Antiproliferative effects of lectins from Canavalia ensiformis and Canavalia brasiliensis in human leukemia cell lines

AbstractThe antiproliferative activity of lectins Canavalia ensiformis (ConA) and Canavalia brasiliensis (ConBr) were studied using human leukemia MOLT-4 and HL-60 cell lines. It was revealed that both ConA and ConBr were markedly cytotoxic to cells using MTT and NAC assays. The IC50 values were approximately 3 and 20μg/mL for ConA and ConBr, respectively, for both MOLT-4 and HL-60 cells. However, in normal human peripheral blood lymphocytes, the lectins were not cytotoxic, even when tested at concentrations as high as 200μg/ml. Using comet assay, the lectins produced a rate of DNA damage exceeding 80% in MOLT-4 and HL-60 cells. Fluorescence analysis revealed the morphology characteristic of apoptosis, with low concentrations of apoptotic bodies and fragmented DNA (5μg/ml). Flow cytometric analysis demonstrated an accumulation of cells in the sub-G1 cell cycle that is characteristic of DNA fragmentation, and a decrease in membrane integrity at high concentrations. Lastly, we evaluated the alterations in mitochondrial potential that reduced after treatment with lectins. Our results indicate that ConA and ConBr inhibited cell proliferation selectively in tumor cells and that apoptosis was the main death mechanism. Therefore, lectins can be considered a class of molecules with a high antitumor activity potential

Allergenicity reduction of cow's milk proteins using latex peptidases

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 30 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.Fil: Oliveira, João P. B.. Universidade Estadual do Ceará; BrasilFil: Candreva, Ángela María. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Estudios Inmunológicos y Fisiopatológicos. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Instituto de Estudios Inmunológicos y Fisiopatológicos; ArgentinaFil: Rizzo, Gaston Pascual. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Estudios Inmunológicos y Fisiopatológicos. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Instituto de Estudios Inmunológicos y Fisiopatológicos; ArgentinaFil: Ramos, Márcio V.. Universidade Estadual do Ceará; BrasilFil: Oliveira, Jefferson S.. Universidade Estadual do Ceará; BrasilFil: Oliveira, Hermógenes D.. Universidade Federal Do Piaui.; BrasilFil: Ary, Maria B.. Universidade Estadual do Ceará; BrasilFil: Docena, Guillermo H.. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Estudios Inmunológicos y Fisiopatológicos. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Instituto de Estudios Inmunológicos y Fisiopatológicos; ArgentinaFil: Freitas, Cleverson D. T.. Universidade Federal Do Ceara; Brasi

Insights into milk-clotting activity of latex peptidases from <i>Calotropis procera</i> and <i>Cryptostegia grandiflora</i>

Latex fractions from Calotropis procera, Cryptostegia grandiflora, Plumeria rubra, and Himatanthus drasticus were assayed in order to prospect for new plant peptidases with milk-clotting activities, for use as rennet alternatives. Only C. procera and C. grandiflora latex fractions exhibited proteolytic and milk-clotting activities, which were not affected by high concentrations of NaCl and CaCl2. However, pre-incubation of both samples at 75 °C for 10 min eliminated completely their activities. Both proteolytic fractions were able to hydrolyze k-casein and to produce peptides of 16 kDa, a similar SDS-PAGE profile to commercial chymosin. RP-HPLC and mass spectrometry analyses of the k-casein peptides showed that the peptidases from C. procera or C. grandiflora hydrolyzed k-casein similar to commercial chymosin. The cheeses made with both latex peptidases exhibited yields, dry masses, and soluble proteins similar to cheeses prepared with commercial chymosin. In conclusion, C. procera and C. grandiflora latex peptidases with the ability to coagulate milk can be used as alternatives to commercial animal chymosin in the cheese manufacturing process.Centro de Investigación de Proteínas Vegetale

Insights into milk-clotting activity of latex peptidases from <i>Calotropis procera</i> and <i>Cryptostegia grandiflora</i>

Latex fractions from Calotropis procera, Cryptostegia grandiflora, Plumeria rubra, and Himatanthus drasticus were assayed in order to prospect for new plant peptidases with milk-clotting activities, for use as rennet alternatives. Only C. procera and C. grandiflora latex fractions exhibited proteolytic and milk-clotting activities, which were not affected by high concentrations of NaCl and CaCl2. However, pre-incubation of both samples at 75 °C for 10 min eliminated completely their activities. Both proteolytic fractions were able to hydrolyze k-casein and to produce peptides of 16 kDa, a similar SDS-PAGE profile to commercial chymosin. RP-HPLC and mass spectrometry analyses of the k-casein peptides showed that the peptidases from C. procera or C. grandiflora hydrolyzed k-casein similar to commercial chymosin. The cheeses made with both latex peptidases exhibited yields, dry masses, and soluble proteins similar to cheeses prepared with commercial chymosin. In conclusion, C. procera and C. grandiflora latex peptidases with the ability to coagulate milk can be used as alternatives to commercial animal chymosin in the cheese manufacturing process.Centro de Investigación de Proteínas Vegetale

Insights into milk-clotting activity of latex peptidases from <i>Calotropis procera</i> and <i>Cryptostegia grandiflora</i>

Latex fractions from Calotropis procera, Cryptostegia grandiflora, Plumeria rubra, and Himatanthus drasticus were assayed in order to prospect for new plant peptidases with milk-clotting activities, for use as rennet alternatives. Only C. procera and C. grandiflora latex fractions exhibited proteolytic and milk-clotting activities, which were not affected by high concentrations of NaCl and CaCl2. However, pre-incubation of both samples at 75 °C for 10 min eliminated completely their activities. Both proteolytic fractions were able to hydrolyze k-casein and to produce peptides of 16 kDa, a similar SDS-PAGE profile to commercial chymosin. RP-HPLC and mass spectrometry analyses of the k-casein peptides showed that the peptidases from C. procera or C. grandiflora hydrolyzed k-casein similar to commercial chymosin. The cheeses made with both latex peptidases exhibited yields, dry masses, and soluble proteins similar to cheeses prepared with commercial chymosin. In conclusion, C. procera and C. grandiflora latex peptidases with the ability to coagulate milk can be used as alternatives to commercial animal chymosin in the cheese manufacturing process.Centro de Investigación de Proteínas Vegetale

Latex peptidases of Calotropis procera for dehairing of leather as an alternative to environmentally toxic sodium sulfide treatment

Dehairing of crude leather is a critical stage performed at the beginning of its processing to obtain industrially useful pieces. Tanneries traditionally apply a chemical process based on sodium sulfide. Since this chemical reactive is environmentally toxic and inefficiently recycled, innovative protocols for reducing or eliminating its use in leather depilation are welcomed. Therefore, latex peptidases from Calotropis procera (CpLP) and Cryptostegia grandiflora (CgLP) were assayed for this purpose. Enzyme activity on substrates representative of skin such as hide powder azure (UHPA), elastin (UE), azocollagen (UAZOCOL), keratin (UK), and epidermis (UEP) was determined, while depilation activity was assayed on cow hide. Only CpLP was active against keratin (13.4 UK) and only CgLP was active against elastin (0.12 UE). CpLP (93.0 UHPA, 403.6 UAZOCOL, 36.3 UEP) showed higher activity against the other substrates than CgLP (47.6 UHPA, 261.5 UAZOCOL, 8.5 UEP). In pilot assays, CpLP (0.05% w/v with sodium sulfite 0.6% w/v as activator) released hairs from cow hide pieces. Macroscopic and microscopic analyses of the hide revealed that the dehairing process was complete and the leather structure was preserved. The proteolytic system of C. procera is a suitable bioresources to be exploited by tanneries.Fil: Lopez, Laura Maria Isabel. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Instituto Nacional de Tecnología Industrial. Centro de Investigación y Desarrollo del Cuero. Provincia de Buenos Aires. Gobernación. Comision de Investigaciones Centíficas. Centro de Investigación y Desarrollo del Cuero; ArgentinaFil: Viana, Carolina A.. Universidade Estadual Do Ceara; BrasilFil: Errasti, María Eugenia. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Instituto Nacional de Tecnología Industrial. Centro de Investigación y Desarrollo del Cuero. Provincia de Buenos Aires. Gobernación. Comision de Investigaciones Centíficas. Centro de Investigación y Desarrollo del Cuero; ArgentinaFil: Garro, María Laura. Instituto Nacional de Tecnología Industrial. Centro de Investigación y Desarrollo del Cuero. Provincia de Buenos Aires. Gobernación. Comision de Investigaciones Centíficas. Centro de Investigación y Desarrollo del Cuero; ArgentinaFil: Martegani, José Eduardo. Instituto Nacional de Tecnología Industrial. Centro de Investigación y Desarrollo del Cuero. Provincia de Buenos Aires. Gobernación. Comision de Investigaciones Centíficas. Centro de Investigación y Desarrollo del Cuero; ArgentinaFil: Mazzilli, German Ariel. Instituto Nacional de Tecnología Industrial. Centro de Investigación y Desarrollo del Cuero. Provincia de Buenos Aires. Gobernación. Comision de Investigaciones Centíficas. Centro de Investigación y Desarrollo del Cuero; ArgentinaFil: Freitas, Cléverson D. T.. Universidade Estadual Do Ceara; BrasilFil: Araújo, Ídila M. S.. EMBRAPA Agrotropical; BrasilFil: da Silva, Rafaela O.. Universidade Estadual Do Ceara; BrasilFil: Ramos, Márcio V.. Universidade Estadual Do Ceara; Brasi

Allergenicity reduction of cow's milk proteins using latex peptidases

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 30 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.Instituto de Estudios Inmunológicos y Fisiopatológico

Seeds of Amazonian Fabaceae as a source of new lectins

Seeds from fifty native Amazonian Fabaceae species (representing subfamilies Caesalpinioideae, Mimosoideae and Faboideae) were screened for the presence of new lectins. Their crude protein extracts were assayed for hemagglutinating activity (HA). The protein fractions of Anadenanthera peregrina, Dimorphandra caudata, Ormosia lignivalvis and Swartzia laevicarpa exhibited HA, and this activity was inhibited by galactose or lactose but not by glucose or mannose. The crude extract of S. laevicarpa exhibited HA activity only after ion exchange chromatography, and its lectin was further purified by affinity chromatography on immobilized lactose. Despite the large number of lectins that have been reported in leguminous plants, this is the first description of lectins in the genera Anadenanthera, Dimorphandra and Ormosia. The study of lectins from these genera and from Swartzia will contribute to the understanding of the evolutionary relationships of legume lectins in terms of their protein processing properties and structures