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1.
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
2.
Exp Parasitol ; 204: 107723, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31299265

RESUMO

Toxoplasmosis, caused by apicomplexan parasite Toxoplasma gondii, is a common food-borne disease in humans. Undercooked meat is a potential source of T. gondii infection. As meat of chicken or rabbit is consumed worldwide, tools such as ELISA for the detection of infection of this parasite in rabbits and chickens are much-needed. To search diagnostic antigens of T. gondii special for rabbits and chickens, we conducted two dimensional electrophoresis (2-DE), Western blotting and mass spectrometry (MS) with T. gondii tachyzoite proteins. When probed with rabbit or chicken anti-T. gondii sera, about 60 positive spots among over 500 visible protein spots were detected. In subsequent mass spectrometric analysis, microneme 4 (MIC4) and a putative rhoptry protein are of diagnositic value among the 13 spots selectively picked from the equivalent gel. This study encourages further validation of these candidate antigens for the development of immunologic tools for the detection of T. gondii infection in chickens and rabbits.


Assuntos
Antígenos de Protozoários/análise , Parasitologia de Alimentos , Carne/parasitologia , Proteínas de Protozoários/imunologia , Toxoplasma/imunologia , Toxoplasmose Animal/diagnóstico , Animais , Western Blotting , Galinhas , Biologia Computacional , Soros Imunes/imunologia , Immunoblotting , Proteínas de Membrana/imunologia , Distribuição Normal , Coelhos , Testes Sorológicos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Espectrometria de Massas em Tandem , Toxoplasmose Animal/imunologia , Eletroforese em Gel Diferencial Bidimensional
3.
Life Sci ; 231: 116541, 2019 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-31216441

RESUMO

AIMS: The most frequent cancers among women worldwide. The mortality of cervical cancer has declined significantly primarily due to the widespread use of Pap smear tests as a screening test and therapeutic vaccination. However, cervical cancer still remains a severe disease among the female population, as the prognosis of metastatic cervical cancer is very poor. KEY METHODS: In this study, we performed 2D-DIGE and MALDI-TOF/TOF MS to analyze differentially expressed proteins between HeLa and invasive HeLa-I5 cells.. KEY FINDINGS: According to our proteomics data, 68 differentially expressed proteins between the HeLa and HeLa-I5 cells were identified. One of these differentially expressed proteins, Progesterone receptor membrane component 1 (PGRMC1), was selected as a candidate for further studies. To correlate the role of PGRMC1 with cellular migration and cancer progression, small interfering RNA (siRNA) was used to knockdown the expression of PGRMC1. Similar function of PGRMC1 was also observed in two other cervical cancer lines, CaSki and ME-180. SIGNIFICANCE: PGRMC1 plays an essential role in regulating cancer progression and metastasis of cervical cancer cells, thus serving as a potential therapeutic target for cervical cancer.


Assuntos
Proteínas de Membrana/metabolismo , Receptores de Progesterona/metabolismo , Neoplasias do Colo do Útero/metabolismo , Linhagem Celular Tumoral , Movimento Celular/fisiologia , Proliferação de Células/fisiologia , Feminino , Células HeLa , Humanos , Invasividade Neoplásica , Proteômica/métodos , RNA Interferente Pequeno/genética , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Eletroforese em Gel Diferencial Bidimensional/métodos , Neoplasias do Colo do Útero/genética , Neoplasias do Colo do Útero/patologia
4.
Medicine (Baltimore) ; 98(25): e16117, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31232959

RESUMO

The study aimed to find novel effect biomarkers for occupational benzene exposure and chronic benzene poisoning (CBP), which might also provide clues to the mechanism of benzene toxicity.We performed a comparative serological proteome analysis between healthy control workers with no benzene exposure, workers with short-term benzene exposure, workers with long-term benzene exposure, and CBP patients using 2D-DIGE and MALDI-TOF-MS. Two of the differentially expressed proteins were then selected to be validated by immune turbidimetric analysis.A total of 10 proteins were found to be significantly altered between different groups. The identified deferentially expressed proteins were classified according to their molecular functions, biological processes, and protein classes. The alteration of 2 important serum proteins among them, apolipoprotein A-I and transthyretin, were further confirmed.Our findings suggest that the identified differential proteins could be used as biomarkers for occupational benzene exposure and CBP, and they may also help elucidate the mechanisms of benzene toxicity.


Assuntos
Benzeno/toxicidade , Proteômica/métodos , Adulto , Análise de Variância , Benzeno/efeitos adversos , Biomarcadores/sangue , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Exposição Ocupacional/efeitos adversos , Exposição Ocupacional/análise , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Eletroforese em Gel Diferencial Bidimensional/métodos
5.
Microb Cell Fact ; 18(1): 93, 2019 May 28.
Artigo em Inglês | MEDLINE | ID: mdl-31138236

RESUMO

BACKGROUND: Polyhydroxyalkanoates (PHAs) have attracted much attention in recent years as natural alternatives to petroleum-based synthetic polymers that can be broadly used in many applications. Pseudomonas putida KT2440 is a metabolically versatile microorganism that is able to synthesize medium-chain-length PHAs (mcl-PHAs). The phenomena that drive mcl-PHAs synthesis and accumulation seems to be complex and are still poorly understood. Therefore, here we determine new insights into cellular responses of Pseudomonas putida KT2440 during biopolymers production using two-dimensional difference gel-electrophoresis (2D-DIGE) followed by MALDI TOF/TOF mass spectrometry. RESULTS: The maximum mcl-PHAs content in Pseudomonas putida KT2440 cells was 24% of cell dry weight (CDW) and was triggered by nitrogen depletion. Proteomic analysis allowed the detection of 150 and 131 protein spots differentially regulated at 24 h and 48 h relative to the cell growth stage (8 h), respectively. From those, we successfully identified 84 proteins that had altered expression at 24 h and 74 proteins at 48 h of the mcl-PHAs synthesis process. The protein-protein interactions network indicated that the majority of identified proteins were functionally linkage. The abundance of proteins involved in carbon metabolism were significantly decreased at 24 h and 48 h of the cultivations. Moreover, proteins associated with ATP synthesis were up-regulated suggesting that the enhanced energy metabolism was necessary for the mcl-PHAs accumulation. Furthermore, the induction of proteins involved in nitrogen metabolism, ribosome synthesis and transport was observed. Our results indicate that mcl-PHAs accumulated in the bacterial cells changed the protein abundance involved in stress response and cellular homeostasis. CONCLUSIONS: The presented data allow us to investigate time-course proteome rearrangement in response to nitrogen limitation and biopolyesters accumulation. Our results have pointed out novel proteins that might take part in cellular responses of mcl-PHA-accumulated bacteria. The study provides an additional knowledge that could be helpful to improve the efficiency of the bioprocess and make it more economically feasible.


Assuntos
Proteínas de Bactérias/metabolismo , Poli-Hidroxialcanoatos/biossíntese , Proteoma/metabolismo , Pseudomonas putida/metabolismo , Carbono/metabolismo , Homeostase , Nitrogênio/metabolismo , Poliésteres/metabolismo , Proteômica/métodos , Estresse Fisiológico , Eletroforese em Gel Diferencial Bidimensional/métodos
6.
Int J Mol Sci ; 20(8)2019 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-30991638

RESUMO

The cerebellum contains a circadian clock, generating internal temporal signals. The daily oscillations of cerebellar proteins were investigated in mice using a large-scale two-dimensional difference in gel electrophoresis (2D-DIGE). Analysis of 2D-DIGE gels highlighted the rhythmic variation in the intensity of 27/588 protein spots (5%) over 24 h based on cosinor regression. Notably, the rhythmic expression of most abundant cerebellar proteins was clustered in two main phases (i.e., midday and midnight), leading to bimodal distribution. Only six proteins identified here to be rhythmic in the cerebellum are also known to oscillate in the suprachiasmatic nuclei, including two proteins involved in the synapse activity (Synapsin 2 [SYN2] and vesicle-fusing ATPase [NSF]), two others participating in carbohydrate metabolism (triosephosphate isomerase (TPI1] and alpha-enolase [ENO1]), Glutamine synthetase (GLUL), as well as Tubulin alpha (TUBA4A). Most oscillating cerebellar proteins were not previously identified in circadian proteomic analyses of any tissue. Strikingly, the daily accumulation of mitochondrial proteins was clustered to the mid-resting phase, as previously observed for distinct mitochondrial proteins in the liver. Moreover, a number of rhythmic proteins, such as SYN2, NSF and TPI1, were associated with non-rhythmic mRNAs, indicating widespread post-transcriptional control in cerebellar oscillations. Thus, this study highlights extensive rhythmic aspects of the cerebellar proteome.


Assuntos
Cerebelo/metabolismo , Relógios Circadianos , Regulação da Expressão Gênica , Proteoma/análise , Proteoma/genética , Animais , Cerebelo/química , Ritmo Circadiano , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteômica , RNA Mensageiro/análise , RNA Mensageiro/genética , Eletroforese em Gel Diferencial Bidimensional
7.
BMC Plant Biol ; 19(1): 21, 2019 Jan 11.
Artigo em Inglês | MEDLINE | ID: mdl-30634904

RESUMO

BACKGROUND: Rapeseed (Brassica napus, B. napus) is an important oil seed crop in the world. Previous studies showed that seed germination vigor might be correlated with seed oil content in B. napus, but the regulation mechanism for seed germination has not yet been explained clearly. Dissecting the regulation mechanism of seed germination and germination vigor is necessary. RESULTS: Here, proteomic and genomic approaches were used to analyze the germination process in B. napus seeds with different oil content. The identification of 165 differentially expressed proteins (DEPs) in the germinating seeds of B. napus with high and low oil content was accomplished by two-dimensional fluorescence difference in gel electrophoresis (2D-DIGE). The comparative proteomic results revealed that seeds with high oil content had higher metabolic activity, especially for sulfur amino acid metabolism. Thirty-one unique genes were shown to be significantly changed during germination between the seeds with high and low oil content, and thirteen of these genes were located within the confidence interval of germination-related quantitative trait locus (QTLs), which might play an important role in regulating seed germination vigor. CONCLUSIONS: The present results are of importance for the understanding of the regulation mechanism for seed germination vigor in B. napus.


Assuntos
Brassica napus/metabolismo , Brassica napus/fisiologia , Genômica/métodos , Germinação/fisiologia , Óleos Vegetais/metabolismo , Proteômica/métodos , Sementes/metabolismo , Sementes/fisiologia , Brassica napus/genética , Locos de Características Quantitativas/genética , Sementes/genética , Eletroforese em Gel Diferencial Bidimensional
8.
Biochim Biophys Acta Proteins Proteom ; 1867(1): 47-56, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-29753087

RESUMO

OBJECTIVE: We investigated effects of salazosulfapyridine (SASP) on the protein profile of cell surface (CS)-proteins of SW982, a human synovial sarcoma cell line, using biotinylation of CS-proteins and 2-dimensional fluorescence difference gel electrophoresis (2D-DIGE). METHODS: SW982 cells were treated with SASP and its metabolites, sulfapyridine (SP) and 5-aminosalicylic acid (5ASA). Then the cells were treated with a membrane-impermeable biotinylating reagent. Biotinylated CS-proteins were isolated using NeutrAvidin-bound beads. CS-proteins affected by the drugs were detected by 2D-DIGE and subjected to mass spectrometry. RESULTS: By the 2D-DIGE analysis, in total 576 spots were detected, 29 out of which showed more than ±1.5-fold different intensity in the SASP-, SP-, and 5ASA-treated cells, compared to non-treated cells (p < 0.05). Interestingly, 7 out of the 29 spots changed their intensity only by SASP and 17 spots changed their intensity only by SP. We identified 9 protein from 15 out of the 29 spots, most of which were evidenced to exist on the cell surface by flow cytometry. CONCLUSION: We found novel effects of SASP and its metabolites on SW982 cells by the combination of biotinylation of cell surface proteins and 2D-DIGE analysis. These data would help understanding of anti-rheumatic actions of SASP. Furthermore, the combination would be a useful method for the analysis of CS-proteins in various conditions.


Assuntos
Proteínas de Membrana/efeitos dos fármacos , Sarcoma Sinovial/metabolismo , Sulfassalazina/farmacologia , Biotinilação/métodos , Linhagem Celular Tumoral , Humanos , Proteínas de Membrana/análise , Proteínas de Membrana/metabolismo , Mesalamina/farmacologia , Sarcoma Sinovial/patologia , Sulfapiridina/farmacologia , Sulfassalazina/metabolismo , Eletroforese em Gel Diferencial Bidimensional/métodos
9.
Biochim Biophys Acta Proteins Proteom ; 1867(1): 28-37, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-29883687

RESUMO

Cancer cells can reprogram their metabolic machinery to survive. This altered metabolism, which is distinct from the metabolism of normal cells, is thought to be a possible target for the development of new cancer therapies. In this study, we constructed a screening system that focuses on bioenergetic profiles (specifically oxygen consumption rate and extracellular acidification rate) and characteristic proteomic changes. Thus, small molecules that target cancer-specific metabolism were investigated. We screened the chemical library of RIKEN Natural Products Depository (NPDepo) and found that unantimycin A, which was recently isolated from the fraction library of microbial metabolites, and NPL40330, which is derived from a chemical library, inhibit mitochondrial respiration. Furthermore, we developed an in vitro reconstitution assay method for mitochondrial electron transport chain using semi-intact cells with specific substrates for each complex of the mitochondrial electron transport chain. Our findings revealed that NPL40330 and unantimycin A target mitochondrial complexes I and III, respectively.


Assuntos
Descoberta de Drogas/métodos , Neoplasias/metabolismo , Proteômica/métodos , Animais , Descoberta de Drogas/tendências , Avaliação Pré-Clínica de Medicamentos/métodos , Avaliação Pré-Clínica de Medicamentos/tendências , Complexo de Proteínas da Cadeia de Transporte de Elétrons/efeitos dos fármacos , Células HeLa , Humanos , Compostos Macrocíclicos/farmacologia , Mitocôndrias/efeitos dos fármacos , Neoplasias/tratamento farmacológico , Fenótipo , Marcadores de Fotoafinidade , Bibliotecas de Moléculas Pequenas , Eletroforese em Gel Diferencial Bidimensional/métodos
10.
Biochim Biophys Acta Proteins Proteom ; 1867(1): 57-61, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-29883688

RESUMO

Protein phosphorylation is one of the most common post-translational modifications in eukaryotes and can regulate diverse properties of proteins. Protein kinases are encoded by more than 500 genes in higher eukaryotes and play central roles in various cellular signaling pathways. Consequently, genetic abnormalities of protein kinases have been implicated in many diseases. To fully understand the complex phosphorylation-mediated signaling networks, it is important to globally identify and functionally characterize in vivo substrates of individual protein kinases. Advances in electrophoresis-based phosphoproteomic technologies such as two-dimensional difference gel electrophoresis (2D-DIGE) following immobilized metal affinity chromatography (IMAC) and phosphate-affinity Phos-tag PAGE have enabled efficient and detailed analysis of protein kinase substrates. Here, we describe physiological functions of the newly identified substrates of several disease-related protein kinases including ERK, PKD and PINK1.


Assuntos
Fosfoproteínas/metabolismo , Proteínas Quinases/metabolismo , Eletroforese em Gel Diferencial Bidimensional/métodos , Animais , Cromatografia de Afinidade/métodos , Humanos , Fosforilação , Processamento de Proteína Pós-Traducional , Proteômica/métodos , Transdução de Sinais/fisiologia , Espectrometria de Massas em Tandem/métodos
11.
Artigo em Inglês | MEDLINE | ID: mdl-30392560

RESUMO

Cancer results from the accumulation of genomic alterations. As the genome is functionally translated to the proteome and regulates tumor cell behavior, proteomics studies are expected to further the current understanding of the molecular mechanisms underlying carcinogenesis and cancer progression. Biomarkers are potential tools to classify cancers for therapy, predict responses to treatments, and support treatment-related decision-making. Biomarker development has been actively pursued in oncology by proteomic approaches. Two-dimensional difference gel electrophoresis (2D-DIGE) is a proteomics technique based on two-dimensional polyacrylamide gel electrophoresis (2D-PAGE). In 2D-DIGE, protein samples are labeled with distinct fluorescent dyes before fractionation via 2D-PAGE. 2D-DIGE offers advantages to identify biomarker candidates, including reproducibility, high sensitivity, comprehensiveness, and high throughput. 2D-DIGE has contributed to the establishment of tissue biomarkers, which potentially facilitate precision medicine. 2D-DIGE is thus expected to yield major advancements in cancer biomarker identification and development.


Assuntos
Biomarcadores Tumorais/química , Eletroforese em Gel Diferencial Bidimensional/métodos , Eletroforese em Gel Diferencial Bidimensional/tendências , Biomarcadores Tumorais/fisiologia , Eletroforese em Gel Bidimensional/métodos , Eletroforese em Gel Bidimensional/tendências , Humanos , Proteômica/métodos
12.
Methods Mol Biol ; 1855: 229-247, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30426421

RESUMO

Two-dimensional difference gel electrophoresis (2D DIGE) is a modified form of 2D electrophoresis (2D E) that allows one to compare two or three protein samples simultaneously on the same gel. The proteins in each sample are covalently tagged with different color fluorescent dyes that are designed to have no effect on the relative migration of proteins during electrophoresis. Proteins that are common to the samples appear as "spots" with a fixed ratio of fluorescent signals, whereas proteins that differ between the samples have different fluorescence ratios. With conventional imaging systems, DIGE is capable of reliably detecting as little as 0.2 fmol of protein, and protein differences down to ± 15%, over a ~10,000-fold protein concentration range. DIGE combined with digital image analysis therefore greatly improves the statistical assessment of proteome variation. Here we describe a protocol for conducting DIGE experiments, which takes 2-3 days to complete. We have further improved upon 2D DIGE by introducing in-gel equilibration to improve protein retention during transfer between the first and second dimensions of electrophoresis and by developing a fluorescent gel imaging system with a millionfold dynamic range.


Assuntos
Proteínas/isolamento & purificação , Eletroforese em Gel Diferencial Bidimensional/métodos , Corantes Fluorescentes/química , Coloração e Rotulagem
13.
Methods Mol Biol ; 1888: 127-139, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30519944

RESUMO

Recent improvements in technologies such as omics analysis have enabled us to acquire a large amount of data regarding the biological changes in cells treated with bioactive small molecules. Using such data, a variety of profiling methods have been established for target identification of such bioactive compounds. In this chapter, we describe a proteomic profiling system, ChemProteoBase, based on proteome analysis using two-dimensional difference gel electrophoresis. This system compares the similarities in protein expression of 296 spots detected in the gel among the test compounds.


Assuntos
Descoberta de Drogas/métodos , Proteoma , Proteômica/métodos , Eletroforese em Gel Diferencial Bidimensional , Linhagem Celular , Análise de Dados , Corantes Fluorescentes , Humanos , Bibliotecas de Moléculas Pequenas , Coloração e Rotulagem
14.
Meat Sci ; 148: 127-136, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30388477

RESUMO

Thirty Nellore crossbred male sheep (Ovis aries) were divided into two groups of 15 animals each and subjected to either pre-slaughter electrical stunning followed by slaughter (ST) or traditional halal slaughter without stunning (NST) to investigate the changes in blood biochemical parameters, meat quality and proteomic profile. Higher (P < .05) pH, water holding capacity and Warner-Bratzler shear force were observed in meat from stunned sheep. Quantitative proteomic approach using DIGE was employed to find a panel of protein markers that could differentiate ST and NST muscle proteome. Comparison of muscle proteome of ST and NST samples by 2D-DIGE and MALDI-TOF/TOF MS analysis revealed 46 significant (P < .05) differentially expressed proteins. Our analysis revealed changes in the abundance of proteins involved in catalytic, structural, and stress related process. Current study has demonstrated variation meat quality and identified important proteins that correlate with meat texture and pre-slaughter stress in sheep that are slaughtered without and with electrical stunning.


Assuntos
Matadouros , Anestesia/veterinária , Manipulação de Alimentos/métodos , Carne Vermelha/análise , Bem-Estar do Animal , Animais , Qualidade dos Alimentos , Islamismo , Masculino , Proteínas Musculares/análise , Proteômica , Carneiro Doméstico , Estresse Fisiológico , Eletroforese em Gel Diferencial Bidimensional/métodos
15.
Anal Bioanal Chem ; 410(24): 6219-6235, 2018 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-30058028

RESUMO

Nitrogen (N) serves as a macronutrient that is essential to plant growth and development, and significantly influences storage protein and starch biosyntheses and, ultimately, grain yield and quality. In this study, we performed the first comparative proteomic analysis of developing wheat grains under high-N conditions using 2D-DIGE and tandem mass spectrometry. High-N fertilizer application caused significant increases in ear number, ear grain number, and grain yield. 2D-DIGE identified 142 differentially accumulated proteins (DAPs) during grain development in the elite Chinese bread wheat cultivar Zhongmai 175, of which 132 (93%) were identified by MALDI-TOF/TOF-MS, representing 92 unique proteins. These proteins are involved mainly in energy, N and protein metabolism, carbon metabolism, and starch biosynthesis. Subcellular localization prediction and fluorescence confocal microscopic analysis showed that the DAPs identified were localized mainly in the cytosol and chloroplast. Principal component analysis (PCA) revealed a greater proteomic difference among grain developmental periods than between the high-N and control groups. Protein-protein interaction analysis highlighted a complex network centered around enzymes involved in energy, N and protein metabolism, and starch biosynthesis. Six key DAP genes showed expression patterns consistent with their protein accumulation trends during grain development. A putative metabolic pathway was proposed, with synergistic regulatory networks of grain storage protein and starch biosyntheses in response to high-N application.


Assuntos
Grão Comestível/metabolismo , Fertilizantes , Nitrogênio/metabolismo , Proteínas de Plantas/metabolismo , Amido/metabolismo , Triticum/metabolismo , Eletroforese em Gel Diferencial Bidimensional/métodos , Vias Biossintéticas , Grão Comestível/química , Grão Comestível/crescimento & desenvolvimento , Fertilizantes/análise , Proteínas de Plantas/análise , Mapas de Interação de Proteínas , Proteômica/métodos , Amido/análise , Triticum/química , Triticum/crescimento & desenvolvimento
17.
Artigo em Inglês | MEDLINE | ID: mdl-29353015

RESUMO

Pseudechis (black snakes) is an Australasian elapid snake genus that inhabits much of mainland Australia, with two representatives confined to Papua New Guinea. The present study is the first to analyse the venom of all 9 described Pseudechis species (plus one undescribed species) to investigate the evolution of venom composition and functional activity. Proteomic results demonstrated that the typical Pseudechis venom profile is dominated by phospholipase A2 toxins. Strong cytotoxicity was the dominant function for most species. P. porphyriacus, the most basal member of the genus, also exhibited the most divergent venom composition, being the only species with appreciable amounts of procoagulant toxins. The relatively high presence of factor Xa recovered in P. porphyriacus venom may be related to a predominantly amphibian diet. Results of this study provide important insights to guide future ecological and toxinological investigations.


Assuntos
Venenos Elapídicos/metabolismo , Hydrophiidae/fisiologia , Modelos Moleculares , Proteínas de Répteis/metabolismo , Animais , Austrália , Coagulantes/química , Coagulantes/metabolismo , Coagulantes/toxicidade , Bases de Dados de Proteínas , Venenos Elapídicos/química , Venenos Elapídicos/genética , Venenos Elapídicos/toxicidade , Eletroforese em Gel de Poliacrilamida , Evolução Molecular , Hydrophiidae/crescimento & desenvolvimento , Conformação Molecular , Nova Guiné , Fosfolipases A2/química , Fosfolipases A2/genética , Fosfolipases A2/metabolismo , Fosfolipases A2/toxicidade , Filogenia , Proteômica/métodos , Proteínas de Répteis/química , Proteínas de Répteis/genética , Proteínas de Répteis/toxicidade , Especificidade da Espécie , Eletroforese em Gel Diferencial Bidimensional
18.
Biol Trace Elem Res ; 185(1): 78-88, 2018 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-29340859

RESUMO

Chromium (Cr) is a highly toxic, common heavy metal used in industrial production. There are two types of Cr in nature: hexavalent chromium (Cr(VI)) and chromium trichloride (Cr(III)). Cr(III) is involved in the metabolism of sugars and lipids, whereas Cr(VI) is absorbed through the respiratory tract and skin and generates free radicals that result in secondary toxicity. Cr(VI) leads to cancer in the occupational population and is therefore recognized as a human carcinogen by the International Agency for Research on Cancer. The specific mechanism underlying Cr-induced carcinogenesis is complex. In this study, two-dimensional fluorescence difference gel electrophoresis and matrix-assisted laser desorption ionization-time-of-flight/time-of-flight mass spectrometry-based techniques were performed to analyze differentially expressed proteins between Beas-2B human bronchial epithelial cells and Cr(VI)-transformed Beas-2B cells. Many differentially expressed proteins were identified in the cells after malignant transformation, including serine/threonine kinase 11, endothelial nitric oxide synthase 3, apolipoprotein A1, vinculin, and lamin A/C. These proteins are involved in many signaling and metabolic pathways, including apoptosis, autophagy, the PI3K/Akt signaling pathway, focal adhesion, cell motility, and actin cytoskeleton rearrangement.


Assuntos
Transformação Celular Neoplásica/metabolismo , Cromo/toxicidade , Proteômica/métodos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Eletroforese em Gel Diferencial Bidimensional/métodos , Animais , Linhagem Celular , Transformação Celular Neoplásica/induzido quimicamente , Transformação Celular Neoplásica/genética , Humanos , Masculino , Camundongos , Camundongos Nus , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética
19.
Toxicol Mech Methods ; 28(5): 335-344, 2018 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-29228856

RESUMO

The potent neurotoxin saxitoxin produced by both marine and freshwater phytoplankton causes paralytic shellfish poisoning syndrome. The toxicity and mode of action of the acute exposure of high-dose saxitoxin have been intensively studied for decades; however, the potential risk of exposure of low-dose saxitoxin remained to be uncovered. Here we present a proteomics study of murine neuroblastoma N2A cell with low-dose saxitoxin exposure (1 nM and 10 nM, 24-h intoxication). Differential proteins were profiled by two-dimensional fluorescence difference gel electrophoresis (2D-DIGE) coupled with matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS). A total of 9 proteins, including 14-3-3 beta (1433B), alpha enolase (ENO1) and cofilin 2 (CFL2), were altered by the low-dose saxitoxin exposure. We further validated the expressions of 1433B, ENO1 and CFL2 by Western blot analysis and the enzyme-linked immunosorbent assay. These 9 proteins involve cell apoptotic pathways, cell skeleton maintenance, membrane potentials and mitochondrial functions. Modulation of these 9 proteins by low-dose saxitoxin exposure could correlate to the reports on genotoxicity and neurotoxicity induced by saxitoxin. This study also suggested other potential risks of saxitoxin.


Assuntos
Neuroblastoma/metabolismo , Neurônios/efeitos dos fármacos , Proteoma/efeitos dos fármacos , Saxitoxina/toxicidade , Proteínas 14-3-3/metabolismo , Animais , Western Blotting , Linhagem Celular Tumoral , Cofilina 2/metabolismo , Relação Dose-Resposta a Droga , Ensaio de Imunoadsorção Enzimática , Camundongos , Neuroblastoma/patologia , Neurônios/metabolismo , Neurônios/patologia , Fosfopiruvato Hidratase/metabolismo , Proteoma/metabolismo , Proteômica/métodos , Medição de Risco , Transdução de Sinais/efeitos dos fármacos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Testes de Toxicidade/métodos , Eletroforese em Gel Diferencial Bidimensional
20.
Methods Mol Biol ; 1664: 3-14, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29019120

RESUMO

Two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) continues to be one of the most versatile and widely used techniques to study the proteome of a biological system. In particular, a modified version of 2D-PAGE, two-dimensional difference gel electrophoresis (2D-DIGE), which uses differential labeling of protein samples with up to three fluorescent tags, offers greater sensitivity and reproducibility over conventional 2D-PAGE gels for differential quantitative analysis of protein expression between experimental groups. Both these methods have distinct advantages in the separation and identification of thousands of individual proteins species including protein isoforms and post-translational modifications. This review will discuss the principles of 2D-PAGE and 2D-DIGE including limitations to the methods. 2D-PAGE and 2D-DIGE continue to be popular methods in bioprocessing-related research (particularly on recombinant Chinese hamster ovary cells), which will also be discussed in the review chapter.


Assuntos
Eletroforese em Gel Bidimensional , Eletroforese em Gel Diferencial Bidimensional , Animais , Eletroforese em Gel Bidimensional/métodos , Humanos , Proteoma , Proteômica/métodos , Eletroforese em Gel Diferencial Bidimensional/métodos
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