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1.
Science ; 221(4613): 855-8, 1983 Aug 26.
Artigo em Inglês | MEDLINE | ID: mdl-6348945

RESUMO

The complete nucleotide sequence of the diphtheria tox228 gene encoding the nontoxic serologically related protein CRM228 has been determined. A comparison of the predicted amino acid sequence with the available amino acid sequences from the wild-type toxin made it possible to deduce essentially the entire nucleotide sequence of the wild-type tox gene. The signal peptide of pro-diphtheria toxin and the putative tox promoter have been identified, a highly symmetrical nucleotide sequence downstream of the toxin gene has been detected; this region may be the corynebacteriophage beta attachment site (attP). The cloned toxin gene was expressed at a low level in Escherichia coli.


Assuntos
Toxina Diftérica/genética , Sequência de Bases , Clonagem Molecular , Escherichia coli/genética , Regulação da Expressão Gênica , Genes , Genes Bacterianos , Conformação de Ácido Nucleico , Óperon
2.
J Clin Invest ; 86(6): 2117-24, 1990 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-1979339

RESUMO

The interference of the recombinant HIV-1 glycoproteins gp160 and gp120 with the CD3/T cell antigen receptor (TcR)-mediated activation process has been investigated in the CD4+ diphtheria toxoid-specific human P28D T cell clone. Both glycoproteins clearly inhibit the T cell proliferation induced in an antigen-presenting cell (APC)-free system by various cross-linked monoclonal antibodies specific for the CD3 molecule or the TcR alpha chain (up to 80% inhibition). Biochemical studies further demonstrate that exposure of the T cell clone to both glycoproteins (gps) specifically inhibits the CD3/TcR phospholipase C (PLC) transduction pathway, without affecting the CD3/TcR cell surface expression. Thus, inositol phosphate production, phosphatidic acid turnover, intracellular free calcium, and intracellular pH increase induced by CD3/TcR-specific MAbs are specifically impaired in gps-treated P28D T cells. Addition of purified soluble CD4 prevents binding of gps to T cells and overcomes all observed inhibitions. Maximal inhibitions are obtained for long-term exposure of the T cell clone to gps (16 h). No early effect of gps is observed. By contrast, gp160 and gp120 fail to suppress the CD2-triggered functional and biochemical P28D T cell responses. These results demonstrate that, in addition to their postulated role in the alteration of the interaction between CD4 on T lymphocytes and MHC class II molecules on APC, soluble HIV-1 envelope glycoproteins may directly and specifically impair the CD3/TcR-mediated activation of PLC in uninfected T cells via the CD4 molecule.


Assuntos
Antígenos de Diferenciação de Linfócitos T/fisiologia , Linfócitos T CD4-Positivos/fisiologia , Produtos do Gene env/farmacologia , Proteína gp120 do Envelope de HIV/farmacologia , HIV-1/imunologia , Fosfatidilinositóis/fisiologia , Receptores de Antígenos de Linfócitos T/fisiologia , Transdução de Sinais/efeitos dos fármacos , Antígenos CD/fisiologia , Antígenos CD2 , Complexo CD3 , Cálcio/fisiologia , Células Clonais , Humanos , Concentração de Íons de Hidrogênio , Técnicas In Vitro , Ativação Linfocitária/efeitos dos fármacos , Receptores Imunológicos/fisiologia
3.
Nat Biotechnol ; 16(8): 748-52, 1998 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-9702773

RESUMO

We describe the rational design of immunosuppressive peptides without relying on information regarding their receptors or mechanisms of action. The design strategy uses a variety of topological and shape descriptors in combination with an analysis of molecular dynamics trajectories for the identification of potential drug candidates. This strategy was applied to the development of immunosuppressive peptides with enhanced potency. The lead compounds were peptides, derived from the heavy chain of HLA class I, that modulate immune responses in vitro and in vivo. In particular, a peptide derived from HLA-B2702, amino acids 75-84 (2702.75-84) prolonged skin and heart allograft survival in mice. The biological activity of the rationally designed peptides was tested in a heterotopic mouse heart allograft model. The molecule predicted to be most potent displayed an immunosuppressive activity approximately 100 times higher than the lead compound.


Assuntos
Desenho Assistido por Computador , Desenho de Fármacos , Sobrevivência de Enxerto/efeitos dos fármacos , Imunossupressores , Peptídeos , Animais , Simulação por Computador , Sequência Consenso , Avaliação Pré-Clínica de Medicamentos , Transplante de Coração , Antígenos de Histocompatibilidade Classe I , Humanos , Imunossupressores/química , Imunossupressores/farmacologia , Imunossupressores/uso terapêutico , Ligantes , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBA , Biblioteca de Peptídeos , Peptídeos/química , Peptídeos/farmacologia , Peptídeos/uso terapêutico , Conformação Proteica , Linfócitos T Citotóxicos/imunologia , Transplante Homólogo/imunologia
4.
Oncogene ; 18(30): 4357-63, 1999 Jul 29.
Artigo em Inglês | MEDLINE | ID: mdl-10439043

RESUMO

The p16-cyclin D-pRB-E2F pathway is frequently deregulated in human tumors. This critical regulatory pathway controls the G1/S transition of the mammalian cell cycle by positive and negative regulation of E2F-responsive genes required for DNA replication. To assess the value of the transcription factors E2Fs as targets for antiproliferative strategies, we have initiated a program aiming to develop inhibitors targeting specifically these proteins in vitro and in vivo. The cellular activity of E2F is the result of the heterodimeric association of two families of proteins, E2Fs and DPs, which then bind DNA. Here, we use a two hybrid approach to isolate from combinatorial libraries peptide aptamers that specifically interact with E2Fs DNA binding and dimerization domains. One of these is a potent inhibitor of E2F binding activity in vitro and in mammalian fibroblasts, blocks cells in G1, and the free variable region from this aptamer has the same effect. Our experiments argue that the variable region of this aptamer is structured, and that it functions by binding E2F with a motif that resembles a DP heterodimerization region, and blocking E2F's association with DP. These results show that cell proliferation can be inhibited using genetically-selected synthetic peptides that specifically target protein-protein interaction motifs within cell cycle regulators. These results also emphasize the critical role of the E2F pathway for cell proliferation and might allow the design of novel antiproliferative agents targeting the cyclin/CDK-pRB-E2F pathway.


Assuntos
Proteínas de Transporte , Proteínas de Ciclo Celular , Divisão Celular , Proteínas de Ligação a DNA , Inibidores do Crescimento/farmacologia , Peptídeos/farmacologia , Fatores de Transcrição/antagonistas & inibidores , Sequência de Aminoácidos , Animais , Linhagem Celular , Cricetinae , Relação Dose-Resposta a Droga , Desenho de Fármacos , Fatores de Transcrição E2F , Fibroblastos , Imunofluorescência , Fase G1 , Humanos , Modelos Biológicos , Dados de Sequência Molecular , Proteína 1 de Ligação ao Retinoblastoma , Fase S , Tiorredoxinas/química , Fator de Transcrição DP1
5.
Gene ; 55(2-3): 255-63, 1987.
Artigo em Inglês | MEDLINE | ID: mdl-2444498

RESUMO

Tripartite fusion proteins comprising the nontoxic mutant protein CRM228 of diphtheria toxin (DT), the hepatitis B virus surface antigen (HBsAg), and beta-galactosidase were obtained by expression of hybrid genes from the pR promoter of bacteriophage lambda and purification by affinity chromatography. The antigenicity and immunogenicity of the individual protein constituents were analyzed. A major neutralizing epitope of DT was inactivated by the HBsAg insertion into the DT B fragment. The fusion proteins elicited antibodies reactive with 22 nm HBsAg particles. This suggests a novel approach towards the use of DT mutants as immunogenic carriers of heterologous antigens.


Assuntos
Toxina Diftérica/genética , Antígenos de Superfície da Hepatite B/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes/genética , Animais , Anticorpos Antibacterianos/biossíntese , Anticorpos Antibacterianos/imunologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/imunologia , Toxina Diftérica/imunologia , Epitopos/imunologia , Genes Sintéticos , Cobaias , Anticorpos Anti-Hepatite B/biossíntese , Anticorpos Anti-Hepatite B/imunologia , Antígenos de Superfície da Hepatite B/imunologia , Proteínas Recombinantes de Fusão/imunologia , beta-Galactosidase/genética
6.
Gene ; 41(1): 103-11, 1986.
Artigo em Inglês | MEDLINE | ID: mdl-3009269

RESUMO

The tox228 gene encoding the non-toxic, immunologically cross-reactive CRM228 mutant diphtheria toxin (DT) has been cloned downstream of the PR promoter and the cro translational initiation region of bacteriophage lambda carried by plasmid pCQV2 (Queen, 1983). Efficient transcription but no appreciable amount of a translational product corresponding to complete DT could be detected in Escherichia coli hosts. Deletion of 320 bp from the C-terminal region of the B-fragment of DT, and fusion of the truncated tox228 gene to lacZ yielded several hybrid beta-galactosidases (beta Gal) in an E. coli lon- strain in addition to beta Gal. The various DT fragments fused to beta Gal were immunologically reactive and were identified with antibodies specifically directed against the A- or the B-fragment of DT. Antibodies raised against the DT-beta Gal fusion proteins in guinea pigs cross-reacted with wild-type DT and its B-fragment and protected Vero cells in tissue culture against the lethal action of DT. Immunized guinea pigs survived upon injection of a five-fold lethal dose of wild-type DT.


Assuntos
Bacteriófago lambda/genética , Toxina Diftérica/genética , Genes Bacterianos , Genes , Regiões Promotoras Genéticas , Proteínas do Envelope Viral/genética , Sequência de Aminoácidos , Anticorpos , Complexo Antígeno-Anticorpo , Sequência de Bases , Clonagem Molecular , Reações Cruzadas , Enzimas de Restrição do DNA , Toxina Diftérica/imunologia , Mutação , Plasmídeos , Biossíntese de Proteínas , Proteínas do Envelope Viral/imunologia , Proteínas Virais de Fusão
7.
AIDS Res Hum Retroviruses ; 8(4): 469-78, 1992 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-1376136

RESUMO

Cellular immunogenicity of env gp160, nef p27, and gag p55 proteins of human immunodeficiency virus type 1 (HIV-1) was studied in mice immunized with vaccinia virus recombinants. Proliferative responses of spleen cells were comparable against env gp160, nef p27, and gag p25 recombinant proteins. No specific activity was observed against gag p18 protein. Env, nef, and gag-specific T-cell lines were generated by repeated stimulation of immune spleen cells with recombinant HIV-1 proteins. They were CD4 positive, proliferative, and also cytotoxic against HIV-transfected target cells. Specificity of the T-cell response against nef and gag protein was analyzed with synthetic peptides. Peptides nef 15, nef 16, and gag AM-30 were, respectively, reactive in nef- and gag-specific proliferative and cytolytic assays. The three peptides described have a relatively conserved amino acid sequence among HIV isolates and appear broadly immunoreactive among species.


Assuntos
Linfócitos T CD4-Positivos/imunologia , Produtos do Gene env/imunologia , Produtos do Gene gag/imunologia , Produtos do Gene nef/imunologia , HIV-1/imunologia , Precursores de Proteínas/imunologia , Sequência de Aminoácidos , Animais , Linfócitos T CD4-Positivos/citologia , Divisão Celular , Homólogo 5 da Proteína Cromobox , Epitopos , Antígenos HIV/imunologia , Proteína gp160 do Envelope de HIV , Imunidade Celular , Camundongos , Dados de Sequência Molecular , Baço/citologia , Vacinas Virais/administração & dosagem , Produtos do Gene gag do Vírus da Imunodeficiência Humana , Produtos do Gene nef do Vírus da Imunodeficiência Humana
8.
AIDS Res Hum Retroviruses ; 8(6): 1117-23, 1992 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-1503824

RESUMO

Three groups of four rhesus macaques were immunized twice, one month apart with purified recombinant HIV-1LAI gp160 in the presence of either alum, incomplete Freund's adjuvant (IFA), or SAF-1. Two months later, the animals were injected twice again with a synthetic peptide with the sequence of the principal neutralization determinant (PND) of the HIV-1LAI isolate mixed with the same adjuvants. All animals received a booster injection of gp160 and PND peptide at 6 months. This regimen of immunization induced in the SAF-1 and IFA groups a high-titer neutralizing antibody response that declined progressively over the course of the following 6 months. In contrast, only a weak response was observed in the alum group. Neutralizing antibody titers varied as anti-PND titers, suggesting that they were principally targeted to the PND. A shortened immunization protocol comprising two injections of gp160 at 0 and 1 month followed by one injection of PND peptide at 3 months is suggested as optimal for the induction of high titers of HIV-1 neutralizing antibodies in primates.


Assuntos
Produtos do Gene env/imunologia , Anticorpos Anti-HIV/biossíntese , Proteína gp120 do Envelope de HIV/imunologia , HIV-1/imunologia , Fragmentos de Peptídeos/imunologia , Precursores de Proteínas/imunologia , Adjuvantes Imunológicos , Sequência de Aminoácidos , Animais , Adjuvante de Freund/imunologia , Proteína gp160 do Envelope de HIV , Imunização , Cinética , Macaca mulatta , Dados de Sequência Molecular , Testes de Neutralização
11.
C R Seances Acad Sci D ; 289(14): 1021-4, 1979 Nov 26.
Artigo em Francês | MEDLINE | ID: mdl-232859

RESUMO

Linear unintegrated DNA of Schmidt-Ruppin Rous sarcoma virus, subgroup D (SR-RSV-D), was digested with SmaI restriction endonuclease, analyzed by agarose gel electrophoresis, "blotted" by Southern's method and hybridized with a viral 32P cDNA. SmaI cleaves this DNA at five sites, two of which are localized at the ends of the provirus. Src and env genes seem not to be restrictied by SmaI cleavage.


Assuntos
Vírus do Sarcoma Aviário/análise , Enzimas de Restrição do DNA , DNA Viral , Desoxirribonucleases de Sítio Específico do Tipo II , Animais , Embrião de Galinha , Fibroblastos , Genes Virais , Peso Molecular , Hibridização de Ácido Nucleico
12.
Proc Natl Acad Sci U S A ; 77(5): 3014-8, 1980 May.
Artigo em Inglês | MEDLINE | ID: mdl-6248882

RESUMO

Chicken cells of chicken helper factor-positive (chf+) phenotype were infected with a cloned (envE-free) Rous sarcoma virus, subgroup D, and examined for the presence of parent and recombinant proviruses by transfection in chicken and turkey cells, respectively. It was found that most parent virus DNA is integrated into the host cell genome during the first 18 hr after infection, and no significant integration occurs between 18 and 72 hr after infection. On the other hand, no recombinant virus DNA was detected at 18 hr, although both unintegrated and integrated (provirus) forms of this DNA occurred 72 hr after infection. Recombination proviruses were also found in chronically virus-infected chf+ cells but not in chf- cells lacking virus-related RNA. Our results show that recombinants between the exogenous virus and endogenous chf gene can be cloned from the DNA of the host cell by transfection and suggest that a second replicative cycle of the virus is required to generate such recombinants.


Assuntos
Vírus do Sarcoma Aviário/genética , Genes Virais , Vírus Auxiliares/genética , Recombinação Genética , Animais , Linhagem Celular , Galinhas , Clonagem Molecular/métodos , Transfecção , Replicação Viral
13.
Infect Immun ; 57(10): 3221-5, 1989 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-2506133

RESUMO

Previous studies provided indirect evidence that in Corynebacterium diphtheriae regulation of diphtheria toxin gene (tox) transcription by iron is mediated by a bacterial repressor. By performing in vitro protein-DNA binding experiments, we establish that a corynebacterial Fe2+-sensitive protein, named DtoxR, can bind to a palindromic motif present in the tox promoter region. Binding of this factor prevents the interaction of the transcription initiation machinery with presumptive critical promoter elements, providing evidence that DtoxR is responsible for the repression of toxinogenesis observed in iron-containing growth medium.


Assuntos
Corynebacterium diphtheriae/genética , Proteínas de Ligação a DNA/fisiologia , Toxina Diftérica/genética , Genes Bacterianos , Ferro/fisiologia , Proteínas Repressoras/fisiologia , Fatores de Transcrição/fisiologia , Transcrição Gênica , Ligação Competitiva , Proteínas de Transporte/fisiologia , Corynebacterium diphtheriae/fisiologia , Regulação da Expressão Gênica , Proteínas de Ligação ao Ferro , Regiões Promotoras Genéticas , Proteínas de Ligação a Transferrina
14.
Vaccine ; 7(2): 132-6, 1989 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-2473577

RESUMO

The diphtheria toxin (DT) secreted by Corynebacterium diphtheriae is used after formolization as an efficient vaccine against diphtheria. In an attempt to evaluate its capacity to present heterologous peptide sequence in a recognized form, we created in-phase insertion in the gene encoding the non-toxic mutant protein CRM228 of DT. The sequence chosen for insertion was the synthetic DNA fragment encoding a poliovirus neutralization epitope. Tripartite fusion proteins comprising the mutant DT, the poliovirus peptide and beta-galactosidase were obtained in E. coli and purified by affinity chromatography. These fusion proteins reacted both with antibodies directed against the DT and a poliovirus specific monoclonal antibody. Moreover, these hybrid toxins induced protective antibodies against the lethal effect of DT and neutralizing antibodies against poliovirus. We conclude that the modification of highly immunogenic DT may provide a means for the presentation of foreign peptide sequences to the immune system.


Assuntos
Toxina Diftérica/imunologia , Epitopos/imunologia , Poliovirus/imunologia , Proteínas Virais de Fusão/imunologia , Animais , Western Blotting , Cromatografia de Afinidade , Clonagem Molecular , Eletroforese em Gel de Poliacrilamida , Masculino , Plasmídeos , Coelhos , Proteínas Virais de Fusão/análise , Proteínas Virais de Fusão/biossíntese
15.
Nucleic Acids Res ; 13(9): 3147-59, 1985 May 10.
Artigo em Inglês | MEDLINE | ID: mdl-3923442

RESUMO

The expression of the diphtheria tox228 gene encoding the nontoxic, serologically related CRM228 mutant diphtheria toxin has been analyzed in Corynebacterium diphtheriae and Escherichia coli. The diphtheria toxin promoter has been used to direct the expression of beta-galactosidase in E.coli, and the efficiency of promotion has been compared to that obtained with the lac promoter. Expression in C.diphtheriae is known to be dependent on the absence of iron, and we present for the first time direct evidence that this regulation occurs at the level of transcription. The 5' end of toxin mRNA maps at the same position in C.diphtheriae and E.coli, suggesting identical sequences to be recognized by C.diphtheriae and E.coli RNA polymerase. The diphtheria toxin promoter carries at position -34 a TTGATT sequence closely related to the E.coli -35 consensus sequence and in the -14 to -8 region a set of overlapping sequences with complete or partial homology to the E.coli -10 consensus sequence.


Assuntos
Corynebacterium diphtheriae/genética , Toxina Diftérica/genética , Escherichia coli/genética , Óperon , Sequência de Bases , RNA Polimerases Dirigidas por DNA/metabolismo , Regulação da Expressão Gênica , Óperon Lac , Mutação , RNA Mensageiro/análise , Transcrição Gênica
16.
Microb Pathog ; 4(5): 345-57, 1988 May.
Artigo em Inglês | MEDLINE | ID: mdl-3071655

RESUMO

The nucleotide sequence of a 4.8 kilobase (kb) HindIII fragment from pWR100, the virulence plasmid of Shigella flexneri 5, was determined and analysed. This fragment encodes polypeptides b (62 kilodalton, kD) and c (43 kD) which have already been described as two of the four immunogenic polypeptides of Shigellae. The nucleotide sequence revealed that in addition to the ipaB and ipaC genes encoding polypeptides b and c, a third complete open reading frame was found within the fragment. The gene, named ippI, encoded a 17 kD polypeptide. The deduced amino acids sequence of polypeptides b and c showed no signal peptide but presence of highly hydrophobic domains compatible with a transmembraneous location. The surprising A and T richness of the three genes as compared with the Escherichia coli and Shigella genomes, resulted in a biased codon usage, and raises the question of the origin of the sequences.


Assuntos
Antígenos de Bactérias/genética , DNA Bacteriano/genética , Shigella flexneri/genética , Sequência de Aminoácidos , Sequência de Bases , Clonagem Molecular , Códon/genética , Genes Bacterianos , Immunoblotting , Dados de Sequência Molecular , Peptídeos/genética , Peptídeos/imunologia , Plasmídeos , Mapeamento por Restrição , Shigella flexneri/imunologia , Shigella flexneri/patogenicidade , Virulência
17.
Mol Pharmacol ; 57(4): 679-86, 2000 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-10727512

RESUMO

Many therapeutic drugs are excluded from entering the brain, due to their lack of transport through the blood-brain barrier (BBB). To overcome this problem, we have developed a novel method in which short, naturally derived peptides (16-18 amino acids) cross the cellular membranes of the BBB with high efficiency and without compromising its integrity. The antineoplastic agent doxorubicin (dox) was coupled covalently to two peptides, D-penetratin and SynB1. The ability of dox to cross the BBB was studied using an in situ rat brain perfusion technique and also by i.v. injection in mice. In the brain perfusion studies, we first confirmed the very low brain uptake of free radiolabeled dox because of the efflux activity of P-glycoprotein at the BBB. By contrast, we have demonstrated that when dox is coupled to either the D-penetratin or SynB1 vectors, its uptake was increased by a factor of 6, suggesting that the vectorized dox bypasses P-glycoprotein. Moreover, using a capillary depletion method, we have shown that vectorization of dox led to a 20-fold increase in the amount of dox transported into brain parenchyma. Intravenous administration of vectorized dox at a dose of 2.5 mg/kg in mice led to a significant increase in brain dox concentrations during the first 30 min of postadministration, compared with free dox. Additionally, vectorization led to a significant decrease of dox concentrations in the heart. In summary, our results establish that the two peptide vectors used in this study enhance the delivery of dox across the BBB.


Assuntos
Antineoplásicos/farmacocinética , Barreira Hematoencefálica , Doxorrubicina/análogos & derivados , Doxorrubicina/farmacocinética , Peptídeos/metabolismo , Animais , Transporte Biológico , Doxorrubicina/metabolismo , Portadores de Fármacos , Feminino , Masculino , Camundongos , Camundongos Nus , Ratos , Ratos Sprague-Dawley
18.
Immunotechnology ; 1(3-4): 189-96, 1995 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-9373347

RESUMO

BACKGROUND: Various systems have been described for the expression of recombinant monoclonal antibodies for therapeutical applications. Insect cells offer great advantages with respect to post-translational modifications, stability, yields and applications. OBJECTIVES: To construct plasmid cassette transfer vectors in order to express chimeric, humanized or human antibodies in insect cells using baculovirus expression system. STUDY DESIGN: Two transfer vectors, pBHuC kappa and pBHuC gamma 1, were designed. They contain a viral promoter (polyhedrin or p10 promoters, respectively), a signal peptide sequence and a human immunoglobulin light chain C kappa gene or heavy chain C gamma 1 sequence, respectively. Restriction sites have been introduced to allow insertion of rearranged variable genes, after amplification by polymerase chain reaction. RESULTS: Recombinant baculoviruses expressing complete immunoglobulins have been generated by a double-recombination event between baculovirus DNA and the loaded cassette transfer vectors. CONCLUSION: Our genetic cassette approach makes this system a very flexible and convenient one for the rapid production of therapeutic monoclonal antibodies with heavy and light chains of any human isotype. Specific variable regions selected by the antibody phage display technology can be easily transferred in these vectors to obtain a complete antibody.


Assuntos
Anticorpos Monoclonais/biossíntese , Baculoviridae , Vetores Genéticos , Mutagênese Insercional/métodos , Animais , Sequência de Bases , Clonagem Molecular , Ensaio de Imunoadsorção Enzimática , Humanos , Cadeias Pesadas de Imunoglobulinas/genética , Cadeias Leves de Imunoglobulina/genética , Região Variável de Imunoglobulina/genética , Células Jurkat , Dados de Sequência Molecular , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes/biossíntese , Spodoptera
19.
J Physiol (Paris) ; 84(4): 273-7, 1990.
Artigo em Inglês | MEDLINE | ID: mdl-2079663

RESUMO

The mechanism by which diphtheria toxin (DT) crosses the endosomal membrane to exert its biological activity in the cell cytoplasm is still poorly understood. By measuring the change in conductance of planar lipid bilayers induced by cyanogen bromide peptides of fragment B of DT, we have identified a domain that could be involved in the pH-dependent membrane interaction of DT. Moreover, infrared spectroscopy has allowed us to demonstrate that, at low pH, in the presence of a lipid bilayer, this domain is mainly helical with the axis of the helices oriented parallel to the lipid acyl chains. On the basis of these results, we have designed mutants of DT which should provide information about the molecular mechanism of the DT membrane translocation process.


Assuntos
Toxina Diftérica/química , Fragmentos de Peptídeos/química , Toxina Diftérica/farmacologia , Bicamadas Lipídicas/metabolismo , Membranas/metabolismo , Mutação , Espectrofotometria Infravermelho
20.
Eur J Biochem ; 268(5): 1304-14, 2001 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-11231282

RESUMO

The pAntp peptide, corresponding to the third helix of the Antennapedia homeodomain, is internalized by a receptor-independent process into eucaryotic cells. The precise mechanism of entry remains unclear but the interaction between the phospholipids of plasma membrane and pAntp is probably involved in the translocation process. In order to define the role of peptide-lipid interaction in this mechanism and the physico-chemical properties that are necessary for an efficient cellular uptake, we have carried out an Ala-Scan mapping. The peptides were labeled with a fluorescent group (7-nitrobenz-2-oxo-1,3-diazol-4-yl-; NBD) and their cell association was measured by flow cytometry. Furthermore, we determined the fraction of internalized peptide by using a dithionite treatment. Comparison between cell association and cell uptake suggests that the affinity of pAntp for the plasma membrane is required for the import process. To further investigate which are the physico-chemical requirements for phospholipid-binding of pAntp, we have determined the surface partition coefficient of peptides by titrating them with phospholipid vesicles having different compositions. In addition, we estimated by circular dichroism the conformation adopted by these peptides in a membrane-mimetic environment. We show that the phospholipid binding of pAntp depends on its helical amphipathicity, especially when the negative surface charge density of phospholipid vesicles is low. The cell uptake of pAntp, related to lipid-binding affinity, requires a minimal hydrophobicity and net charge. As pAntp does not seem to translocate through an artificial phospholipid bilayer, this might indicate that it could interact with other cell surface components or enters into cells by a nonelucidated biological mechanism.


Assuntos
Endocitose , Proteínas de Homeodomínio/química , Proteínas de Homeodomínio/metabolismo , Proteínas Nucleares , Fosfolipídeos/metabolismo , Fatores de Transcrição , Alanina/genética , Alanina/metabolismo , Sequência de Aminoácidos , Substituição de Aminoácidos/genética , Proteína do Homeodomínio de Antennapedia , Membrana Celular/química , Membrana Celular/metabolismo , Dicroísmo Circular , Ditionita/metabolismo , Citometria de Fluxo , Corantes Fluorescentes/metabolismo , Proteínas de Homeodomínio/genética , Humanos , Células K562 , Cinética , Bicamadas Lipídicas/química , Bicamadas Lipídicas/metabolismo , Lipossomos/química , Lipossomos/metabolismo , Micelas , Dados de Sequência Molecular , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Ligação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Dodecilsulfato de Sódio/metabolismo , Eletricidade Estática , Relação Estrutura-Atividade
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