RESUMO
Photochemical reaction dynamics of the primary events in recombinant bacteriorhodopsin (bRrec) was studied by femtosecond laser absorption spectroscopy with 25-fs time resolution. bRrec was produced in an Escherichia coli expression system. Since bRrec was prepared in a DMPC-CHAPS micelle system in the monomeric form, its comparison with trimeric and monomeric forms of the native bacteriorhodopsin (bRtrim and bRmon, respectively) was carried out. We found that bRrec intermediate I (excited state of bR) was formed in the range of 100 fs, as in the case of bRtrim and bRmon. Further processes, namely the decay of the excited state I and the formation of intermediates J and K of bRrec, occurred more slowly compared to bRtrim, but similarly to bRmon. The lifetime of intermediate I, judging from the signal of ΔAESA(470-480 nm), was 0.68 ps (78%) and 4.4 ps (22%) for bRrec, 0.52 ps (73%) and 1.7 ps (27%) for bRmon, and 0.45 ps (90%) and 1.75 ps (10%) for bRtrim. The formation time of intermediate K, judging from the signal of ΔAGSA(625-635 nm), was 13.5 ps for bRrec, 9.8 ps for bRmon, and 4.3 ps for bRtrim. In addition, there was a decrease in the photoreaction efficiency of bRrec and bRmon as seen by a decrease in absorbance in the differential spectrum of the intermediate K by ~14%. Since photochemical properties of bRrec are similar to those of the monomeric form of the native protein, bRrec and its mutants can be considered as a basis for further studies of the mechanism of bacteriorhodopsin functioning.
Assuntos
Bacteriorodopsinas/química , Biopolímeros/química , Cromatografia Líquida de Alta Pressão , Dicroísmo Circular , Proteínas Recombinantes/química , Espectrofotometria UltravioletaRESUMO
Anion-selective opsins slow ChloC and ACR2 were expressed in rat brain cortical neurons by electroporation in utero. It is shown that the light-activated channel ACR2 has pronounced advantages in terms of both the inactivation kinetics and the neuron inhibition intensity, which is associated with a more negative value of the light-activated current reversal potential compared to the slow ChloC channel. The identified properties of opsin ACR2 indicate that it can be used for strictly controlled suppression of neuronal activity in optogenetic experiments, including the expression in the retinal ganglionic cells for reconstituting the OFF-component of their receptive field, which is essential for optogenetic prosthetics of degenerative retina.
Assuntos
Optogenética , Rodopsina/metabolismo , Canal de Ânion 2 Dependente de Voltagem/metabolismo , Animais , Córtex Cerebral/fisiologia , Córtex Cerebral/efeitos da radiação , Eletroporação , Luz , Potenciais da Membrana/fisiologia , Potenciais da Membrana/efeitos da radiação , Neurônios/fisiologia , Neurônios/efeitos da radiação , Optogenética/métodos , Técnicas de Patch-Clamp , Ratos , Rodopsina/genética , Técnicas de Cultura de Tecidos , Canal de Ânion 2 Dependente de Voltagem/genéticaRESUMO
Anionic channelrhodopsin slow ChloC was expressed in the culture of nerve cells and in vivo in mouse brain. We demonstrated ability of slow ChloC to suppress effectively the activity of the neuron in response to the illumination with the visible light. It has been shown for a first time that slow ChloC works equally efficiently in both neuronal culture and in the whole brain being expressed in vivo. Thus, slow ChloC could be considered as an effective optogenetic tool capable in response to light stimulation to inhibit the generation of action potentials in the neuron.
Assuntos
Potenciais de Ação , Encéfalo/metabolismo , Neurônios/metabolismo , Rodopsina/metabolismo , Animais , Encéfalo/citologia , Encéfalo/fisiologia , Células Cultivadas , Luz , Camundongos , Neurônios/fisiologia , Optogenética/métodos , Rodopsina/genética , Rodopsina/efeitos da radiaçãoRESUMO
Eph receptor tyrosine kinases and their ligands, the ephrins, perform an important regulatory function in tissue organization, as well as participate in malignant transformation of cells. Ephrin-A1, a ligand of A class Eph receptors, is a modulator of tumor growth and progression, and the mechanism of its action needs detailed investigation. Here we report on the development of a system for bacterial expression of an ephrin-A1 receptor-binding domain (eA1), a procedure for its purification, and its renaturation with final yield of 50 mg/liter of culture. Functional activity of eA1 was confirmed by immunoblotting, laser scanning confocal microscopy, and flow cytometry. It is shown that monomeric non-glycosylated receptor-binding domain of ephrin-A1 is able to activate cellular EphA2 receptors, stimulating their phosphorylation. Ligand eA1 can be used to study the features of ephrin-A1 interactions with different A class Eph receptors. The created expression cassette is suitable for the development of ligands with increased activity and selectivity and experimental systems for the delivery of cytotoxins into tumor cells that overexpress EphA2 or other class A Eph receptors.
Assuntos
Efrina-A1/genética , Efrina-A1/metabolismo , Escherichia coli/genética , Engenharia Genética/métodos , Receptores da Família Eph/metabolismo , Clonagem Molecular , Efrina-A1/química , Efrina-A1/isolamento & purificação , Escherichia coli/citologia , Expressão Gênica , Células HEK293 , Humanos , Células MCF-7 , Fosforilação , Estrutura Terciária de Proteína , Receptor EphA2/metabolismo , Solubilidade , Água/químicaRESUMO
High-resolution NMR is shown to be applicable for investigation of membrane proteins and membrane-active peptides embedded into lipid-protein nanodiscs (LPNs). (15)N-Labeled K+-channel from Streptomyces lividans (KcsA) and the antibiotic antiamoebin I from Emericellopsis minima (Aam-I) were embedded in LPNs of different lipid composition. Formation of stable complexes undergoing isotropic motion in solution was confirmed by size-exclusion chromatography and (31)P-NMR spectroscopy. The 2D 1H-(15)N-correlation spectra were recorded for KcsA in the complex with LPN containing DMPC and for Aam-I in LPNs based on DOPG, DLPC, DMPC, and POPC. The spectra recorded were compared with those in detergent-containing micelles and small bicelles commonly used in high-resolution NMR spectroscopy of membrane proteins. The spectra recorded in LPN environments demonstrated similar signal dispersion but significantly increased (1)H(N) line width. The spectra of Aam-I embedded in LPNs containing phosphatidylcholine showed significant selective line broadening, thus suggesting exchange process(es) between several membrane-bound states of the peptide. (15)N relaxation rates were measured to obtain the effective rotational correlation time of the Aam-I molecule. The obtained value (approximately 40 nsec at 45 degrees C) is indicative of additional peptide motions within the Aam-I/LPN complex.
Assuntos
Proteínas de Bactérias/química , Lipídeos/química , Espectroscopia de Ressonância Magnética/métodos , Proteínas de Membrana/química , Nanoestruturas/química , Peptídeos/química , Canais de Potássio/química , Hypocreales/química , PeptaibolsRESUMO
Myofibrillar protein tropomyosin (TM) is a normal physiological protein participating in regulation of muscular contraction. It is widely prevalent among living organisms. This explains cross-reactivity of allergic patients to home dust, sea fish, cockroaches, etc. The presence of similar IgE-binding epitopes in TM of different origin is a key factor in development of cross-reactivity (CR). CR to TM is a general biological phenomenon. We consider modified TM as a basic component in design of allergovaccines of a new generation.
Assuntos
Alérgenos/imunologia , Artrópodes/metabolismo , Reações Cruzadas/imunologia , Hipersensibilidade/imunologia , Tropomiosina/imunologia , Alérgenos/administração & dosagem , Alérgenos/efeitos adversos , Alérgenos/biossíntese , Animais , Artrópodes/imunologia , Dessensibilização Imunológica , Humanos , Hipersensibilidade/prevenção & controle , Contração Muscular/fisiologia , Miofibrilas/imunologia , Miofibrilas/metabolismo , Miofibrilas/fisiologia , Proteínas Recombinantes/administração & dosagem , Proteínas Recombinantes/efeitos adversos , Proteínas Recombinantes/imunologia , Tropomiosina/administração & dosagem , Tropomiosina/efeitos adversos , Tropomiosina/biossínteseRESUMO
A full-size human antibody to Ebola virus was constructed by joining genes encoding the constant domains of the heavy and light chains of human immunoglobulin with the corresponding DNA fragments encoding variable domains of the single-chain antibody 4D1 specific to Ebola virus, which was chosen from a combinatorial phage display library of single-strand human antibodies. Two expression plasmids. pCH1 and pCL1, containing the artificial genes encoding the light and heavy chains of human immunoglobulin, respectively, were constructed. Their cotransfection into the human embryonic kidney cell line HEK293T provided the production of a full-size recombinant human antibody. The affinity constant for the antibody was estimated by solid-phase enzyme-linked immunoassay to be 7.7 x 10(7) +/- 1.5 x 10(7) M(-1). Like the parent single-chain antibody 4DI, the resulting antibody bound the nucleoprotein of Ebola virus and did not interact with the proteins of Marburg virus.
Assuntos
Anticorpos Antivirais/biossíntese , Ebolavirus/imunologia , Proteínas Recombinantes/biossíntese , Anticorpos Antivirais/genética , Anticorpos Antivirais/imunologia , Linhagem Celular , Clonagem Molecular , Humanos , Nucleoproteínas/imunologia , Plasmídeos/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Transfecção , Proteínas do Core Viral/imunologiaRESUMO
Potassium voltage-gated Kv1.6 channel, which is distributed primarily in neurons of central and peripheral nervous systems, is of significant physiological importance. To date, several high-affinity Kv1.6-channel blockers are known, but the lack of selective ones among them hampers the studies of tissue localization and functioning of Kv1.6 channels. Here we present an approach to advanced understanding of interactions of peptide toxin blockers with a Kv1.6 pore. It combines molecular modeling studies and an application of a new bioengineering system based on a KcsA-Kv1.6 hybrid channel for the quantitative fluorescent analysis of blocker-channel interactions. Using this system we demonstrate that peptide toxins agitoxin 2, kaliotoxin1 and OSK1 have similar high affinity to the extracellular vestibule of the K+-conducting pore of Kv1.6, hetlaxin is a low-affinity ligand, whereas margatoxin and scyllatoxin do not bind to Kv1.6 pore. Binding of toxins to Kv1.6 pore has considerable inverse dependence on the ionic strength. Model structures of KcsA-Kv1.6 and Kv1.6 complexes with agitoxin 2, kaliotoxin 1 and OSK1 were obtained using homology modeling and molecular dynamics simulation. Interaction interfaces, which are formed by 15-19 toxin residues and 10 channel residues, are described and compared. Specific sites of Kv1.6 pore recognition are identified for targeting of peptide blockers. Analysis of interactions between agitoxin 2 derivatives with point mutations (S7K, S11G, L19S, R31G) and KcsA-Kv1.6 confirms reliability of the calculated complex structure.
Assuntos
Proteínas de Bactérias/antagonistas & inibidores , Proteínas de Bactérias/metabolismo , Modelos Moleculares , Bloqueadores dos Canais de Potássio/metabolismo , Canais de Potássio/metabolismo , Superfamília Shaker de Canais de Potássio/antagonistas & inibidores , Superfamília Shaker de Canais de Potássio/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Relação Dose-Resposta a Droga , Humanos , Canal de Potássio Kv1.6 , Bloqueadores dos Canais de Potássio/química , Bloqueadores dos Canais de Potássio/farmacologia , Canais de Potássio/química , Canais de Potássio/genética , Ligação Proteica/fisiologia , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Superfamília Shaker de Canais de Potássio/química , Superfamília Shaker de Canais de Potássio/genéticaAssuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Imidazóis/uso terapêutico , Neoplasias Pulmonares/tratamento farmacológico , Compostos Organoplatínicos/efeitos adversos , Substâncias Protetoras/uso terapêutico , Idoso , Caproatos , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma de Células Pequenas/tratamento farmacológico , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Compostos Organoplatínicos/administração & dosagem , Resultado do TratamentoRESUMO
Adjuvant activities of granulocyte-macrophage colony-stimulating factor (GM-CSF) and synthetic glucosaminyl-muramyl dipeptide (GMDP) were studied in immunization against type 1 herpes simplex virus (HSV1). Gene encoding the gD HSV1 protein (pDNAgD) was used as an immunogen. Gene encoding GM-CSF in pDNAGM-CSF plasmid, which was developed for eukaryotic expression, and GM-DP were used as immune response modulators. GMDP and plasmid DNA with inserted GM-CSF gene enhanced T-cell immune response to HSV1 after a single injection (pDNAGM-CSF) or 24 h before (GMDP) immunization with the gD HSV1 gene. Both adjuvants increased protective effect of DNA-immunization by a virus gene with 63 up to 100% after injection of two genes and up to 96% after the viral gene was inoculated 24 h after GMDP. These high effects indicate that further investigation of anti-HSV1 DNA-based vaccines used with genetic and peptide adjuvant is prospective.
Assuntos
Acetilmuramil-Alanil-Isoglutamina/análogos & derivados , Acetilmuramil-Alanil-Isoglutamina/imunologia , Fator Estimulador de Colônias de Granulócitos e Macrófagos/imunologia , Herpesvirus Humano 1/imunologia , Imunização , Vacinas de DNA/imunologia , Vacinas Virais/imunologia , Adjuvantes Imunológicos , Animais , Chlorocebus aethiops , Fator Estimulador de Colônias de Granulócitos e Macrófagos/genética , Infecções por Herpesviridae/imunologia , Infecções por Herpesviridae/prevenção & controle , Herpesvirus Humano 1/genética , Imunidade Celular , Camundongos , Camundongos Endogâmicos BALB C , Vacinas de DNA/genética , Células Vero , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/imunologia , Vacinas Virais/genéticaRESUMO
A study was made of the adjuvant effect of the mouse tumor necrosis factor alpha (mTNF alpha) on DNA immunization against the herpes simplex virus type 1 (HSV1). The HSV1 gD gene (pDNAgD) served as an immunogen; mTNF alpha or its gene cloned in an eukaryotic expression vector (pDNAmTNF) were used to modulate the immune response. Double immunization with pDNAgD led to a sixfold increase in the in vitro T-cell response, a high (1:2000) titer of anti-HSV1 antibodies (including virus-neutralizing antibodies), an increase in IgG2a/IgG1 (suggesting a shift of the immune response to the Th1 type), and no change in CD4/CD8 T-cell ratio. A single injection of mTNF alpha along with inactivated HSV1 allowed a twice higher antibody titer and a fourfold higher T-cell response as compared with immunization with HSV1 alone. Double immunization with both pDNAgD and pDNAmTNF increased the titer of anti-HSV1 antibodies and the T-cell response by factors of 8 and 1.5, respectively, as compared with immunization with pDNAgD alone. However, the protective effect was significantly lower with the two plasmids than with pDNAgD (73 vs. 100%). Thus, DNA immunization with pDNAgD induced both B- and T-cell responses and completely protected mice from a lethal doze of HSV1. The adjuvant properties of mTNF alpha and pDNAmTNF need further investigation.
Assuntos
DNA/imunologia , Simplexvirus/imunologia , Fator de Necrose Tumoral alfa/genética , Vacinas de DNA/imunologia , Anticorpos Antivirais/biossíntese , Sequência de Bases , Primers do DNA , Imunidade CelularRESUMO
Plasmids of the pFPCP series have been constructed, containing the whole gene for the filamentous phage main coat protein or its regulatory elements along with unique restrictase sites.
Assuntos
Clonagem Molecular , Colífagos/genética , Vetores Genéticos , Plasmídeos , Sequência de Bases , Enzimas de Restrição do DNA , Regulação da Expressão GênicaRESUMO
Expression of the human leucocyte interferon alpha 2 gene has been studied, cloned into filamentous phages (M13mp8, M13mp9) and plasmid vehicles comprising the regulatory regions and signal sequence of the filamentous phage main coat protein gene (plasmids pFPCP2 and pFPCP8).
Assuntos
Clonagem Molecular , Colífagos/genética , Vetores Genéticos , Sequência de Aminoácidos , Sequência de Bases , Enzimas de Restrição do DNA , Interferon Tipo I/biossíntese , Interferon Tipo I/genética , Plasmídeos , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Recombinação GenéticaRESUMO
Multicopy plasmids of pMCR series have been constructed from pRRN2 (a pBR322 derivative) including plasmids carrying transcription terminators downstream the tet gene and those with opposite orientation of the tet and RNAI genes. The plasmids permit cloning and high expression of genes with strong promoters.
Assuntos
Clonagem Molecular , Regulação da Expressão Gênica , Vetores Genéticos , Plasmídeos , Mapeamento Cromossômico , Enzimas de Restrição do DNA , Escherichia coli/genética , Regiões Promotoras Genéticas , RepliconRESUMO
Chemical-enzymatic synthesis and cloning in Escherichia coli of double-stranded DNAs, coding for simple and complex antigenic determinants of foot-and-mouth disease virus (FMDV) strain A22, have been carried out. The simple antigenic determinants are a part of the viral coat protein VP1 (amino acid sequence 131-152 or 131-160) whereas the complex antigenic determinants comprise additionally the amino acid sequence 200-213 of VP1 linked to N-terminus of simple antigenic determinants through a tetrapeptide spacer Pro-Pro-Ser-Pro. Recombinant DNAs containing genes for antigenic determinants of FMDV fused with C-terminus of gene for human tumor necrosis factor (hrTNF) have been constructed. Expression of the hybrid genes and properties of the proteins coded were studied. All recombinant proteins were shown to interact specifically with polyclonal antibodies both against hrTNF and FMDV strain A22. The recombinant proteins produced by bacteria are perspective for study as a vaccine against FMDV.
Assuntos
Aphthovirus/genética , Epitopos/genética , Regulação Bacteriana da Expressão Gênica , Regulação Viral da Expressão Gênica , Genes Virais , Sequência de Aminoácidos , Antígenos Virais , Aphthovirus/imunologia , Sequência de Bases , Western Blotting , Clonagem Molecular , DNA Viral/genética , Eletroforese em Gel de Poliacrilamida , Escherichia coli/genética , Genes Bacterianos , Dados de Sequência Molecular , Plasmídeos , Mapeamento por RestriçãoRESUMO
Using recombinant DNA technology, construction and bacterial expression of genes was carried out which code for hybrid proteins, human tumor necrosis factor and hepatitis B core protein fused to immunogenic epitopes of foot-and-mouth disease virus, strains A22 and O1-194. Hybrids of tumor necrosis factor with foot-and-mouth disease antigenic determinants protected laboratory animals against the experimental challenge with a homologous strain of foot-and-mouth disease virus. Hybrid protein that contained immunogenic regions of two strains, A22 and O1-194, protected animals against infection with both A and O serotypes. Hybrid proteins based on hepatitis B virus core antigen retained the ability to assemble into core-like particles.
Assuntos
Aphthovirus/genética , Epitopos/biossíntese , Antígenos do Núcleo do Vírus da Hepatite B/genética , Fator de Necrose Tumoral alfa/genética , Animais , Aphthovirus/imunologia , Sequência de Bases , DNA Complementar/genética , Epitopos/genética , Cobaias , Humanos , Immunoblotting , Dados de Sequência Molecular , Sondas de Oligonucleotídeos , Plasmídeos , Reação em Cadeia da Polimerase , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/genéticaRESUMO
RecA-independent recombinations accompanying the processes of plasmids preparation and cloning into Escherichia coli cells are induced within the short direct and inverted repeats of several types.
Assuntos
Clonagem Molecular , Recombinação Genética , Sequência de Bases , DNA Recombinante , Escherichia coli/genética , Dados de Sequência Molecular , PlasmídeosRESUMO
Functional activity of macrophages and intensity of T cell immune response in mice were studied after intravaginal and intraperitoneal infection with herpes simplex virus type 1 and DNA vaccination in combination with adjuvant treatment (recombinant granulocyte-macrophage colony-stimulating factor and glucosaminylmuramyl dipeptide). DNA vaccination induced a virus-specific T cell immune response with no macrophagic inflammatory reaction. Infection with herpes simplex virus type 1 was accompanied by sustained inflammation, but not by the T cell immune response.
Assuntos
Herpes Simples/sangue , Herpesvirus Humano 1/metabolismo , Macrófagos/metabolismo , Vacinas de DNA/farmacologia , Acetilmuramil-Alanil-Isoglutamina/análogos & derivados , Acetilmuramil-Alanil-Isoglutamina/uso terapêutico , Animais , Movimento Celular , Fator Estimulador de Colônias de Granulócitos e Macrófagos/uso terapêutico , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Vacinas de DNA/metabolismoRESUMO
The authors' findings permit a conclusion that the risk of ethmozine overdosage leading to undesirable side effects (dryness in the mouth, noise in the ears, a 'net' in eyes; giddiness, nausea, vomiting) is very high when routine ethmozine doses are administered to patients with grave (Stages II-III) impairments of liver function; this is explained by (1) reduced rate of ethmozine biotransformation, this resulting in a heightened concentration of the drug in the blood, and (2) by an increase of the drug free fraction concentration due to its reduced ability to bind with the blood plasma proteins. This necessitates a pharmacokinetic monitoring of such patients prescribed ethmozine and a correction of the drug dose, if necessary.