RESUMO
We evaluated the effect of the total macerate (TM) and seed oil (SO) of mature Carica candamarcensis fruits, on the release of Matrix metalloproteinase 9 (MMP9) and the phosphorylation of MAPK in neutrophils. The antioxidant capacity of these extracts was evaluated by ABTS assay. Neutrophils stimulated with different dilutions of TM or SO were analyzed for cytotoxicity, MMP9 release, and MAPK phosphorylation, using trypan blue exclusion assays, zymography, and immunoblotting, respectively. Both extracts show antioxidant activity, being higher in TM; none presented cytotoxic effect. The 5% and 2.5% dilutions of TM significantly reduced MMP9 release, and all decreased MAPK phosphorylation. SO significantly increased the release o f MMP9 and MAPK phosphorylation, the effect being greater when they were prestimulated with lipopolysaccharide.TM may have anti - inflammatory potential, while SO could have a priming effect that needs to be confirmed
Evaluamos el efecto del macerado total (MT) y aceite de semillas (AV) de frutos maduros de Carica candamarcensis , en la liberación de Matriz metaloproteinasa 9 (MMP9) y la fosfor ilación de MAPK en neutrófilos. La capacidad antioxidante de estos extractos se evaluó por ensayo ABTS. En neutrófilos estimulados con diferentes diluciones de MT o AV se analizó la citotoxicidad, liberación de MMP9 y fosforilación de MAPK, mediante ensayo s de exclusión con azul de tripano, zimografía e inmunotransferencia, respectivamente. Ambos extractos muestran actividad antioxidante, siendo mayor en MT; ninguno presentó efecto citotóxico. Las diluciones 5% y 2,5% de MT redujeron significativamente la l iberación de MMP9, y todas disminuyeron la fosforilación de MAPK. El AV incrementó significativamente la liberación de MMP9 y la fosforilación de MAPK, el efecto fue mayor cuando se preestimularon con lipopolisacárido. El MT puede tener potencial antiinfla matorio, mientras que el AV podría tener un efecto "priming" que necesita ser corroborado.
Assuntos
Humanos , Extratos Vegetais/farmacologia , Carica/enzimologia , Neutrófilos/efeitos dos fármacos , Fosforilação , Proteína Quinase 1 Ativada por Mitógeno , Metaloproteinase 9 da Matriz/metabolismo , Látex/análiseRESUMO
Papaya is a fleshy fruit that undergoes fast ethylene-induced modifications. The fruit becomes edible, but the fast pulp softening is the main factor that limits the post-harvest period. Papaya fast pulp softening occurs due to cell wall disassembling coordinated by ethylene triggering that massively expresses pectinases. In this work, RNA-seq analysis of ethylene-treated and non-treated papayas enabled a wide transcriptome overview that indicated the role of ethylene during ripening at the gene expression level. Several families of transcription factors (AP2/ERF, NAC, and MADS-box) were differentially expressed. ACO, ACS, and SAM-Mtase genes were upregulated, indicating a high rate of ethylene biosynthesis after ethylene treatment. The correlation among gene expression and physiological data demonstrated ethylene treatment can indeed simulate ripening, and regulation of changes in fruit color, aroma, and flavor could be attributed to the coordinated expression of several related genes. Especially about pulp firmness, the identification of 157 expressed genes related to cell wall metabolism demonstrated that pulp softening is accomplished by a coordinated action of several different cell wall-related enzymes. The mechanism is different from other commercially important fruits, such as strawberry, tomato, kiwifruit, and apple. The observed behavior of this new transcriptomic data confirms ethylene triggering is the main event that elicits fast pulp softening in papayas.
Assuntos
Carica/metabolismo , Etilenos/metabolismo , Frutas/metabolismo , Carica/enzimologia , Carica/genética , Parede Celular/metabolismo , Etilenos/farmacologia , Frutas/efeitos dos fármacos , Frutas/enzimologia , Expressão Gênica/genética , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Genes de Plantas , Proteínas de Plantas/metabolismo , Biologia de Sistemas/métodos , Fatores de Transcrição/metabolismo , Transcriptoma/efeitos dos fármacosRESUMO
Papaya fruit, widely consumed around the world, is mechanically and structurally affected by several enzymatic processes during ripening, where pectin methylesterase plays a key role. Hence, the aim of this work was to evaluate possible correlations among physicochemical changes, mechanical parameters, viscoelastic behavior, and enzyme activity of pectin methylesterase to provide information about the softening phenomenon by applying the Maxwell and Peleg models. Mechanical parameters were estimated by texture profile analysis, enzyme activity by Michaelis-Menten parameters, and viscoelastic behavior by relaxation test responses fitted to these models. The Maxwell model described properly mechanical changes during ripening, displaying a better adjustment (R2 > 0.97) than the Peleg model (0.80 < R2 < 0.84). Pearson correlation analysis (P ≤ 0.01) indicated an inversely proportional relation among firmness, total soluble solids, and the first elastic element of the Maxwell model. Besides, the PME Michaelis-Menten affinity constant showed a correlation between the first elastic element and the first viscoelastic element of the Maxwell model. Findings of this work pointed out that the first Maxwell elastic element could explain structural changes as papaya ripening advance, associated with pectin methylesterase activity, cell wall disruption, and cell assembling into the tissue. PRACTICAL APPLICATION: Mechanical and viscoelastic behavior of papaya fruit tissue were described by the Maxwell model associating both viscous and elastic elements to the softening process. The results provide background and practical knowledge to describe structural changes during the ripening process of papaya depending on its enzymatic activity. Outcomes could be further applied to understand changes in other fruits or food matrixes that soften during postharvest, storage, and food chain supply processes.
Assuntos
Hidrolases de Éster Carboxílico/química , Carica/enzimologia , Frutas/crescimento & desenvolvimento , Proteínas de Plantas/química , Carica/química , Carica/crescimento & desenvolvimento , Parede Celular/química , Parede Celular/enzimologia , Estabilidade Enzimática , Frutas/química , Frutas/enzimologia , Cinética , ViscosidadeRESUMO
The present study evaluated four laticifer fluids as a novel source of peptidases capable of hydrolyzing proteins in cow's milk. The latex peptidases from Calotropis procera (CpLP), Cryptostegia grandiflora (CgLP), and Carica papaya (CapLP) were able to perform total hydrolysis of caseins after 30â¯min at pH 6.5, as confirmed by a significant reduction in the residual antigenicity. Casein hydrolysis by Plumeria rubra latex peptidases (PrLP) was negligible. Moreover, whey proteins were more resistant to proteolysis by latex peptidases; however, heat pretreatment of the whey proteins enhanced the degree of hydrolysis and reduced the residual antigenicity of the hydrolysates. The in vivo assays show that the cow's milk proteins hydrolysed by CgLP and CapLP exhibited no immune reactions in mice allergic to cow's milk, similar to a commercial partially hydrolysed formula. Thus, these peptidases are promising enzymes for the development of novel hypoallergenic formulas for children with a milk allergy.
Assuntos
Caseínas/metabolismo , Hipersensibilidade a Leite/patologia , Peptídeo Hidrolases/metabolismo , Animais , Apocynaceae/enzimologia , Calotropis/enzimologia , Carica/enzimologia , Caseínas/imunologia , Bovinos , Humanos , Hidrólise , Látex/metabolismo , Masculino , Camundongos , Leite/metabolismo , Hipersensibilidade a Leite/imunologia , Hipersensibilidade a Leite/veterinária , Proteínas do Soro do Leite/imunologia , Proteínas do Soro do Leite/metabolismoRESUMO
Carica papaya latex is one of the most studied sources of plant lipases. However, the complexity of the matrix composition makes it difficult to isolate and purify the lipolytic enzymes present in Carica papaya latex. Therefore, diverse strategies have been developed to study the catalytic properties of these enzymes.Recently the first lipase from Carica papaya latex (CpLip1) has been successfully cloned and expressed in order to study their catalytic properties. In order to improve the catalytic properties and increase the potential for its use at industrial scale.In this chapter, a practical protocol to recombinant CpLip1 lipase is given.
Assuntos
Carica/enzimologia , Expressão Gênica , Lipase/metabolismo , Ácidos e Sais Biliares/farmacologia , Carica/genética , Ativação Enzimática/efeitos dos fármacos , Lipase/genéticaRESUMO
We present an efficient approach to the synthesis of a series of glycyrrhetinic acid derivatives. Six derivatives, five of them new compounds, were obtained through chemoenzymatic reactions in very good to excellent yield. In order to find the optimal reaction conditions, the influence of various parameters such as enzyme source, nucleophile:substrate ratio, enzyme:substrate ratio, solvent and temperature was studied. The excellent results obtained by lipase catalysis made the procedure very efficient considering their advantages such as mild reaction conditions and low environmental impact. Moreover, in order to explain the reactivity of glycyrrhetinic acid and the acetylated derivative to different nucleophiles in the enzymatic reactions, molecular docking studies were carried out. In addition, one of the synthesized compounds exhibited remarkable antiviral activity against TKâ¯+â¯and TK- strains of Herpes simplex virus type 1 (HSV-1), sensitive and resistant to acyclovir (ACV) treatment.
Assuntos
Antivirais/farmacologia , Ácido Glicirretínico/farmacologia , Herpesvirus Humano 1/efeitos dos fármacos , Lipase/metabolismo , Simulação de Acoplamento Molecular , Aciclovir/farmacologia , Antivirais/química , Antivirais/metabolismo , Biocatálise , Candida/enzimologia , Carica/enzimologia , Relação Dose-Resposta a Droga , Eurotiales/enzimologia , Ácido Glicirretínico/química , Ácido Glicirretínico/metabolismo , Testes de Sensibilidade Microbiana , Estrutura Molecular , Rhizomucor/enzimologia , Rhizopus/enzimologia , Relação Estrutura-AtividadeRESUMO
PREMISE OF THE STUDY: The red flesh of some papaya cultivars is caused by a recessive loss-of-function mutation in the coding region of the chromoplast-specific lycopene beta cyclase gene (CYC-b). We performed an evolutionary genetic analysis of the CYC-b locus in wild and cultivated papaya to uncover the origin of this loss-of-function allele in cultivated papaya. METHODS: We analyzed the levels and patterns of genetic diversity at the CYC-b locus and six loci in a 100-kb region flanking CYC-b and compared these to genetic diversity levels at neutral autosomal loci. The evolutionary relationships of CYC-b haplotypes were assessed using haplotype network analysis of the CYC-b locus and the 100-kb CYC-b region. KEY RESULTS: Genetic diversity at the recessive CYC-b allele (y) was much lower relative to the dominant Y allele found in yellow-fleshed wild and cultivated papaya due to a strong selective sweep. Haplotype network analyses suggest the y allele most likely arose in the wild and was introduced into domesticated varieties after the first papaya domestication event. The shared haplotype structure between some wild, feral, and cultivated haplotypes around the y allele supports subsequent escape of this allele from red cultivars back into wild populations through feral intermediates. CONCLUSIONS: Our study supports a protracted domestication process of papaya through the introgression of wild-derived traits and gene flow from cultivars to wild populations. Evidence of gene flow from cultivars to wild populations through feral intermediates has implications for the introduction of transgenic papaya into Central American countries.
Assuntos
Carica/genética , Evolução Molecular , Variação Genética , Liases Intramoleculares/genética , Proteínas de Plantas/genética , Alelos , Carica/classificação , Carica/enzimologia , América Central , Fluxo Gênico , Frequência do Gene , Genética Populacional , Haplótipos , Liases Intramoleculares/classificação , Modelos Genéticos , Mutação , Fenótipo , Filogenia , Proteínas de Plantas/classificação , Plastídeos/genéticaRESUMO
Alicyclobacillus spp. are spore forming bacteria that are often related to the deterioration of acidic products such as beverages and citrus juices. After the process of industrial pasteurization, the spore produced by the bacteria can germinate and the microorganism can grow, causing sensory abnormalities in the product. Alternative biopreservatives, such as the antimicrobial compounds, are of considerable importance to the food industry. Papain and bromelain are proteolytic enzymes derived frompapaya and pineapple, respectively. These enzymes are widely used in medicine and in the pharmaceutical and food industries, but while some studies have described their antibacterial action, no studies of the Alicyclobacillus spp. exist. The aimof this studywas to analyze the antibacterial effect of papain and bromelain on Alicyclobacillus spp. through 1) determining minimum inhibitory and bactericidal concentration (MIC and MBC); 2) determining the death time curve of the micro-organism in the presence and absence of enzymes; and 3) investigating the enzymatic mechanism on the microorganism. The antibacterial activity of enzymes in combination with nisin was also evaluated. The results showed that for the Alicyclobacillus acidoterrestris strain, the MIC of papain was 0.98 µg/mL and the MBC was 3.91 µg/mL, while theMIC of bromelain was 62.5 µg/mL and the MBCwas 250 µg/mL. The concentration of 4 ×MIC for both the enzymes was sufficient to eliminate 4 logs of the micro-organism after 24 h of incubation. Through the use of enzyme inhibitors specific for cysteine proteases, it was found that the antibacterial activity of papain and bromelain is not related to its proteolytic activity, butmay be related to other activities, such as amidse and esterase. The synergistic activity of the enzymes revealed a fractional inhibitory concentration (FIC) level of 0.16. Combination with nisin revealed an FIC of 0.25 for papain and 0.19 for bromelain, indicating synergism between both compounds. The application of enzymes in reconstituted orange juice contaminated with A. acidoterrestris was found to be effective, as after 48 h of incubation, at three different temperatures, the initial microbial population was eliminated. This study showed that the enzymes papain and bromelain have an antibacterial effect on A. acidoterrestris.
Assuntos
Alicyclobacillus/efeitos dos fármacos , Antibacterianos/farmacologia , Bromelaínas/farmacologia , Nisina/farmacologia , Papaína/farmacologia , Amidoidrolases/metabolismo , Ananas/enzimologia , Bebidas/microbiologia , Carica/enzimologia , Citrus sinensis/microbiologia , Cisteína Proteases/metabolismo , Inibidores de Cisteína Proteinase/química , Sinergismo Farmacológico , Esterases/metabolismo , Testes de Sensibilidade Microbiana , PasteurizaçãoRESUMO
The proteolytic enzymes from V. cundinamarcensis latex, (P1G10), display healing activity in animal models following various types of lesions. P1G10 or the purified isoforms act as mitogens on fibroblast and epithelial cells by stimulating angiogenesis and wound healing in gastric and cutaneous ulcers models. Based on evidence that plant proteinases act as antitumorals, we verified this effect on a murine melanoma model. The antitumoral effect analyzed mice survival and tumor development after subcutaneous administration of P1G10 into C57BL/6J mice bearing B16F1 low metastatic melanoma. Possible factors involved in the antitumoral action were assessed, i.e., cytotoxicity, cell adhesion and apoptosis in vitro, haemoglobin (Hb), vascular endothelial growth factor (VEGF), tumor growth factor-ß (TGF-ß), tumor necrosis factor-α (TNF-α) content and N-acetyl-glucosaminidase (NAG) activity. We observed that P1G10 inhibited angiogenesis measured by the decline of Hb and VEGF within the tumor, and TGF-ß displayed a non-significant increase and TNF-α showed a minor non-significant reduction. On the other hand, there was an increase in NAG activity. In treated B16F1 cells, apoptosis was induced along with decreased cell binding to extracellular matrix components (ECM) and anchorage, without impairing viability.
Assuntos
Inibidores da Angiogênese/administração & dosagem , Antineoplásicos Fitogênicos/administração & dosagem , Carica/enzimologia , Melanoma Experimental/tratamento farmacológico , Peptídeo Hidrolases/administração & dosagem , Neoplasias Cutâneas/tratamento farmacológico , Inibidores da Angiogênese/farmacologia , Animais , Antineoplásicos Fitogênicos/farmacologia , Apoptose , Adesão Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Masculino , Melanoma Experimental/metabolismo , Melanoma Experimental/patologia , Camundongos , Peptídeo Hidrolases/farmacologia , Proteínas de Plantas/administração & dosagem , Proteínas de Plantas/farmacologia , Neoplasias Cutâneas/metabolismo , Neoplasias Cutâneas/patologia , Fator de Crescimento Transformador beta/metabolismo , Resultado do Tratamento , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Papaya (Carica papaya L.) is a climacteric fleshy fruit that undergoes dramatic changes during ripening, most noticeably a severe pulp softening. However, little is known regarding the genetics of the cell wall metabolism in papayas. The present work describes the identification and characterization of genes related to pulp softening. We used gene expression profiling to analyze the correlations and co-expression networks of cell wall-related genes, and the results suggest that papaya pulp softening is accomplished by the interactions of multiple glycoside hydrolases. The polygalacturonase cpPG1 appeared to play a central role in the network and was further studied. The transient expression of cpPG1 in papaya results in pulp softening and leaf necrosis in the absence of ethylene action and confirms its role in papaya fruit ripening.
Assuntos
Carica/genética , Parede Celular/genética , Frutas/genética , Regulação da Expressão Gênica de Plantas , Proteínas de Plantas/genética , Poligalacturonase/genética , Arabidopsis/enzimologia , Arabidopsis/genética , Carica/enzimologia , Carica/crescimento & desenvolvimento , Parede Celular/enzimologia , Frutas/enzimologia , Frutas/crescimento & desenvolvimento , Perfilação da Expressão Gênica , Redes Reguladoras de Genes , Cinética , Folhas de Planta/enzimologia , Folhas de Planta/genética , Proteínas de Plantas/metabolismo , Poligalacturonase/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMO
The immunomodulatory properties of a mixture of cysteine peptidases (P1G10) obtained from the fruit lattice of Carica candamarcensis were investigated. P1G10 was obtained from fresh latex samples by chromatography in a Sephadex column and initially administered to Swiss mice (n = 5; 1 or 10 mg/kg) via i.p. After 30 min, the mice were injected with carrageenan (0.5 mg/mouse) or heat-killed S. Typhimurium (10(7) CFU/mL; 100°C/30 min) into the peritoneal cavity. Afterwards, two animal groups were i.p. administered with P1G10 (n = 6; 1, 5, or 10 mg/Kg) or PBS 24 hours prior to challenge with live S. Typhimurium (10(7) CFU/mL). P1G10 stimulated the proliferation of circulating neutrophils and lymphocytes, 6 h after injection of carrageenan or heat-killed bacteria, respectively. Furthermore, survival after infection was dose-dependent and reached 60% of the animal group. On the other hand, control mice died 1-3 days after infection. The examination of mRNA transcripts in liver cells 24 h after infection confirmed fold variation increases of 5.8 and 4.8 times on average for IL-1 and COX-2, respectively, in P1G10 pretreated mice but not for TNF-α, IL-10, γ-IFN and iNOS, for which the results were comparable to untreated animals. These data are discussed in light of previous reports.
Assuntos
Carica/enzimologia , Ciclo-Oxigenase 2/metabolismo , Inflamação/metabolismo , Interleucina-1/metabolismo , Peptídeo Hidrolases/química , Salmonelose Animal/metabolismo , Animais , Anti-Infecciosos/química , Proliferação de Células , Regulação Enzimológica da Expressão Gênica , Inflamação/microbiologia , Látex , Fígado/enzimologia , Camundongos , Óxido Nítrico/metabolismo , RNA Mensageiro/metabolismo , Salmonella typhimurium , Células-Tronco , Fatores de TempoRESUMO
The latex from Vasconcellea quercifolia ("oak leaved papaya"), a member of the Caricaceae family, contains at least seven cysteine endopeptidases with high proteolytic activity, which helps to protect these plants against injury. In this study, we isolated and characterized the most basic of these cysteine endopeptidases, named VQ-VII. This new purified enzyme was homogeneous by bidimensional electrophoresis and MALDI-TOF mass spectrometry, and exhibited a molecular mass of 23,984 Da and an isoelectric point >11. The enzymatic activity of VQ-VII was completely inhibited by E-64 and iodoacetic acid, confirming that it belongs to the catalytic group of cysteine endopeptidases. By investigating the cleavage of the oxidized insulin B-chain to establish the hydrolytic specificity of VQ-VII, we found 13 cleavage sites on the substrate, revealing that it is a broad-specificity peptidase. The pH profiles toward p-Glu-Phe-Leu-p-nitroanilide (PFLNA) and casein showed that the optimum pH is about 6.8 for both substrates, and that in casein, it is active over a wide pH range (activity higher than 80 % between pH 6 and 9.5). Kinetic enzymatic assays were performed with the thiol peptidase substrate PFLNA (K m = 0.454 ± 0.046 mM, k cat = 1.57 ± 0.07 s(-1), k cat/K m = 3.46 × 10(3) ± 14 s(-1) M(-1)). The N-terminal sequence (21 amino acids) of VQ-VII showed an identity >70 % with 11 plant cysteine peptidases and the presence of highly conserved residues and motifs shared with the "papain-like" family of peptidases. VQ-VII proved to be a new latex enzyme of broad specificity, which can degrade extensively proteins of different nature in a wide pH range.
Assuntos
Carica/enzimologia , Cisteína Proteases/isolamento & purificação , Cisteína Proteases/metabolismo , Látex/metabolismo , Proteínas de Plantas/isolamento & purificação , Proteínas de Plantas/metabolismo , Sequência de Aminoácidos , Carica/química , Domínio Catalítico , Cisteína Proteases/química , Eletroforese em Gel de Poliacrilamida , Concentração de Íons de Hidrogênio , Insulina/metabolismo , Cinética , Espectrometria de Massas , Dados de Sequência Molecular , Proteínas de Plantas/química , Proteólise , Especificidade por Substrato , TitulometriaRESUMO
A group of cysteine-proteolytic enzymes from C. candamarcensis latex, designated as P1G10 displays pharmacological properties in animal models following various types of lesions. This enzyme fraction expresses in vitro fibrinolytic effect without need for plasminogen activation. Based on this evidence, we assessed by intravital microscopy the effect of P1G10 on recanalization of microvessels after thrombus induction in the ear of hairless mice. Video playback of intravital microscopic images allowed measurement of blood flow velocity (mm/s) during the experimental procedure. Groups treated with 5 or 7.5mg/Kg P1G10 showed thrombolysis between 7-15min, without vessel obstruction. Ex vivo experiments demonstrated that platelet activation by ADP is impaired in a dose dependent manner following treatment with P1G10. The P1G10 action on plasma coagulation also showed that prothrombin time (PT), thrombin time (TT) and activated partial thromboplastin time (aPTT, µg/uL) are increased in a dose dependent manner. In addition, P1G10 displayed fibrinogenolytic and fibrinolytic activities, both in a dose dependent manner. Each of these effects was suppressed by inhibition of the proteolytic activity of the fraction. The antithrombotic action of P1G10 can be explained by proteolytic cleavage of fibrinogen and fibrin, both key factors during formation of a stable thrombus. These results combined with prior evidence suggest that P1G10 has potential as thrombolytic agent.
Assuntos
Carica/enzimologia , Cisteína Proteases/farmacologia , Fibrinolíticos/farmacologia , Trombose/tratamento farmacológico , Animais , Coagulação Sanguínea/efeitos dos fármacos , Masculino , Camundongos , Camundongos Pelados , Agregação Plaquetária/efeitos dos fármacos , Ratos , Ratos Wistar , Trombose/sangueRESUMO
CMS1MS2 (CC-Ib) from Carica candamarcensis (Vasconcellea cundinamarcensis) is a cysteine proteinase found as a single polypeptide containing 213 residues of 22,991 Da. The enzyme was purified by three chromatographic steps, two of them involving cationic exchange. Crystals of CMS1MS2 complexed with E-64 were obtained by the hanging drop vapor-diffusion method at 291 K using ammonium sulfate and polyethylene glycol 4000/8000 as precipitant. The complex CMS1MS2-E-64 crystallized in the tetragonal space group P4(1)2(1)2 with unit-cell parameters; a = b = 73.64, c = 118.79 Å. The structure was determined by Molecular Replacement and refined at 1.87 Å resolution to a final R factor of 16.2 % (R (free) = 19.3 %). Based on the model, the structure of CMS1MS2 (PDB 3IOQ) ranks as one of the least basic cysteine isoforms from C. candamarcensis, is structurally closer to papain, caricain, chymopapain and mexicain than to the other cysteine proteinases, while its activity is twice the activity of papain towards BAPNA substrate. Two differences, one in the S2 subsite and another in the S3 subsite of CMS1MS2 may contribute to the enhanced activity relative to papain. In addition, the model provides a structural basis for the sensitivity of CMS1MS2 to inhibition by cystatin, not shown by other enzymes of the group, e.g., glycyl endopeptidase and CMS2MS2.
Assuntos
Carica/enzimologia , Cisteína Proteases/química , Cristalografia por Raios X , Cisteína Proteases/metabolismo , Isoenzimas/química , Isoenzimas/metabolismo , Modelos Moleculares , Conformação ProteicaRESUMO
Previous studies demonstrated that proteinases from latex of C. candamarcensis act as mitogens on fibroblast and epithelial cells and a subsequent report showed their protective, angiogenic and wound healing effects on gastric ulcers. In this study, we present evidence of skin healing activity by the group of proteinases known as P1G10. By using a hairless mouse model, we compared the healing effect following topical application of various concentrations of P1G10. The data confirm that healing actions take place between 0.1 and 1%, without adverse local irritation or systemic toxicological action after a prolonged period of use. The wound healing effect is unaltered when P1G10 is previously inhibited with iodoacetamide. The low permeation of the hydrosoluble formulation Polawax(®) supports the maintenance of the drug at the site of application. These results extend the healing properties of these groups of enzymes in situations of dermatological trauma and open the way to future clinical applications.
Assuntos
Cisteína Endopeptidases/farmacologia , Glicoproteínas/farmacologia , Látex/química , Fitoterapia , Pele/efeitos dos fármacos , Cicatrização/efeitos dos fármacos , Cicatrização/fisiologia , Animais , Peso Corporal/efeitos dos fármacos , Carica/enzimologia , Cisteína Endopeptidases/uso terapêutico , Dextranos , Eletroforese em Gel de Poliacrilamida , Feminino , Géis , Glicoproteínas/uso terapêutico , Masculino , Camundongos , Camundongos PeladosRESUMO
Cysteine proteinases from the Caricaceae belong to the C1 family of the CA clan and display papain-like structured, the archetype enzyme for this group of proteins. Carica candamarcensis, also named Vasconcellea cundinamarcensis, a member of Caricaceae family common to many areas in South America, contains cysteine proteinases with proteolytic activity five to eight-fold higher than those from latex of Carica papaya. The cysteine protease CMS2MS2 from C. candamarcensis latex has been shown to enhance proliferation of L929 fibroblast and to activate the extracellular signal-regulated protein kinase (ERK). In this study, the cDNA cloning, expression and evaluation of biological activity of a CMS2MS2-like protein from C. candamarcensis is reported. The 650 bp fragment was cloned in bacteria and the DNA sequence confirmed a cysteine-proteinase similar to CMS2MS2. The recombinant protein is 30 kDa, induces a mitogenic response, and enhances ERK1/2 phosphorylation, like the non-recombinant enzyme, but lacks either amidase or caseinolytic activity. The mitogenic activity of this protein and its lack of proteolytic activity underscore a potential for use in wound healing treatment.
Assuntos
Carica/enzimologia , Cisteína Proteases/fisiologia , Mitógenos/fisiologia , Proteínas de Plantas/fisiologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Clonagem Molecular , Cisteína Proteases/química , Cisteína Proteases/farmacologia , Camundongos , Mitógenos/química , Mitógenos/farmacologia , Dados de Sequência Molecular , Proteínas de Plantas/química , Proteínas de Plantas/farmacologia , Estrutura Terciária de Proteína , Alinhamento de Sequência , Análise de Sequência de DNA , Análise de Sequência de Proteína , Cicatrização/efeitos dos fármacosRESUMO
Carica candamarcensis is a species from the Caricaceae family whose immature fruit contains latex with large amounts of cysteine proteinases. In prior studies, we isolated two of these enzymes displaying mitogenic activity when incubated with L929 fibroblastic cells. One of the fractions containing these enzymes (P1G10) was shown to enhance wound healing of skin and to accelerate healing of chemically induced gastric ulcer. In this study we evaluate the effect of P1G10 on heat-induced, third-degree burn using a rodent model. The results show that 0.1% P1G10 accelerates epithelisation while the effect of 1% or 0.01% P1G10 is not significantly different to 1% silver sulphadiazine, 2% papain or the hydrosoluble vehicle used as control. In a double-blind randomised experiment comparing the healing response of 0.1%, 1% and the vehicle alone, we confirmed the enhanced healing property of P1G10. Histological analysis of burn-tissue sections following treatment with P1G10 support these observations. These results extend the healing properties of these groups of enzymes to a different type of trauma and open the way to future clinical applications.
Assuntos
Queimaduras/tratamento farmacológico , Carica/enzimologia , Fitoterapia/métodos , Cicatrização/efeitos dos fármacos , Animais , Queimaduras/patologia , Relação Dose-Resposta a Droga , Avaliação Pré-Clínica de Medicamentos/métodos , Eletroforese em Gel de Poliacrilamida/métodos , Feminino , Masculino , Camundongos , Camundongos Pelados , Extratos Vegetais/administração & dosagem , Extratos Vegetais/uso terapêutico , Distribuição AleatóriaRESUMO
The cysteine protease CMS2MS2 from Carica candamarcensis latex has been shown to enhance proliferation of L929 fibroblast and to activate the extracellular signal-regulated protein kinase (ERK). In experiments with CMS2MS2 irreversibly inhibited by E-64, the proliferative effect on fibroblasts remains unaffected. ERK phosphorylation mediated by CMS2MS2 was abolished in the presence of PD 98059 or U0126, both MAPK cascade inhibitors. In addition, these inhibitors suppress the mitogenic activity of intact CMS2MS2 or CMS2MS2-E-64. Furthermore, ERK phosphorylation and the mitogenic effect are partially suppressed by a phospholipase C (PLC) inhibitor. These data suggest that the mitogenic effect of CMS2MS2 on fibroblasts is independent of its proteolytic activity, requires ERK phosphorylation, and involves activation of PLC.
Assuntos
Carica/enzimologia , Proliferação de Células , Cisteína Proteases/farmacologia , Fibroblastos/citologia , Fibroblastos/enzimologia , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Mitógenos/farmacologia , Animais , Butadienos/farmacologia , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Ativação Enzimática , Flavonoides/farmacologia , Leucina/análogos & derivados , Leucina/farmacologia , Camundongos , Nitrilas/farmacologia , Inibidores de Proteases/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Fosfolipases Tipo C/metabolismoRESUMO
Pulp softening is one of the most remarkable changes during ripening of papaya (Carica papaya) fruit and it is a major cause for post-harvest losses. Although cell wall catabolism has a major influence on papaya fruit, quality information on the gene products involved in this process is limited. A full-length polygalacturonase cDNA (cpPG) was isolated from papaya pulp and used to study gene expression and enzyme activity during normal and ethylene-induced ripening and after exposure of the fruit to 1-MCP. Northern-blot analysis demonstrated that cpPG transcription was strongly induced during ripening and was highly ethylene-dependent. The accumulation of cpPG transcript was paralleled by enzyme activity, and inversely correlated to the pulp firmness. Preliminary in silico analysis of the cpPG genomic sequence revealed the occurrence of putative regulatory motifs in the promoter region that may help to explain the effects of plant hormones and non-abiotic stresses on papaya fruit firmness. This newly isolated cpPG is an important candidate for functional characterization and manipulation to control the process of pulp softening during papaya ripening.
Assuntos
Carica/enzimologia , Frutas/enzimologia , Regulação da Expressão Gênica de Plantas , Expressão Gênica , Genes de Plantas , Poligalacturonase/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Northern Blotting , Carica/genética , Clonagem Molecular , Ciclopropanos , DNA Complementar , Etilenos , Frutas/genética , Dados de Sequência Molecular , Proteínas de Plantas , Poligalacturonase/genética , Regiões Promotoras Genéticas , RNA Mensageiro/metabolismoRESUMO
Papaya (Carica papaya) is a very important crop in many tropical countries but it is highly susceptible to parasitic diseases, physiological disorders, mechanical damage and fruit overripening. Here we report a study on ACC oxidase cosuppression and its effects on papaya fruit ripening. Papaya ACC oxidase was isolated using PCR and embriogenic cells transformed by biolistic using the CaMV 35S promoter to drive the expression of the PCR fragment in sense orientation. Fifty transgenic lines were recovered and 20 of those were grown under field conditions. Southern analysis showed incorporation of the transgene in different copy numbers in the papaya genome. Fruits were evaluated in terms of texture (firmness), colour development, respiration and ethylene production. A sharp reduction in ethylene and CO2 production was detected, whereas softening and colour development of the peel were also altered. Overall, transgenic fruits showed a delay in ripening rate. A reduction in mRNA level for ACC oxidase in transgenic fruit was clearly detectable by northern blot. More studies are necessary before this technology can be used to extend the shelf life of papaya fruit.