RESUMEN
We described the complete genome sequence of a novel baculovirus isolate of species Buzura suppressaria nucleopolyhedrovirus, called by isolate CNPSo-25. The occlusion bodies were found to be polyhedral in shape and to contain virions with singly embedded nucleocapsids. The size of the genome is 121,377 bp with a G+C content of 36.7%. We annotated 131 ORFs that cover 90.42% of the genome. Moreover, phylogenetic inference indicated that CNPSo-25 is a member of genus Alphabaculovirus that clustered together with two other Chinese isolates of the same species. We called the virus by Biston suppressaria nucleopolyhedrovirus isolate CNPSo-25 (BisuNPV-CNPSo-25), as Buzura was placed inside the lepidopteran genus Biston. As expected, we detected intra-population variability in the virus sample when the novel isolate was compared to the Chinese isolates: 292 single nucleotide variants were found in the genome, with 181 affecting the protein product. The closest representatives of other species to BisuNPV-CNPSo-25 was found to be Sucra jujuba nucleopolyhedrovirus and Hyposidra talaca nucleopolyhedrovirus, two other virus isolates of geometrid caterpillars. The study of baculovirus genomes is of importance for the development of tools for insect pest biological control and biotechnology.
Asunto(s)
Genoma Viral , Genómica , Mariposas Nocturnas/virología , Nucleopoliedrovirus/clasificación , Nucleopoliedrovirus/genética , Animales , Composición de Base , Genes Virales/genética , Nucleopoliedrovirus/aislamiento & purificación , Filogenia , Análisis de Secuencia de ADN , Té , Virión , Secuenciación Completa del GenomaRESUMEN
A strain of Adoxophyes honmai resistant to Adoxophyes honmai nucleopolyhedrovirus (AdhoNPV) was established from a field-collected colony by repeated selection. Fifth-instar larvae of this resistant strain (R-strain) had over 66 666-fold greater resistance in terms of 50â% lethal concentration values to oral infection of AdhoNPV than non-selected strain larvae (susceptible for AdhoNPV; S2-strain). In this study, the mechanism of resistance to AdhoNPV was determined in R-strain larvae. An assessment of viral genome replication in AdhoNPV-infected S2- and R-strain larvae by quantitative PCR showed no viral genome replication occurring in R-strain larvae. Transcription of AdhoNPV ie-1, vp39 and polyhedrin genes was also not detected in R-strain midgut cells. Besides, a fluorescent brightener had no effect on AdhoNPV infection in either S2- or R-strain. However, binding and fusion of occlusion-derived virus with R-strain were significantly lower than those of S2-strain. These findings suggest that R-strain Adoxophyeshonmai larvae possess a midgut-based resistance to oral infection by AdhoNPV in which midgut epithelial cells are infected less efficiently.
Asunto(s)
Sistema Digestivo/virología , Lepidópteros/virología , Nucleopoliedrovirus/fisiología , Replicación Viral , Animales , Camellia sinensis/parasitología , Sistema Digestivo/citología , Células Epiteliales/virología , Genoma Viral , Nucleopoliedrovirus/genética , Transcripción GenéticaRESUMEN
The tea slug moth Iragoidae fasciata (Lepidoptera, Eucleidae) is one of the main insect pests that attack tea bushes. A new nucleopolyhedrovirus (NPV) called Iragoidae fasciata NPV (IrfaNPV) was recently isolated from diseased larvae. An 11,626 bp fragment of the viral genomic DNA containing the polyhedrin gene and other 12 genes was cloned and sequenced. Gene comparison and phylogenetic analysis showed that IrfaNPV is a member of the Group I NPVs. However, the genomic organization of IrfaNPV is highly distinct. In addition, electron microscopy analysis showed that IrfaNPV is a single nucleocapsid NPV (SNPV). An inoculation assay showed that IrfaNPV is semi-permissive in the Trichoplusia ni cell line Tn-5Bl-4. Bioassays on lethal concentration (LC(50)) and lethal time (LT(50)) were conducted to test the susceptibility of I. fasciata larvae to the virus.
Asunto(s)
Mariposas Nocturnas/virología , Nucleopoliedrovirus/aislamiento & purificación , Animales , Camellia sinensis/parasitología , Línea Celular , Larva/virología , Datos de Secuencia Molecular , Nucleocápside/genética , Nucleocápside/ultraestructura , Nucleopoliedrovirus/clasificación , Nucleopoliedrovirus/genética , Nucleopoliedrovirus/ultraestructura , FilogeniaRESUMEN
Garlic cultivars in Brazil are infected by a complex of viruses and for some virus species, such as the allexivirus, purification of the virions is sometimes cumbersume. To overcome this problem, recombinant expression of viral proteins in heterologous systems is an alternative method for producing antibodies. The capsid gene from Garlic virus C (GarV-C), an Allexivirus, was inserted into the genome of Autographa californica multiple nucleopolyhedrovirus (AcMNPV) generating the recombinant virus vSynGarV-C. The recombinant protein expression was confirmed by SDS-PAGE and western-blot of extracts from recombinant virus infected insect cells, where a protein band of approximately 32KDa was observed only in extracts from recombinant infected cells. This protein corresponded to the predicted size of the capsid protein of the GarV-C. A rabbit polyclonal antibody was raised against this protein, shown to be specific for the GarV-C protein in western-blot and dot-Elisa, however with a low titer.
Asunto(s)
Proteínas de la Cápside/biosíntesis , Flexiviridae/genética , Animales , Anticuerpos Antivirales/aislamiento & purificación , Western Blotting , Brasil , Proteínas de la Cápside/química , Proteínas de la Cápside/genética , Línea Celular , Electroforesis en Gel de Poliacrilamida , Ajo/virología , Insectos , Peso Molecular , Nucleopoliedrovirus/genética , Conejos , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/genéticaRESUMEN
We earlier documented the involvement of novel Sp-family-like protein factors in transcription from the Autographa californica nucleopolyhedrovirus (AcNPV) polyhedrin (polh) gene promoter [Ramachandran et al. (2001) J. Biol. Chem. 276: 23440-23449]. These zinc-dependent Sp-like factors bind to two putative Sp-factor-binding motifs, present within the AcSp sequence upstream of the polh promoter, with very high affinity (K(d) = 2.1 x 10(-12) M). Like other polh-promoter-associated host transcription factors, these Sp-like factors display tolerance to high ion concentrations up to even 3 M NaCl. An electrophoretic mobility shift assay demonstrated a probable cross-talk between the Spodoptera frugiperda (Sf9) Sp-family-like proteins and the TFIID complex. In complementary experiments, specific replacements of the Sp-factor-binding motifs with TATA-like elements resulted in expression of a luciferase reporter gene to almost the same level as that obtained with a wild-type native construct. Our results point to the possibility of the involvement of TFIID and Sf9 Sp protein interaction in transcription from the baculovirus polyhedrin promoter.
Asunto(s)
Proteínas de Insectos , Nucleopoliedrovirus/genética , Factor de Transcripción Sp1/fisiología , Spodoptera/virología , Proteínas Virales/genética , Animales , Secuencia de Bases , Sitios de Unión , Extractos Celulares/análisis , Células Cultivadas , Clonación Molecular , Reactivos de Enlaces Cruzados/metabolismo , ADN Viral/metabolismo , Relación Dosis-Respuesta a Droga , Ensayo de Cambio de Movilidad Electroforética , Genes Reporteros , Genes Virales , Vectores Genéticos , Luciferasas/metabolismo , Datos de Secuencia Molecular , Mutación , Proteínas Nucleares/metabolismo , Proteínas de la Matriz de Cuerpos de Oclusión , Plásmidos , Unión Proteica , Cloruro de Sodio/farmacología , Spodoptera/citología , Spodoptera/metabolismo , Rayos Ultravioleta , Proteínas Estructurales Virales/metabolismoRESUMEN
The complete nucleotide sequence of Ectropis obliqua nucleopolyhedrovirus (EcobNPV), which infects the tea looper caterpillar, was determined and analyzed. The double stranded circular genome is composed of 131,204 bp and is 37.6% G+C rich. The analysis predicted 126 putative, minimally overlapping open reading frames (ORFs) with 150 or more nucleotides that together compose 89.8% of the genome. The remaining 10.2% constitute non-coding and three homologous regions. Comparison with previously sequenced baculoviruses indicated that three ORFs were unique to EcobNPV, while the remaining 123 ORFs shared identity with other baculovirus genes. In addition to two bro homologues, three other repeat ORFs, including dbp, p26, and odv-e66, were identified. Phylogenetic analysis indicated that each member of the paired ORFs was acquired independently. Gene parity plot analysis and percent identity of gene homologues suggested that EcobNPV is a Group II NPV, although its genomic organization was highly distinct.
Asunto(s)
Genoma Viral/genética , Lepidópteros/virología , Nucleopoliedrovirus/genética , Animales , Composición de Base , Lepidópteros/fisiología , Nucleopoliedrovirus/clasificación , Sistemas de Lectura Abierta/genética , Filogenia , Especificidad de la Especie , Té/parasitologíaRESUMEN
Primary hypomagnesaemia is composed of a heterogeneous group of disorders characterized by renal or intestinal Mg(2+) wasting, often associated with disturbances in Ca(2+) excretion. We identified a putative dominant-negative mutation in the gene encoding the Na(+), K(+)-ATPase gamma-subunit (FXYD2), leading to defective routing of the protein in a family with dominant renal hypomagnesaemia.
Asunto(s)
Túbulos Renales Distales/metabolismo , Deficiencia de Magnesio/genética , Magnesio/metabolismo , ATPasa Intercambiadora de Sodio-Potasio/deficiencia , Empalme Alternativo , Secuencia de Aminoácidos , Animales , Células COS , Chlorocebus aethiops , Cromosomas Humanos Par 11/genética , ADN Complementario/genética , Genes Dominantes , Vectores Genéticos , Humanos , Deficiencia de Magnesio/sangre , Mamíferos/metabolismo , Ratones , Datos de Secuencia Molecular , Nucleopoliedrovirus/genética , Subunidades de Proteína , Transporte de Proteínas , Ratas , Proteínas Recombinantes de Fusión/metabolismo , Alineación de Secuencia , ATPasa Intercambiadora de Sodio-Potasio/química , ATPasa Intercambiadora de Sodio-Potasio/genética , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Especificidad de la Especie , Spodoptera/citología , Spodoptera/metabolismo , TransfecciónRESUMEN
Phosphorylation of serine 51 residue on the alpha-subunit of eukaryotic initiation factor 2 (eIF2alpha) inhibits the guanine nucleotide exchange (GNE) activity of eIF2B, presumably, by forming a tight complex with eIF2B. Inhibition of the GNE activity of eIF2B leads to impairment in eIF2 recycling and protein synthesis. We have partially purified the wild-type (wt) and mutants of eIF2alpha in which the serine 51 residue was replaced with alanine (51A mutant) or aspartic acid (51D mutant) in the baculovirus system. Analysis of these mutants has provided novel insight into the role of 51 serine in the interaction between eIF2 and eIF2B. Neither mutant was phosphorylated in vitro. Both mutants decreased eIF2alpha phosphorylation occurring in hemin and poly(IC)-treated reticulocyte lysates due to the activation of double-stranded RNA-dependent protein kinase (PKR). However, addition of 51D, but not 51A mutant eIF2alpha protein promoted inhibition of the GNE activity of eIF2B in hemin-supplemented rabbit reticulocyte lysates in which relatively little or no endogenous eIF2alpha phosphorylation occurred. The 51D mutant enhanced the inhibition in GNE activity of eIF2B that occurred in hemin and poly(IC)-treated reticulocyte lysates where PKR is active. Our results show that the increased interaction between eIF2 and eIF2B protein, occurring in reticulocyte lysates due to increased eIF2alpha phosphorylation, is decreased significantly by the addition of mutant 51A protein but not 51D. Consistent with the idea that mutant 51D protein behaves like a phosphorylated eIF2alpha, addition of this partially purified recombinant subunit, but not 51A or wt eIF2alpha, increases the interaction between eIF2 and 2B proteins in actively translating hemin-supplemented lysates. These findings support the idea that phosphorylation of the serine 51 residue in eIF2alpha promotes complex formation between eIF2alpha(P) and eIF2B and thereby inhibits the GNE activity of eIF2B.
Asunto(s)
Factor 2B Eucariótico de Iniciación/metabolismo , Factor 2 Eucariótico de Iniciación/metabolismo , Factores de Intercambio de Guanina Nucleótido/antagonistas & inhibidores , Serina/metabolismo , Alanina/genética , Animales , Ácido Aspártico/genética , Baculoviridae/genética , Sistema Libre de Células/metabolismo , Factor 2 Eucariótico de Iniciación/biosíntesis , Factor 2 Eucariótico de Iniciación/genética , Factor 2 Eucariótico de Iniciación/inmunología , Factor 2B Eucariótico de Iniciación/antagonistas & inhibidores , Factores de Intercambio de Guanina Nucleótido/metabolismo , Hemina/metabolismo , Humanos , Masculino , Nucleopoliedrovirus/genética , Nucleopoliedrovirus/metabolismo , Fosforilación , Poli I-C/metabolismo , Conejos , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Reticulocitos/metabolismo , Spodoptera/genética , Spodoptera/metabolismoRESUMEN
Spodoptera frugiperda (Sf9) cells are widely employed for high-level expression of heterologous recombinant genes from baculovirus vectors. Using a plasmid library encoding cDNA of Sf9 cells we have identified here the Spodoptera frugiperda analog of the proprotein convertase furin which plays an important role in posttranslational protein processing. Spodoptera frugiperda furin (Sfurin) is closest related to Drosophila melanogasterfurin with which it shares an extended cysteine-rich domain, whereas mammalian furin shows high homology only in the catalytic domain. Mammalian furin and Sfurin were further compared by expression from baculovirus vectors. Substrate specificity and inhibitor profiles are identical for Sfurin and mammalian furin, whereas calcium-dependence, pH-optimum, and thermostability differ. Cleavage of recombinant influenza virus hemagglutinin was significantly enhanced in Sf9 cells after overexpression of Sfurin.
Asunto(s)
Spodoptera/enzimología , Subtilisinas/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Calcio/metabolismo , Bovinos , Clonación Molecular , Cisteína/análisis , ADN Complementario , Estabilidad de Enzimas , Furina , Hemaglutininas Virales/química , Hemaglutininas Virales/genética , Concentración de Iones de Hidrógeno , Hidrólisis , Virus de la Influenza A/genética , Datos de Secuencia Molecular , Nucleopoliedrovirus/genética , Homología de Secuencia de Aminoácido , Inhibidores de Serina Proteinasa/farmacología , Subtilisinas/química , Subtilisinas/genética , TemperaturaRESUMEN
A cDNA coding for the murine proprotein convertase-1 (mPC1 also known as mPC3 or mSPC3) was inserted into the Autographa californica nuclear polyhedrosis virus. Following infection of Spodoptera frugiperda cells, the recombinant N-glycosylated protein is secreted into the cell culture medium from which it can be purified to homogeneity as a fully enzymatically active enzyme. Two major secreted molecular forms of mPC1 with apparent molecular weights of 85 and 71 kDa, respectively, and a minor one of 75 kDa are immunodetected in the medium. Automated NH2-terminal sequencing reveals that all three forms result from processing at the predicted zymogen activation site whereas both the 75- and the 71-kDa forms are truncated at their COOH-terminus. Labeling by an active-site titrant demonstrates that the 85-kDa form is optimally labeled at near neutral pH whereas the COOH-truncated forms are optimally labeled at acidic pH. Additionally it is shown that the 85-kDa mPC1 is transformed into the COOH-truncated forms following in vitro incubation at acidic pH levels and in presence of calcium. Concomitantly, the transformation from 85 to 71 kDa is accompanied by a 10- to 40-fold increase in enzymatic activity upon assaying at pH 6.0. The 71-kDa form can be recovered after purification at a level of 1 to 1.5 mg per liter of cell culture medium and is enzymatically stable only in the pH range from 5.0 to 6.5. Cells treated with tunicamycin show a drastically reduced secretion of the convertase in the medium but are not affected by swainsonine and deoxymannojirimycin. Finally, the 85-kDa secreted mPC1 is shown to be sulfated.
Asunto(s)
Ácido Aspártico Endopeptidasas/aislamiento & purificación , Proproteína Convertasa 1 , 1-Desoxinojirimicina/farmacología , Animales , Ácido Aspártico Endopeptidasas/biosíntesis , Ácido Aspártico Endopeptidasas/genética , Ácido Aspártico Endopeptidasas/metabolismo , Células CHO , Calcio/fisiología , Línea Celular , Cricetinae , Cricetulus , ADN Complementario/genética , Inhibidores Enzimáticos/farmacología , Vectores Genéticos/genética , Glicosilación/efectos de los fármacos , Glicosiltransferasas/antagonistas & inhibidores , Concentración de Iones de Hidrógeno , Ratones , Nucleopoliedrovirus/genética , Proproteína Convertasas , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/aislamiento & purificación , Proteínas Recombinantes de Fusión/metabolismo , Spodoptera/citología , Sulfatos/metabolismo , Swainsonina/farmacología , Tunicamicina/farmacología , Virus Vaccinia/genéticaRESUMEN
Mammalian dihydroorotate dehydrogenase (EC 1.3.99.11), the fourth enzyme of pyrimidine de novo synthesis is located in the mitochondrial inner membrane with functional connection to the respiratory chain. From the cDNA of rat liver dihydroorotate dehydrogenase cloned in our laboratory the first complete sequence of a mammalian enzyme was deduced. Two hydrophobic stretches centered around residues 20 and 357, respectively, and a short N-terminal mitochondrial targeting sequence of 10 amino acids was proposed. A recombinant baculovirus containing the rat liver cDNA for dihydroorotate dehydrogenase was constructed and used for virus infection and protein expression in Trichoplusia ni cells. The targeting of the recombinant protein to mitochondria of the insect cells was monitored by activity determination of dihydroorotate dehydrogenase in subcellular compartments in comparison to succinate dehydrogenase activity (EC 1.3.5.1), which is a specific marker enzyme of the inner mitochondrial membrane. The results of subcellular distribution were verified by Western blotting with anti-dihydroorotate dehydrogenase immunoglobulins. The activity of the recombinant enzyme in the mitochondria of infected insect cells was found to be about 570-fold above the level of dihydroorotate dehydrogenase in rat liver mitochondria. By cation exchange chromatography of the Triton X-114 solubilisate of mitochondria, dihydroorotate dehydrogenase was purified to give a specific activity of 15 U/mg at pH 8.0. This was a marked progress over the six-step purification procedure of the enzyme from rat liver which resulted in a specific activity of 0.7 U/mg at pH 8.0. The characteristic flavin absorption spectrum obtained with the recombinant enzyme gave strong evidence that the rodent enzyme is a flavoprotein. By enzyme kinetic studies K(m) values for dihydroorotate and ubiquinone were 6.4 and 9.9 microM with the recombinant enzyme, and were 5.0 and 19.7 microM, respectively, with the rat liver enzyme. After expression of only truncated forms of human dihydroorotate dehydrogenase, the present successful generation of the complete rodent enzyme using insect cells and the efficient procedure will promote structure and function studies of the eukaryotic dihydroorotate dehydrogenases in comparison to the microbial enzyme.
Asunto(s)
Mitocondrias/química , Mariposas Nocturnas/metabolismo , Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH , Oxidorreductasas/genética , Ratas/genética , Secuencia de Aminoácidos , Animales , Western Blotting , Línea Celular , Cromatografía por Intercambio Iónico , ADN Complementario/genética , Dihidroorotato Deshidrogenasa , Vectores Genéticos/genética , Humanos , Cinética , Hígado/química , Datos de Secuencia Molecular , Mariposas Nocturnas/citología , Nucleopoliedrovirus/genética , Ácido Orótico/análogos & derivados , Ácido Orótico/metabolismo , Oxidorreductasas/biosíntesis , Oxidorreductasas/aislamiento & purificación , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/aislamiento & purificación , Spodoptera/citología , Spodoptera/metabolismo , Especificidad por Sustrato , Ubiquinona/metabolismoRESUMEN
We have reported previously that the heavy chain of ferritin is required for iron incorporation by ceruloplasmin (J.-H. Guo, M. Abedi, and S. D. Aust (1996) Arch. Biochem. Biophys. 335(1)). The purpose of this study was to determine how many heavy chains were required for ceruloplasmin to interact with ferritin such that iron loading occurred. The cDNA sequences encoding the heavy and light chains of rat liver ferritin were cloned into the baculovirus transfer vector pA-cUW51 under the control of polyhedrin and p10 promoters, respectively, which was then incorporated by homologous recombination into the infections Autographa californica nuclear polyhedrosis virus genome. Both ferritin chains were expressed and assembled into two heteropolymers following the infection of insect cells by recombinant virus, which were separated by DEAE-Sepharose chromatography. The percentage of heavy (H) and light (L) chains making up the two heteropolymers, determined by gel scanning following the resolution of chains on SDS-PAGE, were equivalent to 1 H and 23 L chains and 2 H and 22 L chains. The maximal extent of iron loading was observed using 1 mol of rat ceruloplasmin per mole of H chain in the two heteropolymers. The extent of iron incorporation decreased with additional ceruloplasmin. Iron incorporation into rat liver ferritin, found to contain 10 H chains, increased as the molar ratio of ceruloplasmin to ferritin increased to 4:1 and remained the same up to 8:1. Iron loading into horse spleen ferritin, found to have one H chain, appeared similar to that for recombinant ferritin, having only one H chain. Therefore, we propose that the optimal molar ratio of ceruloplasmin to ferritin depends upon the numbers of H chain making up the ferritin molecule for the maximal incorporation of iron into ferritin. These results also suggest that the iron loading channel is contained within a single H chain subunit.
Asunto(s)
Ceruloplasmina/metabolismo , Ferritinas/metabolismo , Hierro/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Animales , Apoferritinas/aislamiento & purificación , Apoferritinas/metabolismo , Línea Celular , ADN Complementario/genética , Electroforesis en Gel de Poliacrilamida , Ferritinas/química , Ferritinas/genética , Ferritinas/aislamiento & purificación , Caballos , Cinética , Hígado/química , Nucleopoliedrovirus/genética , Conformación Proteica , Multimerización de Proteína , Ratas , Bazo/química , Spodoptera/citologíaRESUMEN
A number of mutants with single amino acid replacements were generated in the highly conserved ATP-binding cassette (ABC)-signature region (amino acids 531-543) of the N-terminal half of the human multidrug resistance (MDR1) protein. The cDNA variants were inserted into recombinant baculoviruses and the MDR1 proteins were expressed in Spodoptera frugiperda (Sf9) insect cells. The level of expression and membrane insertion of the MDR1 variants was examined by immunostaining, and MDR1 function was followed by measuring drug-stimulated ATPase activity. We found that two mutations, L531R and G534V, practically eliminated MDR1 expression; thus these amino acid replacements seem to inhibit the formation of a stable MDR1 protein structure. The MDR1 variants G534D and I541R were expressed at normal levels with normal membrane insertion, but showed a complete loss of drug-stimulated ATPase activity, while mutant R538M yielded full protein expression but with greatly decreased ATPase activity. Increasing the ATP concentration did not restore MDR1 ATPase activity in these variants. Some amino acid replacements in the ABC-signature region (K536I, K536R, I541T and R543S) affected neither the expression and membrane insertion nor the ATPase function of MDR1. We found no alteration in the drug-sensitivity of ATP cleavage in any of the MDR1 variants that had measurable ATPase activity. These observations suggest that the ABC-signature region is essential for MDR1 protein stability and function, but alterations in this region do not seem to modulate MDR1-drug interactions directly.
Asunto(s)
Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/genética , Transportadoras de Casetes de Unión a ATP/genética , Adenosina Trifosfato/metabolismo , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/química , Transportadoras de Casetes de Unión a ATP/química , Secuencia de Aminoácidos , Animales , Sitios de Unión , Bloqueadores de los Canales de Calcio/farmacología , Secuencia de Consenso , ADN Complementario/genética , Resistencia a Múltiples Medicamentos/genética , Fluoresceínas/farmacología , Vectores Genéticos , Humanos , Cinética , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Nucleopoliedrovirus/genética , Proteínas Recombinantes de Fusión/metabolismo , Rodamina 123 , Rodaminas/farmacología , Spodoptera/citología , Relación Estructura-Actividad , Valinomicina/farmacología , Verapamilo/farmacologíaRESUMEN
Pemphigus vulgaris (PV) is mediated by autoantibodies to desmoglein 3, the pemphigus vulgaris antigen (PVA). PVA and an extracellular domain of PVA-Ig fusion protein (PV-Ig) can completely adsorb the blister-causing Abs from PV patient sera, suggesting that the extracellular segment of PVA might be sufficient to induce pathogenic Abs. To test this, we immunized rabbits with either PVA or its extracellular domain (EPVA) expressed in insect cells in our laboratory. When Igs were passively transferred from these rabbits into neonatal mice, anti-PVA, but not the anti-EPVA, induced blisters. To understand the basis for their differential pathogenic effects, we examined the properties of these sera. Both sera showed comparable ELISA titers and indirect immunofluorescence reactivity against monkey esophagus, a source of native PVA. Moreover, EPVA, like PVA adsorbed blister-causing Abs from sera of PV patients and rabbits immunized with PVA. In contrast, when IgG preparations were incubated with fura-2-AM (acetyloxymethyl ester)-loaded human keratinocytes in culture, only IgG from anti-PVA serum induced intracellular calcium mobilization. These data showed that PVA but not EPVA can elicit Abs that induced blisters in neonatal mice and mediate intracellular signaling through calcium mobilization.
Asunto(s)
Autoanticuerpos/biosíntesis , Enfermedades Autoinmunes/inmunología , Vesícula/etiología , Cadherinas/inmunología , Epítopos/inmunología , Pénfigo/inmunología , Fragmentos de Péptidos/inmunología , Proteínas Recombinantes de Fusión/inmunología , Secuencia de Aminoácidos , Animales , Animales Recién Nacidos , Autoanticuerpos/sangre , Autoanticuerpos/inmunología , Enfermedades Autoinmunes/sangre , Vesícula/inmunología , Cadherinas/química , Cadherinas/genética , Calcio/metabolismo , Línea Celular , ADN Complementario/genética , Desmogleína 3 , Epítopos/química , Epítopos/genética , Humanos , Inmunización Pasiva , Técnicas de Inmunoadsorción , Queratinocitos/efectos de los fármacos , Queratinocitos/metabolismo , Ratones , Ratones Endogámicos BALB C , Datos de Secuencia Molecular , Mariposas Nocturnas/citología , Nucleopoliedrovirus/genética , Pénfigo/sangre , Fragmentos de Péptidos/química , Fragmentos de Péptidos/genética , Pliegue de Proteína , Estructura Terciaria de Proteína , Conejos , Proteínas Recombinantes de Fusión/químicaRESUMEN
DNA encoding the coat protein (P3) of a Scottish isolate of potato leafroll virus (PLRV) was inserted into the genome of Autographa californica nucleopolyhedrovirus (AcNPV) such that the coat protein was expressed either in an unmodified form or with the addition of the amino acid sequence MHHHHHHGDDDDKDAMG at the N terminus (P3-6H). Insect cells infected with these recombinant baculoviruses accumulated substantial amounts of P3 and P3-6H. P3 could not be recovered from cell extracts unless it was denatured in SDS but a proportion of the P3-6H was recoverable in a soluble form in non-denaturing conditions. Immunogold labelling of sections of infected cells showed that P3 accumulated in nuclei in large amorphous bodies. In contrast, although much of the P3-6H also accumulated in nuclei, it formed virus-like particles (VLP) which were often grouped in close-packed, almost cystalline arrays. When electron microscope grids coated with antibodies to PLRV were floated on cell extracts containing P3-6H, VLP were trapped which were indistinguishable from PLRV particles trapped from extracts of PLRV-infected plants. The VLP co- sedimented in sucrose gradients with PLRV particles which suggests that the VLP contained RNA. VLP collected from sucrose density gradient fractions contained protein which reacted with nickel chelated to nitrilotriacetic acid, a histidine-specific reagent. Cells infected with either recombinant baculovirus also synthesized a protein, with an Mr of about 17000, which was shown to be the translation product of the P4 gene which is in the +1 reading frame within the coat protein gene. This protein was also found in the nuclear fraction of infected cells but was more readily soluble than was P3.
Asunto(s)
Cápside/genética , Luteovirus/genética , Ensamble de Virus , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Cápside/metabolismo , Línea Celular , ADN Viral , Expresión Génica , Vectores Genéticos/genética , Histidina , Luteovirus/fisiología , Luteovirus/ultraestructura , Datos de Secuencia Molecular , Nucleopoliedrovirus/genética , Conejos , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Solanum tuberosum/virología , Spodoptera/citología , Virión/metabolismo , Virión/ultraestructuraRESUMEN
Two novel peptides were purified from the venom of the scorpion Pandinus imperator, and were named Pi2 and Pi3. Their complete primary structures were determined and their blocking effects on Shaker B K+ channels were studied. Both peptides contain 35 amino acids residues, compacted by three disulfide bridges, and reversibly block the Shaker B K+ channels. They have only one amino acid changed in their sequence, at position 7 (a proline for a glutamic acid). Whereas peptide Pi2, containing the Pro7, binds the Shaker B K+ channels with a Kd of 8.2 nm, peptide Pi3 containing the Glu7 residue has a much lower affinity of 140 nm. Both peptides are capable of displacing the binding of 125I-noxiustoxin to brain synaptosome membranes. Since these two novel peptides are about 50% identical to noxiustoxin, the present results support previous data published by our group showing that the amino-terminal region of noxiustoxin, and also the amino-terminal sequence of the newly purified homologues: Pi2, and Pi3, are important for the recognition of potassium channels.
Asunto(s)
Canales de Potasio/metabolismo , Venenos de Escorpión/aislamiento & purificación , Escorpiones/química , Secuencia de Aminoácidos , Animales , Unión Competitiva , Línea Celular , ADN Complementario/genética , Vectores Genéticos/genética , Cinética , Datos de Secuencia Molecular , Nucleopoliedrovirus/genética , Canales de Potasio/efectos de los fármacos , Ratas , Venenos de Escorpión/química , Venenos de Escorpión/metabolismo , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Canales de Potasio de la Superfamilia Shaker , Spodoptera/citología , Relación Estructura-ActividadRESUMEN
NKG2-C is a member of the recently discovered NKG2 family of genes and proteins, which are preferentially expressed on human natural killer (NK) cells. These potential NK cell receptors belong to a larger class of type II transmembrane proteins with a C-type lectin domain. We show here that NKG2-C is expressed as a 36-kDa glycoprotein by translation in vitro, recombinant expression and immunoprecipitation from a human NK cell clone. Further, a recombinant soluble NKG2-C-receptor binds specifically to K562 cells, which are target cells for NK cell killing, and to RPMI 8866 cells, which are feeder cells for NK cells; several other hematopoietic cell lines tested do not show any binding. The binding structures on the surface of K562 cells disappear, concomitant with a loss in susceptibility to killing when the cells are induced to differentiate with phorbol ester and Ca2+ ionophore. Our data suggest the presence of specific target molecules for NKG2-C on K562 cells, since overall glycosylation, Lewis X and Lewis Y structures, as well as the mucin-like CD43 molecule, do not change following induction of the cells. We propose that NKG2-C mediates a specific interaction of NK cells and their target cells with functional importance for NK cell killing.
Asunto(s)
Células Asesinas Naturales/inmunología , Leucemia Mielógena Crónica BCR-ABL Positiva/patología , Glicoproteínas de Membrana/fisiología , Proteínas de la Membrana/metabolismo , Proteínas de Neoplasias/metabolismo , Receptores Inmunológicos/fisiología , Animales , Calcio/metabolismo , Diferenciación Celular/efectos de los fármacos , Línea Celular , Citotoxicidad Inmunológica , ADN Complementario/genética , Perros , Glicosilación , Humanos , Ionomicina/farmacología , Células Asesinas Naturales/metabolismo , Leucemia Mielógena Crónica BCR-ABL Positiva/metabolismo , Glicoproteínas de Membrana/genética , Subfamília C de Receptores Similares a Lectina de Células NK , Nucleopoliedrovirus/genética , Receptores Inmunológicos/genética , Receptores de Células Asesinas Naturales , Proteínas Recombinantes de Fusión/metabolismo , Spodoptera/citología , Acetato de Tetradecanoilforbol/farmacología , Células Tumorales Cultivadas/efectos de los fármacos , Células Tumorales Cultivadas/metabolismoRESUMEN
A full-length cDNA from the parasitic nematode Brugia pahangi encoding a secreted homolog of glutathione peroxidase in which the codon for the active site selenocysteine is substituted naturally by a cysteine codon has been expressed in Spodoptera frugiperda (insect) cells via Autographa californica nuclear polyhedrosis virus (baculovirus). The recombinant protein was glycosylated and secreted from the cells in tetrameric form. The purified protein showed glutathione peroxidase activity with a range of organic hydroperoxides, including L-alpha-phosphatidylcholine hydroperoxide, but no significant activity against hydrogen peroxide. Glutathione was the only thiol tested that served as a substrate for the enzyme, which showed no activity with the thioredoxin system (thioredoxin, thioredoxin reductase, and NADPH). No glutathione-conjugating activity was detected against a range of electrophilic compounds that are common substrates for glutathione S-transferases. The apparent (pseudo)m for glutathione was determined as 4.9 mM at a fixed concentration of linolenic acid hydroperoxide (3 microM). The enzyme showed low affinity for hydroperoxide substrates (apparent Km for linolenic acid hydroperoxide and L-alpha-phosphatidylcholine hydroperoxide of 3.8 and 9.7 mM, respectively at a fixed glutathione concentration of 3 mM).
Asunto(s)
Brugia pahangi/enzimología , Glutatión Peroxidasa/metabolismo , Proteínas del Helminto/metabolismo , Selenio/metabolismo , Animales , Secuencia de Bases , Brugia pahangi/genética , Células Cultivadas , Glutatión Peroxidasa/genética , Glutatión Peroxidasa/aislamiento & purificación , Glicosilación , Proteínas del Helminto/genética , Proteínas del Helminto/aislamiento & purificación , Concentración de Iones de Hidrógeno , Ácidos Linolénicos/metabolismo , Peróxidos Lipídicos/metabolismo , Datos de Secuencia Molecular , Nucleopoliedrovirus/genética , Fosfatidilcolinas/metabolismo , Procesamiento Proteico-Postraduccional , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Spodoptera/citología , Especificidad por SustratoRESUMEN
Difficulties in purifying the plasma enzyme lecithin-cholesterol acyltransferase (LCAT) have hampered detailed studies of its (patho)physiological role in lipoprotein metabolism and of structure-function relationships. Potentially, baculovirus-driven expression systems offer a powerful means to produce significant amounts of LCAT. Accordingly, full-length LCAT cDNA was cloned into pVL 1392, a high-level expression derivative of Autographa californica nuclear polyhedrosis virus (AcNPV), and the resultant plasmid was co-transfected into Trichoplusia ni insect cells (High 5 line) with a linearized viral DNA using lipofectin. Such viral DNA had a lethal mutation and grew only when recombined with a pVL1392-type rescue plasmid; cells infected with recombinant Autographa californica LCAT virus changed from a fibroblast-like morphology to rounded, but lacked the polyhedrin occlusion bodies characteristic of wild-type AcNPV infections. Enzymically active recombinant LCAT (rLCAT), sensitive to sulphydryl reagents, was secreted in the late phase of infection (36-48 h) but was absent with wild-type infections. The secreted protein had an apparent molecular mass of 53 kDa by SDS/PAGE, lower than that of native plasma LCAT; it was susceptible to endoglycosidase H digestion and was bound by concanavalin A, suggesting that precursor high-mannose N-glycan chains had not undergone full maturation to complex types. Pretreatment of the cells with tunicamycin to inhibit the first step of N-glycosylation led to intracellular accumulation of immature rLCAT (approximately 46-48 kDa) and a marked reduction in enzyme secreted. We conclude that the baculovirus gene-expression system will permit production of biologically active normal and mutant forms of LCAT protein.
Asunto(s)
Nucleopoliedrovirus/genética , Fosfatidilcolina-Esterol O-Aciltransferasa/metabolismo , Animales , Línea Celular , Clonación Molecular , ADN Complementario , Humanos , Mariposas Nocturnas , Fosfatidilcolina-Esterol O-Aciltransferasa/genética , Pruebas de PrecipitinaRESUMEN
Two overlapping cDNA clones encoding pigeon liver carnitine acetyltransferase (EC 2.3.1.7) (CAT) were isolated from a pigeon liver lambda gt11 cDNA library by gene amplification using oligonucleotide primers based on the N-terminal amino acid sequence of the enzyme. The two clones, which represent the 5' and 3' ends of the gene, were spliced together to form a single cDNA construct containing the entire coding sequence for CAT, with an in-frame TGA stop codon 42 bases before the first ATG start site and a 3'-untranslated segment of 1057 bases. The largest open reading frame of 1942 nucleotides predicted a polypeptide of 627 amino acids and a molecular mass of 71.1 kDa. The N-terminus and four internal peptides from the amino acid sequence of pigeon breast muscle CAT were identified in the predicted sequence of the liver cDNA clone. The identity of the CAT cDNA was confirmed by heterologous expression of active recombinant CAT (rCAT) in insect cells using the baculovirus expression system. Western blots of rCAT from infected insect cell lysates and immunodetection with a rabbit anti-CAT polyclonal serum showed an immunoreactive protein band similar in size to native CAT from pigeon breast muscle. Like the native enzyme, rCAT was capable of acylating carnitine with a preference for small-chain acyl-CoAs of carbon chain lengths C2-C4.