RESUMO
Kalanchoe pinnata L. plants bearing an artificial CP1 gene encoding the cecropin P1 antimicrobial peptide have been obtained. The presence of the CP1 gene in the plant genome has been confirmed by PCR. Cecropin P1 synthesis in transgenic plants has been shown by MALDI mass spectrometry and Western blotting. The obtained plants have been highly resistant to bacterial and fungal phytopathogens, and their extracts have demonstrated antimicrobial activity towards human and animal pathogens. It has been shown that transgenic plants bearing the CP1 gene can be colonized by the beneficial associative microorganisms Methylovorus mays.
Assuntos
Anti-Infecciosos/metabolismo , Proteínas de Bactérias , Betaproteobacteria/genética , Expressão Gênica , Kalanchoe , Peptídeos , Plantas Geneticamente Modificadas , Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/genética , Kalanchoe/genética , Kalanchoe/metabolismo , Peptídeos/genética , Peptídeos/metabolismo , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/metabolismoRESUMO
The bacterial cell wall muramyl dipeptides MDP and glucosaminyl-MDP (GMDP) are powerful immunostimulators but their binding target remains controversial. We previously reported expression cloning of GMDP-binding polypeptides and identification of Y-box protein 1 (YB-1) as their sole target. Here we show specific binding of GMDP to recombinant YB-1 protein and subcellular colocalization of YB-1 and GMDP. GMDP binding to YB-1 upregulated gene expression levels of NF-κB2, a mediator of innate immunity. Furthermore, YB-1 knockdown abolished GMDP-induced Nfkb2 expression. GMDP/YB-1 stimulation led to NF-κB2 cleavage, transport of activated NF-κB2 p52 to the nucleus, and upregulation of NF-κB2-dependent chemokine Cxcr4 gene expression. Therefore, our findings identify YB-1 as new target for muramyl peptide signaling.
Assuntos
Acetilmuramil-Alanil-Isoglutamina/análogos & derivados , Bactérias/metabolismo , Parede Celular/metabolismo , Imunidade Inata , Proteína 1 de Ligação a Y-Box/metabolismo , Acetilmuramil-Alanil-Isoglutamina/metabolismo , Animais , Sequência de Bases , Sítios de Ligação , Células Cultivadas , Primers do DNA , CamundongosRESUMO
Structure-function relationship studies for novel GMDP-peptide mimic, RVPPRYHAKISPMVN (RN-peptide) were performed on array of truncated and 'Ala-scan' analogues. The shortest peptide fragment possessing detectable affinity towards anti-GMDP-antibodies was demonstrated to be PRYH. RN-peptide analogues lacking up to 8 residues at C-terminus were found to retain adjuvant activity with the minimal active peptide being RVPPRYH. Evaluation of Ala-scan analogues highlighted that adjuvant activity is most critically dependent on both arginine residues, but also is sensitive to substitution of K9, I10, S11 and M13 amino acid residues, the functional importance of which was additionally confirmed by testing peptides truncated at both termini.
Assuntos
Adjuvantes Imunológicos/química , Adjuvantes Imunológicos/farmacologia , Glicopeptídeos/química , Glicopeptídeos/farmacologia , Relação Estrutura-Atividade , Substituição de Aminoácidos , Animais , Anticorpos/sangue , Ensaio de Imunoadsorção Enzimática , Camundongos , Camundongos Endogâmicos BALB C , Ovalbumina/administração & dosagem , Ovalbumina/imunologiaRESUMO
Sandwich EIA for measurement of soluble Fas was developed on the basis of SA-7 and SA-8 monoclonal antibodies to full-length human Fas. The threshold sensitivity of the test system is 0.3 ng/ml. Several isoforms of soluble Fas were identified. The structure of SA-7 and SA-8 antibody epitopes was determined using the peptide phage library. It was shown that SA-7 antibody epitope is determined by amino acid residues 129-134 (CKPNFF), while SA-8 antibody epitope is determined by amino acid residues 94-99 (KAHFSS) of full-length Fas. Hence, sandwich EIA on the basis of SA-7 and SA-8 monoclonal antibodies for detection of soluble Fas in human serum is to detect the following Fas isoforms: FasExo6Del, FasExo4Del, FasExo4,6Del, FasExo4.7Del, and FasExo8Del.
Assuntos
Mapeamento de Epitopos/métodos , Biblioteca de Peptídeos , Receptor fas/imunologia , Humanos , Técnicas ImunoenzimáticasRESUMO
Hemodynamic activity of peptides from differentiation factor HLDF (promyelocytic HL-60 line) was studied on WKY and SHR-SP rats. Intravenous infusion of the test peptides was accompanied by changes in blood pressure and heart rate, which depended on the structure of peptides and functional activity of the organism and differed in normotensive and hypertensive animals.
Assuntos
Pressão Sanguínea/efeitos dos fármacos , Frequência Cardíaca/efeitos dos fármacos , Proteínas de Neoplasias/genética , Peptídeos/farmacologia , Sequência de Aminoácidos , Animais , Infusões Intravenosas , Masculino , Dados de Sequência Molecular , Peptídeos/administração & dosagem , Peptídeos/genética , Ratos , Ratos Endogâmicos SHR , Ratos Endogâmicos WKY , Especificidade da Espécie , Fatores de TempoRESUMO
A number of protected proline-containing dipeptides Boc-Xaa-Pro-OBu(t) were converted via epimerization-free oxidation with RuO4 to dipeptides with an internal pyroglutamic acid residue, Boc-Xaa-Glp-OBu(t). The latter were subjected to oxidative Hoffman-type rearrangement induced by PhI[OC(O)CF3]2 to give N-(aminoacyl)-pyroglutamates. The behavior of these derivatives under basic conditions was studied, and for two such a derivatives an aminoacyl incorporation reaction was observed, producing otherwise poorly accessible 10-membered-ring dilactams derived from 1,4-diaminobutyric and glutamic acids in practicable yields.
Assuntos
Aminobutiratos/química , Ácido Glutâmico/análogos & derivados , Ácido Glutâmico/síntese química , Peptídeos Cíclicos/química , Peptídeos Cíclicos/síntese química , Aminoacilação , Ácido Glutâmico/química , Estrutura Molecular , Ornitina/química , Oxirredução , Ácido Pirrolidonocarboxílico/químicaRESUMO
Effect of the monoclonal antibody (MAb) 5B6 produced to the solubilized preparation of bacteriorhodopsin on the protein photocycle was studied to examine conformational rearrangements on the surface of functioning bacteriorhodopsin molecule. Using the methods of solid phase enzyme immunoassay, peptide phage display, and 1H NMR spectroscopy, we demonstrated that the epitope recognized by MAb 5B6 is the Val69-Pro-Phe-Gly72 fragment of the protein, with the aromatic ring of Phe71 and the methyl groups of Val69 participating in the binding. MAb 5B6 exerted no significant effect on the photocycle of bacteriorhodopsin solubilized in Triton X-100 at pH 6.2 and 7.4, which suggested that, when functioning, bacteriorhodopsin retains the conformation and position of its Val69-Pro-Phe-Gly72 fragment.
Assuntos
Bacteriorodopsinas/química , Bacteriorodopsinas/metabolismo , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Sequência de Aminoácidos , Anticorpos Monoclonais/metabolismo , Bacteriorodopsinas/imunologia , Sítios de Ligação , Epitopos , Glicina/química , Espectroscopia de Ressonância Magnética , Dados de Sequência Molecular , Octoxinol/química , Fragmentos de Peptídeos/imunologia , Fenilalanina/química , Conformação Proteica , Valina/químicaRESUMO
It was shown that the full-size neurotrophic factor from pigment epithelium (PEDF) induces the cell differentiation of the human promyelocyte leukemia cell line HL-60. A structural analysis of PEDF revealed in its C-terminal region a six-membered peptide fragment PEDF-(352-357) (PEDF-6) whose sequence is highly homologous to the 41-46 fragment of the active site of the human leukocyte differentiation factor HLDF (HLDF-6). The biological effect of PEDF and synthetic peptides PEDF-6 and HLDF-6 on the HL-60 cells and the early gastrula ectoderm of Xenopus laevis embryos was studied. On the basis of the structural and functional homologies of HLDF, PEDF, and their homologous peptides and the computer models of the spatial structures of the full-size PEDF and the PEDF with the C-terminal fragment split off tby the cleavage of the Leu380-Thr381 bond in the serpin loop, a hypothesis on the functional role of the serpin loop in PEDF was put forward.
Assuntos
Diferenciação Celular/fisiologia , Proteínas do Olho/fisiologia , Fatores de Crescimento Neural/fisiologia , Proteínas/fisiologia , Serpinas/fisiologia , Sequência de Aminoácidos , Animais , Proteínas do Olho/química , Células HL-60 , Humanos , Dados de Sequência Molecular , Fatores de Crescimento Neural/química , Epitélio Pigmentado Ocular/citologia , Epitélio Pigmentado Ocular/embriologia , Proteínas/química , Homologia de Sequência de Aminoácidos , Serpinas/química , Xenopus laevisRESUMO
Six-membered peptide fragment TGENHR (HLDF-6) was identified in the HL-60 cell culture of human promyelocyte leukemia treated with retinoic acid when studying the differentiation factor HLDF of this cell line. HLDF-6 retains the ability of the full-size factor to induce the differentiation and arrest the proliferation of the starting HL-60 cells. It was shown that the synthetic peptide HLDF-6 has no specific receptors on the surface of the HL-60 cells but can affect the binding of interleukin IL-1 beta, a cytokine involved in proliferation, to the cell surface. It was found on a model of transplantable NSO myeloma that HLDF-6 has an antitumor activity.