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1.
Braz Oral Res ; 33: e043, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31508727

RESUMO

Proteomic techniques have become popular in medicine and dentistry because of their widespread use in analyzing bodily fluids such as blood, saliva, urine, and gingival crevicular fluids as well as hard tissues such as enamel, dentine, and cementum. This review is a guide to proteomic techniques in general dentistry, summarizing techniques and their clinical application in understanding and diagnosing diseases and their use in identifying biomarkers of various diseases.


Assuntos
Proteoma , Proteômica/métodos , Saliva/química , Proteínas e Peptídeos Salivares/química , Biomarcadores/química , Eletroforese em Gel de Poliacrilamida/métodos , Humanos , Espectrometria de Massas/métodos , Neoplasias Bucais/diagnóstico , Síndrome de Sjogren/diagnóstico
2.
Rev Soc Bras Med Trop ; 52: e20180526, 2019 Sep 05.
Artigo em Inglês | MEDLINE | ID: mdl-31508780

RESUMO

INTRODUCTION: Crotalus envenomations cause serious complications and can be fatal without appropriate treatment. Venom isoforms present and inter/intraspecific variations in the venom composition can result in different symptoms presented by bites by snakes from the same species but from different geographical regions. We comparatively evaluated the local and systemic effects caused by Crotalus durissus terrificus (Cdt), C.d. collilineatus (Cdcolli), and C.d. cascavella (Cdcasc) envenomation. METHODS: Venom chromatography was performed. Proteolytic, phospholipase, and LAAO activities were analyzed. Edema, myotoxicity, hepatotoxicity, nephrotoxicity, and coagulation alterations were evaluated. RESULTS: The venom SDS-PAGE analyses found the presence of convulxin, gyroxin, crotoxin, and crotamine in Cdt and Cdcolli venoms. Crotamine was not present in the Cdcasc venom. Cdt, Cdcollli, and Cdcasc venoms had no proteolytic activity. Only Cdcasc and Cdt venoms had phospholipase activity. LAAO activity was observed in Cdcolli and Cdcasc venoms. Cdcolli and Cdcasc venoms caused 36.7% and 13.3% edema increases, respectively. Cdt venom caused a 10% edema induction compared to those by other venoms. All venoms increased TOTAL-CK, MB-CK, and LDH levels (indicating muscle injury) and ALT, AST, GGT, and ALP levels (markers of liver damage) and were able to induce a neuromuscular blockade. Urea and creatinine levels were also altered in both plasma and urine, indicating kidney damage. Only Cdcolli and Cdcasc venoms increased TAPP and TAP. CONCLUSIONS: Together, these results allow us to draw a distinction between local and systemic effects caused by Crotalus subspecies, highlighting the clinical and biochemical effects produced by their respective venoms.


Assuntos
Venenos de Crotalídeos/toxicidade , Crotalus/classificação , Edema/induzido quimicamente , Rim/efeitos dos fármacos , Fígado/efeitos dos fármacos , Fosfatase Alcalina/sangue , Fosfatase Alcalina/efeitos dos fármacos , Animais , Creatina Quinase/sangue , Creatina Quinase/efeitos dos fármacos , Creatinina/sangue , Edema/patologia , Eletroforese em Gel de Poliacrilamida , Rim/patologia , L-Lactato Desidrogenase/sangue , L-Lactato Desidrogenase/efeitos dos fármacos , Fígado/patologia , Camundongos , Modelos Animais , Transaminases/sangue , Transaminases/efeitos dos fármacos , Ureia/sangue
3.
Exp Parasitol ; 204: 107731, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31374185

RESUMO

Neospora caninum is an obligate intracellular parasite related to cases of abortion and fertility impairment in cattle. The control of the parasite still lacks an effective protective strategy and the understanding of key mechanisms for host infection might be crucial for identification of specific targets. There are many proteins related to important mechanisms in the host cell infection cycle such as adhesion, invasion, proliferation and immune evasion. The surface proteins, especially SRS (Surface Antigen Glycoprotein - Related Sequences), have been demonstrated to have a pivotal role in the adhesion and invasion processes, making them potential anti-parasite targets. However, several predicted surface proteins were not described concerning their function and importance in the parasite life cycle. As such, a novel SRS protein, NcSRS57, was described. NcSRS57 antiserum was used to detect SRS proteins by immunofluorescence in parasites treated or not with phosphatidylinositol-specific phospholipase C (PI-PLC). The treatment with PI-PLC also allowed the identification of NcSRS29B and NcSRS29C, which were the most abundant SRS proteins in the soluble fraction. Our data indicated that SRS proteins in N. caninum shared a high level of sequence similarity and were susceptible to PI-PLC. In addition, the description of the SRS members, regarding abundance, function and immunogenicity will be useful in guiding specific methods to control the mechanism of adhesion and invasion mediated by these surface proteins.


Assuntos
Antígenos de Protozoários/metabolismo , Antígenos de Superfície/metabolismo , Neospora/química , Fosfoinositídeo Fosfolipase C/farmacologia , Proteínas de Protozoários/metabolismo , Animais , Antígenos de Protozoários/genética , Antígenos de Protozoários/imunologia , Antígenos de Superfície/genética , Antígenos de Superfície/imunologia , Cercopithecus aethiops , Clonagem Molecular , DNA de Protozoário/isolamento & purificação , Eletroforese em Gel Bidimensional , Eletroforese em Gel de Poliacrilamida , Soros Imunes/imunologia , Soros Imunes/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Microscopia Confocal , Neospora/efeitos dos fármacos , Neospora/genética , Neospora/imunologia , Fosfoinositídeo Fosfolipase C/metabolismo , Proteínas de Protozoários/genética , Proteínas de Protozoários/imunologia , Espectrometria de Massas em Tandem , Fosfolipases Tipo C/metabolismo , Fosfolipases Tipo C/farmacologia , Células Vero
4.
BMC Plant Biol ; 19(1): 333, 2019 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-31370789

RESUMO

BACKGROUND: Wheat grains contain gluten proteins, which harbour immunogenic epitopes that trigger Coeliac disease in 1-2% of the human population. Wheat varieties or accessions containing only safe gluten have not been identified and conventional breeding alone struggles to achieve such a goal, as the epitopes occur in gluten proteins encoded by five multigene families, these genes are partly located in tandem arrays, and bread wheat is allohexaploid. Gluten immunogenicity can be reduced by modification or deletion of epitopes. Mutagenesis technologies, including CRISPR/Cas9, provide a route to obtain bread wheat containing gluten proteins with fewer immunogenic epitopes. RESULTS: In this study, we analysed the genetic diversity of over 600 α- and γ-gliadin gene sequences to design six sgRNA sequences on relatively conserved domains that we identified near coeliac disease epitopes. They were combined in four CRISPR/Cas9 constructs to target the α- or γ-gliadins, or both simultaneously, in the hexaploid bread wheat cultivar Fielder. We compared the results with those obtained with random mutagenesis in cultivar Paragon by γ-irradiation. For this, Acid-PAGE was used to identify T1 grains with altered gliadin protein profiles compared to the wild-type endosperm. We first optimised the interpretation of Acid-PAGE gels using Chinese Spring deletion lines. We then analysed the changes generated in 360 Paragon γ-irradiated lines and in 117 Fielder CRISPR/Cas9 lines. Similar gliadin profile alterations, with missing protein bands, could be observed in grains produced by both methods. CONCLUSIONS: The results demonstrate the feasibility and efficacy of using CRISPR/Cas9 to simultaneously edit multiple genes in the large α- and γ-gliadin gene families in polyploid bread wheat. Additional methods, generating genomics and proteomics data, will be necessary to determine the exact nature of the mutations generated with both methods.


Assuntos
Edição de Genes/métodos , Genes de Plantas/genética , Gliadina/genética , Glutens/genética , Triticum/genética , Proteína 9 Associada à CRISPR , Sistemas CRISPR-Cas , Eletroforese em Gel de Poliacrilamida , Glutens/imunologia , Melhoramento Vegetal/métodos , Plantas Geneticamente Modificadas , Alinhamento de Sequência
5.
J Enzyme Inhib Med Chem ; 34(1): 1439-1450, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31409157

RESUMO

Leishmaniasis is a tropical disease found in more than 90 countries. The drugs available to treat this disease have nonspecific action and high toxicity. In order to develop novel therapeutic alternatives to fight this ailment, pteridine reductase 1 (PTR1) and dihydrofolate reductase-thymidylate synthase (DHF-TS) have been targeted, once Leishmania is auxotrophic for folates. Although PTR1 and DHFR-TS from other protozoan parasites have been studied, their homologs in Leishmania chagasi have been poorly characterized. Hence, this work describes the optimal conditions to express the recombinant LcPTR1 and LcDHFR-TS enzymes, as well as balanced assay conditions for screening. Last but not the least, we show that 2,4 diaminopyrimidine derivatives are low-micromolar competitive inhibitors of both enzymes (LcPTR1 Ki = 1.50-2.30 µM and LcDHFR Ki = 0.28-3.00 µM) with poor selectivity index. On the other hand, compound 5 (2,4-diaminoquinazoline derivative) is a selective LcPTR1 inhibitor (Ki = 0.47 µM, selectivity index = 20).


Assuntos
Inibidores Enzimáticos/farmacologia , Leishmania infantum/enzimologia , Complexos Multienzimáticos/antagonistas & inibidores , Oxirredutases/antagonistas & inibidores , Timidilato Sintase/antagonistas & inibidores , Catálise , Cromatografia de Afinidade , Clonagem Molecular , Avaliação Pré-Clínica de Medicamentos , Eletroforese em Gel de Poliacrilamida , Escherichia coli/genética , Concentração Inibidora 50 , Complexos Multienzimáticos/genética , Complexos Multienzimáticos/isolamento & purificação , Complexos Multienzimáticos/metabolismo , Oxirredutases/genética , Oxirredutases/isolamento & purificação , Oxirredutases/metabolismo , Tetra-Hidrofolato Desidrogenase/genética , Tetra-Hidrofolato Desidrogenase/isolamento & purificação , Tetra-Hidrofolato Desidrogenase/metabolismo , Timidilato Sintase/genética , Timidilato Sintase/isolamento & purificação , Timidilato Sintase/metabolismo
6.
Klin Lab Diagn ; 64(7): 413-416, 2019.
Artigo em Russo | MEDLINE | ID: mdl-31408593

RESUMO

The selective properties of a solution of oligonucleotide specific to IL-6 on the concentration of IL-6 in mixed saliva of patients with oral inflammatory processes were studied using SDS-PAGE by electrophoresis and enzyme immunoassay. The application of these methods showed that in the mixed saliva of patients after rinsing with a solution of an oligonucleotide specific for IL-6, the amount of IL-6 decreases. The ELISA Kit and 20% SDS-PAGE showed the highest sensitivity to determine the concentration of IL-6 in saliva, which should be considered in clinical laboratory practice.


Assuntos
Interleucina-6/análise , Antissépticos Bucais/administração & dosagem , Oligonucleotídeos/administração & dosagem , Saliva/química , Eletroforese em Gel de Poliacrilamida , Ensaio de Imunoadsorção Enzimática , Humanos , Boca , Sensibilidade e Especificidade
7.
Adv Exp Med Biol ; 1140: 541-561, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31347070

RESUMO

Proteomics involves large-scale comprehensive study of specific proteomes which have been widely used in the field of biomarker discovery, drug development, disease diagnosis and therapy. Comprehensive proteomics involve two or more proteomics approaches that are confirmatory, complementary, and/or synergistic. Obstructive sleep apnea (OSA) is a sleep disorder which causes respiratory cessation (due to upper airway collapse). Here we describe a comprehensive MS based label-free quantitative proteomic analysis of the OSA induced rat atria homogenates and matched controls by using 1 dimensional SDS PAGE (1-D PAGE) and 2 dimensional SDS PAGE (2-D PAGE) separation of the proteins, enzymatic digestion and analysis by nanoliquid chromatography tandem-mass spectrometry (LC-MS/MS). The outcomes from the 1D-PAGE and 2D-PAGE studies not only identified dysregulated proteins due to OSA, but also confirmed and complemented each other.


Assuntos
Átrios do Coração/metabolismo , Proteoma/análise , Proteômica , Apneia Obstrutiva do Sono , Animais , Cromatografia Líquida , Eletroforese em Gel de Poliacrilamida , Ratos , Espectrometria de Massas em Tandem
8.
Adv Exp Med Biol ; 1140: 563-574, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31347071

RESUMO

Two-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis (2D SDS PAGE) is a method that separates proteins according to their isoelectric points in the first dimension and molecular masses in the second dimension. Evidence is provided that 2D SDS PAGE is reproducible, robust and compatible with SDS in both dimensions including isoelectric focusing in tube gels, the first dimension. The 2D gel pattern of rat liver microsomes shows more detail and sharper spot outlines when dissolved in SDS buffer with heating than in urea buffer and is better yet when dissolved in a mixture of both buffers. Quantification of 60 proteins in rat liver cytosol over a wide range of pI and MW gave linear plots of spot density versus total protein for loads of 200, 400 and 600 µg protein dissolved in SDS buffer and run in triplicate on 2D gels (Average R2 = 0.987). Examples of biomedical applications are provided in which 2D proteins of interest found by comparing stained or western blotted 2D gel patterns were identified by mass spectrometry (MS).


Assuntos
Western Blotting , Eletroforese em Gel Bidimensional , Espectrometria de Massas , Proteômica/métodos , Animais , Eletroforese em Gel de Poliacrilamida , Focalização Isoelétrica , Microssomos Hepáticos , Ratos
9.
Zhongguo Zhong Yao Za Zhi ; 44(10): 1983-1988, 2019 May.
Artigo em Chinês | MEDLINE | ID: mdl-31355551

RESUMO

In this study,the protein in different Cordyceps samples,which include fresh sample( S1),22 ℃ drying sample( S2),37 ℃ drying sample( S3) and 60 ℃ drying sample( S4),were analyzed by sodium dodecylsupinate-polyacrylamide gel electrophoresis( SDS-PAGE) and two-dimensional electrophoresis( 2-DE). The total protein contents in Cordyceps samples were from 1. 655-4. 493 mg·g~(-1) and the protein contents in fresh sample was the highest. The results of SDS-PAGE showed that the mainly ranges of protein molecular weight of Cordyces samples were 10-100 kDa and the numbers of protein bands were 28 to 41,the fresh sample had the maximum number of protein bands. The 2-DE profiles were analyzed by PDQuest software. The resulted indicated that 488-876 protein spots were detected in different Cordyceps samples and the isoelectric point( pI) was distributed between 4. 5 and 6. 5,the protein molecular weight was distributed in 10-20 kDa and 25-100 kDa,the fresh sample had the maximum number of protein spots. Therefore,the drying process could decrease contents and species of protein in Cordyceps,and the different drying conditions had different effects on protein. These results provide a reference for improving the drying process of Cordyceps.


Assuntos
Cordyceps/química , Dessecação/métodos , Proteínas Fúngicas/análise , Eletroforese em Gel Bidimensional , Eletroforese em Gel de Poliacrilamida , Peso Molecular
10.
Exp Parasitol ; 204: 107722, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31279928

RESUMO

In the present study, we attempted to identify antigens with high sensitivity and specificity for the serological diagnosis of human toxoplasmosis. We investigated soluble proteins from the tachyzoites of the RH strain of Toxoplasma gondii (T. gondii) and excreted/secreted antigens (ESAs) from the peritoneal protein of T. gondii-infected mice. One-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and western blot analysis revealed that in both soluble tachyzoite antigens and ESAs, the antigens located between 25 and 35 kDa had high diagnostic sensitivity. Further analysis of antigenic specificity revealed that the antigens located between 25 and 35 kDa were specifically recognized by the sera of toxoplasmosis patients, but other parasitic diseases were not. The protein spots between 25 and 35 kDa were selected after two-dimensional electrophoresis of both soluble tachyzoite antigens and ESAs. GRA2, GRA7, and triosephosphate isomerase (TPI) were successfully characterized from the protein spots using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectroscopy analysis. We expressed, purified, and evaluated proteins GRA2, GRA7, and TPI. TPI is a novel antigen with potential for the serological diagnosis of toxoplasmosis, and composite recombinant proteins (TPI, GRA2, and GRA7) have great sera diagnostic value for the detection of the disorder.


Assuntos
Anticorpos Antiprotozoários/sangue , Antígenos de Protozoários/imunologia , Imunoglobulina G/sangue , Toxoplasma/imunologia , Toxoplasmose/diagnóstico , Animais , Western Blotting , DNA Complementar/biossíntese , Eletroforese em Gel de Poliacrilamida , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Focalização Isoelétrica , Espectrometria de Massas , Camundongos , Camundongos Endogâmicos ICR , Reação em Cadeia da Polimerase , Proteínas de Protozoários/imunologia , RNA de Protozoário/genética , RNA de Protozoário/isolamento & purificação , Sensibilidade e Especificidade , Toxoplasmose/sangue , Toxoplasmose/imunologia , Triose-Fosfato Isomerase/imunologia , Eletroforese em Gel Diferencial Bidimensional
11.
Int. j. morphol ; 37(2): 773-779, June 2019. tab, graf
Artigo em Espanhol | LILACS | ID: biblio-1002292

RESUMO

La información disponible referente a las características proteómicas del E. granulosus es escasa (no supera los 50 estudios publicados); y nos parece que la identificación proteómica, podría mejorar la comprensión de algunas características bioquímicas e inmunológicas de la Equinococosis Quística (EQ). De tal modo que el proteoma de E. granulosus aún no está bien descrito. Sólo existen reportes de algunas secuencias de proteínas. El objetivo de este manuscrito fue comentar algunos aspectos de la evidencia existente respecto de los estudios del perfil proteómico del E. granulosus. Se recomienda el estudio de al menos el quiste y su pared, el líquido hidatídico y la víscera del hospedero. Para ello, existen metodologías que han sido empleadas para estudiar las características proteómicas de la EQ. Entre ellas, destacan SDS-PAGE, electroforesis bidimensional combinada con Western Blot, inmunoanálisis, y espectrometría de masas mediante técnicas MALDI-TOF. Se han identificado una serie de proteínas en muestras de EQ. Algunas de ellas, asociadas a procesos inmunológicos, de gluconeogénesis, glucogenolisis y glucogénesis. Por otra parte, se ha documentado la liberación de exosomas al líquido hidatídico por parte de los protoescólex y la capa germinativa; estructuras en las que se han identificado factores de virulencia asociados con la supervivencia del quiste. No obstante lo anteriormente señalado, se requiere de múltiples estudios exhaustivos en la materia para comprender mejor la caracterización perfil proteómico del E. granulosus.


The information available regarding the proteomic characteristics of E. granulosus is scarce; and it seems that the proteomic identification could improve the understanding of some biochemical and immunological characteristics of cystic echinococcosis (CE). So, the proteome of E. granulosus is still not well described yet. There are only reports of some protein sequences. The objective of this manuscript was to comment on some aspects of the existing evidence regarding studies of the proteomic profile of E. granulosus. The study of at least the cyst and its wall, the hydatid fluid and the viscera of the host are recommended. There are a series of methodologies that have been used to study the proteomic characteristics of EQ. These include SDS-PAGE, bidimensional electrophoresis combined with Western Blot, immunoassay, and mass spectrometry using MALDI-TOF techniques. A series of proteins have been identified in CE samples. Some of them, associated with immune response, gluconeogenesis, glycogenolysis and glycogenesis. On the other hand, release of exosomes to the hydatid fluid by protoescolex and germinative layer has been documented (associated virulence factors have been identified in these structures). Notwithstanding the foregoing, it requires multiple exhaustive studies in the field to better understand the characterization of the proteomic profile of E. granulosus.


Assuntos
Proteínas/análise , Proteômica/métodos , Echinococcus granulosus/química , Echinococcus granulosus/genética , Echinococcus granulosus/metabolismo , Eletroforese em Gel de Poliacrilamida
12.
Exp Parasitol ; 203: 23-29, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31150654

RESUMO

In Brazil, Leishmania amazonensis is one of the etiological agents of tegumentary leishmaniasis and can cause a wide spectrum of diseases in humans, resulting in cutaneous, mucosal, diffuse, and even visceral leishmaniasis. Besides, this species has also been reported to affect dogs, causing typical symptoms of visceral disease. Unfortunately, the diagnostic of the Leishmania species is not routinely performed due to the difficulties of the available methods. In view of this, different molecular methods have been used in an attempt to solve the problem of diagnosis. Loop-mediated isothermal amplification (LAMP) is a relatively new nucleic acid amplification method, which has been successfully applied in the diagnosis of Leishmania spp. infections. However, this is the first work that standardizes a specific LAMP reaction for L. amazonensis. The set of primers selected were designed from the kDNA minicircle sequence of the L. amazonensis (GenBank: U19810.1). The LAMP assay developed in the present study showed 100% specificity and 89% sensitivity when compared with conventional PCR and was more sensitive than qPCR. In addition, the LAMP reaction developed here was able to amplify a qPCR sample with a parasite load of only 28 parasites in 50 ng of DNA. Consequently, considering the LAMP reaction specific to L. amazonensis and several advantages of the method (such as high efficiency, sensitivity and specificity), we believe that this reaction can be used as a promising diagnostic tool in clinical practice, field studies, and research.


Assuntos
Leishmania mexicana/isolamento & purificação , Leishmaniose Cutânea/diagnóstico , Pele/parasitologia , Animais , Sequência de Bases , Colorimetria , Cricetinae , DNA de Cinetoplasto/química , DNA de Cinetoplasto/genética , DNA de Protozoário/isolamento & purificação , Eletroforese em Gel de Poliacrilamida , Feminino , Leishmania mexicana/genética , Leishmaniose Cutânea/parasitologia , Masculino , Mesocricetus , RNA Ribossômico 18S/química , RNA Ribossômico 18S/genética , Reação em Cadeia da Polimerase em Tempo Real , Sensibilidade e Especificidade , Coloração pela Prata
13.
Adv Exp Med Biol ; 1073: 1-22, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31236837

RESUMO

The proteome represents the total set of proteins produced by an organism or a system at a particular time or state, with proteomics being the study of the proteome. The proteome is a dynamic system wherein proteins are interconnected and serve to facilitate cellular processes in a concurrent and coordinated manner. Over the years, various biochemical and biophysical methods have been developed to elucidate the identities, structures and functions of proteins in order to understand their roles in complex biological systems. The success of proteomic approaches hinges on efficient protein extraction and sample preparation; however, these preliminary steps are often considered a bottleneck in proteomic workflows. Every biological sample is unique and complex, and sample processing needs to be tailored to the nature of the protein sample due to its vulnerability towards post-collection degradation and the complexity of its non-protein constituents. Sample pretreatment steps often employ buffers, solvents, salts and detergents that are not always compatible with the downstream analytical tools. This chapter will provide an overview of sample pretreatment techniques commonly used in conjunction with proteomics tools and discuss protein analysis methods. Such methods include the use of antibody-based techniques, separation sciences (e.g. chromatography, SDS-PAGE), detection methods (e.g. mass spectrometry) and structural techniques (e.g. NMR and X-ray crystallography).


Assuntos
Proteoma , Proteômica/métodos , Anticorpos/química , Cromatografia , Cristalografia por Raios X , Eletroforese em Gel de Poliacrilamida , Espectroscopia de Ressonância Magnética , Espectrometria de Massas
14.
Pestic Biochem Physiol ; 157: 99-107, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31153482

RESUMO

The entmopathogenic fungus Lecaniicillium lecanii is a naturally available biological control and it is considered to be one of the best mycoinsecticide agents against the destructive insect pest Diaphorina citri Kuwayama. The present study aimed to extract and characterize the toxic insecticidal protein from L. lecanii and to assess the toxicity level against the Asian citrus psyllid the vector of Huanglongbing disease (HLB), also called citrus greening. Extracts of a toxic substance from submerged batch culture examined by sodium dodecyl sulfate-poly-acrylamide (SDS-PAGE), had a molecular weight of 45 kDa. The most abundant toxic metabolite was subjected to HPLC to purify and identified it by mass spectrometry. Subsequently, metabolite toxicity was tested against D. citri at three different concentrations (1%, 2%, and 3%). The results showed that the highest concentration had a significant maximum mortality at 120 h post application. Furthermore, we investigated the expression of the GAS1 gene which was previously identified to have a role in pathogenicity in in vivo studies in adult insect psyllids. Results of this study indicated that expression of the virulence factor gene was present at three concentrations of the fungal suspension post inoculation. This is the first study to provide this novel approach for the characterization of fungal mediated synthesis of a cuticle degrading soluble protein against the insect D. citri. The present results provide strong information on the in vivo expression of the GAS1 gene involved in fungal virulence pertaining to penetration of the insect cuticle, but not to inhibiting the growth of the host.


Assuntos
Hemípteros/microbiologia , Hypocreales/metabolismo , Hypocreales/patogenicidade , Animais , Eletroforese em Gel de Poliacrilamida , Hypocreales/genética , Virulência
15.
Biochemistry (Mosc) ; 84(6): 627-636, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31238862

RESUMO

The cytokine TRAIL induces apoptosis in tumor cells of various origin without affecting normal cells. Clinical trials of TRAIL-receptor (DR4 and DR5) agonists (recombinant TRAIL or death receptors antibodies) have largely failed because most human tumors were resistant to them. Currently, a second generation of agents targeted at TRAIL-R with increased efficiency has been developed. To this end, we have developed DR5-B, a variant of TRAIL selectively interacting with DR5. We have developed a new efficient method for production of TRAIL and DR5-B using expression of these proteins in Escherichia coli strain SHuffle B. The proteins were isolated from the cytoplasmic fraction of cells and purified to a high degree of homogeneity using metal-affinity and ion-exchange chromatography. The protein yield was 211 and 173 mg from one liter of cell culture for DR5-B and TRAIL, respectively, which significantly exceeded the results obtained by other methods. DR5-B killed tumor cells of different origin more efficiently and rapidly compared with TRAIL. The resulting preparations can be used for the study of TRAIL signaling pathways and in preclinical and clinical trials as antitumor agents.


Assuntos
Receptores do Ligante Indutor de Apoptose Relacionado a TNF/biossíntese , Ligante Indutor de Apoptose Relacionado a TNF/biossíntese , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Cromatografia de Afinidade/métodos , Cromatografia por Troca Iônica/métodos , Ensaios de Seleção de Medicamentos Antitumorais , Eletroforese em Gel de Poliacrilamida , Escherichia coli/genética , Humanos , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/genética , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/isolamento & purificação , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/farmacologia , Ligante Indutor de Apoptose Relacionado a TNF/genética , Ligante Indutor de Apoptose Relacionado a TNF/isolamento & purificação , Ligante Indutor de Apoptose Relacionado a TNF/farmacologia
16.
Biochemistry (Mosc) ; 84(6): 672-685, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31238867

RESUMO

Mature pore-forming OmpF protein from the outer membrane of Yersinia pseudotuberculosis was expressed in Escherichia coli in the form of inclusion bodies (IBs) under different cultivation conditions. The properties and structural organization of the IBs as well as the structure of the recombinant porin (rOmpF) solubilized from the IBs were investigated using electron microscopy, dynamic light scattering, optical spectroscopy, and specific hydrophobic dyes. The size, shape, and stability of the IBs under denaturing solutions were determined. It was found that the IBs were readily soluble in SDS and more resistant to urea. Dissolution of the IBs in both denaturing agents led to formation of a heterogeneous in size population of oligomeric particles. The IBs contained an intermediate form of the rOmpF with native-like secondary structure and elements of tertiary structure, which was able to penetrate a lipid bilayer and adopt a functionally active conformation. There were no significant differences in the properties and structure between the examined IBs formed at different concentrations of the inducer (IPTG). However, the content of amyloids in the IBs increased with increasing concentration of the inducer. These results contribute to the development of new approaches for the production of active proteins from IBs, as well as biologically and functionally active IBs.


Assuntos
Proteínas da Membrana Bacteriana Externa/metabolismo , Corpos de Inclusão/metabolismo , Porinas/metabolismo , Yersinia pseudotuberculosis/metabolismo , Proteínas da Membrana Bacteriana Externa/química , Dicroísmo Circular , Eletroforese em Gel de Poliacrilamida , Temperatura Alta , Microscopia Eletrônica de Varredura , Porinas/química , Conformação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Solubilidade , Espectrometria de Fluorescência , Espectrofotometria Ultravioleta
17.
Food Chem ; 297: 124995, 2019 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-31253267

RESUMO

The possible interactions between α-zein and Ca2+ in nixtamalization process were analyzed from a multidisciplinary approach, considering the effect of these interactions on the thermal properties of the nixtamalized flour. SDS-PAGE under reducing and non-reducing conditions did not reveal differences between patterns of zeins from nixtamalized and control samples. However, analysis from affinity capillary electrophoresis indicated an increment in protein volume when calcium is added to zein extracted from nixtamalized flour. In addition, the binding constant for the zein-calcium interaction was calculated indicating a higher affinity for calcium by zein from nixtamalized samples. Molecular dynamics simulations indicated that the interaction α-zein-Ca2+ through C-ter was more favorable than Glu48. However, in excess of Ca2+ ions, each site could bind one calcium atom at the same time, confirming that aggregation of α-zein through calcium bridges is possible, expanding the technological applications of this protein.


Assuntos
Cálcio/química , Modelos Teóricos , Zeína/química , Sítios de Ligação , Cálcio/metabolismo , Culinária , Eletroforese em Gel de Poliacrilamida , Farinha/análise , Simulação de Dinâmica Molecular , Termodinâmica , Temperatura de Transição , Zea mays/metabolismo , Zeína/metabolismo
18.
Food Chem ; 297: 124939, 2019 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-31253337

RESUMO

Effects of the brine solution with different NaOH concentrations on the physicochemical properties, intermolecular forces, and gel properties of duck egg whites were studied. The free alkalinity, salt content and surface hydrophobicity of egg whites increased as the pickling time and NaOH concentration increased. The primary intermolecular forces (disulfide bonds and hydrophobic interactions) in the alkali egg white gels were increased firstly and then decreased, resulting from the fact that the proteins were degraded under strong alkali conditions during the late stage of the pickling process. Changes of hardness and springiness of egg white gels were closely related with the interactions of the protein molecules. Thereinto, the change tendency of springiness was in accordance with the changes of hydrogen bond of egg white gels. The colour of the egg white gels obtained from alkali treatment changed from white to amber during pickling.


Assuntos
Clara de Ovo/química , Géis/química , Cloreto de Sódio/química , Hidróxido de Sódio/química , Animais , Patos , Eletroforese em Gel de Poliacrilamida , Ligações de Hidrogênio , Interações Hidrofóbicas e Hidrofílicas , Temperatura Ambiente
19.
BMC Plant Biol ; 19(1): 195, 2019 May 14.
Artigo em Inglês | MEDLINE | ID: mdl-31088366

RESUMO

BACKGROUND: Flavonol synthase (FLS) is the key enzyme responsible for the biosynthesis of flavonols, the most abundant flavonoids, which have diverse pharmaceutical effects. Flavonol synthase has been previously found in other species, but not yet in Ornithogalum caudatum. RESULTS: The transcriptome-wide mining and functional characterisation of a flavonol synthase gene family from O. caudatum were reported. Specifically, a small FLS gene family harbouring two members, OcFLS1 and OcFLS2, was isolated from O. caudatum based on transcriptome-wide mining. Phylogenetic analysis suggested that the two proteins showed the closest relationship with FLS proteins. In vitro enzymatic assays indicated OcFLS1 and OcFLS2 were flavonol synthases, catalysing the conversion of dihydroflavonols to flavonols in an iron-dependent fashion. In addition, the two proteins were found to display flavanone 3ß-hydroxylase (F3H) activity, hydroxylating flavanones to form dihydroflavonols. Unlike single F3H enzymes, the F3H activity of OcFLS1 and OcFLS2 did not absolutely require iron. However, the presence of sufficient Fe2+ was demonstrated to be conducive to successive catalysis of flavanones to flavonols. The qRT-PCR analysis demonstrated that both genes were expressed in the leaves, bulbs, and flowers, with particularly high expression in the leaves. Moreover, their expression was regulated by developmental and environmental conditions. CONCLUSIONS: OcFLS1 and OcFLS2 from O. caudatum were demonstrated to be flavonol synthases with iron-independent flavanone 3-hydroxylase activity.


Assuntos
Oxigenases de Função Mista/metabolismo , Ornithogalum/enzimologia , Oxirredutases/metabolismo , Proteínas de Plantas/metabolismo , Eletroforese em Gel de Poliacrilamida , Flavonóis/metabolismo , Perfilação da Expressão Gênica , Genes de Plantas/genética , Genes de Plantas/fisiologia , Ferro/metabolismo , Redes e Vias Metabólicas , Ornithogalum/genética , Ornithogalum/metabolismo , Análise de Sequência de DNA , Transcriptoma
20.
J Agric Food Chem ; 67(26): 7475-7484, 2019 Jul 03.
Artigo em Inglês | MEDLINE | ID: mdl-31117490

RESUMO

Chia seeds are becoming increasingly common in Europe because of their functional and nutritional properties. Despite this, few studies have focused on the allergic potential and antibody cross-reactivity among storage proteins in chia seed and other plants. The aim of this study was to identify chia seed's immunoglobulin G (IgG) and immunoglobulin E (IgE) binding proteins ( Salvia hispanica L.) and to investigate the antibody cross-reactivity among its storage proteins and those of other seeds. Extracted chia seed proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Immunodetection was performed with commercial antibodies against sesame seed, hazelnut, and peanut and sera from 33 patients with a hazelnut allergy and five with a sesame allergy. Cross-reactivity of certain antibodies with storage proteins of chia seed, sesame seed, and hazelnut was assessed using an enzyme-linked immunosorbent assay (ELISA) inhibition, blot inhibition, and surface plasmon resonance (SPR) spectroscopy. IgG binding proteins were identified at molecular weight (MW) 70, 49, 34, 23, and 20 kDa by applying commercial antibodies. Furthermore, the interaction of chia proteins with sera from sesame-allergic patients led to identify IgE binding proteins at MW 49, 45, 31, 20, and 12 kDa, while IgEs in sera from hazelnut-allergic patients reacted with proteins at MW 300, 140, 49, 45, 31, 20, and 6 kDa. The results of ELISA inhibition and blot inhibition indicated chia seed proteins are similar to sesame seed and hazelnut proteins in the primary structure. The antisesame antibodies' binding to sesame proteins was more strongly inhibited by the chia globulin fraction (GLO) than the antihazelnut antibodies' binding to hazelnut proteins. SPR results confirmed the presence of IgG binding proteins in GLO and the high similarity of epitopes on globulins of chia seed and sesame seed. Thus, chia seed consumption might lead to cross-sensitization in patients with a sesame allergy.


Assuntos
Antígenos de Plantas/imunologia , Arachis/imunologia , Corylus/imunologia , Imunoglobulina E/imunologia , Imunoglobulina G/imunologia , Proteínas de Plantas/imunologia , Salvia/imunologia , Sementes/imunologia , Sesamum/imunologia , Arachis/química , Corylus/química , Reações Cruzadas , Eletroforese em Gel de Poliacrilamida , Hipersensibilidade Alimentar/imunologia , Humanos , Salvia/química , Sementes/química , Sesamum/química
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