RESUMEN
Bacterial infections are a serious cause of high morbidity and mortality worldwide. Over the past decades, the drug resistance of bacterial pathogens has been steadily increasing, while the rate of development of new effective antibacterial drugs remains consistently low. The plant kingdom is sometimes called a bottomless well for the search for new antimicrobial therapies. This is due to the fact that plants are easily accessible and cheap to process, while extracts and components of plant origin often demonstrate a high level of biological activity with minor side effects. The variety of compounds obtained from plant raw materials can provide a wide choice of various chemical structures for interaction with various targets inside bacterial cells, while the rapid development of modern biotechnological tools opens the way to the targeted production of bioactive components with desired properties. The objective of this review is to answer the question, whether antimicrobials of plant origin have a chance to play the role of a panacea in the fight against infectious diseases in the "post-antibiotic era".
Asunto(s)
Antiinfecciosos , Plantas , Plantas/química , Plantas/metabolismo , Antibacterianos/farmacología , Antibacterianos/uso terapéutico , Antibacterianos/química , Antiinfecciosos/química , Antiinfecciosos/metabolismo , Antiinfecciosos/farmacología , BacteriasRESUMEN
Tumor-derived autologous antigenic peptides when bound to endogenous 70 kDa family heat shock proteins (HSP70) are able to induce effective T-cell responses against tumors. However, efficacy of HSPbased vaccines in clinical practical stand point still has a number of certain limitations including an activation of immune responses against alien non-human HSPs. In this study we reconstructed the complexes of human recombinant HSPs70 (human recombinant HSP70A1B and HSC70 mixture; hrHSPs70) with antigenic lowweight peptides derived from mice B16F10 melanoma cell lysate (PepMCL) in vitro and investigated the prophylactic potential of these complexes to activate anti-tumor immunity in melanoma mouse model. Our results demonstrate that the developed prophylactic vaccine elicits melanoma-specific immune responses and anti-tumor effects against melanoma. These results suggest that hrHSPs70 has capability to reconstitute complexes with peptides obtained from tumor cells lysates in vitro and, therefore, can be used for delivery of multiple antigenic peptides into antigen-presenting cells (APCs) to activate effectors cells. Designed in such a way hrHSPs70-based prophylactic vaccines induce immune responses resulting in a significant efficient prevention of tumor growth and metastases.
Asunto(s)
Proteínas de Choque Térmico , Antígenos Específicos del Melanoma , Melanoma/inmunología , Fragmentos de Péptidos , Proteínas Recombinantes de Fusión/inmunología , Animales , Vacunas contra el Cáncer/administración & dosificación , Vacunas contra el Cáncer/inmunología , Modelos Animales de Enfermedad , Femenino , Proteínas HSP70 de Choque Térmico/genética , Proteínas HSP70 de Choque Térmico/aislamiento & purificación , Proteínas HSP70 de Choque Térmico/metabolismo , Proteínas de Choque Térmico/metabolismo , Humanos , Melanoma/mortalidad , Melanoma/patología , Melanoma/terapia , Melanoma Experimental , Antígenos Específicos del Melanoma/química , Antígenos Específicos del Melanoma/inmunología , Antígenos Específicos del Melanoma/metabolismo , Ratones , Fragmentos de Péptidos/química , Fragmentos de Péptidos/inmunología , Fragmentos de Péptidos/metabolismo , Unión Proteica , Proteínas Recombinantes de Fusión/administración & dosificación , Carga Tumoral/inmunologíaRESUMEN
A new chimeric gene ApE1 encoding the receptor-binding domain of the human alpha-fetoprotein fused to a sequence of 22 glutamic acid residues was constructed. A new bacterial producer strain E. coli SHExT7 ApE1 was selected for ApE1 production in a soluble state. A simplified method was developed to purify ApE1 from bacterial biomass. It was shown that the new vector protein selectively interacts with AFP receptors on the tumor cell surface and can be efficiently accumulated in tumor cells. In addition, ApE1 was shown to be stable in storage and during its chemical modification. An increased number of carboxyl groups in the molecule allows the production of cytotoxic compound conjugates with higher drug-loading capacity and enhanced tumor targeting potential.
Asunto(s)
Antineoplásicos/administración & dosificación , Antineoplásicos/farmacocinética , Portadores de Fármacos/administración & dosificación , Portadores de Fármacos/farmacocinética , Proteínas Recombinantes de Fusión/administración & dosificación , Proteínas Recombinantes de Fusión/farmacocinética , alfa-Fetoproteínas/metabolismo , Secuencia de Aminoácidos , Antineoplásicos/química , Sitios de Unión , Línea Celular Tumoral , Clonación Molecular , Portadores de Fármacos/química , Escherichia coli/genética , Fluoresceína-5-Isotiocianato , Humanos , Datos de Secuencia Molecular , Ácido Poliglutámico/química , Ácido Poliglutámico/genética , Ácido Poliglutámico/metabolismo , Unión Proteica , Receptores de Péptidos/metabolismo , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , alfa-Fetoproteínas/química , alfa-Fetoproteínas/genéticaRESUMEN
Molecular chaperones of HSP70 family assists presentation of exogenous antigenic peptides by antigen-presenting cells (APC). HSP70-peptide complexes are powerful immunotherapeutic agents, which enhance cross-presentation of captured antigen in dendritic cells and macrophages. Several clinical trials have shown that HSP-based cancer vaccines possess good efficacy and safety. However, sometime it is impossible to isolate sufficient amount of vaccine. These make us to pay attention for recombinant HSP70-based vaccines and to optimize in vitro complex formation mechanism. Here we have investigated two human recombinant proteins HSP70(HYB) and HSC70. Optimal values of ADP concentration, pH, temperature and peptides excess are determined in this work. We have also shown that proposed complex formation method enriches eluted from HSP70-complexes peptide repertoire compared to in vivo assembled ones.
Asunto(s)
Antígenos de Neoplasias/inmunología , Antígenos/metabolismo , Proteínas HSP70 de Choque Térmico/inmunología , Proteínas HSP70 de Choque Térmico/metabolismo , Péptidos/metabolismo , Antígenos/inmunología , Antígenos de Neoplasias/metabolismo , Vacunas contra el Cáncer/inmunología , Línea Celular Tumoral , Proteínas HSP70 de Choque Térmico/genética , Humanos , Concentración de Iones de Hidrógeno , Péptidos/inmunología , Antígeno Nuclear de Célula en Proliferación/química , Antígeno Nuclear de Célula en Proliferación/inmunología , Antígeno Nuclear de Célula en Proliferación/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Temperatura , Proteínas no Estructurales Virales/química , Proteínas no Estructurales Virales/inmunología , Proteínas no Estructurales Virales/metabolismo , Antígeno gp100 del Melanoma/química , Antígeno gp100 del Melanoma/inmunología , Antígeno gp100 del Melanoma/metabolismoRESUMEN
We investigated ATP-ase and peptide-binding activity of recombinant human heat shock protein HSP70(A1B), HSC70, and two hybrid proteins derived from those. The UV-spectral recorded data was used to characterize conformational rearrangements, which were induced by domain replacement or HSP70-peptide interaction. We have shown that N-terminal domain dramatically affect substrate specificity of C-terminal peptide-binding domain. This proposes new hypothesis for HSP70 chaperone machinery. The linear dependence between ATP-ase activity and peptide complex ratio was found. This relationship could be used for unlabeled peptide-HSP70 complex quantification.
Asunto(s)
Adenosina Trifosfatasas/metabolismo , Proteínas HSP70 de Choque Térmico/metabolismo , Péptidos/metabolismo , Estructura Terciaria de Proteína/genética , Proteínas Recombinantes de Fusión/metabolismo , Adenosina Trifosfatasas/química , Adenosina Trifosfatasas/genética , Adenosina Trifosfatasas/aislamiento & purificación , Adenosina Trifosfato/metabolismo , Secuencia de Aminoácidos , Clonación Molecular , Escherichia coli/genética , Escherichia coli/metabolismo , Ingeniería Genética , Proteínas HSP70 de Choque Térmico/química , Proteínas HSP70 de Choque Térmico/genética , Proteínas HSP70 de Choque Térmico/aislamiento & purificación , Humanos , Cinética , Datos de Secuencia Molecular , Péptidos/síntesis química , Plásmidos , Unión Proteica , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/aislamiento & purificación , Espectrofotometría Ultravioleta , Especificidad por Sustrato , Transformación BacterianaRESUMEN
A human alpha-fetoprotein fragment (AFP) modified with oligocationic homologs of nuclear localization signal was used to construct new target cell-selective DNA-carrier proteins. The new recombinant vectors containing C- or N-terminal polynucleotide-binding domains are able to form stable complexes with single- or double-stranded oligonucleotides and plasmid DNA. Using flow cytometry and fluorescent microscopy, it was shown that such nucleoprotein complexes can be selectively internalized in target cells receptors superexpressing AFP receptors. The results obtained are important both for understanding mechanisms of formation of DNA-protein complexes and for studying their interaction with intracellular molecular targets. The new proteins can be used as a tool for the development of highly selective and efficacious gene-selective antitumour drugs.
Asunto(s)
ADN/administración & dosificación , alfa-Fetoproteínas/genética , Secuencia de Aminoácidos , Línea Celular Tumoral , ADN/química , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Portadores de Fármacos , Colorantes Fluorescentes , Humanos , Ligandos , Datos de Secuencia Molecular , Señales de Localización Nuclear , Oligonucleótidos/administración & dosificación , Oligonucleótidos/química , Plásmidos , Estructura Terciaria de Proteína , Receptores de Péptidos/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , alfa-Fetoproteínas/química , alfa-Fetoproteínas/metabolismoAsunto(s)
Técnicas para Inmunoenzimas , Interferón beta/aislamiento & purificación , Proteínas Recombinantes de Fusión/aislamiento & purificación , beta-Galactosidasa/aislamiento & purificación , Cromatografía Liquida , Humanos , Interferón beta/metabolismo , Plásmidos , beta-Galactosidasa/metabolismoRESUMEN
In vitro MHC restriction of antigen-specific T-cell proliferation to Igk-Ib allotype of Igk chain has been studied in inbred August rats, using polyclonal and monoclonal antibodies to RT-1 molecules. Igk-1b-specific proliferation of immune T cells was completely abrogated by alloantiserum specific for RT-1c (August) molecules. Monoclonal antibodies (MAb) to RT-1B ("1-A-like") molecules inhibited markedly the response (about 70-80% inhibition), while anti-RT-ID ("I-E-like") MAb caused but weak inhibition (about 25%). Thus, the data obtained demonstrate RT-1Bc molecule as a main restriction element of Igk-1b-specific T-cell response.