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- ItemSomente MetadadadosBlocking the Proliferation of Human Tumor Cell Lines by Peptidase Inhibitors from Bauhinia Seeds(Georg Thieme Verlag Kg, 2013-03-01) Nakahata, Adriana Miti [UNIFESP]; Mayer, Barbara; Neth, Peter; Hansen, Daiane [UNIFESP]; Sampaio, Misako Uemura [UNIFESP]; Oliva, Maria Luiza Vilela [UNIFESP]; Universidade Federal de São Paulo (UNIFESP); Max Planck Inst Biochem; Univ MunichIn cancer tumors, growth, invasion, and formation of metastasis at a secondary site play a pivotal role, participating in diverse processes in the development of the pathology, such as degradation of extracellular matrix. Bauhinia seeds contain relatively large quantities of peptidase inhibitors, and two Bauhinia inhibitors were obtained in a recombinant form from the Bauhinia bauhinioides species, B. bauhinoides cruzipain inhibitor, which is a cysteine and serine peptidase inhibitor, and B. bauhinioides kallikrein inhibitor, which is a serine peptidase inhibitor. While recombinant B. bauhinoides cruzipain inhibitor inhibits human neutrophil elastase cathepsin G and the cysteine proteinase cathepsin L, recombinant B. bauhinioides kallikrein inhibitor inhibits plasma kallikrein and plasmin. the effects of recombinant B. bauhinoides cruzipain inhibitor and recombinant B. bauhinioides kallikrein inhibitor on the viability of tumor cell lines with a distinct potential of growth from the same tissue were compared to those of the clinical cytotoxic drug 5-fluorouracil. At 12.5 mu M concentration, recombinant B. bauhinoides cruzipain inhibitor and recombinant B. bauhinioides kallikrein inhibitor were more efficient than 5-fluorouracil in inhibiting MKN-28 and Hs746T (gastric), HCT116 and HT29 (colorectal), SkBr-3 and MCF-7 (breast), and THP-1 and K562 (leukemia) cell lines. Additionally, recombinant B. bauhinoides cruzipain inhibitor inhibited 40% of the migration of Hs746T, the most invasive gastric cell line, while recombinant B. bauhinioides kallikrein inhibitor did not affect cell migration. Recombinant B. bauhinioides kallikrein inhibitor and recombinant B. bauhinoides cruzipain inhibitor, even at high doses, did not affect hMSC proliferation while 5-fluorouracil greatly reduced the proliferation rates of hMSCs. Therefore, both recombinant B. bauhinoides cruzipain inhibitor and recombinant B. bauhinioides kallikrein inhibitor might be considered for further studies to block peptidase activities in order to target specific peptidase-mediated growth and invasion characteristics of individual tumors, mainly in patients resistant to 5-fluorouracil chemotherapy.
- ItemSomente MetadadadosBrazilian red propolis: unreported substances, antioxidant and antimicrobial activities(Wiley-Blackwell, 2011-10-01) Righi, Adne A.; Alves, Thiago R. [UNIFESP]; Negri, Giuseppina [UNIFESP]; Marques, Lucas M.; Breyer, Henrique; Salatino, Antonio; Universidade de São Paulo (USP); Universidade Federal de São Paulo (UNIFESP); Universidade Federal da Bahia (UFBA); Breyer & Cia LTDABACKGROUND: Chloroform, ethyl acetate and methanol extracts of a sample of red propolis from the state of Alagoas (northeast Brazil) were analyzed by gas chromatography-mass spectrometry and high-performance liquid chromatography-diode array detection-electrospray ionization-mass spectrometry. Antimicrobial and antioxidant activities were also obtained.RESULTS: the propolis sample contained low content of narigenin-8-C-hexoside, this being the first report of a C-glycoside in propolis. the main constituent found was characterized as 3,4,2',3'-tetrahydroxychalcone. Other important constituents were the chalcone isoliquiritigenin, the isoflavans (3S)-vestitol, (3S)-7-O-methylvestitol, the pterocarpan medicarpin, the phenylpropenes trans-anethol, methyl eugenol, elimicin, methoxyeugenol and cis-asarone, and the triterpenic alcohols lupeol and alpha- and beta- amyrins. the methanol extract exhibited high antioxidant activities by 2,2-diphenyl-1-picrylhydrazyl and beta-carotene/linoleic acid assay methods, and antimicrobial activity toward Gram-positive and Gram-negative bacteria.CONCLUSION: Structures are suggested for new substances never before seen in any kind of propolis. This is the first report of 3,4,2',3'-tetrahydroxychalcone and a flavone C-glycoside in a propolis sample. (C) 2011 Society of Chemical Industry
- ItemSomente MetadadadosCloning, expression and characterization of Bauhinia variegata trypsin inhibitor BvTI(Walter de Gruyter & Co, 2005-11-01) Souza, A. F. de; Torquato, RJS; Tanaka, A. S.; Sampaio, CAM; Universidade Federal de São Paulo (UNIFESP)A Bauhinia variegata trypsin inhibitor (BvTI) cDNA fragment was cloned into the pCANTAB5E phagemid. the clone pAS 1.1.3 presented a cDNA fragment of 733 bp, including the coding region for a mature BvTI protein comprising 175 amino acid residues. the deduced amino acid sequence for BvTI confirmed it as a member of the Kunitz-type plant serine proteinase inhibitor family. the BvTI cDNA fragment encoding the mature form was cloned into the expression vector, pET-14b, and expressed in E coli BL2I (DE3) pLysS in an active form. in addition, a BvTI mutant form, r(mut)BvTI, with a Pro residue as the fifth amino acid in place of Leu, was produced. the recombinant proteins, rBvTI and r(mut)BvTI, were purified on a trypsin-Sepharose column, yielding 29 and 1.44 mg/I of active protein, respectively, and showed protein bands of approximately 21.5 kDa by SDS-PAGE. Trypsin inhibition activity was comparable for rBvTI (K-I=4 nm) and r(mut)BvTI (K-i=6 nm). Our data suggest that the Leu to Pro substitution at the fifth amino-terminal residue was not crucial for proteinase inhibition.
- ItemSomente MetadadadosThe defensive functions of plant inhibitors are not restricted to insect enzyme inhibition(Elsevier B.V., 2010-02-01) Sumikawa, Joana Tomomi; Brito, Marlon Vilela de; Rodrigues Macedo, Maria Ligia; Uchoa, Adriana F.; Miranda, Antonio [UNIFESP]; Araujo, Ana Paula U.; Silva-Lucca, Rosemeire A.; Sampaio, Misako Uemura; Oliva, Maria Luiza Vilela [UNIFESP]; Universidade Federal de São Paulo (UNIFESP); Univ Fed Mato Grosso; Univ Estadual Fluminense Darcy Ribeiro; Universidade de São Paulo (USP); Univ Estadual Oeste ParanaThree plant proteinase inhibitors BbKI (kallikrein inhibitor) and BbCI (cruzipain inhibitor) from Bauhinia bouhinioides, and a BrTI (trypsin inhibitor) from B. rufa, were examined for other effects in Callosobruchus maculatus development; of these only BrTI affected bruchid emergence. BrTI and BbKI share 81% identities in their primary sequences and the major differences between them are the regions comprising the RGD and RGE motifs in BrTI. These sequences were shown to be essential for BrTI insecticidal activity, since a modified BbKI [that is a recombinant form (BbKIm) with some amino acid residues replaced by those found in BrTI sequence] also strongly inhibited insect development. By using synthetic peptides related to the BrTI sequence, YLEAPVARGDGGLA-NH(2) (RGE) and IVYYPDRGETGL-NH(2) (RGE), it was found that the peptide with an RGE sequence was able to block normal development of C. maculatus larvae (ED(50) 0.16% and LD(50) 0.09%), this being even more effective than the native protein. (C) 2009 Elsevier B.V. All rights reserved.
- ItemSomente MetadadadosLeucaena leucocephala serine proteinase inhibitor: primary structure and action on blood coagulation, kinin release and rat paw edema(Elsevier B.V., 2000-03-07) Oliva, Maria Luiza Vilela [UNIFESP]; Souza-Pinto, José Carlos [UNIFESP]; Batista, Isabel de Fatima Correia [UNIFESP]; Araujo, Mariana da Silva [UNIFESP]; Silveira, Vera Lucia Flor [UNIFESP]; Auerswald, Ennes A.; Mentele, Reini; Eckerskorn, Christoph; Sampaio, Misako Uemura [UNIFESP]; Sampaio, Claudio Augusto Machado [UNIFESP]; Universidade Federal de São Paulo (UNIFESP); LMU; Max Planck Inst BiochemA serine proteinase inhibitor isolated from Leucaena leucocephala seeds (LITI) was purified to homogeneity by acetone fractionation. ion exchange chromatography, gel filtration and reverse phase chromatography (HPLC). SDS-PAGE indicated a protein with M-r 30 000 and two polypeptide chains (alpha-chain, M-r 15 000, and beta-chain, M-r 5000), the sequence being determined by automatic Edman degradation and by mass spectroscopy. LITI is a 174 amino acid residue protein which shows high homology to plant Kunitz inhibitors, especially those double chain proteins purified from the Mimosoideae subfamily. LITI inhibits plasmin (K-i 3.2 X 10(-10) M), human plasma kallikrein (K-i 6.3 X 10(-10) M), trypsin (K-i 1.5 X 10(-8) M) and chymotrypsin (K-i 1.4 X 10(-8) M). Factor XIIa activity is inhibited but K-i was not determined, and factor Xa, tissue kallikrein and thrombin are not inhibited by LITI. the action of LITI on enzymes that participate in the blood clotting extrinsic pathway is confirmed by the prolongation of activated partial thromboplastin time, used as clotting time assay. the inhibition of the fibrinolytic activity of plasmin was confirmed on the hydrolysis of fibrin plates. LlTI inhibits kinin release from high molecular weight kininogen by human plasma kallikrein in vitro and, administered intravenously, causes a decrease in paw edema induced by carrageenin or heat in male Wistar rats. Is addition, lower concentrations of bradykinin were found in limb perfusion fluids of LlTI-treated rats, (C) 2000 Elsevier Science B.V. All rights reserved.
- ItemSomente MetadadadosPrimary sequence determination of a Kunitz inhibitor isolated from Delonix regia seeds(Elsevier B.V., 2001-07-01) Pando, S. C.; Oliva, MLV; Sampaio, CAM; Di Ciero, L.; Novello, J. C.; Marangoni, S.; Universidade Federal de São Paulo (UNIFESP); Universidade Estadual de Campinas (UNICAMP)A serine proteinase inhibitor was purified from Delonix regia seeds a Leguminosae tree of the Caesalpinioideae subfamily. the inhibitor named DrTI, inactivated trypsin and human plasma kallikrein with K-i values 2.19x10(-8) M and 5.25 nM, respectively. Its analysis by SDS-PAGE 10-20% showed that the inhibitor is a protein with a single polypeptide chain of M-r 22 h Da. the primary sequence of the inhibitor was determined by Edman degradation, thus indicating that it contained 185 amino acids and showed that it belongs to the Kunitz type family; however, its reactive site did not contain Arg or Lys at the putative reactive site (position 63, SbTI numbering) or it was displaced when compared to other Kunitz-type inhibitors. (C) 2001 Elsevier B.V. All rights reserved.
- ItemSomente MetadadadosPrimary structure of a Kunitz-type trypsin inhibitor from Enterolobium contortisiliquum seeds(Elsevier B.V., 1996-03-01) Batista, IFC; Oliva, MLV; Araujo, M. S.; Sampaio, M. U.; Richardson, M.; Fritz, H.; Sampaio, CAM; Universidade Federal de São Paulo (UNIFESP); UNIV DURHAM; UNIV MUNICHA trypsin inhibitor was isolated from Enterolobium contortisiliquum seeds. Starting with a saline extract, ECTI (E. contortisiliquum trypsin inhibitor) was purified as a homogeneous protein by acetone precipitation, ion-exchange chromatography (DEAE-Sephadex A-50), gel filtration (Sephadex G-75 and Superose 12) and reversed phase HPLC (mu-Bondapak C-18). the amino acid sequence was determined by automatic degradation and by DABITC/PITC microsequence analysis of the reduced and carboxymethylated protein and also of purified peptides derived from the protein by cleavage with iodosobenzoic acid and by enzymic digestion with trypsin, chymotrypsin and Staphylococcus aureus V8 protease. ECTI contains 174 amino acid residues in two polypeptide chains, an cc-chain consisting of 134 residues and a beta-chain made up of 40 residues. the inhibitor displays a high degree of sequence identity with other Kunitz-type proteinase inhibitors isolated from the Mimosoideae subfamily. the reactive site was identified (by homology) as the arginine-isoleucine peptide bond at position 64-65. ECTI inhibits trypsin and chymotrypsin in the stoichiometric ratio of 1:1 and also Factor XIIa, plasma kallikrein and plasmin, but not thrombin and Factor Xa.
- ItemSomente MetadadadosPrimary structure of Dioclea glabra trypsin inhibitor, DgTI, a Bowman-Birk inhibitor(Academic Press Inc, 1999-08-11) Bueno, N. R.; Fritz, H.; Auerswald, E. A.; Mentele, R.; Sampaio, M.; Sampaio, CAM; Oliva, MLV; Universidade Federal de São Paulo (UNIFESP); Univ MunichA novel serine proteinase inhibitor, DgTI, was purified from Dioclea glabra seeds by acetone precipitation, and ion-exchange and reverse phase chromatography. the inhibitor belongs to the Bowman-Birk family, and its primary sequence, determined by Edman degradation and mass spectrometry, of 67 amino acids is: SSGPCCDRCRCTKSEPPQCQCQDVRLNSC-HSACEACVCSHSMPGLCSCLDITHFCHEPCKSSGD- DED, Although two reactive sites were determined by susceptibility to trypsin (Lys(13) and His(40)), the inhibitory function was assigned only to the first site. the inhibitor forms a 1:1 complex with trypsin, and Ki is 0.5 x 10(-9) M. Elastase, chymotrypsin, kallikreins, factor Xa, thrombin, and plasmin were not inhibited. By its properties, DgTI is a Bowman-Birk inhibitor with structural and inhibitory properties between the class of Bowman-Birk type I (with a fully active second reactive site), and Bowman-Birk type II (devoid of second reactive site). (C) 1999 Academic Press.
- ItemSomente MetadadadosPurification and primary structure determination of two Bowman-Birk type trypsin isoinhibitors from Cratylia mollis seeds(Elsevier B.V., 2006-03-01) Paiva, PMG; Oliva, MLV; Fritz, H.; Coelho, LCBB; Sampaio, CAM; Universidade Federal de São Paulo (UNIFESP); Universidade Federal de Pernambuco (UFPE); LMU MunchenTwo Bowman-Birk type trypsin inhibitors (CmTI1 and CmTI2) were purified from Cratylia mollis seeds by acetone precipitation, ion exchange, gel filtration and reverse-phase chromatography. CmTI1 and CmTI2, with 77 and 78 amino acid residues, respectively, were sequenced in their entirety and show a high structural similarity to Bowman-Birk inhibitors from other Legummosae. the putative reactive sites of CmTI1 are a lysine residue at position 22 and a tyrosine residue at position 49. Different reactive sites, as identified by their alignment with related inhibitors, were found for CmTI2: lysine at position 22 and leucine at position 49. the dissociation constant K-i of the complex with trypsin is 1.4 nM. the apparent molecular mass is 17 kDa without DDT and 11 kDa with reducing agent and heating. (c) 2005 Elsevier B.V. All rights reserved.
- ItemSomente MetadadadosSequence of a new Bowman-Birk inhibitor from Torresea acreana seeds and comparison with Torresea cearensis trypsin inhibitor (TcTI2)(Plenum Publ Corp, 1996-08-01) Tanaka, A. S. [UNIFESP]; Sampaio, M. U. [UNIFESP]; Mentele, R.; Auerswald, E. A.; Sampaio, CAM [UNIFESP]; UNIV MUNICH; Universidade Federal de São Paulo (UNIFESP)TaTI (Torresea acreana trypsin inhibitor), a new member of the Bowman-Birk trypsin inhibitor family, was purified from seeds of Torresea acreana, one of the two known species of Torresea, a Brazilian native Leguminosae of the Papilionoideae subfamily. Purification was performed by acetone fractionation, anion-exchange chromatography, and gel filtration. the TaTI appears as M(r) 7000 in SDS-PAGE under reducing conditions. There are 63 amino acid residues present in the TaTI sequence, which was confirmed by mass spectrometry (8388 daltons). the putative reactive sites residues were Lys-15 and Arg-42 at the first and second site, respectively. the antibodies raised against TcTI2, Torresea cearensis trypsin inhibitor 2, showed a cross-reaction with TaTI, but not with other Bowman-Birk inhibitors purified from Leguminosae. the inhibition constants of TaTI and TcTI2 were comparable when measured against trypsin, chymotrypsin, and factor XIIa, but not on plasmin. the latter was tenfold more effectively inhibited by TcTI2 then by TaTI. Neither TaTI nor TcTI2 affects thrombin, plasma kallikrein, or factor Xa.