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1.
Food Chem ; 309: 125460, 2020 Mar 30.
Artigo em Inglês | MEDLINE | ID: mdl-31732251

RESUMO

The high concentrations of mercury found in Amazon have been intensively studied by the scientific community in the last decades. These mercurial species bind preferentially to proteins. Therefore, this work proposal sought to obtain the fractionation, identification and study of mercury - bound proteins present in samples of muscular and hepatic tissue from fish collected in the reservoir of the Jirau Hydroelectric Power Plant - on the Madeira River. Two-dimensional electrophoresis (2D-PAGE) for protein fractionation, graphite furnace atomic absorption spectrometry (GFAAS) for the quantification of mercury and Mass Spectrometry (ESI-MS/MS) were used for the identification of proteins. Concluding the work with analysis of graphics from the Blast2go program. Two mercury - bound proteins were identified as triosephosphate isomerase A and Protein FAM45A. The data generated by the bioinformatics programs confirm the tendency of these proteins to be linked to mercury and elucity the possibles existing physiological and cellular interactions.


Assuntos
Peixes , Fígado/química , Mercúrio/análise , Músculo Esquelético/química , Animais , Brasil , Eletroforese em Gel Bidimensional , Rios/química , Espectrofotometria Atômica , Espectrometria de Massas em Tandem , Poluentes Químicos da Água/análise
2.
Toxicol Lett ; 318: 22-29, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31634547

RESUMO

An attempt has been made to delineate the role of natural and synthetic retinoid receptor ligands on vimentin expression in the human triple-negative breast cancer cells. The effects of currently synthesized triorganotin derivatives of the general formula R3SnX (R is butyl or phenyl, X is isothiocyanate), which are considered RXR ligands, were investigated in the human MDA-MB-231 breast cancer cell line. Studies were evaluated in the presence and absence of all-trans retinoic acid (ATRA), a natural RAR ligand. Vimentin represents the major protein associated with epithelial-mesenchymal transition (EMT), an essential process when the primary tumour transforms into a malignant one. mRNA and proteomic data obtained in this study, based on the PDQuest software protein evaluation and further quantification of proteins by iTRAQ analysis, suggest that vimentin was significantly reduced in the combination of RAR ligand and RXR ligand treatment. Both tested triorganotin compounds showed similarly reduced expression of vimentin, but tributyltin isothiocyanate (TBT-ITC) proved to be more effective than triphenyltin isothiocyanate (TPT-ITC). Furthermore, the effect of natural (9cRA) and synthetic RXR ligands, both chloride and isothiocyanate derivatives, on vimentin expression was compared.


Assuntos
Antineoplásicos/farmacologia , Neoplasias da Mama/tratamento farmacológico , Proteômica/métodos , Receptores X Retinoide/agonistas , Compostos de Trialquitina/farmacologia , Vimentina/metabolismo , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Regulação para Baixo , Eletroforese em Gel Bidimensional , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Feminino , Humanos , Compostos Orgânicos de Estanho/farmacologia , Receptores X Retinoide/metabolismo , Transdução de Sinais/efeitos dos fármacos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Espectrometria de Massas em Tandem , Tretinoína/farmacologia
3.
Forensic Sci Int ; 303: 109931, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31546160

RESUMO

Clinical and research-based tests in molecular biology require a substantial amount of DNA and RNA, unfortunately, a considerable number of cells is needed for this amount of sample. Blood is one of the best and easiest source of cells, but is not used due to its invasive drawing methods and the needed volume. Another considerable point is the low amount of samples detected in crime scenes. TRI reagent is one of the most available methods for DNA, RNA and protein extraction. However, based on unsuccessful results, this method has not been widely used on blood samples. In this study, for the first time, the use of TRI reagent on micro scale blood volume was reported, resulting in high yield of DNA and RNA with great quality.


Assuntos
DNA/isolamento & purificação , Medicina Legal/métodos , Indicadores e Reagentes , RNA/isolamento & purificação , Adolescente , Adulto , DNA/sangue , Eletroforese em Gel Bidimensional , Feminino , Humanos , Masculino , Reação em Cadeia da Polimerase , RNA/sangue , Adulto Jovem
4.
Cell Biochem Biophys ; 77(4): 335-342, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31489526

RESUMO

Alpha-2-macroglobulin (A2M) is a glycosylated broad spectrum inhibitor of numerous proteases, including those involved in blood coagulation. Glycosylation characteristics can affect protein structure and function. This study compares glycosylation characteristics of A2M in newborn umbilical cord (NUCP) and adult pooled plasmas. Peptide N-Glycosidase F treatment was used to evaluate the total N-glycan content of the molecules. Neuraminidase treatment, and affinity for Ricinus Communis Agglutinin I were used to examine terminal sialic acid and galactose content, respectively. Two-dimensional (2D) electrophoresis was used to determine charge-related isoform profiles and fluorophore-assisted carbohydrate electrophoresis (FACE) was used to characterize N-glycan profiles. Results revealed no difference in total N-glycan mass, however, a statistically significant difference was shown in the change in charge associated with sialic acid loss in the NUCP A2M population. 2D electrophoresis indicated a lower pI range for NUCP A2M isoforms. In addition, NUCP A2M displayed a trend toward higher terminal galactose quantities than adult A2M. FACE revealed an increased abundance of more branched, higher molecular weight glycans in NUCP A2M. These differences in glycan branching and charged residues may impact A2M receptor-based clearance and thus could be responsible for the increased A2M concentration seen in NUCP, and newborns.


Assuntos
Envelhecimento , Polissacarídeos/análise , alfa 2-Macroglobulinas Associadas à Gravidez/metabolismo , Adulto , Eletroforese em Gel Bidimensional , Glicosilação , Humanos , Recém-Nascido , alfa 2-Macroglobulinas Associadas à Gravidez/análise , Isoformas de Proteínas/metabolismo , Cordão Umbilical/metabolismo
5.
Plant Sci ; 287: 110170, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31481192

RESUMO

Protein ubiquitination is a major post-translational modification important for diverse biological processes. In wheat (Triticum aestivum) and Arabidopsis thaliana, STRESS-ASSOCIATED PROTEIN 5 (SAP5) is involved in drought tolerance, acting as an E3 ubiquitin ligase to target DRIP and MBP-1 for degradation. To identify further target proteins of SAP5, we implemented two independent approaches in this work. We used ubiquitylome capture with a di-Gly-Lys antibody-based peptide enrichment and affinity purification with a polyubiquitin antibody coupled with mass spectrometry to elucidate the SAP5-dependent ubiquitylation of its target proteins in response to osmotic stress. Wild type or TaSAP5-overexpressing Arabidopsis line, which was more tolerant to osmotic stress according to our previous study, were used here. We identified HSP90C (chloroplast heat shock protein 90) as a substrate of TaSAP5. Further biochemical experiments indicated that TaSAP5 interacts with HSP90C and mediates its degradation by the 26S proteasome. Our work also demonstrates that ubiquitylome profiling is an effective approach to search for substrates of the TaSAP5 E3 ubiquitin ligase when heterologously expressed in Arabidopsis.


Assuntos
Proteínas de Choque Térmico HSP90/metabolismo , Proteínas de Plantas/metabolismo , Saporinas/metabolismo , Triticum/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Arabidopsis , Eletroforese em Gel Bidimensional , Metabolômica , Plantas Geneticamente Modificadas , Tabaco , Triticum/enzimologia , Ubiquitinação
6.
Planta ; 250(5): 1703-1715, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31414205

RESUMO

MAIN CONCLUSION: The tobacco nectar proteome mainly consists of pathogenesis-related proteins with two glycoproteins. Expression of nectarins was non-synchronous, and not nectary specific. After secretion, tobacco nectar changed from sucrose rich to hexose rich. Floral nectar proteins (nectarins) play important roles in inhibiting microbial growth in nectar, and probably also tailoring nectar chemistry before or after secretion; however, very few plant species have had their nectar proteomes thoroughly investigated. Nectarins from Nicotiana tabacum (NT) were separated using two-dimensional gel electrophoresis and then analysed using mass spectrometry. Seven nectarins were identified: acidic endochitinase, ß-xylosidase, α-galactosidase, α-amylase, G-type lectin S-receptor-like serine/threonine-protein kinase, pathogenesis-related protein 5, and early nodulin-like protein 2. An eighth nectarin, a glycoprotein with unknown function, was identified following isolation from NT nectar using a Qproteome total glycoprotein kit, separation by SDS-PAGE, and identification by mass spectrometry. Expression of all identified nectarins, plus four invertase genes, was analysed by qRT PCR; none of these genes had nectary-specific expression, and none had synchronous expression. The total content of sucrose, hexoses, proteins, phenolics, and hydrogen peroxide were determined at different time intervals in secreted nectar, both within the nectar tube (in vivo) and following extraction from it during incubation at 30 °C for up to 40 h in plastic tubes (in vitro). After secretion, the ratio of hexose to sucrose substantially increased for in vivo nectar, but no sugar composition changes were detected in vitro. This implies that sucrose hydrolysis in vivo might be done by fixed apoplastic invertase. Both protein and hydrogen peroxide levels declined in vitro but not in vivo, implying that some factors other than nectarins act to maintain their levels in the flower, after secretion.


Assuntos
Néctar de Plantas/metabolismo , Proteoma , Proteômica , Tabaco/enzimologia , Eletroforese em Gel Bidimensional , Flores/genética , Flores/metabolismo , Glicoproteínas/genética , Glicoproteínas/metabolismo , Espectrometria de Massas , Proteínas de Membrana/metabolismo , Néctar de Plantas/genética , Proteínas de Plantas/metabolismo , Tabaco/genética
7.
Int J Mol Sci ; 20(16)2019 Aug 08.
Artigo em Inglês | MEDLINE | ID: mdl-31398909

RESUMO

High temperatures seriously limit plant growth and productivity. Investigating heat-responsive molecular mechanisms is important for breeding heat-tolerant crops. In this study, heat-responsive mechanisms in leaves from a heat-sensitive spinach (Spinacia oleracea L.) variety Sp73 were investigated using two-dimensional gel electrophoresis (2DE)-based and isobaric tags for relative and absolute quantification (iTRAQ)-based proteomics approaches. In total, 257 heat-responsive proteins were identified in the spinach leaves. The abundance patterns of these proteins indicated that the photosynthesis process was inhibited, reactive oxygen species (ROS) scavenging pathways were initiated, and protein synthesis and turnover, carbohydrate and amino acid metabolism were promoted in the spinach Sp73 in response to high temperature. By comparing this with our previous results in the heat-tolerant spinach variety Sp75, we found that heat inhibited photosynthesis, as well as heat-enhanced ROS scavenging, stress defense pathways, carbohydrate and energy metabolism, and protein folding and turnover constituting a conservative strategy for spinach in response to heat stress. However, the heat-decreased biosynthesis of chlorophyll and carotenoid as well as soluble sugar content in the variety Sp73 was quite different from that in the variety Sp75, leading to a lower capability for photosynthetic adaptation and osmotic homeostasis in Sp73 under heat stress. Moreover, the heat-reduced activities of SOD and other heat-activated antioxidant enzymes in the heat-sensitive variety Sp73 were also different from the heat-tolerant variety Sp75, implying that the ROS scavenging strategy is critical for heat tolerance.


Assuntos
Resposta ao Choque Térmico , Proteoma , Proteômica , Spinacia oleracea/fisiologia , Antioxidantes/metabolismo , Biologia Computacional/métodos , Eletroforese em Gel Bidimensional , Resposta ao Choque Térmico/genética , Temperatura Alta , Anotação de Sequência Molecular , Fenótipo , Fotossíntese , Folhas de Planta/metabolismo , Proteínas de Plantas/metabolismo , Mapeamento de Interação de Proteínas , Mapas de Interação de Proteínas , Proteômica/métodos , Espécies Reativas de Oxigênio/metabolismo
8.
Biomed Chromatogr ; 33(12): e4686, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31452214

RESUMO

Researchers frequently use two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) prior to mass spectrometric analysis in a proteomics approach. The i2D-PAGE method, which 'inverts' the dimension of protein separation of the conventional 2D-PAGE, is presented in this publication. Protein lysate of Channa striata, a freshwater snakehead fish, was separated based on its molecular weight in the first dimension and its isoelectric point in the second dimension. The first-dimension separation was conducted on a gel-free separation device, and the protein mixture was fractionated into 12 fractions in chronological order of increasing molecular weight. The second-dimension separation featured isoelectric focusing, which further separated the proteins within the same fraction according to their respective isoelectric point. Advantages of i2D-PAGE include better visualisation of the isolated protein, easy identification on protein isoforms, shorter running time, customisability and reproducibility. Erythropoietin standard was applied to i2D-PAGE to show its effectiveness for separating protein isoforms. Various staining methods such as Coomassie blue staining and silver staining are also applicable to i2D-PAGE. Overall, the i2D-PAGE separation method effectively separates protein lysate and is suitable for application in proteomics research.


Assuntos
Eletroforese em Gel Bidimensional/métodos , Isoformas de Proteínas/isolamento & purificação , Focalização Isoelétrica/métodos , Peso Molecular , Isoformas de Proteínas/análise , Isoformas de Proteínas/química , Proteômica/métodos , Proteínas Recombinantes/análise , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Reprodutibilidade dos Testes
9.
Plant Physiol Biochem ; 142: 351-362, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31422174

RESUMO

Cassava is an important tropical crop with strong resistance to drought stress. The chloroplast, the site of photosynthesis, is sensitive to stress, and the drought-response proteins in cassava chloroplasts are worthy of investigation. In this study, cassava leaves were collected for ultra-structure observation from plants subjected to different drought stress conditions. Our results showed that drought stress can promote starch accumulation in cassava chloroplasts. To evaluate changes in chloroplast proteins under different drought conditions, two-dimensional electrophoresis was performed using purified chloroplasts, which resulted in the identification of 26 unique chloroplast proteins responsive to drought stress. These drought-responsive proteins are predominantly related to photosynthesis, carbon and nitrogen metabolism, and amino acid metabolism. Among them, most photosynthesis-related proteins are downregulated, with decreases in photosynthetic parameters upon drought stress. Several proteins associated with carbon and nitrogen metabolism, including rubisco and carbonic anhydrase, were upregulated, which might promote drought tolerance in cassava by enhancing the carbohydrate conversion efficiency and protecting the plant from oxidative stress. Our proteomic data not only provide insight into the complement of proteins in cassava chloroplasts but also further our overall understanding of drought-responsive proteins in cassava chloroplasts.


Assuntos
Cloroplastos/metabolismo , Manihot/metabolismo , Folhas de Planta/metabolismo , Proteoma/metabolismo , Cloroplastos/fisiologia , Cloroplastos/ultraestrutura , Desidratação , Eletroforese em Gel Bidimensional , Manihot/fisiologia , Microscopia Eletrônica de Transmissão , Fotossíntese , Folhas de Planta/fisiologia , Folhas de Planta/ultraestrutura , Proteínas de Plantas/metabolismo , Proteínas de Plantas/fisiologia , Proteoma/fisiologia , Reação em Cadeia da Polimerase em Tempo Real
10.
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 , 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
11.
Toxicol Ind Health ; 35(7): 457-465, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31364504

RESUMO

Solar ultraviolet (UV) radiation is the main factor of photocarcinogenesis, photoaging, and photosensitivity; thus protection from biological damaging UV radiation is a concern. Sunscreens containing UV filters are the most preferred means of photoprotection but the safety and efficacy of UV filters are in question. Benzophenone (BP) and its derivatives, namely, benzophenone 1 (BP1), is commonly used in sunscreens as a UV blocker. The aim of this study was to assess the effects of BP and BP1 on the differential expression of proteins in human keratinocytes (HaCaT cells) under exposure to ultraviolet A radiation. Photosensitive proteins were screened from HaCaT cells by two-dimensional (2-D) gel electrophoresis, and identification of these differentially expressed proteins was performed by matrix-assisted laser desorption/ionization-time-of-flight (MALDI-TOF)/TOF mass spectrometry. Protein identification was performed using the search program MASCOT and a database made of SUMO and GhJMJ12 amino acid sequences. Our results showed that the proteins involved directly or indirectly in apoptosis are 70 kDa heat shock protein, long-chain specific acyl-CoA dehydrogenase, serine/threonine-protein kinase, and FAM78A protein, which were upregulated in comparison to control HaCaT cells. The expressions of binding immunoglobulin protein, podocalyxin-like protein, actin, cytoplasmic, and calreticulin precursors were downregulated. The altered protein expression indicated that cell growth arrest and apoptosis were potential mechanisms of cytotoxicity and genotoxicity of BPs. The results of 2-D gel electrophoresis followed by mass spectrometry showed expression of novel proteins involved in promoting or initiating apoptotic pathways. Hence, we conclude that BPs should be avoided as a UV blocker from sunscreens because of its potential to promote apoptotic proteins in human skin keratinocytes.


Assuntos
Benzofenonas/farmacologia , Queratinócitos/efeitos dos fármacos , Protetores Solares/farmacologia , Raios Ultravioleta , Apoptose/efeitos dos fármacos , Biomarcadores , Eletroforese em Gel Bidimensional , Proteínas de Choque Térmico/efeitos dos fármacos , Humanos , Queratinócitos/metabolismo , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz
12.
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
13.
J Ovarian Res ; 12(1): 64, 2019 Jul 17.
Artigo em Inglês | MEDLINE | ID: mdl-31315664

RESUMO

BACKGROUND: Serous carcinoma, the subtype of ovarian cancer has the highest occurrence and mortality in women. Proteomic profiling using mass spectrometry (MS) has been used to detect biomarkers in tissue s obtained from patients with ovarian cancer. Thus, this study aimed at analyzing the interactome (protein-protein interaction (PPI)) and (MS) data to inspect PPI networks in patients with Low grade serous ovarian cancer. METHODS: For proteome profiling in Low grade serous ovarian cancer, 2DE and mass spectrometry were used. Differentially expressed proteins which had been determined in Low grade serous ovarian cancer and experimental group separately were integrated with PPI data to construct the (QQPPI) networks. RESULTS: Six Hub-bottlenecks proteins with significant centrality values, based on centrality parameters of the network (Degree and between), were found including Transgelin (TAGLN), Keratin (KRT14), Single peptide match to actin, cytoplasmic 1(ACTB), apolipoprotein A-I (APOA1), Peroxiredoxin-2 (PRDX2), and Haptoglobin (HP). DISCUSSION: This study showed these six proteins were introduced as hub-bottleneck protein. It can be concluded that regulation of gene expression can have a critical role in the pathology of Low-grade serous ovarian cancer.


Assuntos
Cistadenocarcinoma Seroso/metabolismo , Cistadenocarcinoma Seroso/patologia , Neoplasias Ovarianas/metabolismo , Neoplasias Ovarianas/patologia , Proteoma , Proteômica , Biologia Computacional/métodos , Eletroforese em Gel Bidimensional , Feminino , Humanos , Gradação de Tumores , Estadiamento de Neoplasias , Proteômica/métodos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz
14.
Plant Foods Hum Nutr ; 74(3): 414-420, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31278561

RESUMO

The amount of cold press oil manufacture is globally rising, which in turn leads to the accumulation of deoiled plant seeds at significant quantities and consequent manufacture of plant protein products. In this study, we made an attempt to analyze the protein profile of black cumin seed protein concentrates prepared by the alkali extraction-acid precipitation technique (AE-IP). The analytical strategy relied on gel-based proteome mapping which included two-dimensional gel electrophoresis followed by matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF/TOF). 14 different protein bands were identified, and in gel-trypsinolysis was carried out for the corresponding gel spots. Using the MASCOT database, current findings on 10 proteins were compared with the existing data. The highest similarity was 46 which was obtained between the highest pI black cumin protein observed here and the cyclin dependent kinase inhibitor of Arabidopsis thaliana. The molecular mass of the intact protein was determined by linear MALDI-TOF/TOF-MS as 23,711.2186 Da. The peptide constructs of this protein have been further studied in order to identify potential biological activity. Matching sequences generated bioactive peptides in silico such as IR, AL, and SL dipeptides during sequential enzymatic digestion with pepsin and trypsin. Since the majority of bioactivity investigations on black cumin seeds have been related to black cumin oil and its oil soluble components, the structure and bioactivities of black cumin proteins deserve further research.


Assuntos
Nigella sativa/metabolismo , Peptídeos/análise , Proteínas de Plantas/análise , Proteoma , Eletroforese em Gel Bidimensional , Peso Molecular , Proteômica , Sementes/metabolismo , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Tripsina/metabolismo
15.
Toxicol Lett ; 313: 150-158, 2019 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-31276768

RESUMO

Ochratoxin A (OTA), one of the most abundant food-contaminating mycotoxins, is a possible carcinogen to humans. We previously demonstrated that long-term (40 weeks) OTA exposure induces the malignant transformation of human gastric epithelium cells (GES-1) in vitro. However, the specific mechanism underlying OTA-induced gastric carcinogenesis is complex. In the present study, we used 2-DE and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI/TOF MS) combined with bioinformatics and immunoblotting to investigate the differentially expressed proteins between GES-1 and OTA-malignant transformed GES-1 cells (OTA-GES-1T cells) in vitro. We found that four differentially expressed proteins were identified after malignant transformation, including actin, cytoplasmic 1 (ACTB), F-actin-capping protein subunit alpha-1 (CAPZA1), Annexin A3 (ANXA3), thioredoxin peroxidase B from red blood cells (TPx-B) and Fibrinogen beta B (Fibrinogen ß). Among the differentially expressed proteins, the effect of Annexin A3 was analyzed by MTT assay, western blot, cell cycle analysis, wound healing assay, Transwell assay, and colony formation assay in OTA-GES-1T cells. The results showed that inhibition of Annexin A3 by siRNA effectively prevented the proliferation, migration, and invasion abilities of OTA-GES-1T cells. Collectively, the results of this study will guide future research on OTA carcinogenicity.


Assuntos
Anexina A3/metabolismo , Carcinógenos/toxicidade , Transformação Celular Neoplásica/induzido quimicamente , Células Epiteliais/efeitos dos fármacos , Mucosa Gástrica/efeitos dos fármacos , Ocratoxinas/toxicidade , Neoplasias Gástricas/induzido quimicamente , Anexina A3/genética , Western Blotting , Linhagem Celular , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/metabolismo , Transformação Celular Neoplásica/patologia , Biologia Computacional , Eletroforese em Gel Bidimensional , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Mucosa Gástrica/metabolismo , Mucosa Gástrica/patologia , Humanos , Invasividade Neoplásica , Proteômica/métodos , Transdução de Sinais/efeitos dos fármacos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Neoplasias Gástricas/genética , Neoplasias Gástricas/metabolismo , Neoplasias Gástricas/patologia
16.
Molecules ; 24(13)2019 Jun 29.
Artigo em Inglês | MEDLINE | ID: mdl-31261846

RESUMO

Honey is a natural sweetener composed mostly of sugars, but it contains also pollen grains, proteins, free amino acids, and minerals. The amounts and proportions of these components depend on the honey type and bee species. Despite the low content of honey protein, they are becoming a popular study object, and have recently been used as markers of the authenticity and quality of honey. Currently, the most popular methods of protein isolation from honey are dialysis against distilled water, lyophilization of dialysate, or various precipitation protocols. In this work, we propose a new method based on saturated phenol. We tested it on three popular polish honey types and we proved its compatibility with both 1D and 2D polyacrylamide gel electrophoresis (PAGE) and MS (mass spectrometry) techniques. The elaborated technique is also potentially less expensive and less time-consuming than other previously described methods, while being equally effective.


Assuntos
Mel/análise , Fenóis/química , Proteínas de Plantas/isolamento & purificação , Brassica napus/metabolismo , Eletroforese em Gel Bidimensional , Fagopyrum/metabolismo , Polônia , Robinia/metabolismo
17.
Methods Mol Biol ; 1934: 21-32, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31256370

RESUMO

Posttranslational modifications (PTMs) are key to the regulation of functional activities of proteins. Quantitative and qualitative information about PTM stages of proteins is crucial for the discovery of disease biomarkers. Fluorescent dyes specifically staining protein PTMs such as phosphorylation and glycosylation enable the specific detection of protein regulations taking place with respect to these modifications. Activity and molecular interactions of many proteins are determined by their extent of phosphorylation. In our search for biomarkers of neurodegenerative diseases such as multiple sclerosis (MS), using an animal model, experimental autoimmune encephalomyelitis (EAE), we have applied the phosphorylation-specific fluorescent dye, ProQ Diamond, to study changes taking place in the phosphoproteome. Subsequent colloidal Coomassie staining of the same gels detects the changes at the whole proteome level. We have detected many changes taking place in the CNS tissue of the EAE animals at the whole proteome as well as at the phosphoproteome level resulting in valuable insights into the pathophysiological mechanism of EAE and MS.


Assuntos
Eletroforese em Gel Bidimensional , Corantes Fluorescentes , Processamento de Proteína Pós-Traducional , Coloração e Rotulagem , Animais , Espectrometria de Massas , Peptídeos , Fosfoproteínas
18.
Parasit Vectors ; 12(1): 339, 2019 Jul 10.
Artigo em Inglês | MEDLINE | ID: mdl-31292008

RESUMO

BACKGROUND: The primary cause of parasitic gastroenteritis in small ruminants in temperate regions is the brown stomach worm, Teladorsagia circumcincta. Host immunity to this parasite is slow to develop, consistent with the ability of T. circumcincta to suppress the host immune response. Previous studies have shown that infective fourth-stage T. circumcincta larvae produce excretory-secretory products that are able to modulate the host immune response. The objective of this study was to identify immune modulatory excretory-secretory proteins from populations of fourth-stage T. circumcincta larvae present in two different host-niches: those associated with the gastric glands (mucosal-dwelling larvae) and those either loosely associated with the mucosa or free-living in the lumen (lumen-dwelling larvae). RESULTS: In this study excretory-secretory proteins from mucosal-dwelling and lumen-dwelling T. circumcincta fourth stage larvae were analysed using comparative 2-dimensional gel electrophoresis. A total of 17 proteins were identified as differentially expressed, with 14 proteins unique to, or enriched in, the excretory-secretory proteins of mucosal-dwelling larvae. One of the identified proteins, unique to mucosal-dwelling larvae, was a putative peroxiredoxin (T. circumcincta peroxiredoxin 1, Tci-Prx1). Peroxiredoxin orthologs from the trematode parasites Schistosoma mansoni and Fasciola hepatica have previously been shown to alternatively activate macrophages and play a key role in promoting parasite induced Th2 type immunity. Here we demonstrate that Tci-Prx1 is expressed in all infective T. circumcincta life-stages and, when produced as a recombinant protein, has peroxidase activity, whereby hydrogen peroxide (H2O2) is reduced and detoxified. Furthermore, we use an in vitro macrophage stimulation assay to demonstrate that, unlike peroxiredoxins from trematode parasites Schistosoma mansoni and Fasciola hepatica, Tci-Prx1 is unable to alternatively activate murine macrophage cells. CONCLUSIONS: In this study, we identified differences in the excretory-secretory proteome of mucosal-dwelling and lumen-dwelling infective fourth-stage T. circumcincta larvae, and demonstrated the utility of this comparative proteomic approach to identify excretory-secretory proteins of potential importance for parasite survival and/or host immune modulation.


Assuntos
Proteínas de Helminto/metabolismo , Peroxirredoxinas/metabolismo , Trichostrongyloidea/metabolismo , Animais , Eletroforese em Gel Bidimensional , Proteínas de Helminto/genética , Interações Hospedeiro-Parasita , Peróxido de Hidrogênio/metabolismo , Enteropatias Parasitárias , Larva/imunologia , Larva/metabolismo , Camundongos , Membrana Mucosa/parasitologia , Peroxirredoxinas/genética , Proteoma/análise , Proteômica , Ovinos/parasitologia , Doenças dos Ovinos/parasitologia , Trichostrongyloidea/imunologia
19.
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
20.
Anal Bioanal Chem ; 411(20): 5115-5126, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31152220

RESUMO

Despite technological advances, two-dimensional electrophoresis (2DE) of biological fluids, such as vitreous, remains a major challenge. In this study, artificial neural network was applied to optimize the recovery of vitreous proteins and its detection by 2DE analysis through the combination of several solubilizing agents (CHAPS, Genapol, DTT, IPG buffer), temperature, and total voltage. The highest protein recovery (94.9% ± 4.5) was achieved using 4% (w/v) CHAPS, 0.1% (v/v) Genapol, 20 mM DTT, and 2% (v/v) IPG buffer. Two iterations were required to achieve an optimized response (580 spots) using 4% (w/v) CHAPS, 0.2% (v/v) Genapol, 60 mM DTT, and 0.5% (v/v) IPG buffer at 35 kVh and 25 °C, representing a 2.4-fold improvement over the standard initial conditions of the experimental design. The analysis of depleted vitreous using the optimized protocol resulted in an additional 1.3-fold increment in protein detection over the optimal output, with an average of 761 spots detected in vitreous from different vitreoretinopathies. Our results clearly indicate the importance of combining the appropriate amount of solubilizing agents with a suitable control of the temperature and voltage to obtain high-quality gels. The high-throughput of this model provides an effective starting point for the optimization of 2DE protocols. This experimental design can be adapted to other types of matrices. Graphical abstract.


Assuntos
Eletroforese em Gel Bidimensional/métodos , Proteômica/métodos , Idoso , Idoso de 80 Anos ou mais , Feminino , Humanos , Masculino , Pessoa de Meia-Idade
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