RESUMO
The adult mammalian cardiomyocyte has a very limited capacity to reenter the cell cycle and advance into mitosis. Therefore, diseases characterized by lost contractile tissue usually evolve into myocardial remodeling and heart failure. Analyzing the cardiac transcriptome at different developmental stages in a large mammal closer to the human than laboratory rodents may serve to disclose positive and negative cardiomyocyte cell cycle regulators potentially targetable to induce cardiac regeneration in the clinical setting. Thus we aimed at characterizing the transcriptomic profiles of the early fetal, late fetal, and adult sheep heart by employing RNA-seq technique and bioinformatic analysis to detect protein-encoding genes that in some of the stages were turned off, turned on, or differentially expressed. Genes earlier proposed as positive cell cycle regulators such as cyclin A, cdk2, meis2, meis3, and PCNA showed higher expression in fetal hearts and lower in AH, as expected. In contrast, genes previously proposed as cell cycle inhibitors, such as meis1, p16, and sav1, tended to be higher in fetal than in adult hearts, suggesting that these genes are involved in cell processes other than cell cycle regulation. Additionally, we described Gene Ontology (GO) enrichment of different sets of genes. GO analysis revealed that differentially expressed gene sets were mainly associated with metabolic and cellular processes. The cell cycle-related genes fam64a, cdc20, and cdk1, and the metabolism-related genes pitx and adipoq showed strong differential expression between fetal and adult hearts, thus being potent candidates to be targeted in human cardiac regeneration strategies.NEW & NOTEWORTHY We characterized the transcriptomic profiles of the fetal and adult sheep hearts employing RNAseq technique and bioinformatic analyses to provide sets of transcripts whose variation in expression level may link them to a specific role in cell cycle regulation. It is important to remark that this study was performed in a large mammal closer to humans than laboratory rodents. In consequence, the results can be used for further translational studies in cardiac regeneration.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Coração/fisiologia , Miocárdio/metabolismo , Regeneração , Transcriptoma , Animais , Ciclina A/genética , Ciclina A/metabolismo , Feminino , Coração/crescimento & desenvolvimento , Masculino , Ovinos , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
The influence of the high genetic variability of hepatitis B virus (HBV) on the sensitivity of serological assays has received little attention so far. A major source of variability is related to viral genotypes and subgenotypes. Their possible influence on diagnosis and prophylaxis is poorly known and has mostly been evaluated for genotypes A, B, C and D. Robust data showing the detection efficiency of HBsAg from genotype F is lacking. This study examined the effect of virus-like particles containing HBsAg from genotypes A and F (particularly, F1b and F4) produced in Pichia pastoris in relation to the anti-HBs antibodies used in the immunoassays for in vitro diagnosis and compared it with that exerted by the G145R S-escape mutant. The results showed that HBsAg detection rates for subgenotypes F1b and F4 differed significantly from those obtained for genotype A and that subgenotype F1b had a major impact on the sensitivity of the immunoassays tested. Prediction of the tertiary structure of subgenotypes F1b and F4 revealed changes inside and outside the major hydrophilic region (aa 101-160) of the HBsAg compared to genotype A and the G145R variant. A phosphorylation site (target for protein kinase C) produced by the G145R substitution might prevent recognition by anti-HBs antibodies. In conclusion, the use of different genotypes or variants for diagnosis could improve the rate of detection of HBV infection. The incorporation of a genotype-F-derived HBsAg vaccine in areas where this genotype is endemic should be evaluated, since this might also affect vaccination efficacy.
Assuntos
Antígenos de Superfície da Hepatite B/genética , Vírus da Hepatite B/isolamento & purificação , Hepatite B/virologia , Sequência de Aminoácidos , Hepatite B/diagnóstico , Antígenos de Superfície da Hepatite B/química , Antígenos de Superfície da Hepatite B/metabolismo , Vírus da Hepatite B/química , Vírus da Hepatite B/classificação , Vírus da Hepatite B/genética , Humanos , Dados de Sequência Molecular , Mutação , Filogenia , Conformação Proteica , Alinhamento de SequênciaRESUMO
Exoinulinases-enzymes extensively studied in recent decades because of their industrial applications-need to be produced in suitable quantities in order to meet production demands. We describe here the production of an acid-stable recombinant inulinase from Aspergillus kawachii in the Pichia pastoris system and the recombinant enzyme's biochemical characteristics and potential application to industrial processes. After an appropriate cloning strategy, this genetically engineered inulinase was successfully overproduced in fed-batch fermentations, reaching up to 840 U/ml after a 72-h cultivation. The protein, purified to homogeneity by chromatographic techniques, was obtained at a 42% yield. The following biochemical characteristics were determined: the enzyme had an optimal pH of 3, was stable for at least 3â¯hâ¯at 55⯰C, and was inhibited in catalytic activity almost completely by Hg+2. The respective Km and Vmax for the recombinant inulinase with inulin as substrate were 1.35â¯mM and 2673⯵mol/min/mg. The recombinant enzyme is an exoinulinase but also possesses synthetic activity (i. e., fructosyl transferase). The high level of production of this recombinant plus its relevant biochemical properties would argue that the process presented here is a possible recourse for industrial applications in carbohydrate processing.
Assuntos
Aspergillus/enzimologia , Proteínas Fúngicas/metabolismo , Glicosídeo Hidrolases/metabolismo , Proteínas Recombinantes/metabolismo , Aspergillus/genética , Estabilidade Enzimática , Fermentação , Proteínas Fúngicas/genética , Proteínas Fúngicas/isolamento & purificação , Glicosídeo Hidrolases/genética , Glicosídeo Hidrolases/isolamento & purificação , Concentração de Íons de Hidrogênio , Microbiologia Industrial/métodos , Inulina/metabolismo , Cinética , Pichia/genética , Especificidade por SubstratoRESUMO
The mosquito iridescent viruses (MIVs) are large icosahedral DNA viruses that replicate and assemble in the cytoplasm of the host. Paracrystalline arrangements of virions that accumulate in the cytoplasm produce an iridescent color that is symptomatic of acute infections. In August 2010, we found larvae of Culex pipiens with these symptoms in suburban ditches around the city of La Plata, Argentina. Electron microscope studies, DNA sequencing, and phylogenetic analysis of the major capsid protein confirmed this as the first record of an MIV in C. Pipiens.
Assuntos
Culex/virologia , Iridovirus , Animais , Sequência de Bases , Iridovirus/classificação , Iridovirus/genética , Iridovirus/isolamento & purificação , Larva/virologia , Filogenia , Alinhamento de Sequência , Análise de Sequência de DNARESUMO
BACKGROUND: Baculoviruses are insect pathogens with important biotechnological applications that transcend their use as biological controllers of agricultural pests. One species, Autographa californica multiple nucleopolhyedrovirus (AcMNPV), has been extensively exploited as a molecular platform to produce recombinant proteins and as a delivery vector for genes in mammals because it can transduce a wide range of mammalian cells and tissues without replicating or producing progeny. METHOD: To investigate if the budded virions of Anticarsia gemmatalis multiple nucleopolhyedrovirus (AgMNPV) species has the same ability, the viral genome was modified by homologous recombination into susceptible insect cells to integrate reporter genes and then it was evaluated on mammalian cell lines in a comparative form with respect to equivalent viruses derived from AcMNPV. Besides, the replicative capacity of AgMNPV´s virions in mammals was determined. RESULTS: The experiments carried out showed that the recombinant variant of AgMNPV transduces and support the expression of delivered genes but not replicates in mammalian cells. CONCLUSION: Consequently, this insect pathogen is proposed as an alternative to non-infectious viruses in humans to explore new approaches in gene therapy and other applications based on the use of mammalian cells.
Assuntos
Terapia Genética , Vetores Genéticos/genética , Nucleopoliedrovírus/genética , Proteínas Recombinantes/genética , Baculoviridae/genética , Técnicas de Transferência de Genes , Genoma Viral/genética , Recombinação Homóloga/genética , Humanos , Vírion/genéticaRESUMO
BACKGROUND: Anticarsia gemmatalis is a pest in South America's soybean crops, which could be controlled by the Multinucleopolyhedrovirus of A. gemmatalis (AgMNPV). Currently, its commercial production is based on infected larvae. However, the possibility of using modified baculoviruses in Integrated Pest Management programs has stimulated an interest to develop alternative multiplication processes. This study evaluated the AgMNPV production in UFL-Ag-286 cells previously deprived Fetal Bovine Serum. RESULTS: Culture media containing 1% FBS during the previous 48 hours achieved a synchronized condition where 90% of cells were found in G0/G1 stage, showing the presence of non-filamentous actin. All characteristics were estimated from cellular viability tests, cell actin detection trials and flow cytometer cell cycle analysis. AgMNPV production was tested by transcript studies and budded viruses (BVs) and occlusion bodies (OBs) yield quantitation. Results showed that the productivity in FBS deprived cells was 9.8 times more in BVs and 3.8 times more in OBs with respect to non-treated cells. CONCLUSIONS: UFL-Ag-286 cells previously deprived in FBS shown to be a better host for AgMNPV propagation, increasing the useful for both in vitro bioinsecticide production and applications such as recombinant protein expression or gene delivery.
Assuntos
Meios de Cultura/química , Nucleopoliedrovírus/fisiologia , Cultura de Vírus/métodos , Animais , Bovinos , Linhagem Celular , Meios de Cultura Livres de Soro , Mariposas/virologia , Soro/químicaRESUMO
Hemolytic uremic syndrome (HUS), principally caused by shiga toxins (Stxs), is associated with Shiga toxin-producing Escherichia coli (STEC) infections. We previously reported Stx2 expression by host cells in vitro and in vivo. As the genes encoding the two Stx subunits are located in bacteriophage genomes, the aim of the current study was to evaluate the role of bacteriophage induction in HUS development in absence of an E. coli O157:H7 genomic background. Mice were inoculated with a non-pathogenic E. coli strain carrying the lysogenic bacteriophage 933W (C600Φ933W), and bacteriophage excision was induced by an antibiotic. The mice died 72 h after inoculation, having developed pathogenic damage typical of STEC infection. As well as renal and intestinal damage, markers of central nervous system (CNS) injury were observed, including aberrant immunolocalization of neuronal nuclei (NeuN) and increased expression of glial fibrillary acidic protein (GFAP). These results show that bacteriophage 933W without an E. coli O157:H7 background is capable of inducing the pathogenic damage associated with STEC infection. In addition, a novel mouse model was developed to evaluate therapeutic approaches focused on the bacteriophage as a new target.
RESUMO
Shiga toxin (Stx) is the principal virulence factor during Shiga toxin-producing Escherichia coli (STEC) infections. We have previously reported the inactivation of bacteriophage encoding Stx after treatment with chitosan, a linear polysaccharide polymer with cationic properties. Cationic antimicrobial peptides (cAMPs) are short linear aminoacidic sequences, with a positive net charge, which display bactericidal or bacteriostatic activity against a wide range of bacterial species. They are promising novel antibiotics since they have shown bactericidal effects against multiresistant bacteria. To evaluate whether cationic properties are responsible for bacteriophage inactivation, we tested seven cationic peptides with proven antimicrobial activity as anti-bacteriophage agents, and one random sequence cationic peptide with no antimicrobial activity as a control. We observed bacteriophage inactivation after incubation with five cAMPs, but no inactivating activity was observed with the random sequence cationic peptide or with the non-alpha helical cAMP Omiganan. Finally, to confirm peptide-bacteriophage interaction, zeta potential was analyzed by following changes on bacteriophage surface charges after peptide incubation. According to our results we could propose that: (1) direct interaction of peptides with phage is a necessary step for bacteriophage inactivation, (2) cationic properties are necessary but not sufficient for bacteriophage inactivation, and (3) inactivation by cationic peptides could be sequence (or structure) specific. Overall our data suggest that these peptides could be considered a new family of molecules potentially useful to decrease bacteriophage replication and Stx expression.
RESUMO
Spodoptera frugiperda (Lepidoptera: Noctuidae) is a major pest in maize crops in Colombia, and affects several regions in America. A granulovirus isolated from S. frugiperda (SfGV VG008) has potential as an enhancer of insecticidal activity of previously described nucleopolyhedrovirus from the same insect species (SfMNPV). The SfGV VG008 genome was sequenced and analyzed showing circular double stranded DNA of 140,913 bp encoding 146 putative ORFs that include 37 Baculoviridae core genes, 88 shared with betabaculoviruses, two shared only with betabaculoviruses from Noctuide insects, two shared with alphabaculoviruses, three copies of own genes (paralogs) and the other 14 corresponding to unique genes without representation in the other baculovirus species. Particularly, the genome encodes for important virulence factors such as 4 chitinases and 2 enhancins. The sequence analysis revealed the existence of eight homologous regions (hrs) and also suggests processes of gene acquisition by horizontal transfer including the SfGV VG008 ORFs 046/047 (paralogs), 059, 089 and 099. The bioinformatics evidence indicates that the genome donors of mentioned genes could be alpha- and/or betabaculovirus species. The previous reported ability of SfGV VG008 to naturally co-infect the same host with other virus show a possible mechanism to capture genes and thus improve its fitness.
Assuntos
Baculoviridae/genética , DNA Viral/química , DNA Viral/genética , Genoma Viral , Spodoptera/virologia , Animais , Baculoviridae/isolamento & purificação , Colômbia , DNA/química , DNA/genética , DNA Circular/química , DNA Circular/genética , Transferência Genética Horizontal , Anotação de Sequência Molecular , Dados de Sequência Molecular , Fases de Leitura Aberta , Recombinação Genética , Análise de Sequência de DNA , Homologia de Sequência , Fatores de Virulência/genéticaRESUMO
The aim of the current study was to search for the presence of genetic variants in the CYP21A2 Z promoter regulatory region in patients with congenital adrenal hyperplasia due to 21-hydroxylase deficiency. Screening of the 10 most frequent pseudogene-derived mutations was followed by direct sequencing of the entire coding sequence, the proximal promoter, and a distal regulatory region in DNA samples from patients with at least one non-determined allele. We report three non-classical patients that presented a novel genetic variant-g.15626A>G-within the Z promoter regulatory region. In all the patients, the novel variant was found in cis with the mild, less frequent, p.P482S mutation located in the exon 10 of the CYP21A2 gene. The putative pathogenic implication of the novel variant was assessed by in silico analyses and in vitro assays. Topological analyses showed differences in the curvature and bendability of the DNA region bearing the novel variant. By performing functional studies, a significantly decreased activity of a reporter gene placed downstream from the regulatory region was found by the G transition. Our results may suggest that the activity of an allele bearing the p.P482S mutation may be influenced by the misregulated CYP21A2 transcriptional activity exerted by the Z promoter A>G variation.
Assuntos
Hiperplasia Suprarrenal Congênita/genética , Alelos , Regiões Promotoras Genéticas , Esteroide 21-Hidroxilase/genética , Feminino , Regulação da Expressão Gênica , Humanos , Masculino , MutaçãoRESUMO
Here, we review current knowledge about pseudouridine synthase and archaeosine transglycosylase (PUA)-domain-containing proteins to illustrate progress in this field. A methodological analysis of the literature about the topic was carried out, together with a 'qualitative comparative analysis' to give a more comprehensive review. Bioinformatics methods for whole-protein or protein-domain identification are commonly based on pairwise protein sequence comparisons; we added comparison of structures to detect the whole universe of proteins containing the PUA domain. We present an update of proteins having this domain, focusing on the specific proteins present in Homo sapiens (dyskerin, MCT1, Nip7, eIF2D and Nsun6), and explore the existence of these in other species. We also analyze the phylogenetic distribution of the PUA domain in different species and proteins. Finally, we performed a structural comparison of the PUA domain through data mining of structural databases, determining a conserved structural motif, despite the differences in the sequence, even among eukaryotes, archaea and bacteria. All data discussed in this review, both bibliographic and analytical, corroborate the functional importance of the PUA domain in RNA-binding proteins.
Assuntos
Pentosiltransferases/metabolismo , Poli U/química , Proteínas de Ligação a RNA/metabolismo , RNA/metabolismo , tRNA Metiltransferases/metabolismo , Sequência de Aminoácidos , Humanos , Dados de Sequência Molecular , Filogenia , Proteínas de Ligação a RNA/química , Homologia de Sequência de Aminoácidos , tRNA Metiltransferases/químicaRESUMO
Shiga toxin (Stx) is considered the main virulence factor in Shiga toxin-producing Escherichia coli (STEC) infections. Previously we reported the expression of biologically active Stx by eukaryotic cells in vitro and in vivo following transfection with plasmids encoding Stx under control of the native bacterial promoter (1,2). Since stx genes are present in the genome of lysogenic bacteriophages, here we evaluated the relevance of bacteriophages during STEC infection. We used the non-pathogenic E. coli C600 strain carrying a lysogenic 933W mutant bacteriophage in which the stx operon was replaced by a gene encoding the green fluorescent protein (GFP). Tracking GFP expression using an In Vivo Imaging System (IVIS), we detected fluorescence in liver, kidney, and intestine of mice infected with the recombinant E. coli strain after treatment with ciprofloxacin, which induces the lytic replication and release of bacteriophages. In addition, we showed that chitosan, a linear polysaccharide composed of d-glucosamine residues and with a number of commercial and biomedical uses, had strong anti-bacteriophage effects, as demonstrated at in vitro and in vivo conditions. These findings bring promising perspectives for the prevention and treatment of haemolytic uremic syndrome (HUS) cases.
RESUMO
Regulation of eukaryotic cell cycle progression requires sequential activation and inactivation of cyclin-dependent kinases (CDKs). Activation of the cyclin B-cdc2 kinase complex is a pivotal step in mitotic initiation and the tyrosine kinase Wee1 is a key regulator of cell cycle sequence during G2/M transition and inhibits mitotic entry by phosphorylating the inhibitory tyrosine 15 on the cdc2 M-phase-inducing kinase. Wee1 degradation is essential for the exit from the G2 phase. In trypanosomatids, little is known about the genes that regulate cyclin B-cdc2 complexes at the G2/M transition of their cell cycle. Although canonical tyrosine kinases are absent in the genome of trypanosomatids, phosphorylation on protein tyrosine residues has been reported in Trypanosoma brucei. Here, we characterized a Wee1-like protein kinase gene from T. brucei. Expression of TbWee1 in a Schizosaccharomyces pombe strain null for Wee1 inhibited cell division and caused cell elongation. This demonstrates the lengthening of G2, which provided cells with extra time to grow before dividing. The Wee1-like protein kinase was expressed in the procyclic and bloodstream proliferative slender forms of T. brucei and the role of Wee1 in cell cycle progression was analyzed by generating RNA interference cell lines. In the procyclic form of T. brucei, the knock-down of TbWee1 expression by RNAi led to inhibition of parasite growth. Abnormal phenotypes showing an increase in the percentage of cells with 1N0K, 0N1K and 2N1K were observed in these RNAi cell lines. Using parasites with a synchronized cell cycle, we demonstrated that TbWee1 is linked to the G2/M phase. We also showed that TbWee1 is an essential gene necessary for proper cell cycle progression and parasite growth in T. brucei. Our results provide evidence for the existence of a functional Wee1 in T. brucei with a potential role in cell division at G2/M.
Assuntos
Proteínas Tirosina Quinases/genética , Trypanosoma brucei brucei/citologia , Trypanosoma brucei brucei/genética , Sequência de Aminoácidos , Divisão Celular , Bases de Dados Genéticas , Regulação para Baixo , Fase G2 , Genoma de Protozoário/genética , Dados de Sequência Molecular , Fenótipo , Fosforilação , Proteínas Tirosina Quinases/química , Proteínas Tirosina Quinases/metabolismo , Schizosaccharomyces/genética , Homologia de Sequência do Ácido Nucleico , Análise de Sobrevida , Trypanosoma brucei brucei/enzimologia , Trypanosoma brucei brucei/crescimento & desenvolvimentoRESUMO
UNLABELLED: Shiga toxins (Stx) are the main agent responsible for the development of hemolytic-uremic syndrome (HUS), the most severe and life-threatening systemic complication of infection with enterohemorrhagic Escherichia coli (EHEC) strains. We previously described Stx2 expression by eukaryotic cells after they were transfected in vitro with the stx2 gene cloned into a prokaryotic plasmid (pStx2). The aim of this study was to evaluate whether mammalian cells were also able to express Stx2 in vivo after pStx2 injection. Mice were inoculated by hydrodynamics-based transfection (HBT) with pStx2. We studied the survival, percentage of polymorphonuclear leukocytes in plasma, plasma urea levels, and histology of the kidneys and the brains of mice. Mice displayed a lethal dose-related response to pStx2. Stx2 mRNA was recovered from the liver, and Stx2 cytotoxic activity was observed in plasma of mice injected with pStx2. Stx2 was detected by immunofluorescence in the brains of mice inoculated with pStx2, and markers of central nervous system (CNS) damage were observed, including increased expression of glial fibrillary acidic protein (GFAP) and fragmentation of NeuN in neurons. Moreover, anti-Stx2B-immunized mice were protected against pStx2 inoculation. Our results show that Stx2 is expressed in vivo from the wild stx2 gene, reproducing pathogenic damage induced by purified Stx2 or secondary to EHEC infection. IMPORTANCE: Enterohemorrhagic Shiga toxin (Stx)-producing Escherichia coli (EHEC) infections are a serious public health problem, and Stx is the main pathogenic agent associated with typical hemolytic-uremic syndrome (HUS). In contrast to the detailed information describing the molecular basis for EHEC adherence to epithelial cells, very little is known about how Stx is released from bacteria in the gut, reaching its target tissues, mainly the kidney and central nervous system (CNS). In order to develop an efficient treatment for EHEC infections, it is necessary to understand the mechanisms involved in Stx expression. In this regard, the present study demonstrates that mammals can synthesize biologically active Stx using the natural promoter associated with the Stx-converting bacteriophage genome. These results could impact the comprehension of EHEC HUS, since local eukaryotic cells transduced and/or infected by bacteriophage encoding Stx2 could be an alternative source of Stx production.
Assuntos
Escherichia coli Êntero-Hemorrágica/metabolismo , Infecções por Escherichia coli/microbiologia , Regiões Promotoras Genéticas , Toxina Shiga II/biossíntese , Toxina Shiga II/genética , Animais , Encéfalo/metabolismo , Encéfalo/microbiologia , Encéfalo/patologia , Escherichia coli Êntero-Hemorrágica/genética , Infecções por Escherichia coli/patologia , Feminino , Humanos , Rim/metabolismo , Rim/microbiologia , Rim/patologia , Fígado/metabolismo , Fígado/microbiologia , Fígado/patologia , Masculino , Camundongos , Camundongos Endogâmicos BALB CRESUMO
Shiga toxins (Stx) are the main virulence factors in enterohemorrhagic Escherichia coli (EHEC) infections, causing diarrhea and hemolytic uremic syndrome (HUS). The genes encoding for Shiga toxin-2 (Stx2) are located in a bacteriophage. The toxin is formed by a single A subunit and five B subunits, each of which has its own promoter sequence. We have previously reported the expression of the B subunit within the eukaryotic environment, probably driven by their own promoter. The aim of this work was to evaluate the ability of the eukaryotic machinery to recognize stx2 sequences as eukaryotic-like promoters. Vero cells were transfected with a plasmid encoding Stx2 under its own promoter. The cytotoxic effect on these cells was similar to that observed upon incubation with purified Stx2. In addition, we showed that Stx2 expression in Stx2-insensitive BHK eukaryotic cells induced drastic morphological and cytoskeletal changes. In order to directly evaluate the capacity of the wild promoter sequences of the A and B subunits to drive protein expression in mammalian cells, GFP was cloned under eukaryotic-like putative promoter sequences. GFP expression was observed in 293T cells transfected with these constructions. These results show a novel and alternative way to synthesize Stx2 that could contribute to the global understanding of EHEC infections with immediate impact on the development of treatments or vaccines against HUS.
Assuntos
Regiões Promotoras Genéticas , Toxina Shiga/genética , Animais , Sequência de Bases , Diferenciação Celular , Linhagem Celular , Chlorocebus aethiops , Cricetinae , Homologia de Sequência do Ácido Nucleico , Células VeroRESUMO
Production of verocytotoxin or Shiga-like toxin (Stx), particularly Stx2, is the basis of hemolytic uremic syndrome, a frequently lethal outcome for subjects infected with Stx2-producing enterohemorrhagic Escherichia coli (EHEC) strains. The toxin is formed by a single A subunit, which promotes protein synthesis inhibition in eukaryotic cells, and five B subunits, which bind to globotriaosylceramide at the surface of host cells. Host enzymes cleave the A subunit into the A(1) peptide, endowed with N-glycosidase activity to the 28S rRNA, and the A(2) peptide, which confers stability to the B pentamer. We report the construction of a DNA vaccine (pStx2DeltaAB) that expresses a nontoxic Stx2 mutated form consisting of the last 32 amino acids of the A(2) sequence and the complete B subunit as two nonfused polypeptides. Immunization trials carried out with the DNA vaccine in BALB/c mice, alone or in combination with another DNA vaccine encoding granulocyte-macrophage colony-stimulating factor, resulted in systemic Stx-specific antibody responses targeting both A and B subunits of the native Stx2. Moreover, anti-Stx2 antibodies raised in mice immunized with pStx2DeltaAB showed toxin neutralization activity in vitro and, more importantly, conferred partial protection to Stx2 challenge in vivo. The present vector represents the second DNA vaccine so far reported to induce protective immunity to Stx2 and may contribute, either alone or in combination with other procedures, to the development of prophylactic or therapeutic interventions aiming to ameliorate EHEC infection-associated sequelae.
Assuntos
Escherichia coli Êntero-Hemorrágica/imunologia , Intoxicação/prevenção & controle , Toxinas Shiga/imunologia , Vacinas de DNA/imunologia , Sequência de Aminoácidos , Animais , Anticorpos Antibacterianos/sangue , Antitoxinas/sangue , Sequência de Bases , Chlorocebus aethiops , Escherichia coli Êntero-Hemorrágica/genética , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Testes de Neutralização , Subunidades Proteicas/genética , Subunidades Proteicas/imunologia , Toxinas Shiga/genética , Análise de Sobrevida , Vacinas de DNA/genética , Células VeroRESUMO
The ecdysteroid UDP-glycosyltransferase (egt) gene of Epinotia aporema granulovirus (EpapGV) was cloned sequenced and its biological activity was assessed. It encodes a protein of 446 amino acids. Direct evidence that the cloned gene encodes an active EGT protein was obtained by transient expression assays in insect cells. The upstream untranslated region of the egt gene exhibits several consensus early promoter elements. Accordingly, the gene is expressed early upon infection of Epinotia aporema larvae and the EGT activity remains high until later times post infection. Sequence analyses indicate the presence of clusters of amino acid residues conserved among all the baculoviral EGTs, although their relation with proper protein folding, ligand binding and catalytic activity remain to be assessed. Phylogenetic trees consistently cluster the granulovirus EGTs separating them clearly from the nucleopolyhedroviruses.
Assuntos
Glucosiltransferases , Granulovirus/enzimologia , Granulovirus/genética , Mariposas/virologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Células Cultivadas , Clonagem Molecular , DNA Viral/análise , Evolução Molecular , Biblioteca Gênica , Glucosiltransferases/química , Glucosiltransferases/genética , Glucosiltransferases/metabolismo , Dados de Sequência Molecular , Filogenia , Análise de Sequência de DNARESUMO
Los genes clonados de las proteínas de nucleocápside, N, de los arenavirus Junín y LCM (choriomeningitis linfocitaria) se insertaron en el vector de expresión pKG4 regulado por el promotor tardío del virus SV40. Cuando estas construcciones se utilizaron para transfectar las líneas celulares BHK-21 (fibroblastos de hamster lactante) y CV-1 (fibroblastos de riñón de mono verde africano) se observó la expresión transiente de un polipéptido de tamaño e inmunoreactividad indistinguible de la proteína N sintetizada durante una infección viral. El análisis por inmunofluorescencia reveló un patrón de distribución intracelular semejante al observado en células infectadas. Este patrón presentó variaciones desde una tinción citoplásmica difusa hasta gránulos citoplásmicos dispersos o concentrados en la zona perinuclear. La asociación de la proteína N con gránulos basófilos es semejante a la descripta en el efecto citópático causado por los arenavirus en las células infectadas, y podría relacionarse con las características fisicoquímicas de la proteina N, que contiene numerosas secuencias de aminoácidos básicos capaces de interactuar con ácidos ribonucleicos celulares