RESUMO
Ozone, UV/ozone, ozone/persulfate (PS) and UV/ozone/PS systems were used to mineralize sulfonamides. Sulfadiazine (SDZ), sulfamerazine (SMR) and sulfamethazine (SMZ) were the target compounds. The novel contribution of this study is its determination of the effects of PS addition, sulfonamide structure, pH and salinity on sulfonamide mineralization in ozone-based systems. The mineralization rate of sulfonamides satisfied pseudo-first-order kinetics. The SMZ mineralization rate constant in ozone, UV/ozone, ozone/PS and UV/ozone/PS systems at pH 5 were 0.0058; 0.0101; 0.0069 and 0.0802 min-1, respectively, and those at pH 7 were 0.0075; 0.0116; 0.0083 and 0.0873 min-1, respectively. The increase in the number of methyl substituents in the heterocyclic group of SMZ and the corresponding increase in the steric hindrance of radical addition, reduced mineralization rates below those of SMR and SDZ. The addition of PS promoted sulfonamide mineralization in the ozone-based systems; conversely, salinity inhibited sulfonamide mineralization.
Assuntos
Ozônio , Poluentes Químicos da Água , Sulfadiazina , Sulfonamidas , Águas Residuárias , Poluentes Químicos da Água/análiseRESUMO
Enterohaemorrhagic Escherichia coli (EHEC) causes life-threatening infections in humans as a consequence of the production of Shiga-like toxins. Lack of a good animal model system currently hinders in vivo study of EHEC virulence by systematic genetic methods. Here we applied the genetically tractable animal, Caenorhabditis elegans, as a surrogate host to study the virulence of EHEC as well as the host immunity to this human pathogen. Our results show that E. coli O157:H7, a serotype of EHEC, infects and kills C. elegans. Bacterial colonization and induction of the characteristic attaching and effacing (A/E) lesions in the intact intestinal epithelium of C. elegans by E. coli O157:H7 were concomitantly demonstrated in vivo. Genetic analysis indicated that the Shiga-like toxin 1 (Stx1) of E. coli O157:H7 is a virulence factor in C. elegans and is required for full toxicity. Moreover, the C. elegans p38 mitogen-activated protein kinase (MAPK) pathway, an evolutionarily conserved innate immune and stress response signalling pathway, is activated in the regulation of host susceptibility to EHEC infection in a Stx1-dependent manner. Our results validate the EHEC-C. elegans interaction as suitable for future comprehensive genetic screens for both novel bacterial and host factors involved in the pathogenesis of EHEC infection.
Assuntos
Caenorhabditis elegans/enzimologia , Caenorhabditis elegans/microbiologia , Escherichia coli O157/patogenicidade , Interações Hospedeiro-Patógeno , Toxina Shiga I/metabolismo , Fatores de Virulência/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/biossíntese , Animais , Caenorhabditis elegans/imunologia , Infecções por Escherichia coli , Mucosa Intestinal/microbiologia , Mucosa Intestinal/patologia , Modelos Animais , Análise de SobrevidaRESUMO
The feasibility of using antigen modifications and synthetic resin-bound peptides to distinguish closely related epitopes has been demonstrated in this report. Epitopes recognized by five monoclonal antibodies (MAbs) specific to Escherichia coli ribosomal protein S13 have been located near the C-terminus and N-terminus of the S13 molecule; these epitopes were characterized by modifications of antigens and by utilization of peptides of increasing length synthesized on aminomethyl crosslinked polystyrene resin. Three of these MAbs react with the C-terminal peptide S13(84-117) which has five Lys residues clustered within the last 16 amino acids. Phthalylation of Lys residues almost eliminated the binding of two MAbs and reduced binding of the third by 50%. Removal of the C-terminal Lys residue(s) at the S13 C-terminus with carboxypeptidase B has no effect on the binding of these three MAbs. A 23-residue peptide corresponding to S13(95-117) was synthesized by a modified Merrifield solid phase method. Samples of resin with peptides of increasing length were obtained after each cycle of amino acid coupling. The peptides were deprotected without hydrolysis of the peptide-resin linkage and used in an enzyme immunoassay to detect the extent of MAb interaction with the lengthening peptides. The epitopes recognized by the two MAbs more sensitive to Lys modification have been localized in S13(97-117). The third MAb binds to S13(98-117) but binds more strongly when the sequence is lengthened. Two MAbs directed toward the N-terminal 22 residues of S13 were similarly characterized. Binding of one MAb, little affected by phthalylation, required the N-terminal residue of S13 to be present in the synthetic peptide. The other MAb, whose binding was inhibited by phthalylation, bound to the synthetic S13(2-22) and bound more strongly to the synthetic S13(1-22).
Assuntos
Antígenos de Bactérias/análise , Epitopos/análise , Mapeamento de Peptídeos , Proteínas Ribossômicas/análise , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais , Antígenos de Bactérias/imunologia , Sítios de Ligação de Anticorpos , Epitopos/imunologia , Proteínas de Escherichia coli , Lisina/metabolismo , Camundongos , Dados de Sequência Molecular , Fragmentos de Peptídeos/síntese química , Fragmentos de Peptídeos/imunologia , Fragmentos de Peptídeos/metabolismo , Anidridos Ftálicos , Resinas Vegetais , Proteínas Ribossômicas/imunologiaRESUMO
The unequivocal identification of immunochemically detected proteins on nitrocellulose blots requires precise alignment of the antibody-stained antigen spot with the protein-stained pattern. We report a method for the staining of proteins on nitrocellulose membranes immediately after electroblotting with Amido Black 10B under mild conditions which do not result in loss of antigenic activity. These membranes can then be probed with immunological detection reagents and the detected antigen correlated with the stained pattern. The procedure is simple, fast and particularly useful for the identification of antibody-labelled spots on blots of high-resolution two-dimensional gels.
Assuntos
Negro de Amido , Anticorpos Monoclonais/imunologia , Especificidade de Anticorpos , Compostos Azo , Técnicas de Imunoadsorção , Colódio , Gelatina , Proteínas Ribossômicas/imunologia , Coloração e RotulagemRESUMO
Maltose-binding protein (MBP) encoded by malE is essential for the energy-dependent translocation of maltose through the cytoplasmic membrane of bacteria. Its property of specific binding to maltose has been used in constructing fusion proteins for easy affinity purification. A monoclonal antibody named MAb SC1D7 was produced against Escherichia coli MBP. This MAb also bound to MBP-containing recombinant proteins in both Western blotting and immunoprecipitation analysis. As a result, this MAb can be a useful probe for tracing MBP-fusion proteins in various applications. Furthermore, intrinsic MBPs from E. coli, Shigella dysenteriae, Salmonella typhimurium, Enterobacter cloacae, and Klebsiella pneumoniae were also detected by this MAb. No reaction was observed with the total proteins from Serratia marcescens, Aeromonas hydrophila and Plesiomonas shigelloides. These observations suggest that the MAb SC1D7-defined epitope is conserved among some enteric bacteria, but not the others. The results strengthen the phylogenetic positions of these closely related bacteria previously placed by other means.
Assuntos
Transportadores de Cassetes de Ligação de ATP , Anticorpos Monoclonais/imunologia , Proteínas de Bactérias/análise , Proteínas de Transporte/análise , Proteínas de Escherichia coli , Escherichia coli/química , Proteínas de Transporte de Monossacarídeos , Proteínas Periplásmicas de Ligação , Animais , Proteínas de Transporte/química , Proteínas de Transporte/imunologia , Enterobacteriaceae/química , Ensaio de Imunoadsorção Enzimática , Proteínas Ligantes de Maltose , Camundongos , Camundongos Endogâmicos BALB CRESUMO
To study the association between anti-Gag and anti-Nef antibody reactivities and their correlations with disease progression, 174 HIV-1/AIDS patients were followed up for 1 year after they received triple therapy. The antibody reactivities were analyzed using a Western blot test with recombinant Gag and Nef proteins. The results showed that decreasing levels of anti-Gag or anti-Nef antibody correlate with disease progression defined by HIV-1 viral loads or T4 cell counts. After receiving triple treatment for 1 year, 8 of 38 (21.1%) Nef antibody-negative patients became positive, while only 9 of 125 (7.2%) Nef antibody-positive persons lost the antibody reactivity (p < 0.01). Therefore, HIV-1 Nef may serve as a clinical marker of disease progression.
Assuntos
Produtos do Gene nef/imunologia , Anticorpos Anti-HIV/sangue , Infecções por HIV/imunologia , HIV-1/fisiologia , Carga Viral , Western Blotting , Contagem de Linfócito CD4 , Progressão da Doença , Produtos do Gene gag/imunologia , HIV-1/isolamento & purificação , Humanos , RNA Viral/sangue , Proteínas Recombinantes de Fusão/metabolismo , Produtos do Gene nef do Vírus da Imunodeficiência HumanaRESUMO
An enzyme immunoassay was developed for monitoring protease reactions of human immunodeficiency virus (HIV). The protease and its substrate, the gag precursor, were generated separately in Escherichia coli. The HIV-1 protease was generated with a glutathione-S-transferase expression system and the gag substrate, named Pin17/24, was prepared with a PinPoint expression system. Pin17/24 consists of an N-terminal peptide, which is biotinylated in E. coli, fused with a C-terminal peptide that contains a protease cleavage site flanked by p17 and p24 segments. Through its biotin in the N-terminal region, Pin17/24 bound to ELISA plates coated with avidin, whereas through its C-terminal region, the same molecule of Pin17/24 could be recognized by an anti-p24 monoclonal antibody. When the protease was added to Pin17/24, the p24 fragment was released from the biotinylated fusion protein and could no longer be retained on the avidin plates, and as a result, binding of the anti-p24 monoclonal antibody decreased. The binding was specific and the reaction was inhibited by a known HIV protease inhibitor. Due to the specific interactions between avidin and biotin, monoclonal antibody and antigen, and the HIV protease and the gag substrate, crude preparations of these reagents can be used readily in the assay. The simplicity and feasibility of this method should be useful for simultaneous monitoring of many enzyme reactions, particularly for screening possible HIV protease inhibitors.
Assuntos
Ensaio de Imunoadsorção Enzimática/métodos , Produtos do Gene gag/metabolismo , Protease de HIV/análise , HIV-1/enzimologia , Animais , Biotina , Escherichia coli , Estudos de Viabilidade , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Proteínas Recombinantes de Fusão/metabolismo , Especificidade por SubstratoRESUMO
Integration of reverse transcribed viral DNA of HIV into host chromosomes is mediated by the viral enzyme, integrase. This enzymatic activity can be monitored in vitro by integration of a small labeled DNA (donor) into a second unlabeled DNA (target). The methodology usually involves isotope labeling and gel electrophoresis. To simplify the measurement, a method mimicking enzyme-linked immunosorbent assay (ELISA) procedures was developed. Fragments of DNA were adsorbed directly on 96-well plates and used as the target DNA. The donor was a synthetic 21-bp DNA duplex of HIV-1 U5 LTR; biotin was incorporated into the 5' end of one strand whose two nucleotides at the 3' end were specifically removed during the integration. As a result of integration, the biotin-labeled donor DNA was joined with the target DNA and became immobilized on plates. These integration products were then measured by binding of avidin-alkaline phosphatase on plates. The method is simple and straightforward and can easily be adapted for high throughput screening of integrase inhibitors.
Assuntos
DNA/metabolismo , Ensaio de Imunoadsorção Enzimática/métodos , Integrase de HIV/análise , HIV-1/enzimologia , Integrase de HIV/genética , Humanos , Proteínas Recombinantes de Fusão/análise , Proteínas Recombinantes de Fusão/genéticaRESUMO
The putative E1 of hepatitis C virus (HCV) was expressed in Escherichia coli using a glutathione-S-transferase (GST) fusion protein system. The full length E1 protein is difficult to express. A series of E1 DNA fragments was generated and used for expression vector construction. Fusion proteins containing the E1 C-terminal region could not be expressed. When this region was truncated, the fusion proteins were synthesized to high levels. The possibility of this C-terminal region hampering the production of fusion protein was further explored. A construct with this segment directly fused to the C-terminus of GST indeed generated no detectable recombinant protein. According to the predicted structure of E1, this region may have membrane-associating properties. The expression results suggest a general approach to facilitate the production of viral membrane proteins in prokaryotes. Furthermore, these recombinant E1 proteins generated as antigens were used for Western blotting with sera from HCV-infected individuals. It was found that E1 is antigenic during HCV natural infection.
Assuntos
Hepacivirus/genética , Proteínas do Envelope Viral/genética , Virologia/métodos , Antígenos Virais/genética , Sequência de Bases , DNA Viral/genética , Escherichia coli/genética , Expressão Gênica , Vetores Genéticos , Hepacivirus/imunologia , Anticorpos Anti-Hepatite/sangue , Hepatite C/imunologia , Anticorpos Anti-Hepatite C , Antígenos da Hepatite C , Dados de Sequência Molecular , Plasmídeos/genética , Proteínas Recombinantes de Fusão/genética , Proteínas do Envelope Viral/imunologiaRESUMO
Sequences of the hepatitis delta virus (HDV) vary to different degrees among isolates. A monoclonal antibody, designated as HP6A1, against the antigen of HDV (HDAg) has been characterized for its specificity. HP6A1 bound to HDAg of isolate 25 (genotype I) that was used for immunization, but not to others of both genotypes I and II. The epitope recognized by HP6A1 was then determined by a phage library displaying various heptapeptides. A consensus peptide deduced has the best match with that of residues 4-10 of HDAg (isolate 25). To confirm the phage mapping result, Escherichia coli recombinant proteins containing different lengths and various segments of HDAg (isolate 25) were constructed. The shortest HDAg segment contained in the fusion protein that reacted with HP6A1 was residues 1-10. When this peptide was added to the N-terminus of a heterologous protein engineered for eucaryotic expression, the fusion protein was detected by HP6A1. It is concluded that HP6A1 recognizes an epitope located at the N-terminus of HDAg (isolate 25). Since viruses of quasi-species exist in natural infections, a question of how different viral strains interact in vivo remains to be explored. The highly specific MAb opens a possibility to examine the fate of one strain in the presence of other related species in a cell transfection system.
Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Antivirais/imunologia , Antígenos Virais/imunologia , Vírus Delta da Hepatite/imunologia , Sequência de Aminoácidos , Anticorpos Monoclonais/biossíntese , Anticorpos Antivirais/biossíntese , Antígenos Virais/genética , Bacteriófago M13/genética , Linhagem Celular , Sequência Consenso , Mapeamento de Epitopos , Imunofluorescência , Humanos , Biblioteca de Peptídeos , Alinhamento de Sequência , TransfecçãoRESUMO
Six monoclonal antibodies (MAbs) were raised in mice against the 26-kDa glutathione S-transferase (GST) of the parasite Schistosoma japonicum. These MAbs were originally selected for their specific binding to the recombinant GST (r-GST) generated in E. coli by an enzyme-linked immunosorbent assay. A further study demonstrated that all these MAbs bound to plate-coated GST affinity-purified from the parasite Schistosoma japonicum. However, in Western blotting analysis only a single monoclonal antibody (MAb Y3D7) yielded positive binding. The binding of MAb Y3D7 on Western blotting was further characterized; specific binding was found on other GST fusion proteins and on the authentic 26-kDa GST but not the 28-kDa GST in the total soluble worm proteins from Schistosoma japonicum. Using protein-A-mediated immunoprecipitation, MAbs Y3D7 and Y5D5 precipitated r-GST while in parallel experiments the remaining MAbs did not generate r-GST precipitation. In an alternative co-precipitation experiment, r-GST was first bound to glutathione (GSH) Sepharose beads and subsequently tested for interaction with the MAbs. In this manner, all MAbs except MAb Y5D5 were co-precipitated with the complexes. Thus, these select MAbs readily reacted with GST although their binding characteristics were different. Because GST has been widely used in the generation of fusion proteins for various purposes and is a potential vaccine candidate in controlling schistosomiasis, these MAbs should prove valuable for their application to molecular biology and parasitology.
Assuntos
Anticorpos Monoclonais/química , Glutationa Transferase/imunologia , Schistosoma japonicum/enzimologia , Schistosoma japonicum/imunologia , Animais , Camundongos , Peso MolecularAssuntos
Anticorpos Monoclonais/química , Complexo Antígeno-Anticorpo , Antígenos/química , Epitopos/química , Sequência de Aminoácidos , Animais , Antígenos/imunologia , Escherichia coli , Imunoglobulina G/química , Indicadores e Reagentes , Camundongos , Dados de Sequência Molecular , Peptídeos/síntese química , Peptídeos/imunologia , Resinas Vegetais , Ribossomos/ultraestruturaRESUMO
To investigate the structure of Escherichia coli ribosomal protein S13 in 30S ribosomal subunits, we have previously generated 22 S13 specific monoclonal antibodies and mapped their specific epitopes to the S13 sequence. The availability of these S13 epitopes in situ has been further examined by incubating these monoclonal antibodies with 30S ribosomal subunits and analyzing formation of monoclonal antibody-linked ribosome dimers by sucrose gradients centrifugation. We have found that none of the 22 monoclonal antibodies makes ribosome dimers individually as do typical antisera. However, one monoclonal antibody, designated AS13-MAb 2, reacts with 30S ribosomal subunits to form immunocomplexes sedimenting faster than subunit monomers. When AS13-MAb 2 is paired with any one of three monoclonal antibodies directed to the S13 C-terminal epitopes, dimer formation is observed. Other pairs of monoclonal antibodies directed to distinct S13 epitopes have been tested similarly for dimer formation. Monoclonal antibody AS13-MAb 22, directed to the N-terminal region of 22 residues, also causes subunits to form typical dimers, but only if paired with one of the three monoclonal antibodies directed to the S13 C-terminal region. The close proximity of the epitopes recognized by AS13-MAbs 2 and 22 has been established by the mutual competition between the antibodies binding to intact 30S subunits. These results corroborate our previous observation, using polyclonal antibodies, that S13 has more than one epitope exposed on 30S subunits. Our finding that sequences on both ends of the S13 molecule are immunochemically accessible provides information about the molecular organization of S13 in situ.
Assuntos
Escherichia coli/metabolismo , Proteínas Ribossômicas/metabolismo , Anticorpos Monoclonais , Epitopos , Proteínas de Escherichia coli , ImunoquímicaRESUMO
To analyze the immunochemical structure of Escherichia coli ribosomal protein S13 and its organization in situ, we have generated and characterized 22 S13-specific monoclonal antibodies. We used a competitive enzyme-linked immunosorbent assay to divide them into groups based on their ability to inhibit binding of one another. The discovery of five groups with distinct binding properties suggested that a minimum of five distinct determinants on S13 are recognized by our monoclonal antibodies. The locations of the epitopes detected by these monoclonal antibodies have been mapped on S13 peptides. Three monoclonal antibodies bind a S13 C-terminal 34-residue segment. All the other 19 monoclonal antibodies bind a S13 N-terminal segment of about 80 residues. The binding sites of these 19 monoclonal antibodies have been further mapped to subfragments of peptides. Two monoclonal antibodies recognized S131-22; three monoclonal antibodies bound to S131-40; the binding sites of three other antibodies have been located in S1323-80, with epitopes possibly associated with residues 40-80. The remaining 11 monoclonal antibodies did not bind to these subfragments. These data provide molecular basis to the structure of S13 epitopes, whose in situ accessibility may reveal the S13 organization on the ribosome.
Assuntos
Epitopos/imunologia , Escherichia coli/análise , Proteínas Ribossômicas/imunologia , Aminoácidos/análise , Animais , Anticorpos Monoclonais/imunologia , Especificidade de Anticorpos , Sítios de Ligação de Anticorpos , Ligação Competitiva , Ensaio de Imunoadsorção Enzimática , Epitopos/análise , Proteínas de Escherichia coli , Hibridomas/imunologia , Iodobenzoatos , Camundongos , Fragmentos de Peptídeos/imunologia , Serina Endopeptidases , TiocianatosRESUMO
The HIV pol sequentially encodes protease (PR), reverse transcriptase (RT), and integrase (IN) from the 5'-3' direction. We explored the significance of this gene arrangement. All six possible gene dispositions were examined. In two situations where PR was removed from the leading place and no two genes were in their original location, viral polyprotein processing was abolished. Processing of the polyprotein did not occur when IN was translocated to the front of PR-RT. However, in the following two arrangements, the polyprotein was processed but only at specific sites. First, PR remained in the leading position while the locations of RT and IN were exchanged; viral polyprotein was processed at a site between the upstream transframe peptide (TF) and PR. Second, PR was placed after RT-IN and located at the distal end of Pol. Processing occurred only at the created junction between TF and RT. These results indicated that cleavage after TF occurred autocatalytically but did not proceed to a second site, which needed an extraneous PR for trans-action. Therefore, arranging Pol in the order of PR-RT-IN warrants the streamline processing of the polyprotein once the autocleavage is initiated.
Assuntos
Produtos do Gene pol/metabolismo , Genes pol , HIV-1/genética , Processamento de Proteína Pós-Traducional , Proteínas Recombinantes/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Western Blotting , Escherichia coli/genética , Escherichia coli/metabolismo , Regulação Viral da Expressão Gênica , Produtos do Gene pol/análise , Produtos do Gene pol/química , Integrase de HIV/química , Integrase de HIV/genética , Integrase de HIV/metabolismo , Protease de HIV/química , Protease de HIV/genética , Protease de HIV/metabolismo , Transcriptase Reversa do HIV/química , Transcriptase Reversa do HIV/genética , Transcriptase Reversa do HIV/metabolismo , HIV-1/enzimologia , Peso Molecular , Fases de Leitura Aberta/genética , Testes de Precipitina , Proteínas Recombinantes/análise , Proteínas Recombinantes/química , Proteínas Recombinantes/genéticaRESUMO
The antigenic structure of Escherichia coli ribosomal protein S3 has been investigated by use of monoclonal antibodies. Six S3-specific monoclonal antibodies secreted by mouse hybridomas have been identified by immunoblotting of two-dimensional ribosomal protein separation gels. By using a competitive enzyme-linked immunosorbent assay, we have divided these monoclonal antibodies into three mutual inhibition groups, members of which are directed to three distinct regions of the S3 molecule. The independence of these monoclonal antibody-defined regions was confirmed by the failure of pairs of monoclonal antibodies from two inhibition groups to block the binding of biotinylated monoclonal antibodies of the third group. To determine the regions recognized by these monoclonal antibodies, chemically cleaved S3 peptides were fractionated by gel filtration and reverse-phase high-performance liquid chromatography. The fractionated peptides were coated on plates and examined for specific interaction with monoclonal antibody by enzyme immunoassay. In this manner, two epitopes have been mapped at the ends of the S3 molecule: one, in the last 22 residues, is recognized by three monoclonal antibodies; and the second, in the first 21 residues, is defined by two monoclonal antibodies. The third S3 epitope, recognized by a single monoclonal antibody, has been localized in a central segment of about 90 residues by gel electrophoresis and immunoblotting. These epitope-mapped monoclonal antibodies are valuable probes for studying S3 structure in situ.
Assuntos
Anticorpos Monoclonais/imunologia , Proteínas de Bactérias/imunologia , Epitopos/imunologia , Escherichia coli/imunologia , Proteínas Ribossômicas/imunologia , Animais , Especificidade de Anticorpos , Sítios de Ligação , Cromatografia em Gel , Hibridomas/imunologia , Immunoblotting , Camundongos , Camundongos Endogâmicos BALB C , Peso Molecular , Fragmentos de Peptídeos/análise , Proteínas Ribossômicas/metabolismoRESUMO
A sensitive method using enzyme immunoassay and sucrose gradient to analyze immunocomplexes of biological particles has been developed. The sensitivity and application of this method were demonstrated by that the in situ accessibility of ribosomal protein epitopes could be easily determined. We used sucrose gradients to separate the ribosome-bound and the free antibodies and traced the antibodies in the gradients by an enzyme-linked immunosorbent assay. Epitopes exposed in situ are bound by specific antibodies, which in turn are detected in sucrose gradients migrating with ribosomes. This method of detecting antibody migration is more sensitive than the conventional means of using A260nm to monitor the antibody-mediated dimerization of ribosomes. Furthermore, an epitope defined by a biotin-labeled monoclonal antibody can be analyzed in the presence of other unlabeled antibodies. Thus, the relationship of different accessible epitopes in situ can be readily examined. Versatility and sensitivity of this method should make it useful in analyzing a variety of immunocomplex systems.
Assuntos
Complexo Antígeno-Anticorpo/análise , Centrifugação com Gradiente de Concentração/métodos , Ensaio de Imunoadsorção Enzimática , Epitopos , Ribossomos/imunologia , Anticorpos Antibacterianos/imunologia , Antígenos de Bactérias/imunologia , Escherichia coli/imunologiaRESUMO
The N-terminal region of the hepatitis-C virus (HCV) core protein is rich in basic residues, while the C-terminal end of the protein comprises of a stretch of hydrophobic amino acids. Between these two extremes is an amphipathic region with two predicted alpha-helical segments. This region embodies Leu or hydrophobic residues in positions of heptad repeats and is possibly capable of self-association. To investigate this possibility, the core sequence was divided into two fragments and expressed separately as recombinant proteins. Recombinant proteins with the N-terminal fragment remained as monomers even at high concentrations in SDS/PAGE. Recombinant protein with the C-terminal fragment appeared largely monomeric on denaturing gels but some oligomers were also detected. Furthermore, proline mutations in either one of the predicted alpha helices adversely affected the observed oligomerization. The self-association capacity of the core protein C-terminal region was further supported by results from a yeast two-hybrid system. To affirm our conclusion, a peptide covering the heptad repeats and the predicted alpha helices was synthesized. Data from mass spectrometry and gel-filtration chromatography concluded that this peptide readily self-associated into the homodimer. Therefore, our results suggest that the oligomerization motifs of the HCV core protein may not be limited to the previously suggested N-terminal region.
Assuntos
Proteínas do Core Viral/química , Sequência de Aminoácidos , Sequência de Bases , Biopolímeros , Primers do DNA , Hepacivirus/química , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Saccharomyces cerevisiae/genética , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Proteínas do Core Viral/genéticaRESUMO
Solavetivone (1), cytotoxic to OVCAR-3 cells with an IC(50) value of 0.1 mM, has been isolated from Solanum indicum. In addition, a novel solafuranone (2) and three known compounds, scopoletin, N-(p-trans-coumaroyl)tyramine, and N-trans-feruloyltyramine, were isolated for the first time from this plant. The structures of the above compounds were established by means of spectroscopic and X-ray analyses.
Assuntos
Antineoplásicos Fitogênicos/isolamento & purificação , Furanos/isolamento & purificação , Solanaceae/química , Antineoplásicos Fitogênicos/química , Antineoplásicos Fitogênicos/farmacologia , Cromatografia , Cristalografia por Raios X , Ensaios de Seleção de Medicamentos Antitumorais , Feminino , Furanos/química , Humanos , Concentração Inibidora 50 , Espectroscopia de Ressonância Magnética , Medicina Tradicional Chinesa , Conformação Molecular , Estrutura Molecular , Neoplasias Ovarianas , Raízes de Plantas/química , Plantas Medicinais/química , Espectrofotometria Infravermelho , Espectroscopia de Infravermelho com Transformada de Fourier , Estereoisomerismo , Relação Estrutura-Atividade , Células Tumorais Cultivadas/efeitos dos fármacosRESUMO
Human immunodeficiency virus (HIV) vaccines targeted at blocking HIV-CD4 interactions are expected to be less affected by the sequence heterogeneity of HIV than those targeted at variable regions of the envelope outercoat glycoprotein, gp120. All potential CD4 binding sites identified thus far in HIV are localized in the C-terminal region of gp120. In this study we demonstrate that the N-terminal region of gp120 also contains conserved residues critical for binding to CD4 and that gp120-CD4 interactions can be blocked by an antiserum with binding specificity to an N-terminal region of gp120. These results suggest that not all potential CD4 binding sites are present in the C-terminal region of gp120 and that an alternative HIV vaccine development strategy may have to include the N-terminal gp120 region as a component to raise effective CD4-blocking antibodies.