Mechanism of action by Bauhinia ssp inhibitor and synthetic peptides related from primary structure on pest development

O objetivo do trabalho foi investigar o mecanismo de ação dos inibidores de proteases e de compostos derivados da estrutura primária destes inibidores para dar subsídios ao desenvolvimento de produtos pesticidas não agressivos ao meio ambiente e que possam ser aplicados à planta e à semente, de modo a protegê-las contra o ataque de organismos predadores, particularmente os bruquídeos. São objetivos específicos: 1. Isolar o inibidor de tripsina (BrTI) das sementes de Bauhinia rufa e estudar a influência do inibidor e de peptídeos derivados no desenvolvimento do bruquídeo, Callosobruchus maculatus e comparar a ação com a de outros inibidores de proteases; 2. Investigar o mecanismo de ação da proteína e dos peptídeos isolando os compostos afetados pela ação do inibidor e dos peptídeos derivados, que prejudicam o desenvolvimento larval; 3. Investigar a localização dos compostos que apresentarem atividade inseticida na larva de C. maculatus por microscopia confocal 4. Analisar o ciclo celular das células do trato digestório das larvas de C. maculatus alimentadas com o peptídeo tóxico por citometria de fluxo..BV UNIFESP: Teses e dissertaçõe

Primary sequence determination of the glycosilated inhibitor isolated from Bauhinia rufa seeds

Objetivo principal: Isolar e caracterizar a glicoproteina que apresenta atividade inibitoria de elastase detectada no extrato das sementes de Bauhinia rufa. Metodos: O inibidor foi purificado por precipitacao por acetona (80 por cento v/v), cromatografia de afinidade Con A Sepharose, cromatografia de troca ionica (HiTrap Q e ResourceTM Q em sistema FPLC), cromatografia de filtracao em gel SuperdexTm 200 (sistema FPLC) e cromatografia de fase reversa em coluna C1$ (sistema HPLC). gBrEl foi analisado por SDS PAGE para analise da massa molecular e deteccao de carboidratos. Resultados: pela analise eletroforetica o inibidor com atividade anti elastasica apresentou-se como uma cadeia unica com uma massa molecular aproximadamente de 20 kDa e pela coloracao com reagente de Schiff confirmou a presenca de carboidratos na estrutura da proteina. gBrEl inibe elastase pancreatica de porco e nao inibe elastase de neutrofilo humana, calicreina plasmatica humana, calicreina pancreatica de porco e tripsina. Atraves da analise por sequenciamento, a proteina e composta por 143 residuos de aminoacidos e o sitio de glicosilacao foi detectado em Asn 38 apresenta Mr. 1170 por espectrometria de massa. Discussao: A metedologia empregada para a purificacao do inibidor de elastase obteve rendimento de 21 e purificacao de cerca de 306 vezes. A constante de inibicao da elastase e de 6,18.10-8 M e a estequiometria de reacao e 1:2. Atraves da analise de similariedade (FASTA program) gBrEl apresenta-se muito similar aos inibidores da familia Kunitz de plantas e pela comparacao conservativa do sitio reativo, o...(au)BV UNIFESP: Teses e dissertaçõe

Purification and characterization of a new trypsin inhibitor from Dimorphandra mollis seeds

A second trypsin inhibitor (DMTI-II) was purified from the seed of Dimorphandra mollis (Leguminosae-Mimosoideae) by ammonium sulfate precipitation (30-60%), gel filtration, and ion-exchange and affinity chromatography. A molecular weight of 23 kDa was estimated by gel filtration on a Superdex 75 column SDS-PAGE under reduced conditions showed that DMTI-II consisted of a single polypeptide chain, although isoelectric focusing revealed the presence of three isoforms. the dissociation constant of 1.7 x 10(-9) M with bovine trypsin indicated a high affinity between the inhibitor and this enzyme. the inhibitory activity was stable over a wide pH range and in the presence of DTT. the N-terminal sequence of DMTI-II showed a high degree of homology with other Kunitz-type inhibitors.Univ Fed Mato Grosso do Sul, Dept Ciencias Nat Tres Lagoas, CEUL, BR-79603011 Tres Lagoas, MS, BrazilUniv Estadual Campinas, Dept Bioquim, Inst Biol, Campinas, SP, BrazilEscola Paulista Med, Dept Bioquim, BR-04023 São Paulo, BrazilUniv Estadual Norte Fluminense, Ctr Biociencias & Biotecnol, Campo Dos Goytacazes, RJ, BrazilEscola Paulista Med, Dept Bioquim, BR-04023 São Paulo, BrazilWeb of Scienc

Eleusine coracana Gaertn.

原著和名: シコクビエ科名: イネ科 = Gramineae採集地: 高知県 香美郡 土佐山田町 北組 (土佐 土佐山田町 北組)採集日: 1984/9/8採集者: 萩庭丈壽整理番号: JH039976国立科学博物館整理番号: TNS-VS-98997

The defensive functions of plant inhibitors are not restricted to insect enzyme inhibition

Three 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.Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)FAP/FADA (UNIFESP)Universidade Federal de São Paulo, Escola Paulista Med, Dept Bioquim, BR-04044020 São Paulo, BrazilUniv Fed Mato Grosso, Dept Nat Sci, BR-79603011 Tres Lagoas, MS, BrazilUniv Estadual Fluminense Darcy Ribeiro, Lab Prot & Peptide Biochem, CBB, BR-28015620 Campos Dos Goytacazes, RJ, BrazilUniversidade Federal de São Paulo, Dept Biophys, BR-04044020 São Paulo, BrazilUniv São Paulo, Inst Fis Sao Carlos, Sao Carlos, SP, BrazilUniv Estadual Oeste Parana, Ctr Engn & Ciencias Exatas, BR-85903000 Toledo, PR, BrazilUniversidade Federal de São Paulo, Escola Paulista Med, Dept Bioquim, BR-04044020 São Paulo, BrazilUniversidade Federal de São Paulo, Dept Biophys, BR-04044020 São Paulo, BrazilWeb of Scienc

Effect of steroid hormones (E₂ and P₄) on the viability of leiomyoma and myometrial adjacent cells.

<p>(A) Leiomyoma cells (5 × 10³) and myometrial adjacent cells were treated with E₂ (100 nmol/L) and P4 (100 nmol/L) for 24 h in 96-well microtiter plates. Cell viability was assessed by the MTT reduction test. (B) Phase contrast–confluent culture of leiomyoma and myometrial adjacent cells after treat with E₂ (100 nmol/L) and P₄ (100 nmol/L). The statistical significance was evaluated using one-way ANOVA followed by the Tukey's test. A p-value of ≤0.05 was considered to indicate significance (*). These experiments were performed with cultured primary cells from specimens collected from patients.</p

Detection of signaling phosphoproteins by Immunoblot analysis in leiomyoma and myometrial adjacent cells.

<p>Leiomyoma and myometrial adjacent cells (5 x 10⁵) were treated with E₂ (100 nmol/L) and P₄ (100 nmol/L) for 16 h in 6-well microtiter plates; lysate proteins were separated by 10% SDS-PAGE and electro-transferred to nitrocellulose membranes. Membranes were blocked and incubated with rabbit primary antibodies, (A and C) anti-phospho-Src (Tyr-416), anti-Src, anti-phospho-FAK (Tyr-397), anti-FAK, anti- phospho-Erk1/2 MAPK, anti-Erk1/2 MAPK, (E) anti-p130Cas Y165, (F) anti-p130Cas Y410, (H and J) anti-phospho-Akt, anti-Akt, and anti-β-actin. (B, D, G, I, and L) Graph bars represent the densitometric analyses of the immunoblotting results. The results are represented as band intensities in arbitrary units relative to the respective total phopho-proteins load and total control (β-actin) load. Antibody binding was visualized by chemiluminescence, and the relative levels of these proteins were determined by the densitometric analyses. These experiments were performed with cultured primary cells from specimens collected from patients (*p<0.01).</p

Immunophenotype of leiomyoma and myometrial cells from women with myoma uterine.

<p>Adhered and spread cells after tissue disaggregation in collagenase following with primary explants. Cells are shown under phase contrast microscopy and indirect immunofluorescence for phalloidin, fibronectin-integrin β1/CD29, vimentin, and DAPI (blue, for nuclei). (A-B) Phase contrast in the confluent culture of leiomyoma cells after three days. (A) Low density (B) high density (magnification, ×400). Mycoplasma contamination was not observed in any of the processed tissues. (C) Log-phase growth rate by cell counting–growth characteristics of leiomyoma cells, myometrial adjacent cells, and co-cultured myometrial adjacent (as feeders) with leiomyoma cells (on plastic surface). (D-F) Analysis of myometrial markers by confocal microscopy; vimentin and fibronectin integrin β1/CD29. (D) Cytoskeletal organization (Phalloidin, Alexa-594-red); (E) integrin β1/CD29 (FITC-488, green); and (F) Co-localization integrin β1(FITC-488, green) and vimentin (Alexa-594-red). Bar, 10 μm.</p

Cell viability by the MTT assay.

<p>(A and B) Leiomyoma and myometrial adjacent cells (1 × 10⁴) were maintained in serum deprivation with different concentrations of FBS (0%-10%) for 24 and 48 h in 96-well microtiter plates. (C) Cell viability of uterine leiomyoma cells and myometrial adjacent cells cultured in two concentrations of FBS (2% and 10%); cells (5 × 10³) were seed on plastic and on a collagen type I coated plates. (D and E) Phase contrast of confluent culture of leiomyoma cells and myometrial adjacent cells on collagen type I coated plates. The morphology of leiomyoma cells is not altered; these cells were less spread than the myometrial adjacent cells when seeded onto collagen type I-coated plates. Mycoplasma contamination was not observed in any of the processed tissues.</p

Characteristics of Samples Used in the Study.

<p>Characteristics of Samples Used in the Study.</p