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1.
Phytopathology ; 110(5): 1056-1066, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-32043419

RESUMO

The fungal pathogen Fusarium graminearum causes Fusarium head blight (FHB) on wheat, barley, and other grains. FHB results in yield reductions and contaminates grain with trichothecene mycotoxins, which threaten food safety and food security. Innovative mechanisms for controlling FHB are urgently needed. We have previously shown that transgenic tobacco and citrus plants expressing a modified thionin (Mthionin) exhibited enhanced resistance toward several bacterial pathogens. The aim of this study was to investigate whether overexpression of Mthionin could be similarly efficacious against F. graminearum, and whether transgenic expression of Mthionin impacts the plant microbiome. Transgenic Arabidopsis plants expressing Mthionin were generated and confirmed. When challenged with F. graminearum, Mthionin-expressing plants showed less disease and fungal biomass in both leaves and inflorescences compared with control plants. When infiltrated into leaves, macroconidia of F. graminearum germinated at lower rates and produced less hyphal growth in Arabidopsis leaves expressing Mthionin. Moreover, marker genes related to defense signaling pathways were expressed at significantly higher levels after F. graminearum infection in Mthionin transgenic Arabidopsis plants. However, Mthionin expression did not appreciably alter the overall microbiome associated with transgenic plants grown under controlled conditions; across leaves and roots of Mthionin-expressing and control transgenic plants, only a few bacterial and fungal taxa differed, and differences between Mthionin transformants were of similar magnitude compared with control plants. In sum, our data indicate that Mthionin is a promising candidate to produce transgenic crops for reducing FHB severity and ultimately mycotoxin contamination.


Assuntos
Arabidopsis , Fusarium , Tioninas , Doenças das Plantas , Plantas Geneticamente Modificadas
2.
Plant Physiol Biochem ; 140: 55-67, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31082659

RESUMO

Plants express various antimicrobial peptides including thionins to protect themselves against pathogens. It was recently found that, in addition to four thionin genes, Arabidopsis contains 67 thionin-like (ThiL) genes including six pseudogenes. It is known that thionins have antimicrobial activity and are part of the plant defense system, however, nothing is known about ThiL genes. In this study, we present a bioinformatic analysis of the (ThiL) gene family in Arabidopsis. We identified 15 different motifs which positioned the ThiL peptides in four groups. A comparison of amino acid sequences showed that the ThiL peptides are actually more similar to the acidic domain of thionin proproteins than to the thionin domain. We selected 10 ThiL genes to study the expression and possible function in the Arabidopsis plant. RT-PCR and promoter:GUS fusions showed that most genes were expressed at a very low level but in several organs and at different developmental stages. Some genes were also expressed in syncytia induced by the beet cyst nematode Heterodera schachti in roots while others were downregulated in syncytia. Some overexpression lines supported lower number of nematodes that developed on the roots after inoculation. Two of the genes resulted in a strong hypersensitive response when infiltrated into leaves of Nicotiana benthamiana. These results indicate that ThiL genes might be involved in the response to biotic stress. ThiL genes have been expanded in the Brassicales and specifically the Brassicaceae. The most extreme example is the CRP2460 subfamily that contains 28 very closely related genes from Arabidopsis which are mostly the result of tandem duplications.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Arabidopsis/parasitologia , Proteínas de Arabidopsis/genética , Biologia Computacional/métodos , Regulação da Expressão Gênica de Plantas/genética , Regulação da Expressão Gênica de Plantas/fisiologia , Folhas de Planta/genética , Folhas de Planta/metabolismo , Regiões Promotoras Genéticas/genética , Tioninas/genética , Tioninas/metabolismo
3.
Plant Biotechnol J ; 17(11): 2184-2198, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31001872

RESUMO

Apple exhibits S-RNase-mediated self-incompatibility. Although the cytotoxic effect of S-RNase inside the self-pollen tube has been studied extensively, the underlying defence mechanism in pollen tube in Rosaceae remains unclear. On exposure to stylar S-RNase, plant defence responses are activated in the pollen tube; however, how these are regulated is currently poorly understood. Here, we show that entry of both self and non-self S-RNase into pollen tubes of apple (Malus domestica) stimulates jasmonic acid (JA) production, in turn inducing the accumulation of MdMYC2 transcripts, a transcription factor in the JA signalling pathway widely considered to be involved in plant defence processes. MdMYC2 acts as a positive regulator in the pollen tube activating expression of MdD1, a gene encoding a defence protein. Importantly, MdD1 was shown to bind to the RNase activity sites of S-RNase leading to inhibition of enzymatic activity. This work provides intriguing insights into an ancient defence mechanism present in apple pollen tubes where MdD1 likely acts as a primary line of defence to inhibit S-RNase cytotoxicity prior to self/non-self recognition.


Assuntos
Malus/genética , Proteínas de Plantas/genética , Tubo Polínico/crescimento & desenvolvimento , Ribonucleases , Autoincompatibilidade em Angiospermas , Tioninas/genética
4.
Food Microbiol ; 82: 504-514, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31027812

RESUMO

As a result of the rapidly growing human population, reducing post-harvest crop losses of cereals due to microbial pests has major importance. Plant defensins have the potential to fulfil these demands, being highly specific and efficient antimicrobial agents. Hence, this study aimed to extract and characterise a peptide from cowpea seeds and investigate its antifungal performance. After extraction and partial purification, N-terminal sequencing was used to identify the primary peptide in the extract as cowpea-thionin II. Antifungal activity in vitro was found against Fusarium culmorum (MIC = 50 µg/mL), but Aspergillus niger and Penecillium expansum showed an MIC > 500 µg/mL. The extract was resistant against heat treatment (100 °C, 15 min) but lost its antifungal activity in presence of cations (Na+, K+, Ca2+ and Mg2+, respectively). Membrane permeabilization of fungal hyphae was evident at 25 µg/mL, while induction of oxidative stress only had minor contribution to the antifungal performance. The extract did not induce haemolysis at all concentrations tested (up to 200 µg/mL). Finally, it was successfully used to protect stored wheat grains from fungal spoilage (determined via ergosterol content) when applied at 100 µg/mL. In conclusion, the defensin Cp-thionin II showed the potential for future application as food bio-preservative.


Assuntos
Antifúngicos/farmacologia , Conservantes de Alimentos/farmacologia , Fungos/efeitos dos fármacos , Proteínas de Plantas/farmacologia , Tioninas/farmacologia , Vigna/química , Antifúngicos/química , Antifúngicos/isolamento & purificação , Cátions , Permeabilidade da Membrana Celular/efeitos dos fármacos , Defensinas/química , Defensinas/isolamento & purificação , Defensinas/farmacologia , Grão Comestível/microbiologia , Ergosterol/análise , Ergosterol/metabolismo , Microbiologia de Alimentos , Conservantes de Alimentos/química , Conservantes de Alimentos/isolamento & purificação , Fungos/metabolismo , Fungos/fisiologia , Temperatura Alta , Hifas/efeitos dos fármacos , Hifas/metabolismo , Hifas/fisiologia , Testes de Sensibilidade Microbiana , Proteínas de Plantas/química , Proteínas de Plantas/isolamento & purificação , Estabilidade Proteica , Sementes/química , Tioninas/química , Tioninas/isolamento & purificação
5.
Phytopathology ; 109(1): 27-35, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-30028233

RESUMO

Thionins are a family of antimicrobial peptides. We performed in silico expression analyses of the 44 rice (Oryza sativa) thionins (OsTHIONs). Modulated expression levels of OsTHIONs under different treatments suggest their involvement in many processes, including biotic, abiotic, and nutritional stress responses, and in hormone signaling. OsTHION15 (LOC_Os06g32600) was selected for further characterization based on several in silico analyses. OsTHION15 in O. sativa subsp. indica 'KDML 105' was expressed in all of the tissues and organs examined, including germinating seed, leaves, and roots of seedlings and mature plants, and inflorescences. To investigate the antimicrobial activity of OsTHION15, we produced a recombinant peptide in Escherichia coli Rosetta-gami (DE3). The recombinant OsTHION15 exhibited inhibitory activities toward rice-pathogenic bacteria such as Xanthomonas oryzae pv. oryzae and Pectobacterium carotovorum pv. atroseptica, with minimum inhibitory concentrations of 112.6 and 14.1 µg ml-1, respectively. A significant hyphal growth inhibition was also observed toward Fusarium oxysporum f. sp. cubense and Helminthosporium oryzae. In addition, we demonstrated the in planta antibacterial activity of this peptide in Nicotiana benthamiana against X. campestris pv. glycines. These activities suggest the possible application of OsTHION15 in plant disease control.


Assuntos
Oryza/genética , Doenças das Plantas/genética , Tioninas/genética , Oryza/microbiologia , Pectobacterium carotovorum/patogenicidade , Doenças das Plantas/microbiologia , Xanthomonas/patogenicidade
6.
Biosens Bioelectron ; 117: 168-174, 2018 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-29894854

RESUMO

The loading capacity of thionin (Thi) on shortened multi-walled carbon nanotubes (S-MWCNTs) and acidified multi-walled carbon nanotubes (A-MWCNTs) was compared. Two DNA probe fragments were designed for hybridization with microRNA-21 (miR-21), the microRNAs (miRNAs) model analyte. DNA probe 1 (P1) was assembled on Au nanoparticles (AuNPs) modified electrode. MiR-21 was captured by the pre-immobilized P1. A signal nanoprobe was synthesized by loading large amount of Thi on S-MWCNTs with covalently bonded probe 2 (P2). Owing to the large effective surface area of MWCNTs, fast electron shuttle of MWCNTs, high-loaded Thi on S-MWCNTs, and the increased conductivity from AuNPs, after signal probe hybridized with miR-21, it gave rise to a magnified current response on electrode. The increased electrochemical current enabled us to quantitatively detect miR-21. Expensive bioreagents and labeled target/detection DNA or miRNAs were avoided in this strategy. The operation complexity and assay cost were also reduced.


Assuntos
Técnicas Biossensoriais/métodos , Técnicas Eletroquímicas , MicroRNAs/análise , Nanotubos de Carbono/química , Tioninas/química , Técnicas Biossensoriais/instrumentação , Eletrodos
7.
Food Microbiol ; 73: 111-121, 2018 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-29526196

RESUMO

Plant defensins are small, cysteine-rich antimicrobial peptides of the immune system found in several organs during plant development. A synthetic peptide, KT43C, a linear analogue of the native Cp-thionin II found in cowpea seeds, was evaluated for its antifungal potential. It was found that KT43C displayed antifungal activity against Fusarium culmorum, Penicillium expansum and Aspergillus niger. Like native plant defensins, KT43C showed thermostability up to 100 °C and cation sensitivity. The synthetic peptide decreased the fungal growth without inducing morphogenic changes in the fungal hyphae. Non-inhibitory concentrations of the peptide induced permeabilization of the fungal membrane. In addition, high concentrations of KT43C induced the production of reactive oxygen species in the granulated cytoplasm. To investigate potential applications, the peptide was used as an additive in the preparation of dough which did not contain yeast. This peptide delayed the development of fungal growth in the dough by 2 days. Furthermore, KT43C did not induce red blood cell lysis up to a concentration of 200 µg.ml-1. These results highlight the potential for the use of synthetic antimicrobial defensins for shelf-life extension of food products.


Assuntos
Antifúngicos/farmacologia , Defensinas/farmacologia , Aditivos Alimentares/farmacologia , Tioninas/farmacologia , Vigna/química , Antifúngicos/síntese química , Antifúngicos/química , Aspergillus niger/efeitos dos fármacos , Aspergillus niger/crescimento & desenvolvimento , Pão/microbiologia , Defensinas/biossíntese , Defensinas/química , Aditivos Alimentares/síntese química , Aditivos Alimentares/química , Fusarium/efeitos dos fármacos , Fusarium/crescimento & desenvolvimento , Penicillium/efeitos dos fármacos , Penicillium/crescimento & desenvolvimento , Tioninas/síntese química , Tioninas/química
8.
Anal Biochem ; 547: 37-44, 2018 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-29452105

RESUMO

Sensitive and accurate detection of tumor markers is critical to early diagnosis, point-of-care and portable medical supervision. Alpha fetoprotein (AFP) is an important clinical tumor marker for hepatocellular carcinoma (HCC), and the concentration of AFP in human serum is related to the stage of HCC. In this paper, a label-free electrochemical aptasensor for AFP detection was fabricated using AFP-aptamer as the recognition molecule and thionin/reduced graphene oxide/gold nanoparticles (TH/RGO/Au NPs) as the sensor platform. With high electrocatalytic property and large specific surface area, RGO and Au NPs were employed on the screen-printed carbon electrode to load TH molecules. The TH not only acted as a bridging molecule to effectively capture and immobilize AFP-aptamer, but as the electron transfer mediator to provide the electrochemical signal. The AFP detection was based on the monitoring of the electrochemical current response change of TH by the differential pulse voltammetry. Under optimal conditions, the electrochemical responses were proportional to the AFP concentration in the range of 0.1-100.0 µg/mL. The limit of detection was 0.050 µg/mL at a signal-to-noise ratio of 3. The proposed method may provide a promising application of aptamer with the properties of facile procedure, low cost, high selectivity in clinic.


Assuntos
Aptâmeros de Nucleotídeos/química , Ouro/química , Grafite/química , Nanopartículas Metálicas/química , Tioninas/química , alfa-Fetoproteínas/análise , Humanos , Sensibilidade e Especificidade
9.
Plant Cell Environ ; 40(11): 2628-2643, 2017 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-28452058

RESUMO

Aphids are phloem-feeding insects that cause yield loss on a wide range of crops, including cereals such as barley. Whilst most aphid species are limited to one or few host species, some are able to reproduce on many plants belonging to different families. Interestingly, aphid probing behaviour can be observed on both host and non-host species, indicating that interactions take place at the molecular level that may impact host range. Here, we aimed to gain insight into the interaction of barley with aphid species differing in their ability to infest this crop by analysing transcriptional responses. Firstly, we determined colonization efficiency, settlement and probing behaviour for the aphid species Rhopalosiphum padi, Myzus persicae and Myzus cerasi, which defined host, poor-host and non-host interactions, respectively. Analyses of barley transcriptional responses revealed gene sets differentially regulated upon the different barley-aphid interactions and showed that the poor-host interaction with M. persicae resulted in the strongest regulation of genes. Interestingly, we identified several thionin genes strongly up-regulated upon interaction with M. persicae, and to a lesser extent upon R. padi interaction. Ectopic expression of two of these genes in Nicotiana benthamiana reduced host susceptibility to M. persicae, indicating that thionins contribute to defences against aphids.


Assuntos
Afídeos/fisiologia , Resistência à Doença/efeitos dos fármacos , Perfilação da Expressão Gênica , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Hordeum/genética , Hordeum/parasitologia , Doenças das Plantas/genética , Doenças das Plantas/parasitologia , Tioninas/farmacologia , Animais , Afídeos/patogenicidade , Análise por Conglomerados , Genes de Plantas , Hordeum/efeitos dos fármacos , Folhas de Planta/efeitos dos fármacos , Folhas de Planta/genética , Plantas Geneticamente Modificadas , Reprodutibilidade dos Testes , Especificidade da Espécie , Tabaco/genética , Transcrição Genética/efeitos dos fármacos , Transcriptoma/genética , Virulência/efeitos dos fármacos
10.
Biopolymers ; 108(3)2017 May.
Artigo em Inglês | MEDLINE | ID: mdl-28073158

RESUMO

Many Fusarium species are able to cause severe infections in plants as well as in animals and humans. Therefore, the discovery of new antifungal agents is of paramount importance. CaThi belongs to the thionins, which are cationic peptides with low molecular weights (∼5 kDa) that have toxic effects against various microorganisms. Herein, we study the mechanism of action of CaThi and its combinatory effect with fluconazole (FLC) against Fusarium solani. The mechanism of action of CaThi was studied by growth inhibition, viability, plasma membrane permeabilization, ROS induction, caspase activation, localization, and DNA binding capability, as assessed with Sytox green, DAB, FITC-VAD-FMK, CaThi-FITC, and gel shift assays. The combinatory effect of CaThi and FLC was assessed using a growth inhibition assay. Our results demonstrated that CaThi present a dose dependent activity and at the higher used concentration (50 µg mL-1 ) inhibits 83% of F. solani growth, prevents the formation of hyphae, permeabilizes membranes, induces endogenous H2 O2 , activates caspases, and localizes intracellularly. CaThi combined with FLC, at concentrations that alone do not inhibit F. solani, result in 100% death of F. solani when combined. The data presented in this study demonstrate that CaThi causes death of F. solani via apoptosis; an intracellular target may also be involved. Combined treatment using CaThi and FLC is a strong candidate for studies aimed at improved targeting of F. solani. This strategy is of particular interest because it minimizes selection of resistant microorganisms.


Assuntos
Antifúngicos/farmacologia , Peptídeos Catiônicos Antimicrobianos/farmacologia , Fluconazol/farmacologia , Tioninas/farmacologia , Antifúngicos/química , Peptídeos Catiônicos Antimicrobianos/química , Capsicum/química , Membrana Celular/efeitos dos fármacos , Permeabilidade da Membrana Celular/efeitos dos fármacos , Frutas/química , Fusarium/efeitos dos fármacos , Fusarium/patogenicidade , Humanos , Hifas/efeitos dos fármacos , Hifas/patogenicidade , Tioninas/química
11.
Angew Chem Int Ed Engl ; 55(47): 14552-14556, 2016 11 14.
Artigo em Inglês | MEDLINE | ID: mdl-27529162

RESUMO

Heterodimeric peptides linked by disulfide bonds are attractive drug targets. However, their chemical assembly can be tedious, time-consuming, and low yielding. Inspired by the cellular synthesis of pro-insulin in which the two constituent peptide chains are expressed as a single-chain precursor separated by a connecting C-peptide, we have developed a novel chemically cleavable bis-linker tether which allows the convenient assembly of two peptide chains as a single "pro"-peptide on the same solid support. Following the peptide cleavage and post-synthetic modifications, this bis-linker tether can be removed in one-step by chemical means. This method was used to synthesize a drug delivery-cargo conjugate, TAT-PKCi peptide, and a two-disulfide bridged heterodimeric peptide, thionin (7-19)-(24-32R), a thionin analogue. To our knowledge, this is the first report of a one-pot chemically cleavable bis-linker strategy for the facile synthesis of cross-bridged two-chain peptides.


Assuntos
Tioninas/síntese química , Dissulfetos/química , Sistemas de Liberação de Medicamentos , Estrutura Molecular , Tioninas/química
12.
Anal Quant Cytopathol Histpathol ; 38(2): 59-69, 2016 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-27386626

RESUMO

BACKGROUND: Prostate cancer is a disease of disrupted cell genomes. Quantification of DNA from cytology preparations can yield prognostic information about tissue biological behaviors; however, this process is very labor-intensive to perform. Quantitative digital pathology can measure the structural chromatin changes associated with neoplasia and can enable prognostic and predictive assays based on imaging of sectioned prostate tissue. OBJECTIVE: To design an automated system to recognize and localize cell nuclei in images of stained sectioned tissue (first step in enabling quantitative digital pathology). STUDY DESIGN: Images of Feulgen-thionin-stained prostate cancer tissue microarray constructed from the surgical specimens of 33 prostate cancer patients were acquired for this study. We implemented a new image segmentation technique to overcome tissue complexity, cell clusters, background noise, image and tissue inhomogeneities, and other imaging issues that introduce uncertainties into the segmentation method and developed a fully automated system to localized prostate cell nuclei. RESULTS: We applied our algorithm on our dataset and obtained a 96.6% true-positive rate and a 12% false-positive rate. CONCLUSION: In this paper we present a new method to automatically localize thionin-stained prostate cancer cells, enabling the extraction of various nuclear and cell sociology features with high precision.


Assuntos
Núcleo Celular/patologia , Interpretação de Imagem Assistida por Computador/métodos , Neoplasias da Próstata/patologia , Coloração e Rotulagem/métodos , Algoritmos , Automação Laboratorial , Núcleo Celular/química , Corantes , DNA/análise , Reações Falso-Positivas , Humanos , Masculino , Valor Preditivo dos Testes , Prognóstico , Neoplasias da Próstata/química , Neoplasias da Próstata/cirurgia , Reprodutibilidade dos Testes , Corantes de Rosanilina , Tioninas , Análise Serial de Tecidos
13.
Peptides ; 78: 109-18, 2016 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-26939717

RESUMO

ß-Defensins are members of the antimicrobial peptide superfamily that are produced in various species from different kingdoms, including plants. Plant defensins exhibit primarily antifungal activities, unlike those from animals that exhibit a broad-spectrum antimicrobial action. Recently, immunomodulatory roles of mammal ß-defensins have been observed to regulate inflammation and activate the immune system. Similar roles for plant ß-defensins remain unknown. In addition, the regulation of the immune system by mammalian ß-defensins has been studied in humans and mice models, particularly in immune cells, but few studies have investigated these peptides in epithelial cells, which are in intimate contact with pathogens. The aim of this work was to evaluate the effect of the chemically synthesized ß-defensin γ-thionin from Capsicum chinense on the innate immune response of bovine mammary epithelial cells (bMECs) infected with Staphylococcus aureus, the primary pathogen responsible for bovine mastitis, which is capable of living within bMECs. Our results indicate that γ-thionin at 0.1 µg/ml was able to reduce the internalization of S. aureus into bMECs (∼50%), and it also modulates the innate immune response of these cells by inducing the mRNA expression (∼5-fold) and membrane abundance (∼3-fold) of Toll-like receptor 2 (TLR2), as well as by inducing genes coding for the pro-inflammatory cytokines TNF-α and IL-1ß (∼14 and 8-fold, respectively) before and after the bacterial infection. γ-Thionin also induces the expression of the mRNA of anti-inflammatory cytokine IL-10 (∼12-fold). Interestingly, the reduction in bacterial internalization coincides with the production of other antimicrobial products by bMECs, such as NO before infection, and the secretion into the medium of the endogenous antimicrobial peptide DEFB1 after infection. The results from this work support the potential use of ß-defensins from plants as immunomodulators of the mammalian innate immune response.


Assuntos
Capsicum/química , Células Epiteliais/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Fatores Imunológicos/farmacologia , Staphylococcus aureus/efeitos dos fármacos , Tioninas/farmacologia , Sequência de Aminoácidos , Animais , Bovinos , Sobrevivência Celular/efeitos dos fármacos , Células Epiteliais/citologia , Células Epiteliais/imunologia , Células Epiteliais/microbiologia , Feminino , Regulação da Expressão Gênica/imunologia , Imunidade Inata , Fatores Imunológicos/isolamento & purificação , Interleucina-10/genética , Interleucina-10/imunologia , Interleucina-1beta/genética , Interleucina-1beta/imunologia , Glândulas Mamárias Animais/citologia , Glândulas Mamárias Animais/efeitos dos fármacos , Glândulas Mamárias Animais/imunologia , Glândulas Mamárias Animais/microbiologia , Extratos Vegetais/química , Cultura Primária de Células , RNA Mensageiro/genética , RNA Mensageiro/imunologia , Transdução de Sinais , Staphylococcus aureus/fisiologia , Tioninas/isolamento & purificação , Receptor 2 Toll-Like/genética , Receptor 2 Toll-Like/imunologia , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/imunologia , beta-Defensinas/biossíntese , beta-Defensinas/imunologia , beta-Defensinas/metabolismo
14.
BMC Microbiol ; 16: 12, 2016 Jan 27.
Artigo em Inglês | MEDLINE | ID: mdl-26819228

RESUMO

BACKGROUND: Thionins are a family of plant antimicrobial peptides (AMPs), which participate in plant defense system against pathogens. Here we describe some aspects of the CaThi thionin-like action mechanism, previously isolated from Capsicum annuum fruits. Thionin-like peptide was submitted to antimicrobial activity assays against Candida species for IC50 determination and synergism with fluconazole evaluation. Viability and plasma membrane permeabilization assays, induction of intracellular ROS production analysis and CaThi localization in yeast cells were also investigated. RESULTS: CaThi had strong antimicrobial activity against six tested pathogenic Candida species, with IC50 ranging from 10 to 40 µg.mL(-1). CaThi antimicrobial activity on Candida species was candidacidal. Moreover, CaThi caused plasma membrane permeabilization in all yeasts tested and induces oxidative stresses only in Candida tropicalis. CaThi was intracellularly localized in C. albicans and C. tropicalis, however localized in nuclei in C. tropicalis, suggesting a possible nuclear target. CaThi performed synergistically with fluconazole inhibiting all tested yeasts, reaching 100% inhibition in C. parapsilosis. The inhibiting concentrations for the synergic pair ranged from 1.3 to 4.0 times below CaThi IC50 and from zero to 2.0 times below fluconazole IC50. CONCLUSION: The results reported herein may ultimately contribute to future efforts aiming to employ this plant-derived AMP as a new therapeutic substance against yeasts.


Assuntos
Antifúngicos/farmacologia , Candida/efeitos dos fármacos , Capsicum/química , Fluconazol/farmacologia , Tioninas/farmacologia , Candida/crescimento & desenvolvimento , Sinergismo Farmacológico , Frutas/química , Testes de Sensibilidade Microbiana
15.
Molecules ; 20(12): 22170-87, 2015 Dec 11.
Artigo em Inglês | MEDLINE | ID: mdl-26690401

RESUMO

There is a strong drive worldwide to discover and exploit the therapeutic potential of a large variety of plants. In this work, an alcoholic extract of Helleborus purpurascens (family Ranunculaceae) was investigated for the identification of amino acids and peptides with putative antiproliferative effects. In our work, a separation strategy was developed using solvents of different polarity in order to obtain active compounds. Biochemical components were characterized through spectroscopic (mass spectroscopy) and chromatographic techniques (RP-HPLC and GC-MS). The biological activity of the obtained fractions was investigated in terms of their antiproliferative effects on HeLa cells. Through this study, we report an efficient separation of bioactive compounds (amino acids and peptides) from a plant extract dependent on solvent polarity, affording fractions with unaffected antiproliferative activities. Moreover, the two biologically tested fractions exerted a major antiproliferative effect, thereby suggesting potential anticancer therapeutic activity.


Assuntos
Aminoácidos/química , Antineoplásicos/química , Helleborus/química , Proteínas de Plantas/química , Tioninas/química , Aminoácidos/isolamento & purificação , Aminoácidos/farmacologia , Antineoplásicos/isolamento & purificação , Antineoplásicos/farmacologia , Butanóis , Sobrevivência Celular/efeitos dos fármacos , Cromatografia Líquida de Alta Pressão , Etanol , Dicloretos de Etileno , Células HeLa , Humanos , Extratos Vegetais/química , Proteínas de Plantas/isolamento & purificação , Proteínas de Plantas/farmacologia , Solventes , Tioninas/isolamento & purificação , Tioninas/farmacologia
16.
J Biol Chem ; 290(29): 18056-67, 2015 Jul 17.
Artigo em Inglês | MEDLINE | ID: mdl-26013828

RESUMO

Thionins are plant-specific antimicrobial peptides that have been isolated from the endosperm and leaves of cereals, from the leaves of mistletoes, and from several other plant species. They are generally basic peptides with three or four disulfide bridges and a molecular mass of ~5 kDa. Thionins are produced as preproproteins consisting of a signal peptide, the thionin domain, and an acidic domain. Previously, only mature thionin peptides have been isolated from plants, and in addition to removal of the signal peptide, at least one cleavage processing step between the thionin and the acidic domain is necessary to release the mature thionin. In this work, we identified a thionin proprotein-processing enzyme (TPPE) from barley. Purification of the enzyme was guided by an assay that used a quenched fluorogenic peptide comprising the amino acid sequence between the thionin and the acidic domain of barley leaf-specific thionin. The barley TPPE was identified as a serine protease (BAJ93208) and expressed in Escherichia coli as a strep tag-labeled protein. The barley BTH6 thionin proprotein was produced in E. coli using the vector pETtrx1a and used as a substrate. We isolated and sequenced the BTH6 thionin from barley to confirm the N and C terminus of the peptide in planta. Using an in vitro enzymatic assay, the recombinant TPPE was able to process the quenched fluorogenic peptide and to cleave the acidic domain at least at six sites releasing the mature thionin from the proprotein. Moreover, it was found that the intrinsic three-dimensional structure of the BTH6 thionin domain prevents cleavage of the mature BTH6 thionin by the TPPE.


Assuntos
Hordeum/enzimologia , Proteínas de Plantas/metabolismo , Serina Proteases/metabolismo , Tioninas/metabolismo , Sequência de Aminoácidos , Hordeum/química , Hordeum/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Proteínas de Plantas/química , Proteínas de Plantas/isolamento & purificação , Conformação Proteica , Proteólise , Alinhamento de Sequência , Serina Proteases/química , Serina Proteases/isolamento & purificação , Tioninas/química
17.
Biomed Res Int ; 2015: 735087, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25815333

RESUMO

Antimicrobial peptides (AMPs) are part of the innate immune defense mechanism of many organisms and are promising candidates to treat infections caused by pathogenic bacteria to animals and humans. AMPs also display anticancer activities because of their ability to inactivate a wide range of cancer cells. Cancer remains a cause of high morbidity and mortality worldwide. Therefore, the development of methods for its control is desirable. Attractive alternatives include plant AMP thionins, defensins, and cyclotides, which have anticancer activities. Here, we provide an overview of plant AMPs anticancer activities, with an emphasis on their mode of action, their selectivity, and their efficacy.


Assuntos
Peptídeos Catiônicos Antimicrobianos/uso terapêutico , Antineoplásicos/uso terapêutico , Imunidade Inata/efeitos dos fármacos , Neoplasias/tratamento farmacológico , Animais , Anti-Infecciosos/uso terapêutico , Ciclotídeos/uso terapêutico , Defensinas/uso terapêutico , Humanos , Imunidade Inata/imunologia , Neoplasias/patologia , Plantas/química , Tioninas/uso terapêutico
18.
Mol Plant Pathol ; 16(8): 870-81, 2015 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-25676661

RESUMO

Thionins are antimicrobial peptides that are involved in plant defence. Here, we present an in-depth analysis of the role of rice thionin genes in defence responses against two root pathogens: the root-knot nematode Meloidogyne graminicola and the oomycete Pythium graminicola. The expression of rice thionin genes was observed to be differentially regulated by defence-related hormones, whereas all analysed genes were consistently down-regulated in M. graminicola-induced galls, at least until 7 days post-inoculation (dpi). Transgenic lines of Oryza sativa cv. Nipponbare overproducing OsTHI7 revealed decreased susceptibility to M. graminicola infection and P. graminicola colonization. Taken together, these results demonstrate the role of rice thionin genes in defence against two of the most damaging root pathogens attacking rice.


Assuntos
Oryza/imunologia , Raízes de Plantas/imunologia , Tioninas/fisiologia , Genes de Plantas , Oryza/genética , Oryza/microbiologia , Raízes de Plantas/microbiologia
19.
Anal Chem ; 86(24): 12064-9, 2014 Dec 16.
Artigo em Inglês | MEDLINE | ID: mdl-25391335

RESUMO

A layered MoS2-thionin composite was prepared by sonicating their mixture in an ionic liquid and gradient centrifugation. Because DNA is rarely present in single-stranded form, either naturally or after PCR amplification, the composite was used for fabrication of a double-stranded DNA (dsDNA) electrochemical biosensor due to stable electrochemical response, intercalation, and electrostatic interaction of thionin with DNA. The linear range over dsDNA concentration from 0.09 ng mL(-1) to 1.9 ng mL(-1) is obtained, and moreover, it is suitable for the detection of single-stranded DNA (ssDNA). The biosensor has been applied to the detection of circulating DNA from healthy human serum, and satisfactory results have been obtained. The constructed DNA electrochemical biosensor shows potential application in the fields of bioanalysis and clinic diagnosis. Furthermore, this work proposes a new method to construct electrochemical biosensors based on MoS2 sheets.


Assuntos
Análise Química do Sangue/instrumentação , DNA/análise , Eletroquímica , Molibdênio/química , Tioninas/química , Técnicas Biossensoriais/instrumentação , DNA/química , Humanos , Limite de Detecção , Microscopia Eletrônica de Varredura
20.
Bioresour Technol ; 169: 277-283, 2014 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-25062539

RESUMO

Prior studies indicated that biodecolorized intermediates of azo dyes could act as electron shuttles to stimulate wastewater decolorization and bioelectricity generation (WD&BG) in microbial fuel cells (MFCs). This study tended to explore whether non-azo textile dyes (i.e., thionin and malachite green) could also own such redox-mediating capabilities for WD&BG. Prior findings mentioned that OH and/or NH2 substitute-containing auxochrome compounds (e.g., 2-aminophenol and 1,2-dihydroxybenzene) could effectively mediate electron transport in MFCs for simultaneous WD&BG. This work clearly suggested that the presence of electron-mediating textile dyes (e.g., thionin and malachite green (MG)) in MFCs is promising to stimulate color removal and bioelectricity generation. That is, using MFCs as operation strategy for wastewater biodecolorization is economically promising in industrial applications due to autocatalytic acceleration of electron-flux for WD&BG in MFCs.


Assuntos
Fontes de Energia Bioelétrica , Corantes/química , Corantes de Rosanilina/química , Têxteis , Tioninas/química , Espectroscopia Dielétrica , Eletricidade , Técnicas Eletroquímicas , Eletrodos , Cinética , Oxirredução
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