RESUMO
PURPOSE: PATRO adults is an ongoing, multicenter, observational, post-marketing surveillance study aimed at investigating the long-term safety (primary endpoint) and effectiveness (secondary endpoint) of the recombinant human growth hormone (rhGH) Omnitrope® during routine clinical practice. This report describes data from Italian participants in PATRO Adults with growth hormone deficiency (GHD), up to August 2017. METHODS: Participants were adults (aged > 18 years) with GHD requiring rhGH therapy and were prescribed Omnitrope®, including those who had previously received another rhGH product. Adverse events (AEs) were evaluated in all study participants. Data were collected on insulin-like growth factor (IGF)-I levels and cardiovascular risk factors, including blood pressure, lipids, and anthropometric parameters. RESULTS: From September 2007 to August 2017, 88 patients (mean age 48.9 years, 58.0% male) were enrolled at 8 sites in Italy. The mean treatment duration with Omnitrope® was 51.5 ± 37 months. AEs occurred in 54 patients; the most common were asthenia (20.5%), headache (14.8%), and arthralgia (13.6%). Serious AEs occurred in 22 patients (25%), including pneumonia (n = 2) and renal failure (n = 2). Neoplasms (2 benign and 1 malignant) developed in three patients, but none were considered to be drug-related. There were no significant changes in fasting glucose or glycosylated hemoglobin (HbA1c) during the study period. Long-term Omnitrope® therapy showed slight positive effects on lipid profile, while no significant changes were observed in body weight and BMI during the study. CONCLUSION: This snapshot analysis of Italian participants in PATRO Adults confirmed the long-term safety and effectiveness of Omnitrope® in adults with GHD.
Assuntos
Transtornos do Crescimento/tratamento farmacológico , Hormônio do Crescimento Humano/administração & dosagem , Hormônio do Crescimento Humano/deficiência , Adulto , Idoso , Feminino , Seguimentos , Transtornos do Crescimento/epidemiologia , Humanos , Itália/epidemiologia , Estudos Longitudinais , Masculino , Pessoa de Meia-Idade , PrognósticoRESUMO
PURPOSE: To investigate the long-term safety (primary endpoint) and effectiveness (secondary endpoint) of the somatropin biosimilar Omnitrope®. METHODS: PATRO Children is an ongoing, multicenter, observational, post-marketing surveillance study. Children who received Omnitrope® for any indication were included. Adverse events (AEs) were evaluated in all study participants. Auxological data, including height standard deviation scores (HSDS) and height velocity standard deviation scores (HVSDS), were used to assess effectiveness. In this snapshot analysis, data from the Italian subpopulation up to August 2017 were reported. RESULTS: A total of 291 patients (mean age 10.0 years, 56.0% male) were enrolled at 19 sites in Italy. The mean duration of Omnitrope® treatment was 33.1 ± 21.7 months. There were 48 AEs with a suspected relationship to the study drug (as reported by the investigator) that occurred in 35 (12.0%) patients, most commonly headache, pyrexia, arthralgia, insulin-like growth factor above normal range, abdominal pain, pain in extremity and acute gastroenteritis. There were no confirmed cases of type 1 or type 2 diabetes; however, two patients (0.7%) had impaired glucose tolerance that was considered Omnitrope® related. The mean HSDS increased from - 2.41 ± 0.73 at baseline (n = 238) to - 0.91 ± 0.68 at 6.5 years (n = 10). The mean HVSDS increased from - 1.77 ± 1.38 at baseline (n = 136) to 0.96 ± 1.13 at 6.5 years (n = 10). CONCLUSIONS: In this sub-analysis of PATRO Children, Omnitrope® appeared to have acceptable safety and effectiveness in the treatment of in Italian children, which was consistent with the earlier findings from controlled clinical trials.
Assuntos
Medicamentos Biossimilares/uso terapêutico , Transtornos do Crescimento/tratamento farmacológico , Hormônio do Crescimento Humano/uso terapêutico , Vigilância de Produtos Comercializados/métodos , Criança , Feminino , Seguimentos , Transtornos do Crescimento/epidemiologia , Humanos , Estudos Longitudinais , Masculino , PrognósticoRESUMO
Pharmacological interventions that increase myofiber size counter the functional decline of dystrophic muscles. We show that deacetylase inhibitors increase the size of myofibers in dystrophin-deficient (MDX) and alpha-sarcoglycan (alpha-SG)-deficient mice by inducing the expression of the myostatin antagonist follistatin in satellite cells. Deacetylase inhibitor treatment conferred on dystrophic muscles resistance to contraction-coupled degeneration and alleviated both morphological and functional consequences of the primary genetic defect. These results provide a rationale for using deacetylase inhibitors in the pharmacological therapy of muscular dystrophies.
Assuntos
Inibidores Enzimáticos/farmacologia , Músculos/enzimologia , Músculos/patologia , Distrofia Muscular Animal/tratamento farmacológico , Animais , Distrofina/genética , Fibrose/patologia , Folistatina/metabolismo , Ácidos Hidroxâmicos/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos mdx , Músculos/efeitos dos fármacos , Distrofia Muscular Animal/genética , Distrofia Muscular Animal/patologia , Fenilbutiratos/farmacologia , Sarcoglicanas/metabolismo , Células Satélites de Músculo Esquelético/citologia , Células Satélites de Músculo Esquelético/enzimologia , Ácido Valproico/farmacologiaRESUMO
DNA mismatch recognition and binding in human cells has been thought to be mediated by the hMSH2 protein. Here it is shown that the mismatch-binding factor consists of two distinct proteins, the 100-kilodalton hMSH2 and a 160-kilodalton polypeptide, GTBP (for G/T binding protein). Sequence analysis identified GTBP as a new member of the MutS homolog family. Both proteins are required for mismatch-specific binding, a result consistent with the finding that tumor-derived cell lines devoid of either protein are also devoid of mismatch-binding activity.
Assuntos
Reparo do DNA , DNA de Neoplasias/metabolismo , Proteínas de Ligação a DNA/metabolismo , Ácidos Nucleicos Heteroduplexes/metabolismo , Sequência de Aminoácidos , Composição de Bases , Sequência de Bases , Clonagem Molecular , Neoplasias Colorretais , Reparo do DNA/genética , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Células HeLa , Humanos , Dados de Sequência Molecular , Peso Molecular , Análise de Sequência , Células Tumorais CultivadasRESUMO
Definition of mechanisms regulating human histone H1 gene transcription during the cell cycle requires the isolation and biochemical characterization of protein factors which interact with specific promoter elements. Two distinct binding activities have been identified in nuclear extracts from HeLa cells and mapped within a 180-base-pair (bp) region of a cell cycle-regulated H1 gene promoter. H1TF1 bound to an H1-specific A + C-rich sequence (AC box), 100 bp upstream of the cap site; H1TF2 interacted with the H1 subtype-specific consensus element and was dependent on the presence of an intact CCAAT box for binding. H1TF2 was purified through a combination of ion-exchange and oligonucleotide affinity chromatographies. Analysis of purified fractions by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and UV crosslinking showed that H1TF2 was a single polypeptide of 47 kilodaltons. This factor was distinct from previously characterized CCAAT-binding proteins in both molecular size and binding properties. Fractions containing H1TF2 activity activated transcription in vitro only if programmed with an H1 DNA template carrying an intact H1TF2-binding site.
Assuntos
Proteínas de Ligação a DNA/isolamento & purificação , Histonas/metabolismo , Sítios de Ligação , Linhagem Celular , Proteínas de Ligação a DNA/metabolismo , Desoxirribonucleotídeos/metabolismo , Regulação da Expressão Gênica , Histonas/genética , Humanos , Regiões Promotoras Genéticas , Transcrição GênicaRESUMO
Expression of the DNA mismatch repair (MMR) pathway was examined in the adult and developing rat brain. Rat homologues of human GTBP and MSH2, which are essential components of the post-replicative DNA MMR system, were identified in nuclear extracts from the adult and developing rat brain. Developmental studies showed that both GTBP and MSH2 levels were higher in nuclei isolated from the embryonic brain (day 16) than adult brain. However, this difference was not as dramatic as the difference in the number of proliferating cells. Levels of thymine DNA glycosylase (TDG), the enzyme which catalyzes the first step in short patch G:T mismatch repair, were also decreased in adult compared to embryonic brain. In the adult brain, MMR proteins were elevated in nuclear extracts enriched for neuronal nuclei. These results suggest that adult brain cells have the capacity to carry out DNA mismatch repair, in spite of a lack of ongoing DNA replication.
Assuntos
Encéfalo/metabolismo , Reparo do DNA , Proteínas de Ligação a DNA/biossíntese , Regulação da Expressão Gênica no Desenvolvimento , Proteínas Proto-Oncogênicas/biossíntese , Animais , Encéfalo/embriologia , Núcleo Celular/metabolismo , Cerebelo/metabolismo , Humanos , Mamíferos , Proteína 2 Homóloga a MutS , Prosencéfalo/metabolismo , RatosAssuntos
Fibroblastos/efeitos dos fármacos , Substâncias de Crescimento/farmacologia , Proteínas Proto-Oncogênicas/biossíntese , Acetato de Tetradecanoilforbol/farmacologia , Animais , Divisão Celular/efeitos dos fármacos , Linhagem Celular , Meios de Cultura , Replicação do DNA/efeitos dos fármacos , Sinergismo Farmacológico , Fator de Crescimento Epidérmico/farmacologia , Fatores de Crescimento de Fibroblastos/farmacologia , Fibroblastos/citologia , Regulação da Expressão Gênica/efeitos dos fármacos , Peptídeos/farmacologia , Fator de Crescimento Derivado de Plaquetas/farmacologia , Proteínas Proto-Oncogênicas c-fos , Ratos , Estimulação Química , Fatores de Crescimento TransformadoresRESUMO
Previous findings have suggested that class IIa histone deacetylases (HDACs) (HDAC4, -5, -7, and -9) are inactive on acetylated substrates, thus differing from class I and IIb enzymes. Here, we present evidence supporting this view and demonstrate that class IIa HDACs are very inefficient enzymes on standard substrates. We identified HDAC inhibitors unable to bind recombinant human HDAC4 while showing inhibition in a typical HDAC4 enzymatic assay, suggesting that the observed activity rather reflects the involvement of endogenous copurified class I HDACs. Moreover, an HDAC4 catalytic domain purified from bacteria was 1,000-fold less active than class I HDACs on standard substrates. A catalytic Tyr is conserved in all HDACs except for vertebrate class IIa enzymes where it is replaced by His. Given the high structural conservation of HDAC active sites, we predicted the class IIa His-Nepsilon2 to be too far away to functionally substitute the class I Tyr-OH in catalysis. Consistently, a Tyr-to-His mutation in class I HDACs severely reduced their activity. More importantly, a His-976-Tyr mutation in HDAC4 produced an enzyme with a catalytic efficiency 1,000-fold higher than WT, and this "gain of function phenotype" could be extended to HDAC5 and -7. We also identified trifluoroacetyl-lysine as a class IIa-specific substrate in vitro. Hence, vertebrate class IIa HDACs may have evolved to maintain low basal activities on acetyl-lysines and to efficiently process restricted sets of specific, still undiscovered natural substrates.
Assuntos
Histona Desacetilases/química , Histona Desacetilases/metabolismo , Vertebrados , Sequência de Aminoácidos , Animais , Sítios de Ligação , Catálise , Ativação Enzimática , Células HeLa , Histidina/genética , Histidina/metabolismo , Histona Desacetilases/classificação , Histona Desacetilases/genética , Humanos , Modelos Moleculares , Mutação/genética , Estrutura Terciária de Proteína , Especificidade por Substrato , Urocordados , Vertebrados/genéticaRESUMO
Mispairs in DNA of guanine with uracil and thymine can arise as a result of deamination of cytosine and 5-methylcytosine, respectively. In humans such mispairs are removed by thymine-DNA glycosylase (TDG). By deleting the carboxy and amino termini of this enzyme we have identified a core region capable of processing G/U but not G/T mispairs. We have further identified two bacterial proteins with strong sequence homology to this core and shown that the homologue from Escherichia coli (dsUDG) can remove uracil from G/U mispairs. This enzyme is likely to act as a back-up to the highly efficient and abundant enzyme uracil-DNA glycosylase (UDG) which is found in most organisms. Pupating insects have been reported to lack UDG activity, but we have identified an enzyme similar to dsUDG in cell lines from three different insect species. These data imply the existence of a family of double-strand-specific uracil-DNA glycosylases which, although they are subservient to UDG in most organisms, may constitute the first line of defence against the mutagenic effects of cytosine deamination in insects.
Assuntos
DNA Glicosilases , Reparo do DNA , Endodesoxirribonucleases/metabolismo , N-Glicosil Hidrolases/metabolismo , Timina DNA Glicosilase , Sequência de Aminoácidos , Animais , Desaminação , Desoxirribonuclease (Dímero de Pirimidina) , Drosophila melanogaster/enzimologia , Endodesoxirribonucleases/classificação , Endodesoxirribonucleases/genética , Escherichia coli/enzimologia , Humanos , Camundongos , Dados de Sequência Molecular , N-Glicosil Hidrolases/classificação , N-Glicosil Hidrolases/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Deleção de Sequência , Homologia de Sequência de Aminoácidos , Serratia marcescens/enzimologia , Timina/metabolismo , Uracila/metabolismo , Uracila-DNA GlicosidaseRESUMO
In this study we used nuclear extracts from centrifugally elutriated cell populations to study histone H1 transcriptional regulation during the cell cycle. Analysis of mutations within the H1 promoter establish that both of the H1 subtype-specific consensus elements participate in induction of transcription upon entry into S phase. The DNA binding activity of H1TF2, which specifically interacts with the H1 proximal subtype-specific element, is increased in S-phase nuclear extracts, whereas no increase in DNA binding is observed for the H1 distal subtype-specific DNA transcription factor H1TF1 or the H2b subtype-specific factor OTF1. These data strongly support the idea that histone gene subtype-specific transcription factors are important for S-phase-dependent expression of histone genes. Further studies of these factors will be important for increased understanding of the transition from G1 to S phase of the mammalian cell cycle.
Assuntos
Regulação da Expressão Gênica , Histonas/genética , Interfase , Regiões Promotoras Genéticas , Transcrição Gênica , Animais , Centrifugação , Células HeLa , Histonas/biossíntese , Humanos , Modelos Genéticos , Mutação , Sequências Reguladoras de Ácido Nucleico , Fatores de Transcrição/metabolismoRESUMO
The herpes simplex virus type 1 immediate-early protein ICP4 plays an essential role in the regulation of the expression of all viral genes. It is the major trans activator of early and late genes and also has a negative regulatory effect on immediate-early gene transcription. ICP4 is a sequence-specific DNA-binding protein and has always been purified in a dimeric form. The part of the protein that consists of the entire highly conserved region 2 and of the distal portion of region 1 retains the ability to specifically associate with DNA and to form homodimers in solution. In an attempt to map the dimerization domain of ICP4, we used a dimerization trap assay, in which we screened deletion fragments of this 217-amino-acid stretch for sequences that could confer dimerization properties on a heterologous cellular transcription factor (LFB1), which binds to its cognate DNA sequence only as a dimer. The analysis of these chimeric proteins expressed in vitro ultimately identified a stretch of 34 amino acids (343 to 376) that could still confer DNA-binding activity on the LFB1 reporter protein and thus apparently contained the ICP4 dimerization motif. Consistent with this result, a truncated ICP4 protein containing amino acids 343 to 490, in spite of the complete loss of DNA-binding activity, appeared to retain the capacity to form a heterodimer with a longer ICP4 peptide after coexpression in an in vitro translation system.
Assuntos
Herpesvirus Humano 1/genética , Proteínas Imediatamente Precoces/genética , Sequência de Aminoácidos , Sequência de Bases , DNA Viral/genética , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Escherichia coli/genética , Herpesvirus Humano 1/química , Proteínas Imediatamente Precoces/química , Dados de Sequência Molecular , Conformação Proteica , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Deleção de Sequência , Transativadores/química , Transativadores/genéticaRESUMO
The hepatitis C virus non-structural protein 3 (NS3) possesses a serine protease activity in the N-terminal one-third, whereas RNA-stimulated NTPase and helicase activities reside in the C-terminal portion. In this study, an N-terminal hexahistidine-tagged full-length NS3 polypeptide was expressed in Escherichia coli and purified to homogeneity by conventional chromatography. Detailed characterization of the helicase activity of NS3 is presented with regard to its binding and strand release activities on different RNA substrates. On RNA double-hybrid substrates, the enzyme was shown to perform unwinding activity starting from an internal ssRNA region of at least 3 nt and moving along the duplex in a 3' to 5' direction. In addition, data are presented suggesting that binding to ATP reduces the affinity of NS3 for ssRNA and increases its affinity for duplex RNA. Furthermore, we have ascertained the capacity of NS3 to specifically interact with and resolve the stem-loop RNA structure (SL I) within the 3'-terminal 46 bases of the viral genome. Finally, our analysis of NS3 processive unwinding under single cycle conditions by addition of heparin in both helicase and RNA-stimulated ATPase assays led to two conclusions: (i) NS3-associated helicase acts processively; (ii) most of the NS3 RNA-stimulated ATPase activity may not be directly coupled to translocation of the enzyme along the substrate RNA molecule.
Assuntos
Hepacivirus/enzimologia , RNA Helicases/metabolismo , Proteínas não Estruturais Virais/metabolismo , Regiões 3' não Traduzidas/genética , Adenosina Trifosfatases/metabolismo , Trifosfato de Adenosina/metabolismo , Sequência de Bases , Heparina/farmacologia , Histidina/metabolismo , Hidrólise , Dados de Sequência Molecular , RNA Helicases/química , RNA de Cadeia Dupla/genética , RNA de Cadeia Dupla/metabolismo , RNA Viral/metabolismo , Proteínas não Estruturais Virais/química , Proteínas não Estruturais Virais/genéticaRESUMO
Nonstructural protein 3 (NS3) of hepatitis C virus contains a bipartite structure consisting of an N-terminal serine protease and a C-terminal DEXH box helicase. To investigate the roles of individual amino acid residues in the overall mechanism of unwinding, a mutational-functional analysis was performed based on a molecular model of the NS3 helicase domain bound to ssDNA, which has largely been confirmed by a recently published crystal structure of the NS3 helicase-ssDNA complex. Three full-length mutated NS3 proteins containing Tyr(392)Ala, Val(432)Gly and Trp(501)Ala single substitutions, respectively, together with a Tyr(392)Ala/Trp(501)Ala double-substituted protein were expressed in Escherichia coli and purified to homogeneity. All individually mutated forms showed a reduction in duplex unwinding activity, single-stranded polynucleotide binding capacity and polynucleotide-stimulated ATPase activity compared to wild-type, though to different extents. Simultaneous replacement of both Tyr(392) and Trp(501) with Ala completely abolished all these enzymatic functions. On the other hand, the introduced amino acid substitutions had no influence on NS3 intrinsic ATPase activity and proteolytic efficiency. The results obtained with Trp(501)Ala and Val(432)Gly single-substituted enzymes are in agreement with a recently proposed model for NS3 unwinding activity. The mutant phenotype of the Tyr(392)Ala and Tyr(392)Ala/Trp(501)Ala enzymes, however, represents a completely novel finding.
Assuntos
Proteínas não Estruturais Virais/química , Adenosina Trifosfatases/metabolismo , DNA de Cadeia Simples/metabolismo , Modelos Moleculares , Mutação , RNA Viral/química , RNA Viral/metabolismo , Relação Estrutura-Atividade , Proteínas não Estruturais Virais/fisiologiaRESUMO
MOTIVATION: In many fields of pattern recognition, combination has proved efficient to increase the generalization performance of individual prediction methods. Numerous systems have been developed for protein secondary structure prediction, based on different principles. Finding better ensemble methods for this task may thus become crucial. Furthermore, efforts need to be made to help the biologist in the post-processing of the outputs. RESULTS: An ensemble method has been designed to post-process the outputs of discriminant models, in order to obtain an improvement in prediction accuracy while generating class posterior probability estimates. Experimental results establish that it can increase the recognition rate of protein secondary structure prediction methods that provide inhomogeneous scores, even though their individual prediction successes are largely different. This combination thus constitutes a help for the biologist, who can use it confidently on top of any set of prediction methods. Moreover, the resulting estimates can be used in various ways, for instance to determine which areas in the sequence are predicted with a given level of reliability. AVAILABILITY: The prediction is freely available over the Internet on the Network Protein Sequence Analysis (NPS@) WWW server at http://pbil.ibcp.fr/NPSA/npsa_server.ht ml. The source code of the combiner can be obtained on request for academic use.
Assuntos
Redes Neurais de Computação , Estrutura Secundária de Proteína , Sequência de Aminoácidos , Simulação por Computador , Bases de Dados Factuais , Modelos Moleculares , Dados de Sequência Molecular , Reconhecimento Automatizado de PadrãoRESUMO
We have previously indicated that epidermal growth factor (EGF) plays a fundamental role in the proliferation control of EL2 rat fibroblast line. It is shown here that transforming growth factor beta (TGF beta) stimulates both DNA synthesis and proliferation of EL2 cells, while exerting an inhibitory effect on the growth of murine NIH-3T3 fibroblasts. We also report the effect of TGF beta and EGF on c-fos expression in EL2 cells, as compared to that of TGF beta in NIH-3T3 fibroblasts. In EL2 cells EGF induces a transient c-fos expression at both mRNA and protein level, as previously observed in NIH-3T3 fibroblasts treated with platelet-derived or fibroblast growth factor (PDGF, FGF). Conversely, TGF beta induces in EL2 cells a sustained expression of fos mRNA and protein, which are still detectable at least 24 and 7 hr after treatment respectively. In NIH-3T3 fibroblasts TGF beta causes a sustained fos RNA expression, which is not associated, however, with detectable fos protein. We conclude that in fibroblasts stimulated by mitogens c-fos expression may be differentially modulated, depending of the growth factor and the cell line. This is seemingly due to differential regulation of fos gene expression, not only at the transcriptional and/or post-transcriptional level (transient or sustained fos RNA induction by EGF or TGF beta in EL2 cells), but also at the translational level (fos protein(s) induction by TGF beta in EL2 but not NIH-3T3 fibroblasts, possibly related to the stimulatory vs inhibitory effect of this factor on the growth of the former vs the latter line).
Assuntos
Divisão Celular/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Substâncias de Crescimento/farmacologia , Peptídeos/farmacologia , Proteínas Proto-Oncogênicas/genética , Animais , Linhagem Celular , DNA/biossíntese , DNA/efeitos dos fármacos , Proteínas de Ligação a DNA/genética , Fator de Crescimento Epidérmico/farmacologia , Fibroblastos , Genes/efeitos dos fármacos , Imunoensaio , Cinética , Camundongos , Hibridização de Ácido Nucleico , Proteínas Proto-Oncogênicas/biossíntese , Proteínas Proto-Oncogênicas c-fos , RNA Mensageiro/biossíntese , Ratos , Fatores de Crescimento TransformadoresRESUMO
Exposure of a number of quiescent murine and human cell lines to low-graded doses of cycloheximide (CXM) results in a pattern of protein synthesis consisting of enhanced and induced species. This pattern is reminiscent of but not identical to that observed after several stress treatments [V. Sorrentino et al. (1985) J. Cell. Physiol. 125, 313]. A pattern identical to that seen after exposure to CXM is synthesized when cells are exposed to an hypertonic growth medium resulting in a full and reversible block of the initiation of polypeptide chain. This suggests that this kind of response is triggered by a reduction of overall protein synthesis rather than by a slow-down of the elongation step. Analysis of the synthesis of histones and ribosomal proteins during these two nonphysiological treatments (CXM or high salt) shows that these classes of proteins are neither stimulated nor preferentially retained. In contrast, greatly enhanced levels of steady-state histone mRNAs have been observed which have been translated in a reticulocyte lysate system, but are not apparently translated in vivo.
Assuntos
Biossíntese de Proteínas , Solução Salina Hipertônica/farmacologia , Cloreto de Sódio/farmacologia , Animais , Linhagem Celular , Cicloeximida/farmacologia , Histonas/biossíntese , Humanos , Ponto Isoelétrico , Cinética , Camundongos , Peso Molecular , RNA Mensageiro/metabolismo , Proteínas Ribossômicas/biossíntese , Equilíbrio HidroeletrolíticoRESUMO
The irreversible inhibition of overall protein synthesis induced by diphtheria toxin in mammalian cells is accompanied by a peculiar pattern of synthesized protein species during the early stages of cell intoxication. This pattern closely resembles patterns of synthesized protein species observed in cells treated with reversible inhibitors of protein synthesis acting at different levels. Synthesized proteins are in the Mr range of 70-100 and 20-40 kDa. A 50-kDa protein, the most represented species, exhibits a peptide map identical to that of the 50-kDa species observed in cells treated with cycloheximide and hypertonic medium. The synthesis of these proteins is apparently regulated at both transcriptional and translational levels.
Assuntos
Toxina Diftérica/farmacologia , Biossíntese de Proteínas , Relação Dose-Resposta a Droga , Regulação da Expressão Gênica/efeitos dos fármacos , Células HeLa , Humanos , Peso Molecular , Mapeamento de Peptídeos , Polirribossomos/metabolismo , RNA Mensageiro/genéticaRESUMO
Whether ribosomal-protein synthesis in Xenopus laevis is autogenously controlled at the translational level as is known to occur in prokaryotes has been studied. For this purpose ribosomal (r) proteins were prepared from X. laevis ribosomal subunits and group fractionated by ion-exchange chromatography. They were then added to an in vitro translation system directed by an oocyte mRNA fraction which contains template activity for r proteins. The synthesized radioactive products were analyzed by 2D gel electrophoresis and compared with controls. Similarly in vivo experiments were performed by microinjection of the fractionated proteins into the cytoplasm of Xenopus oocytes followed by incubation with [35S]methionine for different times. In all the experiments no evident effect of r proteins on the translation of their own mRNA was observed.
Assuntos
Proteínas Ribossômicas/biossíntese , Xenopus laevis/embriologia , Animais , Sistema Livre de Células , Regulação da Expressão Gênica , Biossíntese de Proteínas , Proteínas Ribossômicas/genéticaRESUMO
Infection with the hepatitis C virus (HCV) is the major cause of non-A, non-B hepatitis worldwide. The viral genome, a positive-sense, single-stranded, 9.6-kb long RNA molecule, is translated into a single polyprotein of about 3,000 amino acids. The viral polyprotein is proteoytically processed to yield all the mature viral gene products. The genomic order of HCV has been determined to be C-->E1-->E2-->p7-->NS2-->NS3-->NS4A-->NS4B-->NS5A++ +-->NS5B. C, E1, and E2 are the virion structural proteins. Whereas the function of p7 is currently unknown, NS2 to NS5B are thought to be the nonstructural proteins. Generation of the mature nonstructural proteins relies on the activity of viral proteinases. Cleavage at the NS2-NS3 junction is accomplished by a metal-dependent autocatalytic proteinase encoded within NS2 and the N-terminus of NS3. The remaining downstream cleavages are effected by a serine proteinase contained also within the N-terminal region of NS3. NS3, in addition, contains an RNA helicase domain at its C-terminus. NS3 forms a heterodimeric complex with NS4A. The latter is a membrane protein that acts as a cofactor of the proteinase. Although no function has yet been attributed to NS4B, NS5A has been recently suggested to be involved in mediating the resistance of the HCV to the action of interferon. Finally, the NS5B protein has been shown to be the viral RNA-dependent RNA polymerase. This article reviews the current understanding of the structure and the function of the various HCV nonstructural proteins with particular emphasis on their potential as targets for the development of novel antiviral agents and vaccines.
Assuntos
Antivirais/farmacologia , Hepacivirus/imunologia , Vacinas contra Hepatite Viral/farmacologia , Proteínas não Estruturais Virais/imunologia , Proteínas Virais/imunologia , Hepacivirus/efeitos dos fármacos , Humanos , RNA Viral/fisiologia , Sensibilidade e Especificidade , Proteínas não Estruturais Virais/química , Proteínas não Estruturais Virais/efeitos dos fármacos , Proteínas Virais/química , Proteínas Virais/efeitos dos fármacosRESUMO
The hepatitis C virus is the major causative agent of nonA-nonB hepatitis worldwide. Although this virus cannot be cultivated in cell culture, several of its features have been elucidated in the past few years. The viral genome is a single-stranded, 9.5kb long RNA molecule of positive polarity. The viral genome is translated into a single polyprotein of about 3000 amino acids. The virally encoded polyprotein undergoes proteolytic processing by a combination of cellular and viral proteolytic enzymes in order to yield all the mature viral gene products. The gene order of HCV has been determined to be C-E1-E2-p7-NS2-NS3-NS4A-NS4B-NS5A-NS5B. The mature structural proteins, C, E1 and E2 have been shown to arise from the viral polyprotein via proteolytic processing by host signal peptidases. Conversely, generation of the mature nonstructural proteins relies on the activity of viral proteases. Thus, cleavage at the NS2/NS3 junction is accomplished by a metal-dependent autoprotease encoded within NS2 and the N-terminus of NS3. The remaining cleavages downstream from this site are effected by a serine protease contained within the N-terminal region of NS3. Besides the protease domain, NS3 also contains an RNA helicase domain at its C-terminus. NS3 forms a heterodimeric complex with NS4A. The latter is a membrane protein that has been shown to act as a cofactor of the protease. Whereas the NS5B protein has been shown to be the viral RNA-dependent RNA polymerase, no function has yet been attributed to NS4B and NS5A. The latter is a cytoplasmic phosphoprotein and appears to be involved in mediating the resistance of the hepatitis C virus to the action of interferon.