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1.
Sci Rep ; 14(1): 5188, 2024 03 02.
Artigo em Inglês | MEDLINE | ID: mdl-38431723

RESUMO

Total knee arthroplasty (TKA) is associated with substantial blood loss and tranexamic acid (TXA) effectively reduces postoperative bleeding. Although it is known that there is no difference between intravenous or intra-articular (IA) injection, the general interest is directed towards topical hemostatic agents regarding thromboembolic events in high-risk patients. This study aimed to compare the blood conservation effects of IA MPH powder and TXA in patients undergoing primary TKA. We retrospectively analyzed 103 patients who underwent primary TKA between June 2020 and December 2021. MPH powder was applied to the IA space before capsule closure (MPH group, n = 51). TXA (3 g) was injected via the drain after wound closure (TXA group, n = 52). All patients underwent drain clamping for three postoperative hours. The primary outcome was the drain output, and the secondary outcomes were the postoperative hemoglobin (Hb) levels during the hospitalization period and the perioperative blood transfusion rates. An independent Student's t-test was used to determine differences between the two groups. The drain output in the first 24 h after surgery was significantly higher in the MPH group than in the TXA group. The postoperative Hb levels were significantly lower in the MPH group than in the TXA group. In patients with simultaneous bilateral TKA, there was a significant difference in the blood transfusion volumes and the rates between groups. It is considered that IA MPH powder cannot replace IA TXA because of an inferior efficacy in reducing blood loss and maintaining postoperative Hb levels in the early postoperative period after primary TKA. Moreover, in the case of simultaneous bilateral TKA, we do not recommend the use of IA MPH powder because it was notably less effective in the field of transfusion volume and rate.


Assuntos
Antifibrinolíticos , Artroplastia do Joelho , Ácido Tranexâmico , Humanos , Artroplastia do Joelho/efeitos adversos , Pós , Antifibrinolíticos/uso terapêutico , Estudos Retrospectivos , Hemorragia Pós-Operatória/prevenção & controle , Hemorragia Pós-Operatória/induzido quimicamente , Injeções Intra-Articulares , Administração Intravenosa , Perda Sanguínea Cirúrgica/prevenção & controle
2.
Jt Dis Relat Surg ; 35(1): 3-11, 2024 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-38108160

RESUMO

OBJECTIVES: The study aimed to evaluate the efficacy of a continuous closed irrigation system (CCIS) after open debridement for patients with intractable septic ankle arthritis. PATIENTS AND METHODS: The retrospective study analyzed the intractable septic arthritis of 12 (6 males, 6 females; mean age: 64.1±14.7 years; range, 33 to 80 years) patients managed by CCIS between July 2015 and July 2020. All patients had previously undergone operations to treat septic ankle arthritis without resolution of the infection. After open debridement, the CCIS was usually equipped with two outflow tubes and one inflow tube. Saline inflow was about 1 L per day. RESULTS: The mean follow-up period was 30.8±14.9 (range, 15 to 70) months. The CCIS was maintained for a mean of 5.1±2.1 (range, 3 to 7) days. The mean number of operations the patients had previously undergone was 2.83±1.5 (range, 1 to 6). For 11 (91.6%) out of 12 patients, infection did not recur after one-time CCIS, and laboratory test results remained normal. Six patients had previously undergone total ankle replacement arthroplasty. These patients underwent antibiotics-mixed cement arthroplasty after CCIS. For five of six with infected total ankle replacement arthroplasty, infection did not recur after CCIS. However, one patient without the removal of both implants experienced recurrence at the same site after four postoperative months. In the reoperation, after the removal of both implants and the application of antibiotics-mixed cement arthroplasty, the infection was cleared. CONCLUSION: Use of CCIS after open debridement for intractable septic ankle arthritis is a good treatment option since it is relatively simple and safe, with good results.


Assuntos
Tornozelo , Artrite Infecciosa , Masculino , Feminino , Humanos , Pessoa de Meia-Idade , Idoso , Estudos Retrospectivos , Desbridamento/métodos , Artrite Infecciosa/cirurgia , Artrite Infecciosa/tratamento farmacológico , Antibacterianos/uso terapêutico
3.
Antioxidants (Basel) ; 12(3)2023 Mar 03.
Artigo em Inglês | MEDLINE | ID: mdl-36978887

RESUMO

One of the causes of sarcopenia is that homeostasis between anabolism and catabolism breaks down due to muscle metabolism changes. Rutin has shown antioxidant and anti-inflammatory effects in various diseases, but there are few studies on the effect on muscle loss with aging. The effect of rutin on muscle loss was evaluated using dexamethasone-induced muscle loss C2C12 myoblast and mouse model. In the group treated with dexamethasone, the muscle weight of gastrocnemius (GA), tibialis anterior (TA), and extensor digitorum longus (EDL) in the mouse model were significantly decreased (p < 0.0001 in GA, p < 0.0001 in TA, and p < 0.001 in EDL) but recovered (p < 0.01 in GA, p < 0.0001 in TA, and p < 0.01 in EDL) when treated with rutin. MAFbx, MuRF1, and FOXO3 protein expression of C2C12 myoblast were significantly increased (p < 0.01 in MAFbx, p < 0.01 in MuRF1, and p < 0.01 in FOXO3) when treated with dexamethasone, but it was recovered (p < 0.01 in MAFbx, p < 0.01 in MuRF1, and p < 0.01 in FOXO3) when rutin was treated. In addition, MAFbx and FOXO3 protein expression in GA of mouse model was significantly increased (p < 0.0001 in MAFbx and p < 0.001 in FOXO3) when treated with dexamethasone, but it was also recovered (p < 0.01 in MAFbx and p < 0.001 in FOXO3) when rutin was treated. The present study shows that rutin blocks the FOXO3/MAFbx and FOXO3/MuRf1 pathways to prevent protein catabolism. Therefore, rutin could be a potential agent for muscle loss such as sarcopenia through the blocking ubiquitin-proteasome pathway associated with catabolic protein degradation.

4.
Nutrients ; 14(14)2022 Jul 14.
Artigo em Inglês | MEDLINE | ID: mdl-35889851

RESUMO

Sarcopenia refers to a decline in muscle mass and strength with age, causing significant impairment in the ability to carry out normal daily functions and increased risk of falls and fractures, eventually leading to loss of independence. Maintaining protein homeostasis is an important factor in preventing muscle loss, and the decrease in muscle mass is caused by an imbalance between anabolism and catabolism of muscle proteins. Although ß-sitosterol has various effects such as anti-inflammatory, protective effect against nonalcoholic fatty liver disease (NAFLD), antioxidant, and antidiabetic activity, the mechanism of ß-sitosterol effect on the catabolic pathway was not well known. ß-sitosterol was assessed in vitro and in vivo using a dexamethasone-induced muscle atrophy mice model and C2C12 myoblasts. ß-sitosterol protected mice from dexamethasone-induced muscle mass loss. The thickness of gastrocnemius muscle myofibers was increased in dexamethasone with the ß-sitosterol treatment group (DS). Grip strength and creatine kinase (CK) activity were also recovered when ß-sitosterol was treated. The muscle loss inhibitory efficacy of ß-sitosterol in dexamethasone-induced muscle atrophy in C2C12 myotube was also verified in C2C12 myoblast. ß-sitosterol also recovered the width of myotubes. The protein expression of muscle atrophy F-box (MAFbx) was increased in dexamethasone-treated animal models and C2C12 myoblast, but it was reduced when ß-sitosterol was treated. MuRF1 also showed similar results to MAFbx in the mRNA level of C2C12 myotubes. In addition, in the gastrocnemius and tibialis anterior muscles of mouse models, Forkhead Box O1 (FoxO1) protein was increased in the dexamethasone-treated group (Dexa) compared with the control group and reduced in the DS group. Therefore, ß-sitosterol would be a potential treatment agent for aging sarcopenia.


Assuntos
Sarcopenia , Animais , Dexametasona , Modelos Animais de Doenças , Proteína Forkhead Box O1/metabolismo , Fatores de Transcrição Forkhead/metabolismo , Camundongos , Fibras Musculares Esqueléticas , Músculo Esquelético/metabolismo , Atrofia Muscular/induzido quimicamente , Atrofia Muscular/tratamento farmacológico , Atrofia Muscular/prevenção & controle , Sarcopenia/complicações , Sitosteroides , Ubiquitina-Proteína Ligases/metabolismo
5.
Artigo em Inglês | MEDLINE | ID: mdl-32693679

RESUMO

Benzalkonium chloride (BAC) is a cationic surfactant commonly used as a disinfectant, and is discharged into the aquatic environment by various water sources such as wastewater. BAC may also interact with potentially toxic substances such as persistent organic chemicals. Although studies of BAC contamination toxicity and bioaccumulation have been widely reported, the biochemical responses to BAC toxicity remain incompletely understood, and the detailed molecular mechanisms are largely unknown. In this study, two-dimensional gel electrophoresis (2-DE) and matrix-assisted laser desorption/ionization tandem time-of-flight mass spectrometry-based proteomic approaches were applied to investigate the protein profiles in Oryzias latipes (medaka) chronically exposed to BAC. Fish were exposed to three different concentrations of BAC, 0.05, 0.1, and 0.2 mg/L, for 21 days. A total of 20 proteins involved in the cytoskeleton, the oxidative stress response, the nervous and endocrine systems, signaling pathways, and cellular proteolysis were significantly upregulated by BAC exposure. The proteomic information obtained in the present study will be useful in identification of potential biomarkers for BAC toxicity, and begins to elucidate its molecular mechanisms, providing new insights into the ecotoxicity of BAC.


Assuntos
Compostos de Benzalcônio/toxicidade , Oryzias/metabolismo , Proteoma/metabolismo , Poluentes Químicos da Água/toxicidade , Animais , Biomarcadores/metabolismo , Relação Dose-Resposta a Droga , Ecotoxicologia , Eletroforese em Gel Bidimensional , Dose Letal Mediana , Estresse Oxidativo/efeitos dos fármacos , Proteômica , Transdução de Sinais , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz
6.
Molecules ; 23(8)2018 Aug 17.
Artigo em Inglês | MEDLINE | ID: mdl-30126122

RESUMO

This study identified the volatile organic compounds in the essential oils that are extracted from Chrysanthemum indicum Linné (C. indicum Linné) and investigated the effects of the inhalation of these compounds. We detected a total of 41 volatile organic compounds, including 32 hydrocarbons, four acids, three alcohols, two ketones, and one aldehyde. In a sniffing test, seven types of volatile organic compounds were identified. Furthermore, the volatile organic compounds in C. indicum Linné that were identified were found to be derived from 1,8-cineole and camphor. After inhalation of the essential oils, the subjects' systolic blood pressure and heart rate decreased. This indicates that inhalation of the essential oils extracted from C. indicum Linné provides mental and physical relaxation. We examined the changes in electroencephalogram findings that are observed after C. indicum Linné essential oil inhalation. An increase in theta and alpha waves, which usually appear during relaxation, as well as a decrease in beta and gamma waves, which appear during brain activity such as excessive attention, were noted. These results indicate that C. indicum Linné essential oil inhalation helps to reduce blood pressure and may provide mental and physical relaxation.


Assuntos
Pressão Sanguínea/efeitos dos fármacos , Chrysanthemum/química , Eletroencefalografia , Compostos Orgânicos Voláteis/química , Compostos Orgânicos Voláteis/farmacologia , Administração por Inalação , Ondas Encefálicas/efeitos dos fármacos , Feminino , Frequência Cardíaca , Humanos , Masculino , Óleos Voláteis/química , Óleos Voláteis/farmacologia
7.
Physiol Plant ; 161(2): 273-284, 2017 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-28401568

RESUMO

Chitinases belong to a conserved protein family and play multiple roles in defense, development and growth regulation in plants. Here, we identified a secreted chitinase-like protein, OsCLP, which functions in rice growth. A T-DNA insertion mutant of OsCLP (osclp) showed significant retardation of root and shoot growth. A comparative proteomic analysis was carried out using root tissue of wild-type and the osclp mutant to understand the OsCLP-mediated rice growth retardation. Results obtained revealed that proteins related to glycolysis (phosphoglycerate kinase), stress adaption (chaperonin) and calcium signaling (calreticulin and CDPK1) were differentially regulated in osclp roots. Fura-2 molecular probe staining, which is an intracellular calcium indicator, and inductively coupled plasma-mass spectrometry (ICP-MS) analysis suggested that the intracellular calcium content was significantly lower in roots of osclp as compared with the wild-type. Exogenous application of Ca2+ resulted in successful recovery of both primary and lateral root growth in osclp. Moreover, overexpression of OsCLP resulted in improved growth with modified seed shape and starch structure; however, the overall yield remained unaffected. Taken together, our results highlight the involvement of OsCLP in rice growth by regulating the intracellular calcium concentrations.


Assuntos
Sinalização do Cálcio , Quitinases/metabolismo , Oryza/enzimologia , Raízes de Plantas/crescimento & desenvolvimento , Oryza/crescimento & desenvolvimento , Proteínas de Plantas/metabolismo , Proteômica , Sementes/crescimento & desenvolvimento
8.
Plant Pathol J ; 32(6): 552-562, 2016 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-27904462

RESUMO

Pathogenesis-related proteins play multiple roles in plant development and biotic and abiotic stress tolerance. Here, we characterize a rice defense related gene named "jasmonic acid inducible pathogenesis-related class 10" (JIOsPR10) to gain an insight into its functional properties. Semi-quantitative RT-PCR analysis showed up-regulation of JIOsPR10 under salt and drought stress conditions. Constitutive over-expression JIOsPR10 in rice promoted shoot and root development in transgenic plants, however, their productivity was unaltered. Further experiments exhibited that the transgenic plants showed reduced susceptibility to rice blast fungus, and enhanced salt and drought stress tolerance as compared to the wild type. A comparative proteomic profiling of wild type and transgenic plants showed that overexpression of JIOsPR10 led to the differential modulation of several proteins mainly related with oxidative stresses, carbohydrate metabolism, and plant defense. Taken together, our findings suggest that JIOsPR10 plays important roles in biotic and abiotic stresses tolerance probably by activation of stress related proteins.

9.
PLoS One ; 11(10): e0165149, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27764242

RESUMO

Rice blast disease caused by Magnaporthe oryzae is one of the most devastating fungal diseases of rice and results in a huge loss of rice productivity worldwide. During the infection process, M. oryzae secretes a large number of glycosyl hydrolase proteins into the host apoplast to digest the cell wall and facilitate fungal ingression into host tissues. In this study, we identified a novel arabinofuranosidase-B (MoAbfB) protein that is secreted by M. oryzae during fungal infection. Deletion of MoAbfB from M. oryzae resulted in reduced disease severity in rice. Biochemical assays revealed that the MoAbfB protein exhibited arabinofuranosidase activity and caused degradation of rice cell wall components. Interestingly, pre-treatment of rice with the MoAbfB protein inhibited fungal infection by priming defence gene expression. Our findings suggest that MoAbfB secretion affects M. oryzae pathogenicity by breaking down the host cell wall, releasing oligosaccharides that may be recognized by the host to trigger innate immune responses.


Assuntos
Proteínas Fúngicas/metabolismo , Glicosídeo Hidrolases/metabolismo , Magnaporthe/fisiologia , Oryza/microbiologia , Virulência , Parede Celular/metabolismo , Glicosídeo Hidrolases/genética , Magnaporthe/enzimologia , Microscopia Eletrônica de Varredura , Mutagênese , Doenças das Plantas/imunologia , Doenças das Plantas/microbiologia , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética
10.
Mol Plant Microbe Interact ; 29(4): 299-312, 2016 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-26780420

RESUMO

The Magnaporthe oryzae snodprot1 homolog (MSP1), secreted by M. oryzae, is a cerato-platanin family protein. msp1-knockout mutants have reduced virulence on barley leaves, indicating that MSP1 is required for the pathogenicity of rice blast fungus. To investigate the functional roles of MSP1 and its downstream signaling in rice, recombinant MSP1 was produced in Escherichia coli and was assayed for its functionality. Application of MSP1 triggered cell death and elicited defense responses in rice. MSP1 also induced H2O2 production and autophagic cell death in both suspension-cultured cells and rice leaves. One or more protein kinases triggered cell death, jasmonic acid and abscisic acid enhanced cell death, while salicylic acid suppressed it. We demonstrated that the secretion of MSP1 into the apoplast is a prerequisite for triggering cell death and activating defense-related gene expression. Furthermore, pretreatment of rice with a sublethal MSP1 concentration potentiated resistance to the pathogen. Taken together, our results showed that MSP1 induces a high degree of cell death in plants, which might be essential for its virulence. Moreover, rice can recognize MSP1, resulting in the induction of pathogen-associated molecular pattern-triggered immunity.


Assuntos
Proteínas Fúngicas/metabolismo , Regulação da Expressão Gênica de Plantas , Magnaporthe/fisiologia , Oryza/microbiologia , Doenças das Plantas/microbiologia , Reguladores de Crescimento de Plantas/farmacologia , Arabidopsis/imunologia , Arabidopsis/microbiologia , Arabidopsis/fisiologia , Arabidopsis/ultraestrutura , Autofagia/efeitos dos fármacos , Ciclopentanos/farmacologia , Proteínas Fúngicas/genética , Peróxido de Hidrogênio/metabolismo , Magnaporthe/patogenicidade , Modelos Biológicos , Oryza/imunologia , Oryza/fisiologia , Oryza/ultraestrutura , Oxilipinas/farmacologia , Moléculas com Motivos Associados a Patógenos/imunologia , Doenças das Plantas/imunologia , Imunidade Vegetal , Folhas de Planta/imunologia , Folhas de Planta/microbiologia , Folhas de Planta/fisiologia , Folhas de Planta/ultraestrutura , Plantas Geneticamente Modificadas , Proteínas Recombinantes , Ácido Salicílico/farmacologia , Nicotiana/imunologia , Nicotiana/microbiologia , Nicotiana/fisiologia , Nicotiana/ultraestrutura
11.
Proteomics ; 16(1): 122-35, 2016 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-26460066

RESUMO

Plant growth-promoting rhizobacteria (PGPR) facilitate the plant growth and enhance their induced systemic resistance (ISR) against a variety of environmental stresses. In this study, we carried out integrative analyses on the proteome, transcriptome, and metabolome to investigate Arabidopsis root and shoot responses to the well-known PGPR strain Paenibacillus polymyxa (P. polymyxa) E681. Shoot fresh and root dry weights were increased, whereas root length was decreased by treatment with P. polymyxa E681. 2DE approach in conjunction with MALDI-TOF/TOF analysis revealed a total of 41 (17 spots in root, 24 spots in shoot) that were differentially expressed in response to P. polymyxa E681. Biological process- and molecular function-based bioinformatics analysis resulted in their classification into seven different protein groups. Of these, 36 proteins including amino acid metabolism, antioxidant, defense and stress response, photosynthesis, and plant hormone-related proteins were up-regulated, whereas five proteins including three carbohydrate metabolism- and one amino acid metabolism-related, and one unknown protein were down-regulated, respectively. A good correlation was observed between protein and transcript abundances for the 12 differentially expressed proteins during interactions as determined by qPCR analysis. Metabolite analysis using LC-MS/MS revealed highly increased levels of tryptophan, indole-3-acetonitrile (IAN), indole-3-acetic acid (IAA), and camalexin in the treated plants. Arabidopsis plant inoculated P. polymyxa E681 also showed resistance to Botrytis cinerea infection. Taken together these results suggest that P. polymyxa E681 may promote plant growth by induced metabolism and activation of defense-related proteins against fungal pathogen.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/crescimento & desenvolvimento , Arabidopsis/microbiologia , Paenibacillus/fisiologia , Arabidopsis/genética , Arabidopsis/fisiologia , Proteínas de Arabidopsis/análise , Proteínas de Arabidopsis/genética , Resistência à Doença , Regulação da Expressão Gênica de Plantas , Genômica , Metabolômica , Doenças das Plantas/genética , Doenças das Plantas/microbiologia , Raízes de Plantas/genética , Raízes de Plantas/crescimento & desenvolvimento , Raízes de Plantas/microbiologia , Raízes de Plantas/fisiologia , Brotos de Planta/genética , Brotos de Planta/crescimento & desenvolvimento , Brotos de Planta/microbiologia , Brotos de Planta/fisiologia , Proteômica , Simbiose , Transcriptoma
12.
J Nanosci Nanotechnol ; 15(2): 1691-4, 2015 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-26353715

RESUMO

A combined radio frequency sputtering/sol-gel combustion method was investigated in order to obtain optimum process condition for fabrication of a Titanium (Ti)/Titanium oxide (TiO2) films electrode of transparent conductive oxide-less dye-sensitized solar cells (TCO-less DSCs), Experimentally, the substrate temperature was changed from R.T. to 500 °C, and it was found that there existed an optimum value for efficient performance of the cell. The porous Ti layer with low sheet resistance (-2.5 Ω/sq.) can be prepared by substrate temperature 250 °C under RF power 300 W and Ar 8 mTorr. The efficiency (η) of the cell was 6.52% [FF: 0.76, VOC: 0.72 V, JSC: 11.91 mA/cm2].

13.
Proteomics ; 15(10): 1706-16, 2015 May.
Artigo em Inglês | MEDLINE | ID: mdl-25545850

RESUMO

Seed coat color is an important attribute determining consumption of soybean seeds. Soybean cultivar Mallikong (M) has yellow seed coat while its naturally mutated cultivar Mallikong mutant (MM), has brown colored seed coat. We used integrated proteomics and metabolomics approach to investigate the differences between seed coats of M and MM during different stages of seed development (4, 5, and 6 weeks after flowering). 2DE profiling of total seed coat proteins from three stages showed 178 differentially expressed spots between M and MM of which 172 were identified by MALDI-TOF/TOF. Of these, 62 were upregulated and 105 were downregulated in MM compared with M, while five spots were detected only in MM. Proteins involved in primary metabolism showed downregulation in MM suggesting energy in MM might be utilized for proanthocyanidin biosynthesis via secondary metabolic pathways that leads to the development of brown seed coat color. Besides, downregulation of two isoforms of isoflavone reductase indicated reduced isoflavones in seed coat of MM that was confirmed by quantitative estimation of total and individual isoflavones using HPLC. We propose that low isoflavones level in MM may offer a high substrate for proanthocyanidin production that results in the development of brown seed coat in MM.


Assuntos
Glycine max/metabolismo , Metabolômica/métodos , Pigmentação , Proteômica/métodos , Sementes/metabolismo , Análise por Conglomerados , Eletroforese em Gel Bidimensional , Ontologia Genética , Modelos Biológicos , Proteínas de Plantas/metabolismo , Análise de Componente Principal , Proteoma/metabolismo , Metabolismo Secundário
14.
Plant Pathol J ; 30(4): 343-54, 2014 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-25506299

RESUMO

Rice blast disease caused by Magnaporthe oryzae is one of the most serious diseases of cultivated rice (Oryza sativa L.) in most rice-growing regions of the world. In order to investigate early response genes in rice, we utilized the transcriptome analysis approach using a 300 K tilling microarray to rice leaves infected with compatible and incompatible M. oryzae strains. Prior to the microarray experiment, total RNA was validated by measuring the differential expression of rice defense-related marker genes (chitinase 2, barwin, PBZ1, and PR-10) by RT-PCR, and phytoalexins (sakuranetin and momilactone A) with HPLC. Microarray analysis revealed that 231 genes were up-regulated (>2 fold change, p < 0.05) in the incompatible interaction compared to the compatible one. Highly expressed genes were functionally characterized into metabolic processes and oxidation-reduction categories. The oxidative stress response was induced in both early and later infection stages. Biotic stress overview from MapMan analysis revealed that the phytohormone ethylene as well as signaling molecules jasmonic acid and salicylic acid is important for defense gene regulation. WRKY and Myb transcription factors were also involved in signal transduction processes. Additionally, receptor-like kinases were more likely associated with the defense response, and their expression patterns were validated by RT-PCR. Our results suggest that candidate genes, including receptor-like protein kinases, may play a key role in disease resistance against M. oryzae attack.

15.
Proteomics ; 14(20): 2307-18, 2014 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-25047395

RESUMO

Necrotrophic fungal pathogen Cochliobolus miyabeanus causes brown spot disease in rice leaves upon infection, resulting in critical rice yield loss. To better understand the rice-C. miyabeanus interaction, we employed proteomic approaches to establish differential proteomes of total and secreted proteins from the inoculated leaves. The 2DE approach after PEG-fractionation of total proteins coupled with MS (MALDI-TOF/TOF and nESI-LC-MS/MS) analyses led to identification of 49 unique proteins out of 63 differential spots. SDS-PAGE in combination with nESI-LC-MS/MS shotgun approach was applied to identify secreted proteins in the leaf apoplast upon infection and resulted in cataloging of 501 unique proteins, of which 470 and 31 proteins were secreted from rice and C. miyabeanus, respectively. Proteins mapped onto metabolic pathways implied their reprogramming upon infection. The enzymes involved in Calvin cycle and glycolysis decreased in their protein abundance, whereas enzymes in the TCA cycle, amino acids, and ethylene biosynthesis increased. Differential proteomes also generated distribution of identified proteins in the intracellular and extracellular spaces, providing a better insight into defense responses of proteins in rice against C. miyabeanus. Established proteome of the rice-C. miyabeanus interaction serves not only as a good resource for the scientific community but also highlights its significance from biological aspects.


Assuntos
Ascomicetos/fisiologia , Interações Hospedeiro-Patógeno , Oryza/microbiologia , Doenças das Plantas/microbiologia , Proteínas de Plantas/metabolismo , Proteômica/métodos , Eletroforese em Gel Bidimensional , Eletroforese em Gel de Poliacrilamida , Oryza/metabolismo , Proteínas de Plantas/análise , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz
16.
Fungal Biol ; 118(5-6): 433-43, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24863472

RESUMO

Rhizoctonia solani (R. solani), a soil-borne necrotrophic pathogen, causes various plant diseases. Rhizoctonia solani is a mitosporic fungus, the sclerotium of which is the primary inoculum and ensures survival of the fungus during the offseason of the host crop. Since the fungus does not produce any asexual or sexual spores, understanding the biology of sclerotia is important to examine pathogen ecology and develop more efficient methods for crop protection. Here, one- and two-dimensional gel electrophoresis (1-DE and 2-DE, respectively) were used to examine protein regulation during the maturation of fungal sclerotia. A total of 75 proteins (20 proteins from 1-DE using matrix-assisted laser desorption/ionization (MALDI)-time of flight (TOF) mass spectrometry (MS) and 55 proteins from 2-DE using MALDI-TOF MS or MALDI-TOF/TOF MS) were differentially expressed during sclerotial maturation. The identified proteins were classified into ten categories based on their biological functions, including genetic information processing, carbohydrate metabolism, cell defense, amino acid metabolism, nucleotide metabolism, cellular processes, pathogenicity and mycotoxin production, and hypothetical or unknown functions. Interestingly, two vacuole function-related proteins were highly up-regulated throughout sclerotial maturation, which was confirmed at the transcript level by reverse transcriptase polymerase chain reaction (RT-PCR) analysis. These findings contribute to our understanding of the biology of R. solani sclerotia.


Assuntos
Proteínas Fúngicas/genética , Rhizoctonia/genética , Eletroforese em Gel Bidimensional , Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Dados de Sequência Molecular , Doenças das Plantas/microbiologia , Proteômica , Rhizoctonia/química , Rhizoctonia/metabolismo
17.
Methods Mol Biol ; 1072: 563-72, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24136547

RESUMO

Substantial evidences implicate that sample preparation and protein extraction in proteomic studies of plant-pathogen interactions are critical to understand cross talk between host and pathogen. Therefore, interest is growing in applying proteomics techniques to investigate simultaneously secreted proteins from rice and pathogen. We have found, however, that most proteins of interest are low abundant so that proper prefractionation or extraction of secreted proteins from extracellular space (ECS) in the rice leaf is required to excavate relevant protein. This chapter describes the preparation of sample and extraction procedure to enrich the proteins interested before separation by 2-DE or LC-MS/MS. This method significantly increases the sensitivity of proteomic comparisons.


Assuntos
Interações Hospedeiro-Patógeno , Oryza/metabolismo , Oryza/microbiologia , Proteínas de Plantas/metabolismo , Proteoma/metabolismo , Proteômica/métodos , Fracionamento Químico , Fenóis/química , Doenças das Plantas/microbiologia , Folhas de Planta/metabolismo , Folhas de Planta/microbiologia , Proteínas de Plantas/isolamento & purificação , Polietilenoglicóis/química , Ribulose-Bifosfato Carboxilase/isolamento & purificação , Xanthomonas/fisiologia
18.
Proteomics ; 14(4-5): 593-610, 2014 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-24323464

RESUMO

Rice proteomics has progressed at a tremendous pace since the year 2000, and that has resulted in establishing and understanding the proteomes of tissues, organs, and organelles under both normal and abnormal (adverse) environmental conditions. Established proteomes have also helped in re-annotating the rice genome and revealing the new role of previously known proteins. The progress of rice proteomics had recognized it as the corner/stepping stone for at least cereal crops. Rice proteomics remains a model system for crops as per its exemplary proteomics research. Proteomics-based discoveries in rice are likely to be translated in improving crop plants and vice versa against ever-changing environmental factors. This review comprehensively covers rice proteomics studies from August 2010 to July 2013, with major focus on rice responses to diverse abiotic (drought, salt, oxidative, temperature, nutrient, hormone, metal ions, UV radiation, and ozone) as well as various biotic stresses, especially rice-pathogen interactions. The differentially regulated proteins in response to various abiotic stresses in different tissues have also been summarized, indicating key metabolic and regulatory pathways. We envision a significant role of rice proteomics in addressing the global ground level problem of food security, to meet the demands of the human population which is expected to reach six to nine billion by 2040.


Assuntos
Oryza/genética , Proteínas de Plantas/biossíntese , Proteômica , Abastecimento de Alimentos , Humanos , Espectrometria de Massas , Oryza/metabolismo , Proteínas de Plantas/metabolismo
19.
Proteomics ; 13(14): 2176-9, 2013 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-23576416

RESUMO

Ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) is the most abundant plant leaf protein, hampering deep analysis of the leaf proteome. Here, we describe a novel protamine sulfate precipitation (PSP) method for the depletion of RuBisCO. For this purpose, soybean leaf total proteins were extracted using Tris-Mg/NP-40 extraction buffer. Obtained clear supernatant was subjected to the PSP method, followed by 13% SDS-PAGE analysis of total, PS-supernatant and -precipitation derived protein samples. In a dose-dependent experiment, 0.1% w/v PS was found to be sufficient for precipitating RuBisCO large and small subunits (LSU and SSU). Western blot analysis confirmed no detection of RuBisCO LSU in the PS-supernatant proteins. Application of this method to Arabidopsis, rice, and maize leaf proteins revealed results similar to soybean. Furthermore, 2DE analyses of PS-treated soybean leaf displayed enriched protein profile for the protein sample derived from the PS-supernatant than total proteins. Some enriched 2D spots were subjected to MALDI-TOF-TOF analysis and were successfully assigned for their protein identity. Hence, the PSP method is: (i) simple, fast, economical, and reproducible for RuBisCO precipitation from the plant leaf sample; (ii) applicable to both dicot and monocot plants; and (iii) suitable for downstream proteomics analysis.


Assuntos
Protaminas/química , Proteômica/métodos , Ribulose-Bifosfato Carboxilase/isolamento & purificação , Precipitação Química , Eletroforese em Gel Bidimensional , Eletroforese em Gel de Poliacrilamida , Folhas de Planta/química , Folhas de Planta/enzimologia , Proteoma/análise , Ribulose-Bifosfato Carboxilase/química , Glycine max/química , Glycine max/enzimologia , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz
20.
Proteomics ; 13(12-13): 1901-12, 2013 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-23512849

RESUMO

Xanthomonas oryzae pv. oryzae (Xoo) causes bacterial blight disease in rice, and that severely affects yield loss (upto 50%) of total rice production. Here, we report a proteomics investigation of Xoo (compatible race K3)-secreted proteins, isolated from its in vitro culture and in planta infected rice leaves. 2DE coupled with MALDI-TOF-MS and/or nLC-ESI-MS/MS approaches identified 139 protein spots (out of 153 differential spots), encoding 109 unique proteins. Identified proteins belonged to multiple biological and molecular functions. Metabolic and nutrient uptake proteins were common up to both in vitro and in planta secretomes. However, pathogenicity, protease/peptidase, and host defense-related proteins were highly or specifically expressed during in planta infection. A good correlation was observed between protein and transcript abundances for nine proteins secreted in planta as per semiquantitative RT-PCR analysis. Transgenic rice leaf sheath (carrying PBZ1 promoter::GFP cell death reporter), when used to express a few of the identified secretory proteins, showed a direct activation of cell death signaling, suggesting their involvement in pathogenicity related with secretion effectors. This work furthers our understanding of rice bacterial blight disease, and serves as a resource for possible translation in generating disease resistant rice plants for improved seed yield.


Assuntos
Proteínas de Bactérias/metabolismo , Oryza/microbiologia , Plantas Geneticamente Modificadas/microbiologia , Xanthomonas/genética , Proteínas de Bactérias/classificação , Proteínas de Bactérias/genética , Biologia Computacional , Eletroforese em Gel Bidimensional , Interações Hospedeiro-Patógeno , Espectrometria de Massas , Doenças das Plantas/microbiologia , Proteínas de Plantas/metabolismo , Xanthomonas/metabolismo
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