RESUMO
Currently, chemical bifunctional cross-linkers are regarded as promising therapeutic agents capable of affecting cell metabolism. Depending on the nature of the active groups and on the length of their mediating spacer, these cross-linkers have been shown to influence mitochondrial functions, the cell cycle and cell death. The current study was aimed to assay cellular effects of a cross-linker with 'zero'-length spacer, 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC). When added to cultures of transformed cells, EDC induced a G2/M blockade followed by cell death. Analysis of the molecular targets revealed that alteration of the cell cycle was caused by EDC-induced interchain cross-linking within double-stranded DNA. Administration of EDC to animals with experimental tumors increased their life span. The analysis of tumor cells from EDC-treated mice showed up-regulation of p21/WAF1, disturbance of tumor cell cytokinesis and, hence, cell death. Thus, both in vitro and in vivo, EDC exhibits cytotoxic activity, which may be of potential therapeutic use.
Assuntos
Morte Celular/efeitos dos fármacos , Citocinese/efeitos dos fármacos , Etildimetilaminopropil Carbodi-Imida/farmacologia , Animais , Linhagem Celular , Chlorocebus aethiops , Reagentes de Ligações Cruzadas , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Relação Dose-Resposta a Droga , Humanos , Masculino , Camundongos , Camundongos Endogâmicos DBARESUMO
Studies indicating an important role of the TNF-receptor family in control of cell proliferation, differentiation, and death have drastically increased in number in recent years. The main function of many members of this family is cell death triggering, and this is apparently the only function for some of them. Studies on the molecular mechanisms of cell death activated by members of the TNF-receptor family revealed and identified proteins directly or indirectly associated with TNF receptors. Pathways of cytotoxic signal transduction by some members of the TNF-Rs family based on currently proven protein-protein interactions and the role of distinct proteins in these processes are summarized in this review.
Assuntos
Apoptose/fisiologia , Receptores do Fator de Necrose Tumoral/fisiologia , Transdução de Sinais/fisiologia , Fator de Necrose Tumoral alfa/fisiologia , Morte Celular/fisiologia , Divisão Celular/fisiologia , HumanosRESUMO
A modification of protein-protein interactions can be considered to be a way to regulate cell death. Chemical cross-linking agents have been traditionally used for protein complexing. This study has been undertaken to test a possibility to induce and(or) to modify cell death by a homobifunctional cross-linker dimethyl suberimidate (DMS). It was shown that the protein cross-linking by DMS resulted in a death of transformed cells by apoptosis. DMS-induced apoptosis was accompanied by cell cycle perturbations and down-regulation of p21/Waf1 mRNA expression. The RT-PCR analysis of bcl-2 family genes revealed the engagement of mitochondria in DMS-induced cytotoxicity. Then, the influence of DMS treatment on TNF-dependent and Fas-mediated apoptosis was investigated. Cell pre-incubation with DMS resulted in their increasing sensitivity for the TNF cytotoxic effect, though activities of anti-Fas cytotoxic antibodies were inhibited. The effects observed are probably due to cross-linking of TNF-receptors. Thus, this study first demonstrated that a chemical cross-linker DMS in capable of inducing apoptosis in transformed cells and modifying TNF-dependent and Fas-mediated apoptosis.
Assuntos
Apoptose/efeitos dos fármacos , Reagentes de Ligações Cruzadas/farmacologia , Dimetil Suberimidato/farmacologia , Animais , Apoptose/genética , Linhagem Celular Transformada , Inibidor de Quinase Dependente de Ciclina p21 , Ciclinas/efeitos dos fármacos , Ciclinas/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Genes bcl-2/efeitos dos fármacos , Humanos , Camundongos , Células Tumorais Cultivadas , Fator de Necrose Tumoral alfa/farmacologia , Receptor fas/genéticaRESUMO
Physical association of proteins that underlies cytotoxic signal induction and transduction suggests a possibility of regulating cell response by modifying protein-protein interactions. For protein complexing, chemical cross-linking agents have been traditionally used. However, the ability of various cross-linkers to induce and modify cell responses, cell death in particular, is still obscure. We have undertaken the investigation to test the apoptosis-inducing and modifying properties of the homobifunctional cross-linkers-dimethyl suberimidate (DMS) and 1,5-bis(succinimido-oxycarbonyloxy)pentane (BSOCOP). The functional groups of these cross-linkers are different but both are able to interact with available amino groups. It was shown that bifunctional cross-linkers, unlike their monofunctional analogues, are capable of inducing cell death in transformed cells, thus indicating the crucial role of cross-linking in cell killing. DMS- and BSOCOP-treated cells were shown to undergo cell death by apoptosis, though the signaling pathways were distinct. DMS inhibited bcl-X(L) and bak but not bax gene expression, while BSOCOP potentiated bax mRNA synthesis immediately after application. Cell pre-incubation with DMS, but not with BSOCOP, resulted in an increasing sensitivity to TNF, although activities of anti-Fas cytotoxic antibodies were then inhibited. Thus, this study has demonstrated for the first time that chemical cross-linkers are capable of inducing apoptosis by themselves and modifying the TNF-dependent and Fas-mediated cell death that may have potential therapeutic significance.
Assuntos
Apoptose , Reagentes de Ligações Cruzadas/farmacologia , Animais , Morte Celular , Linhagem Celular , Dimetil Suberimidato/farmacologia , Relação Dose-Resposta a Droga , Células HeLa , Humanos , Proteínas de Membrana/biossíntese , Camundongos , Pentanos/farmacologia , Proteínas Proto-Oncogênicas/biossíntese , Proteínas Proto-Oncogênicas c-bcl-2/biossíntese , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais , Fatores de Tempo , Células Tumorais Cultivadas , Fator de Necrose Tumoral alfa/metabolismo , Células U937 , Proteína Killer-Antagonista Homóloga a bcl-2 , Proteína X Associada a bcl-2 , Proteína bcl-X , Receptor fas/metabolismoRESUMO
We investigated the mode of TNF-dependent death of L929 murine fibroblasts and the influence of overexpression of bcl-2 family genes on this process. Based on morphological and biochemical data it has been shown that L929 cells died after TNF treatment by apoptosis irrespective of TNF dose and protein synthesis inhibition. Analysis of bcl-2 family gene transfectants revealed a down-regulation of TNF-induced apoptosis by bcl-2 and bclX overexpression, and an up-regulation by bax gene.
Assuntos
Apoptose/efeitos dos fármacos , Fibroblastos/patologia , Fator de Necrose Tumoral alfa/farmacologia , Animais , Apoptose/genética , Linhagem Celular , Fibroblastos/efeitos dos fármacos , Fibroblastos/fisiologia , Regulação da Expressão Gênica , Genes bcl-2 , CamundongosRESUMO
Soluble liver proteins (SLP) from old and gamma-irradiated young rats were studied with respect to their carbonyl content, the rates of autolysis and degradation by proteinase K, and their antigenicity for mice and compared with SLP from non-irradiated young animals. A significant increase in the carbonyl level was found in SLP from old and gamma-irradiated young rats as compared to SLP from intact young rats. The rates of SLP autolysis and proteolysis by proteinase K were increased in the same animal groups but did not correlate the carbonyl level. At the same time, whereas the antigenicity for mice of SLP from old rats was significantly higher than that of SLP from young rats, the antigenicity of SLP from gamma-irradiated rats did not differ from non-irradiated animals. Enrichment of the diet with antioxidant and vitamin supplements (AVS) during one month before the irradiation caused a decrease in the radiation-induced carbonyl level in rat SLP. However, this raised antioxidant level in animal diet did not influence the rates of SLP autolysis and degradation by proteinase K and also did not alter the antigenicity of these proteins. The data allow us to suggest that the increase in autolysis, degradation by the exogenous proteinase, and antigenicity of SLP from old rats are determined not only by carbonyl formation in these proteins due to action of oxygen radicals but also by other age-specific protein modifications.
Assuntos
Fígado/química , Fígado/efeitos da radiação , Proteínas/química , Proteínas/metabolismo , Fatores Etários , Animais , Carbono/química , Endopeptidases/metabolismo , Raios gama , Soros Imunes , Masculino , Camundongos , Camundongos Endogâmicos , Proteínas/imunologia , Ratos , Ratos Wistar , SolubilidadeRESUMO
A panel of monoclonal antibodies (MAbs) directed against lipopolysaccharide (LPS) of Salmonella serogroups A, B, and D was generated. Nine most productive hybrid clones were selected from several fusions of mouse myeloma cells with splenocytes from BALB/c mice, immunized with the corresponding heat-killed bacteria. The MAbs were characterized by enzyme immunoassay, Western blot analysis, and dot-immunoblotting with LPS and whole bacteria of Salmonella serogroups A-E and some other representatives of the Enterobacteriaceae family. Seven MAbs were reactive with the sole Salmonella strain used as an immunogen; one MAb, SD:10D9H, reacted with the five major serogroups of Salmonella species (A, B, D, E1, and E2); and one MAb, SA:5D12A, reacted with Salmonella serogroups A-E and a rough strain of S. cholerae-suis. None of the MAbs reacted with LPS of E. coli 055:B5 or whole bacteria of E. coli K12, Klebsiella pneumoniae, or Proteus vulgaris. The typical ladder-like patterns of bands were observed after immunoblotting of MAbs against electrophoretically resolved LPS from Salmonella serogroups A-E, which thus confirmed their LPS-directed specificity. MAbs affinity constants were determined by noncompetitive enzyme immunoassay using serial dilutions of both LPS as antigen (coating the plate) and antibodies. On the base of the results obtained, the presumed epitopes for each of the MAbs were discussed. The usefulness of MAbs generated for diagnostic and protective purposes was declared.
Assuntos
Anticorpos Monoclonais/química , Epitopos/imunologia , Lipopolissacarídeos/imunologia , Antígenos O/imunologia , Salmonella/imunologia , Animais , Anticorpos Monoclonais/biossíntese , Afinidade de Anticorpos , Especificidade de Anticorpos , Epitopos/química , Feminino , Immunoblotting , Camundongos , Camundongos Endogâmicos BALB C , Salmonella/química , Salmonella/classificação , SorotipagemRESUMO
Peptides modelling transmembrane segments C, D, E and G of bacteriorhodopsin were obtained by solid phase method using the conventional Boc strategy. Protected peptides were assembled on PAM polystyrene support. Side chain protecting groups were: Tos for Arg, Bzl for Thr and Ser, cHx for Asp and Glu, Bzl(Cl2) for Tyr, For for Trp, Z(Cl) for Lys. Syntheses were performed on a modernized Beckman 990 synthesizer in the automatic mode. Double couplings by a preformed hydroxybenzotriazole ester were used for all residues. Qualitative and quantitative ninhydrine tests were used to monitor coupling efficiency. Removal of protecting groups and peptide cleavage were achieved by hydrogen fluoride, containing p-cresol and p-thiocresol as scavengers. Preparative reverse phase HPLC was used for purification. Peptide structure and homogeneity were confirmed by amino acid analysis, 1H-NMR and analytical HPLC.
Assuntos
Bacteriorodopsinas/química , Peptídeos/síntese química , Sequência de Aminoácidos , Membrana Celular/química , Cromatografia Líquida de Alta Pressão , Indicadores e Reagentes , Dados de Sequência Molecular , Conformação ProteicaRESUMO
The fusing activity of the synthetic 23 amino-acid fragment (fusion peptide, FP) of the fusion protein of the Lassa arenavirus membrane was tested in a model liposomal system. The resonance energy transfer between two fluorescent phospholipid probes was monitored in order to detect dioleoylphosphatidylcholine liposome fusion induced by the peptide. Fusion rates were compared at different pH values, ionic strength and calcium concentrations. FP demonstrated fusing activity at pH 4.5-5.5, indicating that the protonated form of the FP is the active one. A transmembrane proton-gradient induced by acidification was not relevant to the fusion process, since its elimination with nigericin did not affect the FP-mediated fusion. Both Ca2+ (8 mM) and the increase of the ionic strength (1 M NaCl) inhibited liposome fusion. The efficacy of liposome fusion depended also on the lipid-to-lipid ratio. Non-linear dependence was observed at a saturation ratio of 10 mol lipid per mol peptide. A model of 'side insertion' is suggested, describing FP interaction with the membrane.
Assuntos
Vírus Lassa/fisiologia , Lipossomos , Proteínas Virais de Fusão/fisiologia , Sequência de Aminoácidos , Cálcio/metabolismo , Cátions Bivalentes , Corantes Fluorescentes , Concentração de Íons de Hidrogênio , Dados de Sequência Molecular , Concentração Osmolar , Fragmentos de Peptídeos/síntese química , Fragmentos de Peptídeos/fisiologia , FosfatidiletanolaminasRESUMO
Several peptides from Lassa virus glyco- and nucleoproteins were predicted as probable T-cell epitopes. Their synthesis was performed by solid phase method. The study of possible protective effect in vivo with Lassa-sensitive CBA mice revealed protective epitope within the 277-303 nucleoprotein region. Further studies reduced the protective epitope structure to the 287-300 nucleoprotein fragment.
Assuntos
Antígenos Virais , Epitopos/química , Vírus Lassa/imunologia , Nucleoproteínas/química , Proteínas Virais/química , Sequência de Aminoácidos , Animais , Camundongos , Camundongos Endogâmicos CBA , Dados de Sequência Molecular , Nucleoproteínas/genética , Linfócitos T/imunologia , Proteínas Virais/genéticaRESUMO
The fusing capacity of lipid membranes of a synthetic 23-member peptide was studied. This hydrophobic peptide represents an analog of a predicted functional site ("fusion peptide") of the GP2 envelope protein of the Lassa virus (family Arenaviridae). Fusion of small monolayer liposomes was detected by the method of resonance energy transfer between the fluorescent derivatives of the lipid, NBD-PE (donor) and Rd-PE (acceptor). Using this peptide, the pH-dependent fusing activity was found in liposomes having different phospholipid composition. The rate and efficiency of liposome fusion increased with a decrease in pH and the lipid/peptide ratio as well as with a temperature increase. The increase in the ionic strength and Ca2+ concentration in the reaction mixture led to the inhibition of the peptide-induced fusion of liposomes. Neither the phospholipid charge, nor the transmembrane proton gradient of liposomes had any appreciable effect on the kinetics of the peptide-induced fusion. Neutralization of the medium in the course of the fusion reaction sharply decelerated, whereas repeated acidification activated this process. This finding suggests that peptide protonation plays a role in fusion reactions. It was suggested that acidification causes conformational changes in the peptide structure, thus activating the peptide-induced fusion of liposomes. The fusing capacity of the predicted Lassa virus fusion peptide is similar to that of viruses characterized by a pH-dependent step at the initial stages of the viral infection.
Assuntos
Vírus Lassa/metabolismo , Lipídeos de Membrana/metabolismo , Peptídeos/metabolismo , Proteínas Virais de Fusão/metabolismo , Sequência de Aminoácidos , Polarização de Fluorescência , Concentração de Íons de Hidrogênio , Cinética , Lipossomos , Dados de Sequência Molecular , Concentração Osmolar , Homologia de Sequência do Ácido Nucleico , Proteínas do Envelope Viral/metabolismoRESUMO
Synthetic peptides corresponding to predicted Lassa virus GP1 glycoprotein B-epitopes were used to study the antigenicity and immunogenicity of the protein. ELISA results showed that guinea pig polyclonal anti-Lassa virus serum bound effectively to peptides corresponding to amino acid residues 119-133 and 164-176 of the GP1 protein. Essentially it did not react to a peptide corresponding to GP1 amino acid residues 234-256. Sera obtained against peptides representing amino acid residues 119-133 and 164-176 reacted with inactivated purified Lassa virus.