bovinos vacinados. Pelos resultados obtidos, concluímos que a rBm86-CG é um antígeno que pode ser usado em uma vacina polivalente, como parte de um programa integrado para o controle de R. (B.) microplus no estado do Mato Grosso do Sul, Brasil.


Assuntos
Animais , Bovinos , Feminino , Masculino , Doenças dos Bovinos/parasitologia , Doenças dos Bovinos/prevenção & controle , Glicoproteínas de Membrana/imunologia , Glicoproteínas de Membrana/uso terapêutico , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/uso terapêutico , Rhipicephalus/imunologia , Infestações por Carrapato/veterinária , Vacinas/imunologia , Infestações por Carrapato/prevenção & controle , Vacinas/uso terapêutico
20.
Molecules ; 16(11): 9298-315, 2011 Nov 07.
Artigo em Inglês | MEDLINE | ID: mdl-22064270

RESUMO

Lectins are a structurally heterogeneous group of highly specific carbohydrate-binding proteins. Due to their great biotechnological potential, lectins are widely used in biomedical research. The purpose of the present study was to evaluate the healing potential of the lectin of Bauhinia variegata (nBVL) and its recombinant isoform (rBVL-1). Following surgical creation of dorsal skin wounds, seven groups of mice were submitted to topical treatment for 12 days with lectin, D-galactose, BSA and saline. The animals were anesthetized and euthanized on POD 2, 7 and 12 in order to evaluate the healing potential of each treatment. The parameters considered included wound size, contraction rate, epithelialization rate and histopathological findings. Wound closure was fastest in animals treated with rBVL-1 (POD 7). nBVL was more effective than the controls. All skin layers were reconstructed and keratin deposition increased. Our findings indicate that the lectin of Bauhinia variegata possesses pro-healing properties and may be employed in the treatment of acute skin wounds.


Assuntos
Bauhinia/química , Lectinas de Plantas/farmacologia , Isoformas de Proteínas/farmacologia , Proteínas Recombinantes/farmacologia , Pele , Cicatrização/efeitos dos fármacos , Animais , Procedimentos Cirúrgicos Dermatológicos , Masculino , Camundongos , Lectinas de Plantas/genética , Isoformas de Proteínas/genética , Distribuição Aleatória , Proteínas Recombinantes/genética , Pele/efeitos dos fármacos , Pele/patologia
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