RESUMEN
The latex of Calotropis procera has previously been reported to contain osmotin. This protein (CpOsm) inhibited phytopathogens and this was mechanistically characterized. Here, the time-course profile of CpOsm transcripts was examined in the salt-stressed cultivated callus of C. procera in order to better understand its role in the physiology of the plant. Stressed callus (80 mM NaCl) showed an unbalanced content of organic compounds (proline and total soluble sugar) and inorganic ions (Na+, Cl-, and K+). Under salt treatment, the transcripts of CpOsm were detected after 12 h and slightly increased to a maximum at day seven, followed by reduction. Interestingly, CpOsm was not detected in the soluble protein fraction recovered from the salt-stressed callus as probed by electrophoresis, dot/Western blotting and mass spectrometry. The results suggested that (1) CpOsm is not constitutive in cultivated cells (laticifer-free tissues); (2) CpOsm transcripts appear under salt-stressed conditions; (3) the absence of CpOsm in the protein fractions of stressed cultivated cells indicated that salt-induced transcripts were not used for protein synthesis and this accounts to the belief that CpOsm may be a true laticifer protein in C. procera. More effort will be needed to unveil this process. In this study we show evidences that CpOsm gene is responsive to salt stress. However the corresponding protein is not produced in cultivated cells. Therefore, presently the hypothesis that CpOsm is involved in abiotic stress is not fully supported.
Asunto(s)
Calotropis/metabolismo , Látex/metabolismo , Proteínas de Plantas/metabolismo , Estrés Fisiológico/fisiología , Calotropis/genéticaRESUMEN
In a previous study, we performed the chemical characterization of a polyvinyl alcohol (PVA) membrane supplemented with latex proteins (LP) displaying wound healing activity, and its efficacy as a delivery system was demonstrated. Here, we report on aspects of the mechanism underlying the performance of the PVA-latex protein biomembrane on wound healing. LP-PVA, but not PVA, induced more intense leukocyte (neutrophil) migration and mast cell degranulation during the inflammatory phase of the cicatricial process. Likewise, LP-PVA induced an increase in key markers and mediators of the inflammatory response (myeloperoxidase activity, nitric oxide, TNF, and IL-1ß). These results demonstrated that LP-PVA significantly accelerates the early phase of the inflammatory process by upregulating cytokine release. This remarkable effect improves the subsequent phases of the healing process. The polyvinyl alcohol membrane was fully absorbed as an inert support while LP was shown to be active. It is therefore concluded that the LP-PVA is a suitable bioresource for biomedical engineering.
Asunto(s)
Calotropis , Portadores de Fármacos , Látex/farmacología , Membranas Artificiales , Proteínas de Plantas/farmacología , Alcohol Polivinílico/química , Piel/efectos de los fármacos , Cicatrización de Heridas/efectos de los fármacos , Heridas Penetrantes/tratamiento farmacológico , Administración Cutánea , Animales , Calotropis/química , Degranulación de la Célula/efectos de los fármacos , Modelos Animales de Enfermedad , Composición de Medicamentos , Mediadores de Inflamación/metabolismo , Interleucina-1beta/metabolismo , Látex/aislamiento & purificación , Activación de Macrófagos/efectos de los fármacos , Mastocitos/efectos de los fármacos , Mastocitos/metabolismo , Mastocitos/patología , Ratones , Infiltración Neutrófila/efectos de los fármacos , Óxido Nítrico/metabolismo , Peroxidasa/metabolismo , Fitoterapia , Proteínas de Plantas/aislamiento & purificación , Plantas Medicinales , Piel/lesiones , Piel/metabolismo , Piel/patología , Factores de Tiempo , Factor de Necrosis Tumoral alfa/metabolismo , Heridas Penetrantes/metabolismo , Heridas Penetrantes/patologíaRESUMEN
CpOsm is an antifungal osmotin/thaumatin-like protein purified from the latex of Calotropis procera. The protein is relatively thermostable and retains its antifungal activity over a wide pH range; therefore, it may be useful in the development of new antifungal drugs or transgenic crops with enhanced resistance to phytopathogenic fungi. To gain further insight into the mechanism of action of CpOsm, its three-dimensional structure was determined, and the effects of the protein on Fusarium solani spores were investigated by atomic force microscopy (AFM). The atomic structure of CpOsm was solved at a resolution of 1.61Å, and it contained 205 amino acid residues and 192 water molecules, with a final R-factor of 18.12% and an Rfree of 21.59%. The CpOsm structure belongs to the thaumatin superfamily fold and is characterized by three domains stabilized by eight disulfide bonds and a prominent charged cleft, which runs the length of the front side of the molecule. Similarly to other antifungal thaumatin-like proteins, the cleft of CpOsm is predominantly acidic. AFM images of F. solani spores treated with CpOsm resulted in striking morphological changes being induced by the protein. Spores treated with CpOsm were wrinkled, and the volume of these cells was reduced by approximately 80%. Treated cells were covered by a shell of CpOsm molecules, and the leakage of cytoplasmic content from these cells was also observed. Based on the structural features of CpOsm and the effects that the protein produces on F. solani spores, a possible mechanism of action is suggested and discussed.
Asunto(s)
Antifúngicos/aislamiento & purificación , Antifúngicos/farmacología , Calotropis/química , Fusarium/efectos de los fármacos , Proteínas de Plantas/química , Proteínas de Plantas/aislamiento & purificación , Esporas Fúngicas/efectos de los fármacos , Algoritmos , Secuencia de Aminoácidos , Antifúngicos/química , Secuencia de Bases , Látex/química , Microscopía de Fuerza Atómica , Datos de Secuencia Molecular , Proteínas de Plantas/farmacología , Tetrahidrofolato DeshidrogenasaRESUMEN
OBJECTIVE AND DESIGN: Laticifer proteins (LP) of Calotropis procera were fractionated by ion-exchange chromatography, and the influence of a sub-fraction (LP(PI)) on the inflammatory response of Swiss mice challenged by Salmonella enterica Ser. Typhimurium was investigated. METHODS: Mice (n = 10) received LP(PI) (30 or 60 mg/kg) in a single inoculum by the intraperitoneal route 24 h before infection. To investigate the relevance of the proteolytic activity, three additional groups were included: the first one received heat-treated LP (30 mg/kg-30 min at 100 °C), the second received LP (30 mg/kg) inactivated by iodoacetamide, and a control group received only phosphate-buffered saline (PBS). RESULTS: The survival rate reached 100 % in mice treated with LP(PI) and was also observed with the other treatment, whereas the PBS group died 1-3 days after infection. The neutrophil infiltration into the peritoneal cavity of pretreated mice was enhanced and accompanied by high bacterial clearance from the bloodstream. Tumor necrosis factor-alpha mRNA transcripts, but not interferon-gamma, were detected early in spleen cells of pretreated mice after infection; however, the nitric oxide contents in the bloodstream were decreased in comparison to the PBS group. CONCLUSIONS: The inflammatory stimulus of C. procera proteins increased phagocytosis and balanced the nitric oxide release in the bloodstream, preventing septic shock induced by Salmonella infection.
Asunto(s)
Calotropis , Látex , Proteínas de Plantas/farmacología , Fiebre Tifoidea/prevención & control , Animales , Modelos Animales de Enfermedad , Inflamación/sangre , Inflamación/inmunología , Recuento de Leucocitos , Masculino , Ratones , Infiltración Neutrófila , Óxido Nítrico/sangre , Ratas , Ratas Wistar , Salmonella enterica , Fiebre Tifoidea/sangre , Fiebre Tifoidea/inmunologíaRESUMEN
The proteins derived from the latex (LP) of Calotropis procera are well known for their anti-inflammatory property. In view of their protective effect reported in the sepsis model, they were evaluated for their efficacy in maintaining coagulation homeostasis in sepsis. Intraperitoneal injection of LP markedly reduced the procoagulation and thrombocytopenia observed in mice infected with Salmonella; while in normal mice, LP produced a procoagulant effect. In order to understand its mechanism of action, the LP was subjected to ion-exchange chromatography, and the three subfractions (LPPI, LPPII, and LPPIII) thus obtained were tested for their proteolytic effect and thrombin- and plasmin-like activities in vitro. Of the three subfractions tested, LPPII and LPPIII exhibited proteolytic effect on azocasein and exhibited procoagulant effect on human plasma in a concentration-dependent manner. Like trypsin and plasmin, these subfractions produced both fibrinogenolytic and fibrinolytic effects that were mediated through the hydrolysis of the Aα, Bß, and γ chains of fibrinogen and α-polymer and γ-dimer of fibrin clot, respectively. This study shows that the cysteine proteases present in the latex of C. procera exhibit thrombin- and plasmin-like activities and suggests that these proteins have therapeutic potential in various conditions associated with coagulation abnormalities.
Asunto(s)
Calotropis , Proteasas de Cisteína/farmacología , Proteínas de Plantas/farmacología , Infecciones por Salmonella/sangre , Sepsis/sangre , Animales , Coagulación Sanguínea/efectos de los fármacos , Proteasas de Cisteína/aislamiento & purificación , Fibrinógeno/metabolismo , Hemostasis/efectos de los fármacos , Humanos , Látex/química , Masculino , Ratones , Tiempo de Tromboplastina Parcial , Proteínas de Plantas/aislamiento & purificación , Recuento de Plaquetas , Tiempo de Protrombina , Salmonella typhimuriumRESUMEN
AIM OF THE STUDY: The latex of Calotropis procera has been used in traditional medicine to treat different inflammatory diseases. The anti-inflammatory activity of latex proteins (LP) has been well documented using different inflammatory models. In this work the anti-inflammatory protein fraction was evaluated in a true inflammatory process by inducing a lethal experimental infection in the murine model caused by Salmonella enterica Subsp. enterica serovar Typhimurium. MATERIALS AND METHODS: Experimental Swiss mice were given 0.2 ml of LP (30 or 60 mg/kg) by the intraperitoneal route 24 h before or after lethal challenge (0.2 ml) containing 10(6) CFU/ml of Salmonella Typhimurium using the same route of administration. RESULTS: All the control animals succumbed to infection within 6 days. When given before bacterial inoculums LP prevented the death of mice, which remained in observation until day 28. Even, LP-treated animals exhibited only discrete signs of infection which disappeared latter. LP fraction was also protective when given orally or by subcutaneous route. Histopathological examination revealed that necrosis and inflammatory infiltrates were similar in both the experimental and control groups on days 1 and 5 after infection. LP activity did not clear Salmonella Typhimurium, which was still present in the spleen at approximately 10(4) cells/g of organ 28 days after challenge. However, no bacteria were detected in the liver at this stage. LP did not inhibit bacterial growth in culture medium at all. In the early stages of infection bacteria population was similar in organs and in the peritoneal fluid but drastically reduced in blood. Titration of TNF-alpha in serum revealed no differences between experimental and control groups on days 1 and 5 days after infection while IL-12 was only discretely diminished in serum of experimental animals on day 5. Moreover, cultured macrophages treated with LP and stimulated by LPS released significantly less IL-1beta. CONCLUSIONS: LP-treated mice did not succumb to septic shock when submitted to a lethal infection. LP did not exhibit in vitro bactericidal activity. It is thought that protection of LP-treated mice against Salmonella Typhimurium possibly involves down-regulation of pro-inflammatory cytokines (other than TNF-alpha). LP inhibited IL-1beta release in cultured macrophages and discretely reduced IL-12 in serum of animals given LP. Results reported here support the folk use of latex to treat skin infections by topic application.
Asunto(s)
Antibacterianos/uso terapéutico , Calotropis/química , Proteínas de Plantas/uso terapéutico , Infecciones por Salmonella/prevención & control , Salmonella typhimurium/efectos de los fármacos , Choque Séptico/prevención & control , Animales , Antibacterianos/aislamiento & purificación , Antibacterianos/farmacología , Células Cultivadas , Recuento de Colonia Microbiana , Citocinas/inmunología , Modelos Animales de Enfermedad , Látex/química , Hígado/efectos de los fármacos , Hígado/patología , Macrófagos Peritoneales/efectos de los fármacos , Macrófagos Peritoneales/inmunología , Masculino , Ratones , Proteínas de Plantas/aislamiento & purificación , Proteínas de Plantas/farmacología , Infecciones por Salmonella/inmunología , Infecciones por Salmonella/microbiología , Infecciones por Salmonella/patología , Salmonella typhimurium/patogenicidad , Choque Séptico/inmunología , Choque Séptico/microbiología , Choque Séptico/patología , Bazo/efectos de los fármacos , Bazo/patologíaRESUMEN
AIM OF THE STUDY: The latex of Calotropis procera has been used in the traditional medicinal system for the treatment of leprosy, ulcers, tumors, piles and diseases of liver, spleen, abdomen and toothache. It comprises of a non-dialyzable protein fraction (LP) that exhibits anti-inflammatory properties and a dialyzable fraction (DF) exhibiting pro-inflammatory properties. The present study was carried out to evaluate the effect of LP sub-fractions on neutrophil functions and nociception in rodent models and to elucidate the mediatory role of nitric oxide (NO). MATERIAL AND METHODS: The LP was subjected to ion exchange chromatography and the effect of its three sub-fractions (LP(PI), LP(PII) and LP(PIII)) thus obtained was evaluated on leukocyte functions in the rat peritonitis model and on nociception in the mouse model. RESULTS: LP sub-fractions exhibit distinct protein profile and produce a significant decrease in the carrageenan and DF induced neutrophil influx and exhibit anti-nociceptive property. The LP and its sub-fractions produced a marked reduction in the number of rolling and adherent leukocytes in the mesenteric microvasculature as revealed by intravital microscopy. The anti-inflammatory effect of LP(PI), the most potent anti-inflammatory fraction of LP, was accompanied by an increase in the serum levels of NO. Further, our study shows that NO is also involved in the inhibitory effect of LP(PI) on neutrophil influx. CONCLUSIONS: Our study shows that LP fraction of Calotropis procera comprises of three distinct sets of proteins exhibiting anti-inflammatory and anti-nociceptive properties of which LP(PI) was most potent in inhibiting neutrophil functions and its effects are mediated through NO production.