RESUMO
BACKGROUND: Treatment of poor prognosis metastatic castration-resistant prostate cancer (mCRPC) includes taxane chemotherapy and androgen receptor pathway inhibitors (ARPI). We sought to determine optimal treatment in this setting. PATIENTS AND METHODS: This multicentre, randomised, open-label, phase II trial recruited patients with ARPI-naive mCRPC and poor prognosis features (presence of liver metastases, progression to mCRPC after <12 months of androgen deprivation therapy, or ≥4 of 6 clinical criteria). Patients were randomly assigned 1 : 1 to receive cabazitaxel plus prednisone (group A) or physician's choice of enzalutamide or abiraterone plus prednisone (group B) at standard doses. Patients could cross over at progression. The primary endpoint was clinical benefit rate for first-line treatment (defined as prostate-specific antigen response ≥50%, radiographic response, or stable disease ≥12 weeks). RESULTS: Ninety-five patients were accrued (median follow-up 21.9 months). First-line clinical benefit rate was greater in group A versus group B (80% versus 62%, P = 0.039). Overall survival was not different between groups A and B (median 37.0 versus 15.5 months, hazard ratio (HR) = 0.58, P = 0.073) nor was time to progression (median 5.3 versus 2.8 months, HR = 0.87, P = 0.52). The most common first-line treatment-related grade ≥3 adverse events were neutropenia (cabazitaxel 32% versus ARPI 0%), diarrhoea (9% versus 0%), infection (9% versus 0%), and fatigue (7% versus 5%). Baseline circulating tumour DNA (ctDNA) fraction above the cohort median and on-treatment ctDNA increase were associated with shorter time to progression (HR = 2.38, P < 0.001; HR = 4.03, P < 0.001). Patients with >30% ctDNA fraction at baseline had markedly shorter overall survival than those with undetectable ctDNA (HR = 38.22, P < 0.001). CONCLUSIONS: Cabazitaxel was associated with a higher clinical benefit rate in patients with ARPI-naive poor prognosis mCRPC. ctDNA abundance was prognostic independent of clinical features, and holds promise as a stratification biomarker.
Assuntos
Neoplasias de Próstata Resistentes à Castração , Antagonistas de Androgênios/uso terapêutico , Androstenos , Protocolos de Quimioterapia Combinada Antineoplásica/efeitos adversos , Benzamidas , Humanos , Masculino , Nitrilas , Feniltioidantoína , Prednisona/efeitos adversos , Prognóstico , Neoplasias de Próstata Resistentes à Castração/tratamento farmacológico , Taxoides/uso terapêutico , Resultado do TratamentoRESUMO
INTRODUCTION: The last decade has seen a remarkable shift in the treatment landscape of advanced prostate cancer, none more so than in the management of metastatic castration-naïve disease. METHODS: This narrative review will examine existing and emerging evidence supporting systemic therapy use for metastatic castration-naïve prostate cancer (mCNPC) and provide guidance on the selection of these agents with respect to optimising patient outcomes. RESULTS: The addition of either docetaxel (chemohormonal approach) or an AR pathway inhibitor (abiraterone, enzalutamide or apalutamide) is a reasonable standard of care option for men commencing long-term ADT for mCNPC. While the issue of disease volume as a predictive biomarker for docetaxel benefit has previously been debated, recent data support consideration of upfront docetaxel in all patients, regardless of metastatic burden. Decisions regarding systemic treatment for men with mCNPC should be based on comprehensive consideration of disease, patient and logistical factors. Multiple novel therapeutics for mCNPC are currently under active investigation. CONCLUSION: The introduction of potent systemic therapy earlier in the mCNPC disease course has resulted in dramatic improvements in clinical outcomes for patients. As the management of mCNPC continues to evolve, the future remains promising, with the expectation of ongoing improvements to patient outcomes and quality of life.
Assuntos
Neoplasias da Próstata/tratamento farmacológico , Antagonistas de Androgênios/uso terapêutico , Antagonistas de Receptores de Andrógenos/uso terapêutico , Antineoplásicos/uso terapêutico , Humanos , Masculino , Metástase Neoplásica , Neoplasias da Próstata/patologiaRESUMO
BACKGROUND: Few prognostic models for overall survival (OS) are available for patients with metastatic castration-resistant prostate cancer (mCRPC) treated with recently approved agents. We developed a prognostic index model using readily available clinical and laboratory factors from a phase III trial of abiraterone acetate (hereafter abiraterone) in combination with prednisone in post-docetaxel mCRPC. PATIENTS AND METHODS: Baseline data were available from 762 patients treated with abiraterone-prednisone. Factors were assessed for association with OS through a univariate Cox model and used in a multivariate Cox model with a stepwise procedure to identify those of significance. Data were validated using an independent, external, population-based cohort. RESULTS: Six risk factors individually associated with poor prognosis were included in the final model: lactate dehydrogenase > upper limit of normal (ULN) [hazard ratio (HR) = 2.31], Eastern Cooperative Oncology Group performance status of 2 (HR = 2.19), presence of liver metastases (HR = 2.00), albumin ≤4 g/dl (HR = 1.54), alkaline phosphatase > ULN (HR = 1.38) and time from start of initial androgen-deprivation therapy to start of treatment ≤36 months (HR = 1.30). Patients were categorized into good (n = 369, 46%), intermediate (n = 321, 40%) and poor (n = 107, 13%) prognosis groups based on the number of risk factors and relative HRs. The C-index was 0.70 ± 0.014. The model was validated by the external dataset (n = 286). CONCLUSION: This analysis identified six factors used to model survival in mCRPC and categorized patients into three distinct risk groups. Prognostic stratification with this model could assist clinical practice decisions for follow-up and monitoring, and may aid in clinical trial design. TRIAL REGISTRATION NUMBERS: NCT00638690.
Assuntos
Acetato de Abiraterona/uso terapêutico , Antineoplásicos/uso terapêutico , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Prednisona/uso terapêutico , Neoplasias de Próstata Resistentes à Castração/tratamento farmacológico , Acetato de Abiraterona/efeitos adversos , Antineoplásicos/efeitos adversos , Intervalo Livre de Doença , Método Duplo-Cego , Humanos , Masculino , Prednisona/efeitos adversos , Modelos de Riscos ProporcionaisRESUMO
BACKGROUND: The role of cytoreductive nephrectomy (CN) for metastatic renal cell carcinoma (mRCC) in the era of targeted therapies is currently undefined. In recent years, neutrophil-to-lymphocyte ratio (NLR) has emerged as a prognostic marker in several cancers, including mRCC. In this multicentre retrospective study, we aim to assess the impact of CN in mRCC and the value of NLR in risk stratification and patient selection. METHODS: Retrospective data from patients with de novo mRCC from four large Australian hospitals were collected. Survival analyses were performed using the Kaplan-Meier method and compared using the log-rank test. Multivariate analyses were performed using the Cox proportional hazards method. RESULTS: Our study identified 91 de novo mRCC patients. Patients who underwent CN (n = 46, 51%) were more likely to be younger (59.0 years vs 64.6 years, P = 0.019) and to have received systemic therapy (91% vs 76%, P = 0.043). Median overall survival (mOS) was significantly improved in patients who underwent CN (23.0 months vs 10.9 months, hazard ratios (HR) 0.33, 95% confidence interval (CI) 0.20-0.55, P < 0.0001). Patients with NLR ≥ 5 also had inferior mOS (6.2 months vs 16.7 months, HR 1.94, 95% CI 1.14-3.29, P = 0.014). CN was associated with substantially improved survival in patients with both NLR < 5 (mOS 31.1 months vs 7.0 months, HR 0.41, 95% CI, 0.18-0.64, P = 0.0009) and NLR ≥ 5 (mOS 10.9 months vs 2.3 months, HR 0.33, 95% CI, 0.11-0.69, P = 0.009). Significant survival benefits associated with CN were maintained in multivariate analyses (HR 0.39, 95% CI 0.22-0.70, P = 0.0014). CONCLUSIONS: CN is associated with significantly improved overall survival in de novo mRCC. The incremental survival benefit associated with CN was seen irrespective of NLR.
Assuntos
Carcinoma de Células Renais/cirurgia , Procedimentos Cirúrgicos de Citorredução , Neoplasias Renais/cirurgia , Nefrectomia , Austrália/epidemiologia , Carcinoma de Células Renais/mortalidade , Feminino , Humanos , Neoplasias Renais/mortalidade , Linfócitos/citologia , Masculino , Pessoa de Meia-Idade , Análise Multivariada , Neutrófilos/citologia , Estudos Retrospectivos , Análise de Sobrevida , Resultado do TratamentoRESUMO
We have produced human immunodeficiency virus type 1 (HIV-1) Nef (a myristylated 206-amino-acid protein) in Saccharomyces cerevisaie and shown that, while Nef is normally found as a predominantly intracellular protein, amounts up to 40 micrograms/ml of Nef are also released into the extracellular medium during stress. By electrophoretic (SDS-PAGE) analysis the extracellular Nef is indistinguishable from intracellular Nef. Conditions of stress that lead to the release of Nef include elevated levels of copper or magnesium ions or growth at elevated temperatures. This release appears to be dependent upon the N-terminal sequences of Nef, including the presence of a myristylation site. Our observations concerning Nef release in yeast suggest new ways in which the behaviour of Nef should be examined in order to gain further insights into the development of AIDS. If the release of Nef is important in the development of AIDS, our work reveals that Nef-associated symptoms may be reduced or delayed by reducing stresses, such as fevers.
Assuntos
Produtos do Gene nef/metabolismo , HIV-1/química , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Cobre/farmacologia , Produtos do Gene nef/química , Temperatura Alta , Magnésio/farmacologia , Dados de Sequência Molecular , Ácido Mirístico , Ácidos Mirísticos/metabolismo , Processamento de Proteína Pós-Traducional , Produtos do Gene nef do Vírus da Imunodeficiência HumanaRESUMO
Various expression vectors containing a cDNA fragment encoding all but the first five amino acids (aa) of the large polyprotein (N-VP2-VP4-VP3-C) of infectious bursal disease virus were transformed into yeasts. In both Saccharomyces cerevisiae and Schizosaccharomyces pombe, co- or post-translational processing of the unfused large polyprotein occurred, generating a stable C-terminal product (VP3) or correct size, but without any detectable N-terminal product (VP2). Furthermore, when the processing of the polyprotein was interrupted, because of an engineered in-frame site-specific insertion of 4 aa, even VP3 (as part of the unprocessed polyprotein) was undetected. VP2 was detected in S. cerevisiae only when fused to yeast pre-sequences at the N terminus, suggesting that in yeast, VP2 or the unprocessed polyprotein, in the absence of its native N terminus or proper protection of its N-terminal aa residues is susceptible to proteolytic degradation. The first 8 aa of a modified pre-sequence of the CUP1 gene product and the pre-pro sequence of MF alpha 1 gene product have been used for stable intra- and extra-cellular production of VP2, respectively.
Assuntos
Regulação Viral da Expressão Gênica , Vírus da Doença Infecciosa da Bursa/genética , Saccharomyces cerevisiae/genética , Schizosaccharomyces/genética , Proteínas Virais/genética , Sequência de Aminoácidos , Sequência de Bases , Western Blotting , Clonagem Molecular , Genes Virais , Dados de Sequência Molecular , Processamento de Proteína Pós-Traducional , Proteínas/genética , Proteínas Virais de Fusão/genética , Proteínas Virais de Fusão/metabolismoRESUMO
Viral protein 2 (VP2) from infectious bursal disease virus and its precursor polyprotein (N-VP2-VP4-VP3-C), in the absence of their native N-terminal region (19 amino acids), required fusion of yeast presequences for their stable synthesis in Saccharomyces cerevisiae [Jagadish et al., Gene 95 (1990) 179-186]. Restoration of the missing 19 aa resulted in stable synthesis of VP2, indicating the significance of the N-terminal region in protein stability.
Assuntos
Vírus da Doença Infecciosa da Bursa/genética , Precursores de Proteínas/biossíntese , Proteínas Recombinantes/biossíntese , Saccharomyces cerevisiae/metabolismo , Proteínas Virais/biossíntese , Sequência de Aminoácidos , Anticorpos Monoclonais , Sequência de Bases , Western Blotting , Análise Mutacional de DNA , Expressão Gênica , Dados de Sequência Molecular , Precursores de Proteínas/química , Precursores de Proteínas/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Saccharomyces cerevisiae/genética , Proteínas Virais/química , Proteínas Virais/genéticaRESUMO
We have sought to obtain a convenient system for the high-level production of secreted proteins in yeast. With the aid of a secretion reporter cassette we examined the secretion of beta-lactamase (Bla) as a model protein and found the highest expression in Saccharomyces cerevisiae using a high-copy-number plasmid. We further developed the high-copy-number plasmid introducing a secretion cassette that has a convenient cloning site coinciding with the sequence encoding the KEX2 cleavage site. Large quantities of correctly-processed product can therefore be obtained. We show that 0.3 g/l of correctly processed beta-lactamase can be obtained in fed-batch cultures without the need for selective media or significant loss of the plasmid.
Assuntos
Vetores Genéticos , Plasmídeos , Processamento de Proteína Pós-Traducional , Saccharomyces cerevisiae/enzimologia , beta-Lactamases/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Clonagem Molecular , Meios de Cultura , DNA Fúngico , Dados de Sequência Molecular , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , beta-Lactamases/genéticaRESUMO
The genome of Australian strain 002-73 of infectious bursal disease virus (IBDV) has been cloned as cDNA fragments into an expression library based on pUR plasmid vectors. Recombinant colonies were selected with a monoclonal antibody specific for the 32-kDa host-protective immunogen of IBDV and fully characterized by nucleotide sequence analysis. The amino acid sequence of several tryptic peptides derived from the native 32-kDa structural protein has confirmed the coding region assignment and nucleotide sequence data. We believe this to be the first published sequence of a birnavirus-encoded protein and these data may provide the basis for an effective subunit vaccine against IBDV.
Assuntos
Vírus da Doença Infecciosa da Bursa , Reoviridae , Proteínas Virais/imunologia , Sequência de Aminoácidos , Sequência de Bases , DNA/genética , DNA Recombinante , Genes Virais , Vírus da Doença Infecciosa da Bursa/genética , RNA Viral/genética , Reoviridae/genética , Proteínas Virais/genética , Vacinas ViraisRESUMO
The biological effects of the HIV-1 accessory protein, Vpr, have been studied in yeast expression systems. In our previous study [1], employing the pCUP1-vpr copper-inducible expression cassette, Vpr was shown to cause growth arrest and structural defects. In this study yeast constitutively expressing vpr, through elevated copy number and/or elevated transcription levels, displayed no growth arrest in fermentative growth conditions while Vpr was produced at much lower levels than in the inducible expression system. However, such cells were respiratory deficient and unable to utilise ethanol or glycerol as the sole carbon source. They exhibited gross mitochondrial dysfunction displayed in the loss of respiratory chain complex I, II, III, IV and citrate synthase activities. The effects on mitochondria required a C-terminal domain of Vpr that contains a conserved amino acid sequence motif HFRIGCRHSRIG. These results suggest that the widely observed phenomenon of 'Vpr-induced growth arrest' in human cells could be due to mitochondrial dysfunction.
Assuntos
Produtos do Gene vpr/fisiologia , HIV-1 , Mitocôndrias/metabolismo , Saccharomyces cerevisiae/crescimento & desenvolvimento , Produtos do Gene vpr/biossíntese , Produtos do Gene vpr/genética , Glutationa Transferase/genética , Humanos , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/farmacologia , Saccharomyces cerevisiae/metabolismo , Transfecção , Produtos do Gene vpr do Vírus da Imunodeficiência HumanaRESUMO
We have studied two isoforms of Nef, Nef-27 and Nef-25, which were produced in E. coli. Nef-25 lacked the first 18 N-terminal residues of Nef-27 and both were nonmyristylated. Nef-27 fuses small unilamellar dipalmitoyl phosphatidylcholine vesicles (SUVs), as indicated by enhanced light scattering of SUVs and lipid mixing using concentration-dependent fluorescence dequenching. Nef-27 also causes the appearance of a shifted isotropic peak in the 31P NMR spectra of these vesicles, suggesting that protein interactions induce nonlamellar lipid structures. Recombinant Nef-25, which lacks only the 18 N-terminal residues of Nef-27, does not fuse vesicles and has little effect on the 31P NMR spectra. On the other hand, synthetic peptides consisting of 18 or 21 of the N-terminal residues of Nef-27 are strongly membrane perturbing, causing vesicle fusion and inducing isotropic peaks in the 31P NMR spectrum. Endogenous fluorescence spectra of the N-terminal peptide (21 residues) with SUVs show that the N-terminal sequence of Nef may achieve these perturbing effects by inserting its hydrophobic side into the lipid bilayer. Theoretical calculations using hydrophobic moment plot analysis indicate that short-length stretches (i.e., six amino acid residues) of the N-terminal sequence may insert into the lipid bilayer as multimeric alpha helices or beta sheets. The above-described membrane activities of Nef-27, which principally reside in its N-terminal domain, may play critical role(s) in certain functional properties of the full-length protein. For example, the fusogenic activity of the N-terminal sequence may be involved in the extracellular release of Nef-27, much of which appears to be associated with small membrane vesicles. The fusion activity may also be relevant to the ability of Nef-27 to downregulate CD4 and IL-2 receptors when this protein is electroporated into cultured lymphocytes, an activity not possessed by Nef-25.
Assuntos
1,2-Dipalmitoilfosfatidilcolina , Produtos do Gene nef/metabolismo , HIV-1/metabolismo , Lipossomos , Fusão de Membrana , Fragmentos de Peptídeos/química , Proteínas Recombinantes/metabolismo , Sequência de Aminoácidos , Antígenos de Bactérias/química , Produtos do Gene nef/biossíntese , Produtos do Gene nef/química , Luz , Espectroscopia de Ressonância Magnética , Dados de Sequência Molecular , Fragmentos de Peptídeos/síntese química , Estrutura Secundária de Proteína , Proteínas Recombinantes/biossíntese , Espalhamento de Radiação , Homologia de Sequência de Aminoácidos , Espectrometria de Fluorescência , Produtos do Gene nef do Vírus da Imunodeficiência HumanaRESUMO
Myristoylated 21- and 25-residue N-terminal peptides of the Nef protein of HIV-1 lysed human erythrocytes and were cytotoxic toward a human CD4+ T cell line, CEM, and primary human peripheral blood mononuclear cells (PBMCs). The corresponding nonmyristoylated N-terminal peptides were only very weakly hemolytic and cytotoxic. A myristoylated peptide consisting of residues 31-50 of Nef was neither hemolytic nor cytotoxic. Alteration of the tryptophan residue at position 13 to a serine did not change the hemolytic and cytotoxic activity. Studies of the ultraviolet fluorescence of the tryptophan at position 5 in the peptide, using an artificial membrane system and fluorescence-quenching agents that inserted into the bilayer at different levels, suggested that myristoylation results in this residue being brought into contact with the upper hydrocarbon region of the lipid bilayer of the cell membrane. This tryptophan is flanked by a number of polar residues that would maintain it in this position, resulting in a considerable increase in disorder in the upper regions of the lipid bilayer, leading to its destabilization and to lysis. The cytotoxic activity of the myristoylated Nef fragments may, in part, explain the killing and deletion of cells, especially in lymphoid tissues, during HIV infection.
Assuntos
Citotoxicidade Imunológica/efeitos dos fármacos , Produtos do Gene nef/farmacologia , HIV-1 , Oligopeptídeos/farmacologia , Sequência de Aminoácidos , Linhagem Celular/efeitos dos fármacos , Testes Imunológicos de Citotoxicidade , HIV-1/química , Hemólise/efeitos dos fármacos , Humanos , Bicamadas Lipídicas/química , Lipossomos , Dados de Sequência Molecular , Ácidos Mirísticos/metabolismo , Oligopeptídeos/síntese química , Espectrometria de Fluorescência , Produtos do Gene nef do Vírus da Imunodeficiência HumanaRESUMO
We have found that the hemolytic and cytotoxic activities of myristoylated Nef N-terminal peptides require a net positive charge in the first seven amino residues of the sequence. The activities are considerably less dependent on the secondary structure of the peptides. Film balance studies showed that both active and inactive peptides interacted with neutral phospholipid monolayers, suggesting that binding to neutral lipids was not a sufficient condition for lytic activity. It was also found that nonmyristoylated N-terminal peptide did not interact to the same extent with the monolayer, indicating that myristoylation was essential for lipid interaction. It is considered that the positively charged residues of the proximate N terminus of Nef interact with acidic lipids of biological membranes, reinforcing the weak membrane-targeting properties of the myristyl chain. Parallels are drawn between this mode of interaction with membranes and that of members of the Src family of proteins, which are also myristoylated and have positively charged residues in their proximate N termini. In particular, these proteins and Nef also have serine residues in their proximal N-terminal regions, which when phosphorylated could neutralize the positive charge and thus provide a mechanism for modulating membrane interaction.
Assuntos
Produtos do Gene nef/química , Produtos do Gene nef/fisiologia , HIV-1/fisiologia , Estrutura Secundária de Proteína , Sequência de Aminoácidos , Animais , Hemólise , Humanos , Fusão de Membrana , Dados de Sequência Molecular , Ácido Mirístico , Peptídeos/química , Peptídeos/fisiologia , Ovinos , Relação Estrutura-Atividade , Produtos do Gene nef do Vírus da Imunodeficiência HumanaRESUMO
A growing body of literature suggests that the HIV accessory proteins Nef and Vpr could be involved in depletion of CD4(+) and non-CD4(+) cells and tissue atrophy, and in delaying the death of HIV-infected cells. Cell depletion is likely to be predominantly a bystander effect because the number of cells dying far outnumbers HIV-infected cells and is not confined to CD4(+) cells. The myristylated N-terminal region of Nef has severe membrane disordering properties, and when present in the extracellular medium causes rapid lysis in vitro of a wide range of CD4(+) and non-CD4(+) cells, suggesting a role for extracellular Nef in the depletion of bystander cells. A direct role for HIV-1 Nef in cytopathicity is supported by studies in HIV-infected Hu Liv/Thy SCID mice, in transgenic mice expressing nef gene alone, and in rhesus macaques infected with SIV/HIV chimeric virus containing HIV-1 nef. The N-terminal region of Nef has been directly implicated in development of simian AIDS. Extracellular Vpr and C-terminal fragments of Vpr cause membrane permeabilization and apoptosis of a wide range of CD4(+) and non-CD4(+) cells, and could also contribute to depletion of bystander cells. A direct in vivo role for Vpr in thymocyte depletion, thymic atrophy, and nephropathy is suggested in studies with vpr transgenic mice. Intracellular Nef and Vpr could help HIV-infected cells evade cell death by inhibiting apoptosis of infected cells and by avoiding virus-specific CTL response. Nef and Vpr are potential targets for therapeutic intervention and vaccine development, and strategies that prevent the death of bystander cells while promoting the early death of HIV-infected cells could arrest or retard progression to AIDS.
Assuntos
Produtos do Gene nef/fisiologia , Produtos do Gene vpr/fisiologia , Infecções por HIV/fisiopatologia , HIV-1/fisiologia , Animais , Apoptose , Linfócitos T CD4-Positivos/citologia , Linfócitos T CD4-Positivos/patologia , Linfócitos T CD4-Positivos/virologia , Sobrevivência Celular , Progressão da Doença , Produtos do Gene nef/genética , Produtos do Gene vpr/genética , Genes nef , Humanos , Macaca mulatta , Camundongos , Camundongos SCID , Camundongos Transgênicos , Síndrome de Imunodeficiência Adquirida dos Símios/fisiopatologia , Produtos do Gene nef do Vírus da Imunodeficiência Humana , Produtos do Gene vpr do Vírus da Imunodeficiência HumanaRESUMO
1. This paper describes a standard procedure for the preparation and purification of RNA from the post-mitochondrial supernatants of a number of eukaryotes. 2. Cytoplasmic RNA was fractionated by NaCl precipitation. The 28S (26S), 18S and 5.8S rRNA, and 9S RNA, in the NaCl insoluble fraction were separated by a two-step sucrose gradient fractionation procedure. Poly(A)-containing mRNA in hen 9S RNA was purified by affinity chromatography. The 5S rRNA and tRNA in the NaCl-soluble fraction were fractionated by gel filtration. 3. Polyacrylamide gel electrophoresis showed that the above RNA species were remarkably stable and homogeneous. Differences were found in the 26-28S rRNA, 5.8S rRNA, and 9S RNA of different eukaryotes, but other cytoplasmic RNA species were identical.
Assuntos
Grão Comestível/genética , Hordeum/genética , RNA/isolamento & purificação , Reticulócitos/análise , Sarcoma 180/genética , Animais , Galinhas , Citoplasma/análise , Eletroforese em Gel de Poliacrilamida , Fígado/análise , Masculino , Camundongos , Ratos , Ratos EndogâmicosRESUMO
Highly conserved sequences present at an identical position near the 3' ends of eukaryotic and prokaryotic 5S rRNAs are complementary to the 5' strand of the m2(6)A hairpin structure near the 3' ends of 18S rRNA and 16S rRNA, respectively. The extent of base-pairing and the calculated stabilities of the hybrids that can be constructed between 5S rRNAs and the small ribosomal subunit RNAs are greater than most, if not all, RNA-RNA interactions that have been implicated in protein synthesis. The existence of complementary sequences in 5S rRNA and small ribosomal subunit RNA, along with the previous observation that there is very efficient and selective hybridization in vitro between 5S and 18S rRNA, suggests that base-pairing between 5S rRNA in the large ribosomal subunit and 18S (16S) rRNA in the small ribosomal subunit might be involved in the reversible association of ribosomal subunits. Structural and functional evidence supporting this hypothesis is discussed.
Assuntos
RNA Ribossômico/metabolismo , Ribossomos/metabolismo , Sequência de Bases , Escherichia coli/metabolismo , Conformação de Ácido Nucleico , RNA Bacteriano/metabolismo , Saccharomyces cerevisiae/metabolismo , Triticum/metabolismoRESUMO
As part of our efforts to produce native molecules of the host protective antigen (VP2) of infectious bursal disease virus (IBDV) in microorganisms, we have restored nucleotide sequences encoding the VP2 N-terminus which were not present in the original clone. In addition to this process, we produced constructs that contained +5, +4, -1, and -2 frameshifts that still allowed expression of the IBDV polyprotein, VP2 and VP3. The size of translation products and examination of the nucleotide sequence lead us to speculate that both +1 frameshifting and internal initiation events lead to the synthesis of IBDV proteins in Escherichia coli.
Assuntos
Vírus da Doença Infecciosa da Bursa/genética , Biossíntese de Proteínas , Proteínas Virais/genética , Antígenos Virais/genética , Sequência de Bases , Clonagem Molecular , Escherichia coli/genética , Dados de Sequência Molecular , Iniciação Traducional da Cadeia Peptídica , Proteínas/genética , RNA Viral/genéticaRESUMO
Birnaviruses typically encode a polyprotein that is the precursor to the structural proteins of the virus and a protein of rare abundance, VP1, that is the putative dsRNA replicase and/or transcriptase. We have reconstructed the VP1 gene of IBDV from a library of cDNA clones and expressed the gene in Escherichia coli and in Saccharomyces cerevisiae. We could not detect an RNA polymerase activity associated with E. coli-derived VP1, and neither could we promote the yeast-derived VP1 to replicate exogenous IBDV dsRNA. However, the yeast-derived VP1 was shown to have an actinomycin-insensitive RNA polymerase activity that can recognise an endogenous template in S. cerevisiae. Our work suggests that further studies on birnavirus replication may be best addressed using an S. cerevisiae expression system.
Assuntos
Escherichia coli/genética , Vírus da Doença Infecciosa da Bursa/genética , RNA Polimerase Dependente de RNA/metabolismo , Saccharomyces cerevisiae/genética , Proteínas Virais/metabolismo , Autorradiografia , Sequência de Bases , Clonagem Molecular , DNA Complementar , Escherichia coli/enzimologia , Vírus da Doença Infecciosa da Bursa/enzimologia , Vírus da Doença Infecciosa da Bursa/metabolismo , Dados de Sequência Molecular , Fases de Leitura Aberta , RNA Viral/metabolismo , Saccharomyces cerevisiae/enzimologia , Proteínas Virais/genética , Replicação ViralRESUMO
An immunodominant region of VP3, one of the two structural proteins of infectious bursal disease virus (IBDV strain 002-73), has been mapped by restriction site-specific deletion analysis and subcloning in Escherichia coli, followed by immunoblot analysis of the synthesized products. The epitope located within 58 amino acids reacted very strongly with a mouse monoclonal antibody (MAb 17/80) raised against IBDV 002-73. This immunodominant region may be useful in serodiagnosis of IBDV infection in poultry.
Assuntos
Antígenos Virais/imunologia , Capsídeo/imunologia , Vírus da Doença Infecciosa da Bursa/imunologia , Sequência de Aminoácidos , Antígenos Virais/genética , Capsídeo/genética , Proteínas do Capsídeo , Clonagem Molecular , Análise Mutacional de DNA , Epitopos , Vírus da Doença Infecciosa da Bursa/genética , Dados de Sequência Molecular , Proteínas/genética , Proteínas/imunologia , Mapeamento por RestriçãoRESUMO
The hepatitis B virus X-protein (HBx) has been expressed in Escherichia coli both as an unfused protein and with an N-terminal hexaHis-containing fusion sequence. Both forms of HBx, after purification, displayed a potent AMP kinase activity, in which HBx phosphorylates AMP to ADP, using ATP as the exclusive phosphate donor. We also found that HBx has previously unreported GTPase and GTP-ADP nucleoside diphosphate kinase activities.