Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 22.301
Filtrar
1.
Food Chem ; 334: 127484, 2021 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-32711263

RESUMO

This study investigated the soymilk coagulation induced by fermented yellow whey (FYW), which is extensively used as a natural tofu coagulant in China. The aggregations involving proteins and isoflavone particles caused by FYW were analyzed using the proteomic technology and high-performance liquid chromatography, respectively. As indicated, the FYW-induced coagulation of soy proteins mainly occurred at pH 5.80-5.90. When the pH of soymilk decreased, the 7S ß, 11S A3 and some of 11S A1a subunits and SBP, Bd, lectin and TA aggregated the earliest, and later did the 11S A4, other 11S A1a, 11S A2 and 11S A1b subunits. The 7S α and α' subunits and TB showed an obvious delay in aggregation. Moreover, isoflavones in the form of aglycones were more likely to coprecipitate with proteins, compared with glycosides. These results could provide an important reference and assistance for future research on the development of traditional FYW-tofu.


Assuntos
Isoflavonas/análise , Lactobacillales/crescimento & desenvolvimento , Agregados Proteicos/fisiologia , Leite de Soja/química , Proteínas de Soja/análise , Soro do Leite/química , Reatores Biológicos , Cromatografia Líquida de Alta Pressão , Eletroforese em Gel Bidimensional , Concentração de Íons de Hidrogênio , Proteômica , Leite de Soja/metabolismo , Soro do Leite/metabolismo
2.
PLoS One ; 15(10): e0229430, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33104727

RESUMO

Many compounds have the potential to harm pancreatic beta-cells; organochlorine pollutants belong to those compounds. In this work, we aimed to find markers of acute toxicity of p,p'-DDT exposure among proteins expressed in NES2Y human pancreatic beta-cells employing 2-D electrophoresis. We exposed NES2Y cells to a high concentration (150 µM, LC96 after 72 hours) of p,p'-DDT for 24 and 30 hours and determined proteins with changed expression using 2-D electrophoresis. We have found 22 proteins that changed their expression. They included proteins involved in ER stress (GRP78, and endoplasmin), mitochondrial proteins (GRP75, ECHM, IDH3A, NDUS1, and NDUS3), proteins involved in the maintenance of the cell morphology (EFHD2, TCPA, NDRG1, and ezrin), and some other proteins (HNRPF, HNRH1, K2C8, vimentin, PBDC1, EF2, PCNA, biliverdin reductase, G3BP1, FRIL, and HSP27). The proteins we have identified may serve as indicators of p,p'-DDT toxicity in beta-cells in future studies, including long-term exposure to environmentally relevant concentrations.


Assuntos
Biomarcadores/metabolismo , DDT/toxicidade , Células Secretoras de Insulina/citologia , Proteômica/métodos , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Eletroforese em Gel Bidimensional , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Células Secretoras de Insulina/efeitos dos fármacos , Células Secretoras de Insulina/metabolismo , Espectrometria de Massas
3.
BMC Bioinformatics ; 21(1): 376, 2020 Aug 31.
Artigo em Inglês | MEDLINE | ID: mdl-32867673

RESUMO

BACKGROUND: Two-dimensional gel electrophoresis (2-DGE) is a commonly used tool for proteomic analysis. This gel-based technique separates proteins in a sample according to their isoelectric point and molecular weight. 2-DGE images often present anomalies due to the acquisition process, such as: diffuse and overlapping spots, and background noise. This study proposes a joint pre-processing framework that combines the capabilities of nonlinear filtering, background correction and image normalization techniques for pre-processing 2-DGE images. Among the most important, joint nonlinear diffusion filtering, adaptive piecewise histogram equalization and multilevel thresholding were evaluated using both synthetic data and real 2-DGE images. RESULTS: An improvement of up to 46% in spot detection efficiency was achieved for synthetic data using the proposed framework compared to implementing a single technique of either normalization, background correction or filtering. Additionally, the proposed framework increased the detection of low abundance spots by 20% for synthetic data compared to a normalization technique, and increased the background estimation by 67% compared to a background correction technique. In terms of real data, the joint pre-processing framework reduced the false positives up to 93%. CONCLUSIONS: The proposed joint pre-processing framework outperforms results achieved with a single approach. The best structure was obtained with the ordered combination of adaptive piecewise histogram equalization for image normalization, geometric nonlinear diffusion (GNDF) for filtering, and multilevel thresholding for background correction.


Assuntos
Eletroforese em Gel Bidimensional/métodos , Bases de Dados de Proteínas , Humanos , Processamento de Imagem Assistida por Computador , Proteínas/análise , Proteômica/métodos , Razão Sinal-Ruído
4.
Exp Parasitol ; 218: 107999, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-32956649

RESUMO

Angiostrongylus cantonensis is the main causative agent of eosinophilic meningoencephalitis (EoM) in humans. Molecular diagnostic methods are essential since the identification of larvae in cerebrospinal fluid (CSF) is extremely rare. To date, the detection of a 31 kDa antigen by Western blotting has been the primary immunodiagnostic method for EoM caused by A. cantonensis. However, cross-reactivity with other parasites has been observed. Therefore, we conducted a comparative analysis using sera from individuals with angiostrongyliasis. We also characterized proteins isolated from different cellular sources of A. cantonensis, Toxocara canis, Schistosoma mansoni, and Strongyloides stercoralis with mass spectrometry. A total of 115 cross-reactive proteins were identified. Three of these proteins, heat shock protein, an intermediate filament protein, and galectin 1, represent potential markers for cross-reactivity. In addition, synthetic peptides were generated from previously identified diagnostic targets and tested against sera from individuals infected with several other parasites. As a result, two other markers of cross-reactivity were identified: peptide #4 derived from the 14-3-3 protein and peptide #12 derived from the Lec-5 protein. In contrast, 34 proteins were exclusively present in the Angiostrongylus extracts and represent promising diagnostic molecules for specific identification of A. cantonensis infection. In particular, cytochrome oxidase subunit I is of great interest as a possible immunodiagnostic target for angiostrongyliasis.


Assuntos
Angiostrongylus cantonensis/imunologia , Antígenos de Helmintos/imunologia , Proteínas de Helminto/imunologia , Meningoencefalite/diagnóstico , Meningoencefalite/parasitologia , Infecções por Strongylida/diagnóstico , Sequência de Aminoácidos , Angiostrongylus cantonensis/química , Animais , Antígenos de Helmintos/sangue , Antígenos de Helmintos/química , Antígenos de Helmintos/isolamento & purificação , Western Blotting , Sequência Conservada , Reações Cruzadas , Eletroforese , Eletroforese em Gel Bidimensional , Proteínas de Helminto/química , Proteínas de Helminto/isolamento & purificação , Humanos , Imunoensaio , Testes Imunológicos , Espectrometria de Massas , Meningoencefalite/imunologia , Infecções por Strongylida/imunologia , Infecções por Strongylida/parasitologia
5.
PLoS One ; 15(7): e0227466, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32678822

RESUMO

Trans-methylation reactions are intrinsic to cellular metabolism in all living organisms. In land plants, a range of substrate-specific methyltransferases catalyze the methylation of DNA, RNA, proteins, cell wall components and numerous species-specific metabolites, thereby providing means for growth and acclimation in various terrestrial habitats. Trans-methylation reactions consume vast amounts of S-adenosyl-L-methionine (SAM) as a methyl donor in several cellular compartments. The inhibitory reaction by-product, S-adenosyl-L-homocysteine (SAH), is continuously removed by SAH hydrolase (SAHH), which essentially maintains trans-methylation reactions in all living cells. Here we report on the evolutionary conservation and post-translational control of SAHH in land plants. We provide evidence suggesting that SAHH forms oligomeric protein complexes in phylogenetically divergent land plants and that the predominant protein complex is composed by a tetramer of the enzyme. Analysis of light-stress-induced adjustments of SAHH in Arabidopsis thaliana and Physcomitrella patens further suggests that regulatory actions may take place on the levels of protein complex formation and phosphorylation of this metabolically central enzyme. Collectively, these data suggest that plant adaptation to terrestrial environments involved evolution of regulatory mechanisms that adjust the trans-methylation machinery in response to environmental cues.


Assuntos
Adenosil-Homocisteinase/metabolismo , Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimologia , Evolução Molecular , Adenosil-Homocisteinase/classificação , Adenosil-Homocisteinase/genética , Sequência de Aminoácidos , Proteínas de Arabidopsis/classificação , Proteínas de Arabidopsis/genética , Eletroforese em Gel Bidimensional , Focalização Isoelétrica , Luz , Filogenia , Folhas de Planta/enzimologia , Processamento de Proteína Pós-Traducional/efeitos da radiação , RNA Mensageiro/metabolismo , Alinhamento de Sequência , Estresse Fisiológico
6.
Poult Sci ; 99(5): 2775-2784, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-32359615

RESUMO

Egg yolk is an important source of nutrients for embryo development. In this study, the egg yolk protein composition at 0, 10, and 18 D of incubation was analyzed by 2-dimensional gel electrophoresis (2-DE) combined with matrix-assisted laser desorption/ionization time-of-flight tandem mass spectrometry. A significant difference in the abundance of 42 protein spots representing 12 proteins were identified (P < 0.05). The 2-DE gel image analysis exhibited that the molecular weight (MW) of 29 protein spots was lower than their theoretical value, in which 14 vitellogenin (VTG) fragments were lower than the theoretical value. There were 13 protein spots showed a higher MW including 5 ovotransferrins with MW of 87.2 kDa. The gene ontology enrichment analysis suggested that biological process of the differentially expressed proteins were mainly involved in lipid transport and lipid localization at 10 and 18 D of incubation. The molecular function of the differentially expressed proteins was involved in nutrient reservoir activity, lipid transporter activity, and antigen binding at 10 D of incubation. At 18 D of incubation, the differentially expressed proteins mainly participated in nutrient reservoir activity and substrate-specific transporter activity. The high abundance of VTGs at 10 D of incubation might participate in lipid localization and lipid transportation to facilitate the yolk nutrient transport to embryo. The low expression of ovotransferrins at 10 D of incubation indicated the chondrogenesis of embryo.


Assuntos
Proteínas Aviárias/metabolismo , Embrião de Galinha/crescimento & desenvolvimento , Galinhas/metabolismo , Proteínas do Ovo/metabolismo , Proteoma/metabolismo , Animais , Galinhas/crescimento & desenvolvimento , Eletroforese em Gel Bidimensional/veterinária , Fertilização , Proteômica , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/veterinária , Espectrometria de Massas em Tandem/veterinária
7.
Curr Protoc Plant Biol ; 5(2): e20107, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32250554

RESUMO

Protein-protein interactions, including oligomerization, are involved in regulation of many cellular processes. Unfortunately, many proteins are expressed at a very low level in vivo, making it challenging to observe oligomerization by size-exclusion chromatography, also known as gel filtration. In this protocol, we present detailed steps to perform blue native polyacrylamide gel electrophoresis (BN-PAGE), a method to study protein oligomers in plants. The article describes protein sample preparation from transgenic Arabidopsis thaliana and running a BN-PAGE gel followed by direct western blotting or, alternatively, two-dimensional sodium dodecyl sulfide-polyacrylamide gel electrophoresis (2D SDS-PAGE). This protocol will be helpful for new researchers conducting related experiments to analyze stable protein interactions including homo- or hetero-oligomerization in plants. © 2020 The Authors.


Assuntos
Proteínas de Membrana , Western Blotting , Eletroforese em Gel Bidimensional , Eletroforese em Gel de Poliacrilamida , Eletroforese em Gel de Poliacrilamida Nativa
8.
Artigo em Inglês | MEDLINE | ID: mdl-32229427

RESUMO

Sclerotinia sclerotiorum is a necrotrophic phytopathogen that has been the subject of several scientific research efforts. Despite the numerous research efforts its proteome remains understudied. This study aimed to identify proteins produced by S. sclerotiorum, thereby increasing the current proteomic knowledge base. Total proteins were extracted from mycelia scraped from five-day old cultures of S. sclerotiorum. The extracted proteins were separated by sodium dodecyl polyacrylamide gel electrophoresis (SDS-PAGE) and were annotated using the AB Sciex TripleToF 6600 mass spectrometer. Exactly 1471 proteins were reproducibly present in all three replicates. All proteins detected were classified based on their molecular and biological functions. To the knowledge of the authors, this is the most comprehensive proteomic study on S. sclerotiorum (judging by the high number of proteins identified).


Assuntos
Ascomicetos/química , Proteínas Fúngicas/análise , Proteoma/análise , Ascomicetos/metabolismo , Dissulfetos/química , Eletroforese em Gel Bidimensional , Ensaios de Triagem em Larga Escala , Espectrometria de Massas , Micélio/química , Oxirredução , Proteômica
9.
Artigo em Inglês | MEDLINE | ID: mdl-32278290

RESUMO

Subunit structures of proteins are essential for their properties and functions, but there is a lack of method for global detection of the status of proteins being monomers or homo-oligomers. In this work, we report on a new method to simultaneously speculate hundreds of monomeric or homo-oligomeric subunit structures of cellular proteins, based on in-depth analysis of native 2D protein maps. Previously we have reported on the analysis of soluble proteins of human bronchial muscle cells (HBSMC) by combining nondenaturing 2DE, grid gel-cutting and quantitative LC-MS/MS. Totally 4323 proteins were detected and for each protein the quantity distribution on the gel was reconstructed as a native 2D map. In this work, this large dataset of maps were further mined with bioinformatic analysis. The native 2D maps of 1901 HBSMC proteins that were detected in at least five out of the grid-cut 972 gel squares were examined and 658 proteins that showed one major quantity-peak distribution were subjected to further analysis. After excluding those that mainly formed hetero-oligomeric structures, the monomeric or homo-oligomeric subunit structures of 505 proteins were speculated. The quotient of the apparent molecular mass of the quantity-peak position on the native 2D map divided by the theoretical molecular mass was calculated for each protein, to speculate the number of monomers which constituted its subunit structure. The suggested composition was then compared with the "Subunit structure" record of the protein in UniProtKB. When the database record included possible interactions with other proteins, their native 2D maps were extracted from the native map dataset, presented together and compared to confirm the prominent subunit structure. With this new approach, the monomeric or homo-oligomeric subunit structures of 401 proteins were speculated. Among them, 162 proteins had the speculated subunit structures coinciding with their database records, and 91 proteins with matched database records as being monomers or homo-oligomers but mismatched at the numbers of the composing monomers. For 148 proteins that did not have database record, their subunit structures were newly speculated. We expect this method, combining nondenaturing 2DE separation with in-depth proteomic and bioinformatic analysis, would suggest a means to achieve large-scale information on monomeric and homo-oligomeric subunit structures of cellular proteins.


Assuntos
Células Musculares/química , Proteínas/química , Proteômica/métodos , Brônquios/citologia , Cromatografia Líquida de Alta Pressão , Bases de Dados de Proteínas , Eletroforese em Gel Bidimensional , Humanos , Modelos Estatísticos , Células Musculares/citologia , Eletroforese em Gel de Poliacrilamida Nativa , Multimerização Proteica , Subunidades Proteicas/química , Espectrometria de Massas em Tandem
10.
Nanotechnology ; 31(23): 235605, 2020 Mar 20.
Artigo em Inglês | MEDLINE | ID: mdl-32125281

RESUMO

Intercalation of drug molecules into synthetic DNA nanostructures formed through self-assembled origami has been postulated as a valuable future method for targeted drug delivery. This is due to the excellent biocompatibility of synthetic DNA nanostructures, and high potential for flexible programmability including facile drug release into or near to target cells. Such favourable properties may enable high initial loading and efficient release for a predictable number of drug molecules per nanostructure carrier, important for efficient delivery of safe and effective drug doses to minimise non-specific release away from target cells. However, basic questions remain as to how intercalation-mediated loading depends on the DNA carrier structure. Here we use the interaction of dyes YOYO-1 and acridine orange with a tightly-packed 2D DNA origami tile as a simple model system to investigate intercalation-mediated loading. We employed multiple biophysical techniques including single-molecule fluorescence microscopy, atomic force microscopy, gel electrophoresis and controllable damage using low temperature plasma on synthetic DNA origami samples. Our results indicate that not all potential DNA binding sites are accessible for dye intercalation, which has implications for future DNA nanostructures designed for targeted drug delivery.


Assuntos
Laranja de Acridina/química , Benzoxazóis/química , DNA/química , Substâncias Intercalantes/química , Compostos de Quinolínio/química , Sítios de Ligação , Eletroforese em Gel Bidimensional , Microscopia de Força Atômica , Microscopia de Fluorescência , Modelos Moleculares , Nanoestruturas/química , Conformação de Ácido Nucleico , Imagem Individual de Molécula
11.
BMC Bioinformatics ; 21(1): 117, 2020 Mar 19.
Artigo em Inglês | MEDLINE | ID: mdl-32192430

RESUMO

BACKGROUND: Two-dimensional electrophoresis (2DE) is one of the most widely applied techniques in comparative proteomics. The basic task of 2DE is to identify differential protein expression by quantitative analysis of 2DE images. To reduce the errors of spot quantification in 2DE images, a novel brightness correction method based on gradient interval histogram (GIH) is proposed in this paper. RESULTS: First, GIH equalization is proposed to enhance the protein spot edges, especially the weak protein spots in the 2DE image. Second, to eliminate the overall brightness shift, GIH matching is applied to the 2DE images that need to be compared. Finally, the proposed method is verified by subjective quality evaluation and quantitative analysis of protein spots in real 2DE images. CONCLUSIONS: The experimental results show that the average error of the quantification of corresponding protein spots in the resulting image pairs is less than 3%, which is significantly superior to that of the existing methods.


Assuntos
Eletroforese em Gel Bidimensional , Proteínas/análise , Proteômica/métodos , Algoritmos , Processamento de Imagem Assistida por Computador
12.
Int J Infect Dis ; 96: 73-81, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-32087365

RESUMO

INTRODUCTION: Infective endocarditis (IE) has high mortality, partly due to delayed diagnosis. No biomarker can identify IE in patients with fever and clinical picture of infection. To find putative biomarkers we analyzed serum levels of two proteins found in cardiac valves, fibulin-1 (n=696) and osteoprotegerin (n=689) among patients on clinical suspicion of IE. Proteomic analyses were performed in 24 patients with bacteremia, 12 patients with definite IE and 12 patients with excluded IE. METHODS: Fibulin-1 and osteoprotegerin were studied by enzyme linked immunosorbent assay (ELISA). Proteomic analyses were conducted by 2-dimensional polyacrylamid gel electrophoresis (2D-PAGE) and label-free quantitative liquid chromatography - tandem mass spectrometry (LFQ LC-MS/MS). Controls for 2D 2D-PAGE and LFQ LC-MS/MS had bacteremia and excluded IE. RESULTS: Osteoprotegerin levels were significantly increased in IE patients compared with non-IE patients. Fibulin-1 showed no difference. 2D-PAGE showed significant differences of 6 proteoforms: haptoglobin, haptoglobin-related protein, α-2-macroglobulin, apolipoprotein A-I and ficolin-3. LFQ LC-MS/MS analysis revealed significant level changes of 7 proteins: apolipoprotein L1, complement C1q subcomponent B and C, leukocyte immunoglobulin-like receptor subfamily A member 3, neuropilin-2, multimerin-1 and adiponectin. CONCLUSIONS: The concentration changes in a set of proteoforms/proteins suggest that stress and inflammation responses are perturbed in patients with IE compared to patients with bacteremia without IE.


Assuntos
Endocardite/sangue , Proteoma/metabolismo , Bacteriemia/sangue , Biomarcadores/sangue , Proteínas de Ligação ao Cálcio/sangue , Cromatografia Líquida , Eletroforese em Gel Bidimensional , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Masculino , Osteoprotegerina/sangue , Proteômica , Espectrometria de Massas em Tandem
13.
Parasit Vectors ; 13(1): 93, 2020 Feb 21.
Artigo em Inglês | MEDLINE | ID: mdl-32085718

RESUMO

BACKGROUND: Coccidiosis is caused by Eimeria spp. and can result in severe economic losses to the global poultry industry. Due to anticoccidial drug resistance rapidly developing in the parasites and drug residues in poultry products, efficacious and safe alternative coccidia control measures are needed. The objective of the present study was to identify common protective antigens which may be used as vaccine candidates in the development of subunit, multivalent, cross-protective vaccines against most of the economically important Eimeria species. METHODS: Whole sporozoite proteins of Eimeria acervulina were prepared and analyzed by 2-dimensional gel electrophoresis (2-DE) followed by western blotting using immune sera specific to E. tenella, E. acervulina, or E. necatrix. The protein spots detected by all three immune sera were then excised from the preparative gel and protein ID was performed by MALDI-TOF-MS/MS. RESULTS: Approximately 620 E. acervulina sporozoite protein spots were demonstrated by 2-DE with silver staining, among which 23 protein spots were recognized by immune sera specific to all three Eimeria species. The results showed that 21 putative E. acervulina proteins were identified, which include proteins with known enzymatic properties, and those which are involved in protein translation, transport and trafficking, and ribosomal biogenesis and functions. There is one protein which may be involved in transcription and one heat-shock protein. Two proteins contain predicted domains, but with no apparent functions known. There were 2 protein spots which had no detectable proteins. None of the proteins has a predicted signal peptide or a transmembrane domain; however, 6 of the 21 putative proteins were predicted to be potentially secretory through the non-classical pathway. CONCLUSIONS: Our study identified a diverse group of antigens immunologically common to all three Eimeria species, none of which was previously characterized and tested as a vaccine candidate. Further research on immunogenicity and cross-protective potential of these individual proteins as vaccine candidates will aid the development of vaccines against the most common and pathogenic Eimeria spp.


Assuntos
Antígenos de Protozoários/química , Coccidiose/veterinária , Eimeria/imunologia , Doenças das Aves Domésticas/parasitologia , Animais , Antígenos de Protozoários/imunologia , Galinhas , Coccidiose/imunologia , Coccidiose/parasitologia , Reações Cruzadas , Eimeria/química , Eimeria/classificação , Eimeria/fisiologia , Eletroforese em Gel Bidimensional , Soros Imunes/imunologia , Doenças das Aves Domésticas/sangue , Proteômica , Proteínas de Protozoários/química , Proteínas de Protozoários/imunologia , Espectrometria de Massas em Tandem
14.
BMC Res Notes ; 13(1): 60, 2020 Feb 07.
Artigo em Inglês | MEDLINE | ID: mdl-32028996

RESUMO

OBJECTIVE: Liverworts possess historical adaptive strategies for abiotic stresses because they were the first plants that shifted from water to land. Proteomics is a state-of-the-art technique that can capture snapshots of events occurring at the protein level in many organisms. Herein, we highlight the comparison and optimization of an effective protein extraction and precipitation protocol for two-dimensional gel electrophoresis (2-DE) of liverworts. RESULTS: We compared three different protein extraction methods, i.e.,1.5 M Tris-HCl (pH 8.8), 50 mM Tris-HCl (pH 7.5), and polyvinylpolypyrrolidone (PVPP) extraction, followed by three precipitation methods, i.e., 80% ethanol, 80% acetone, and 20% tricholoroacetic acid (TCA)-acetone, in a liverwort Dumortiera hirsuta. Among these methods, 50 mM Tris-HCl (pH 7.5) extraction, followed by 20% TCA-acetone precipitation, appeared to be more suitable for 2-DE. Furthermore, we performed modifications during protein washing, re-solubilization in rehydration buffer and isoelectric focusing (IEF). The modifications provided us better results in terms of protein yield, resolution, spot numbers, and intensities for 2-DE gels of D. hirsuta and other two liverworts, i.e., Marchantia paleacea and Plagiochasma appendiculatum. Furthermore, we randomly selected spots from the 2-DE gel of D. hirsuta and identified using mass spectrometry, which confirms the applicability of this protocol for liverworts proteomics.


Assuntos
Eletroforese em Gel Bidimensional/métodos , Hepatófitas/metabolismo , Proteínas de Plantas/isolamento & purificação , Ecossistema , Células Germinativas Vegetais/metabolismo
15.
J Biotechnol ; 311: 25-34, 2020 Mar 10.
Artigo em Inglês | MEDLINE | ID: mdl-32057784

RESUMO

Myxococcus xanthus DK1622 is known as a proficient producer of different kinds of secondary metabolites (SM) with various biological activities, including myxovirescin A, myxalamide A, myxochromide A and DKxanthene. Low production of SM in the wild type bacteria makes searching for production optimization methods highly desirable. Identification and induction of endogenous key molecular feature(s) regulating the production level of the metabolites remain promising, while heterologous expression of the biosynthetic genes is not always efficient because of various complicating factors including codon usage bias. This study established proteomic and molecular approaches to elucidate the regulatory roles of the ROK regulatory protein in the modification of secondary metabolite biosynthesis. Interestingly, the results revealed that rok inactivation significantly reduced the production of the SM and also changed the motility in the bacteria. Electrophoretic mobility shift assay using purified ROK protein indicated a direct enhancement of the promoters encoding transcription of the DKxanthene, myxochelin A, and myxalamide A biosynthesis machinery. Comparative proteomic analysis by two-dimensional fluorescence difference in-gel electrophoresis (2D-DIGE) was employed to identify the protein profiles of the wild type and rok mutant strains during early and late logarithmic growth phases of the bacterial culture. Resulting data demonstrated overall 130 differently altered proteins by the effect of the rok gene mutation, including putative proteins suspected to be involved in transcriptional regulation, carbohydrate metabolism, development, spore formation, and motility. Except for a slight induction seen in the production of myxovirescin A in a rok over-expression background, no changes were found in the formation of the other SM. From the outcome of our investigation, it is possible to conclude that ROK acts as a pleiotropic regulator of secondary metabolite formation and development in M. xanthus, while its direct effects still remain speculative. More experiments are required to elucidate in detail the variable regulation effects of the protein and to explore applicable approaches for generating valuable SM in this bacterium.


Assuntos
Proteínas de Bactérias/metabolismo , Myxococcus xanthus/metabolismo , Proteínas de Bactérias/genética , Eletroforese em Gel Bidimensional , Regulação da Expressão Gênica/genética , Regulação da Expressão Gênica/fisiologia , Regulação Bacteriana da Expressão Gênica/genética , Regulação Bacteriana da Expressão Gênica/fisiologia , Lisina/análogos & derivados , Lisina/metabolismo , Myxococcus xanthus/genética , Polienos/metabolismo , Regiões Promotoras Genéticas/genética , Proteômica/métodos , Metabolismo Secundário/genética , Metabolismo Secundário/fisiologia
16.
BMC Genomics ; 21(1): 90, 2020 Jan 29.
Artigo em Inglês | MEDLINE | ID: mdl-31996138

RESUMO

BACKGROUND: Truffles are symbiotic fungi that develop underground in association with plant roots, forming ectomycorrhizae. They are primarily known for the organoleptic qualities of their hypogeous fruiting bodies. Primarily, Tuber magnatum Pico is a greatly appreciated truffle species mainly distributed in Italy and Balkans. Its price and features are mostly depending on its geographical origin. However, the genetic variation within T. magnatum has been only partially investigated as well as its adaptation to several environments. RESULTS: Here, we applied an integrated omic strategy to T. magnatum fruiting bodies collected during several seasons from three different areas located in the North, Center and South of Italy, with the aim to distinguish them according to molecular and biochemical traits and to verify the impact of several environments on these properties. With the proteomic approach based on two-dimensional electrophoresis (2-DE) followed by mass spectrometry, we were able to identify proteins specifically linked to the sample origin. We further associated the proteomic results to an RNA-seq profiling, which confirmed the possibility to differentiate samples according to their source and provided a basis for the detailed analysis of genes involved in sulfur metabolism. Finally, geographical specificities were associated with the set of volatile compounds produced by the fruiting bodies, as quantitatively and qualitatively determined through proton transfer reaction-mass spectrometry (PTR-MS) and gas-chromatography-mass spectrometry (GC-MS). In particular, a partial least squares-discriminant analysis (PLS-DA) model built from the latter data was able to return high confidence predictions of sample source. CONCLUSIONS: Results provide a characterization of white fruiting bodies by a wide range of different molecules, suggesting the role for specific compounds in the responses and adaptation to distinct environments.


Assuntos
Adaptação Biológica , Meio Ambiente , Genômica , Metabolômica , Proteômica , Saccharomycetales/genética , Saccharomycetales/metabolismo , Biologia Computacional , Eletroforese em Gel Bidimensional , Cromatografia Gasosa-Espectrometria de Massas , Genômica/métodos , Metabolômica/métodos , Proteômica/métodos , Transcriptoma , Compostos Orgânicos Voláteis
17.
Mol Biol Rep ; 47(3): 1553-1561, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31925645

RESUMO

There is disputable on the role of nitrilase-like 2 (NIT2) in cancer. Its expression and its relationship with clinicopathological features in tongue squamous cell carcinoma (TSCC) are not yet clear. The purpose of this study is to investigate the expression of NIT2 in TSCC and its correlation with clinicopathological characteristics in TSCC patients. Through proteomic identification, we found that the protein NIT2 was related to the development of TSCC. q-PCR, western blot and immunohistochemistry techniques were applied to detect the expression of NIT2 in TSCC. The relationship between the expression of NIT2 and clinicopathological features was analyzed by Chi square tests. The results showed the expression of NIT2 in TSCC was significantly higher than that in normal tongue tissues (p < 0.05). Univariate and multivariate analysis showed that the positive expression of NIT2 and N classification were associated with decreased disease-free survival rate (DFS) and overall survival (OS) (p < 0.05). The results suggested that NIT2 is overexpressed in TSCC and NIT2 may be a potential therapeutic target for TSCC.


Assuntos
Aminoidrolases/metabolismo , Carcinoma de Células Escamosas/metabolismo , Proteoma/metabolismo , Proteômica/métodos , Neoplasias da Língua/metabolismo , Aminoidrolases/genética , Western Blotting , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/patologia , Estudos de Coortes , Eletroforese em Gel Bidimensional , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Imuno-Histoquímica , Estimativa de Kaplan-Meier , Masculino , Pessoa de Meia-Idade , Prognóstico , Proteoma/genética , Análise Serial de Tecidos , Neoplasias da Língua/genética , Neoplasias da Língua/patologia
18.
Methods Mol Biol ; 2119: 1-13, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31989510

RESUMO

Gel electrophoresis of DNA is one of the most frequently used techniques in molecular biology. Typically, it is used in the following: the analysis of in vitro reactions and purification of DNA fragments, analysis of PCR reactions, characterization of enzymes involved in DNA reactions, and sequencing. With some ingenuity gel electrophoresis of DNA is also used for the analysis of cellular biochemical reactions. For example, DNA breaks that accumulate in cells are analyzed by the comet assay and pulsed-field gel electrophoresis (PFGE). Furthermore, DNA replication intermediates are analyzed with two-dimensional (2D) gel electrophoresis. Moreover, several new methods for analyzing various chromosomal functions in cells have been developed. In this chapter, a brief introduction to these is given.


Assuntos
DNA , Eletroforese em Gel de Campo Pulsado , Eletroforese em Gel Bidimensional , DNA/análise , DNA/química , Quebras de DNA , Replicação do DNA , Eletroforese em Gel de Ágar
19.
Methods Mol Biol ; 2119: 15-24, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31989511

RESUMO

Agarose gel electrophoresis is one of the most straightforward techniques that can be used to differentiate between topoisomers of closed circular DNA molecules. Generally, the products of reactions that monitor the interconversion of DNA between negatively supercoiled and relaxed DNA or positively supercoiled and relaxed DNA can be resolved by one-dimensional gel electrophoresis. However, in more complex reactions that contain both positively and negatively supercoiled DNA, one-dimensional resolution is insufficient. In these cases, a second dimension of gel electrophoresis is necessary. This chapter describes the technique of two-dimensional agarose gel electrophoresis and how it can be used to resolve a spectrum of DNA topoisomers.


Assuntos
DNA Topoisomerases/análise , DNA Super-Helicoidal/análise , Eletroforese em Gel Bidimensional , Eletroforese em Gel de Ágar
20.
Methods Mol Biol ; 2119: 25-42, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31989512

RESUMO

The study of mitochondrial DNA (mtDNA) integrity and how replication, transcription, repair, and degradation maintain mitochondrial function has been hampered due to the inability to identify mtDNA structural forms. Here we describe the use of 2D intact mtDNA agarose gel electrophoresis, or 2D-IMAGE, to identify up to 25 major mtDNA topoisomers such as double-stranded circular mtDNA (including supercoiled molecules, nicked circles, and multiple catenated species) and various forms containing single-stranded DNA (ssDNA) structures. Using this modification of a classical 1D gel electrophoresis procedure, many of the identified mtDNA species have been associated with mitochondrial replication, damage, deletions, and possibly transcription. The increased resolution of 2D-IMAGE allows for the identification and monitoring of novel mtDNA intermediates to reveal alterations in genome replication, transcription, repair, or degradation associated with perturbations during mitochondrial stress.


Assuntos
Replicação do DNA , DNA Mitocondrial , Eletroforese em Gel Bidimensional , Mitocôndrias/metabolismo , Animais , DNA Mitocondrial/análise , DNA Mitocondrial/metabolismo , Eletroforese em Gel de Ágar , Humanos
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA