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The aim of this letter is to share the discussions and proposals made by the VAC2VAC consortium on how to support the deployment of the "Consistency Approach" for quality control of established vaccines and thus facilitate the substitution of in vivo testing. This work answers specific questions about " what does a control strategy according to the consistency testing look like" and " how to submit a control strategy defined according to the consistency testing". Some topics were answered in a very straightforward manner. This was the case when the deployment of the consistency approach and the corresponding changes in vaccines control strategy was supported by the generic application of procedures already described in regulatory guidelines/requirements and related to the establishment or change in the control strategy of vaccines. The application of other procedures required more specific attention and some were deeply debated before reaching a proposal. The key outcomes of this work are that robust science must be used to develop a substitution strategy and generate supportive data packages. And this good science can best occur with good scientific collaboration between the different parties involved. Therefore, early interaction between manufacturers and competent authorities before and during dossier submission is critical to success. The consistency approach, when approved and in place, will ensure vaccine products of assured quality reach the patient in a more efficient manner than when relying on in vivo testing. Adapting the mindset was one of the major hurdles to a progressive vision but there is now consensus between manufacturers and competent authorities to foster the elimination of in vivo testing for routine vaccine release testing.
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The tetanus neurotoxin (TeNT) is one of the most toxic proteins known to man, which prior to the use of the vaccine against the TeNT producing bacteria Clostridium tetani, resulted in a 20% mortality rate upon infection. The clinical detrimental effects of tetanus have decreased immensely since the introduction of global vaccination programs, which depend on sustainable vaccine production. One of the major critical points in the manufacturing of these vaccines is the stable and reproducible production of high levels of toxin by the bacterial seed strains. In order to minimize time loss, the amount of TeNT is often monitored during and at the end of the bacterial culturing. The different methods that are currently available to assess the amount of TeNT in the bacterial medium suffer from variability, lack of sensitivity, and/or require specific antibodies. In accordance with the consistency approach and the three Rs (3Rs), both aiming to reduce the use of animals for testing, in-process monitoring of TeNT production could benefit from animal and antibody-free analytical tools. In this paper, we describe the development and validation of a new and reliable antibody free targeted LC-MS/MS method that is able to identify and quantify the amount of TeNT present in the bacterial medium during the different production time points up to the harvesting of the TeNT just prior to further upstream purification and detoxification. The quantitation method, validated according to ICH guidelines and by the application of the total error approach, was utilized to assess the amount of TeNT present in the cell culture medium of two TeNT production batches during different steps in the vaccine production process prior to the generation of the toxoid. The amount of TeNT generated under different physical stress conditions applied during bacterial culture was also monitored.
Assuntos
Espectrometria de Massas em Tandem , Toxina Tetânica , Técnicas Bacteriológicas , Cromatografia Líquida , Metaloendopeptidases , Toxina Tetânica/análiseRESUMO
Transition to in vitro alternative methods from in vivo in vaccine release testing and characterization, the implementation of the consistency approach, and a drive towards international harmonization of regulatory requirements are most pressing needs in the field of vaccines. It is critical for global vaccine community to work together to secure effective progress towards animal welfare and to ensure that vaccines of ever higher quality can reach the populations in need in the shortest possible timeframe. Advancements in the field, case studies, and experiences from Low and Middle Income Countries (LMIC) were the topics discussed by an international gathering of experts during a recent conference titled "Animal Testing for Vaccines - Implementing Replacement, Reduction and Refinement: Challenges and Priorities". This conference was organized by the International Alliance for Biological Standardization (IABS), and held in Bangkok, Thailand on December 3 and 4 2019. Participants comprised stakeholders from many parts of the world, including vaccine developers, manufacturers and regulators from Asia, Europe, North America, Australia and New Zealand. In interactive workshops and vibrant panel discussions, the attendees worked together to identify the remaining barriers to validation, acceptance and implementation of alternative methods, and how harmonization could be promoted, especially for LMICs.
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Alternativas aos Testes com Animais/métodos , Vacinação/métodos , Vacinas/administração & dosagem , Vacinas/imunologia , Alternativas aos Testes com Animais/normas , Bem-Estar do Animal/normas , Animais , Humanos , Controle de QualidadeRESUMO
Within the Innovative Medicines Initiative 2 (IMI 2) project VAC2VAC (Vaccine batch to vaccine batch comparison by consistency testing), a workshop has been organised to discuss ways of improving the design of multi-centre validation studies and use the data generated for product-specific validation purposes. Moreover, aspects of validation within the consistency approach context were addressed. This report summarises the discussions and outlines the conclusions and recommendations agreed on by the workshop participants.
Assuntos
Conferências de Consenso como Assunto , Estudos Multicêntricos como Assunto , Guias de Prática Clínica como Assunto , Vacinas/uso terapêutico , Estudos de Validação como Assunto , HumanosRESUMO
TIA-1-related (TIAR) protein is a shuttling RNA-binding protein implicated in several steps of RNA metabolism. In the nucleus, TIAR contributes to alternative splicing events, whereas, in the cytoplasm, it acts as a translational repressor on specific transcripts such as adenine and uridine-rich element-containing mRNAs. In addition, TIAR is involved in the general translational arrest observed in cells exposed to environmental stress. This activity is encountered by the ability of TIAR to assemble abortive pre-initiation complexes coalescing into cytoplasmic granules called stress granules. To elucidate these mechanisms of translational repression, we characterized TIAR-containing complexes by tandem affinity purification followed by MS. Amongst the identified proteins, we found the splicing factor ASF/SF2, which is also present in TIA-1 protein complexes. We show that, although mostly confined in the nuclei of normal cells, ASF/SF2 migrates into stress granules upon environmental stress. The migration of ASF/SF2 into stress granules is strictly determined both by its shuttling properties and its RNA-binding capacity. Our data also indicate that ASF/SF2 down-regulates the expression of a reporter mRNA carrying adenine and uridine-rich elements within its 3' UTR. Moreover, tethering of ASF/SF2 to a reporter transcript strongly reduces mRNA translation and stability. These results indicate that ASF/SF2 and TIA proteins cooperate in the regulation of mRNA metabolism in normal cells and in cells having to overcome environmental stress conditions. In addition, the present study provides new insights into the cytoplasmic function of ASF/SF2 and highlights mechanisms by which RNA-binding proteins regulate the diverse steps of RNA metabolism by subcellular relocalization upon extracellular stimuli.
Assuntos
Proteínas de Ligação a Poli(A)/metabolismo , Proteínas de Ligação a RNA/metabolismo , Células 3T3 , Animais , Sítios de Ligação , Células COS , Proteínas de Ligação a Calmodulina/metabolismo , Linhagem Celular , Chlorocebus aethiops , Vaga-Lumes , Imunoglobulinas/metabolismo , Luciferases/metabolismo , Mamíferos , Proteínas de Membrana/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Camundongos , Proteínas de Ligação a Poli(A)/genética , Proteínas de Ligação a RNA/genética , Ratos , Proteínas Recombinantes de Fusão/metabolismo , Antígeno-1 Intracelular de Células T , TransfecçãoRESUMO
During post-harvest storage, potato tubers age as they undergo an evolution of their physiological state influencing their sprouting pattern. In the present study, physiological and biochemical approaches were combined to provide new insights on potato (Solanum tuberosum L. cv. Désirée) tuber ageing. An increase in the physiological age index (PAI) value from 0.14 to 0.83 occurred during storage at 4 degrees C over 270 d. Using this reference frame, a proteomic approach was followed based on two-dimensional electrophoresis. In the experimental conditions of this study, a marked proteolysis of patatin occurred after the PAI reached a value of 0.6. In parallel, several glycolytic enzymes were up-regulated and cellular components influencing protein conformation and the response to stress were altered. The equilibrium between the 20S and 26S forms of the proteasome was modified, the 20S form that recycles oxidized proteins being up-regulated. Two proteins belonging to the cytoskeleton were also differentially expressed during ageing. As most of these changes are also observed in an oxidative stress context, an approach focused on antioxidant compounds and enzymes as well as oxidative damage on polyunsaturated fatty acids and proteins was conducted. All the changes observed during ageing seemed to allow the potato tubers to maintain their radical scavenging activity until the end of the storage period as no accumulation of oxidative damage was observed. These data are interpreted considering the impact of reactive oxygen species on the development and the behaviour of other plant systems undergoing ageing or senescence processes.
Assuntos
Antioxidantes/metabolismo , Tubérculos/crescimento & desenvolvimento , Tubérculos/metabolismo , Proteoma/metabolismo , Solanum tuberosum/crescimento & desenvolvimento , Solanum tuberosum/metabolismo , Ascorbato Peroxidases , Catalase/metabolismo , Regulação para Baixo , Eletroforese em Gel Bidimensional , Esterificação , Sequestradores de Radicais Livres/metabolismo , Glutationa/metabolismo , Cinética , Oxilipinas/metabolismo , Peroxidases/metabolismo , Fenóis/metabolismo , Tubérculos/enzimologia , Carbonilação Proteica , Solanum tuberosum/enzimologia , Superóxido Dismutase/metabolismo , Fatores de Tempo , Regulação para CimaRESUMO
The phytopathogen Rhodococcus fascians induces the development of leafy gall, which is considered to be its ecological niche. To obtain a view of the metabolic changes occurring in R. fascians during this process, an in vitro system was used where bacteria are grown in the presence of a leafy gall extract, a condition mimicking that found by the bacteria in infected plants. Proteins of R. fascians grown for 24 h under these conditions were displayed by two-dimensional polyacrylamide gel electrophoresis. Fifteen polypeptides showing a differential accumulation in response to the inducing conditions were analyzed by mass spectrometry. Two polypeptides potentially linked to the Krebs cycle, a pyruvate dehydrogenase and a fumarate hydratase, were further characterized and shown to be downregulated at the transcriptional level. The identification of these two enzymes suggests that R. fascians may shift its metabolism during the interaction with plants from the Krebs cycle to the glyoxylate shunt.
Assuntos
Interações Hospedeiro-Patógeno , Nicotiana/química , Nicotiana/microbiologia , Extratos Vegetais/metabolismo , Rhodococcus/fisiologia , Proteínas de Bactérias/análise , DNA Bacteriano/química , DNA Bacteriano/genética , Eletroforese em Gel Bidimensional , Perfilação da Expressão Gênica , Espectrometria de Massas , Dados de Sequência Molecular , Extratos Vegetais/isolamento & purificação , Proteoma/análise , Rhodococcus/química , Rhodococcus/metabolismo , Análise de Sequência de DNARESUMO
The role of TGF-beta1 in hydrogen peroxide-induced senescence-like morphogenesis has been described. The aim of this work was to investigate whether TGF-beta1-independent changes in protein synthesis are involved in this morphogenesis and to study possible mechanisms occurring earlier than TGF-beta1 overexpression. Among the multiple TGF-beta1-independent changes in protein neosynthesis, followed or not by posttranslational modifications, identified by proteomic analysis herein, those of ezrin, L-caldesmon, and HSP27 were particularly studied. Rho-GTPase cdc42 was shown to be responsible for p38(MAPK) activation, in turn triggering phosphorylation of L-caldesmon and HSP27. Cdc42 was also shown to be mainly responsible for the increase in TGF-beta1 mRNA level observed at 24 h after treatment with H(2)O(2) and onward. This study further clarified the mechanisms of senescence-like morphogenesis in addition to the previously demonstrated role of TGF-beta1 signaling pathways.
Assuntos
Fibroblastos/metabolismo , Peróxido de Hidrogênio/farmacologia , Fator de Crescimento Transformador beta1/metabolismo , Proteína cdc42 de Ligação ao GTP/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Proteínas de Ligação a Calmodulina/farmacologia , Sobrevivência Celular , Senescência Celular , Eletroforese em Gel Bidimensional , Radicais Livres , Humanos , Peróxido de Hidrogênio/química , Modelos Biológicos , Estresse Oxidativo , Fenótipo , FosforilaçãoRESUMO
Understanding the virus-host interactions that lead to approximately 20% of patients with acute Hepatitis C Virus (HCV) infection to viral clearance is probably a key towards the development of more effective treatment and prevention strategies. Acute hepatitis C infection is usually asymptomatic and therefore rarely diagnosed. Nevertheless, HCV nucleic acid testing carried out on all blood donations detects donors who have resolved their HCV infection after seroconversion. Here we have used SELDI-TOF-MS technology to compare, at a proteomic level, plasma samples respectively from donors with HCV clearance, from donors with chronic HCV infection and from unexposed healthy donors (nâ =â 15 per group). A candidate marker of about 9.4â kDa was detected as differentially expressed in the three groups. After purification we identified by nanoLC-Q-TOF-MS/MS this candidate marker as Apolipoprotein C-III (ApoC-III). The identification was confirmed by western blot analysis. Levels of ApoC-III were then determined in the 45 plasma samples by immunoturbidimetric assay. ApoC-III was found to be higher in donors who had resolved their HCV infection than in donors with chronic infection, results which were consistent with SELDI-TOF-MS data. ApoC-III is the first reported candidate biomarker in plasma associated with the spontaneous resolution of HCV infection.
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Trypanosomes are protozoans showing unique transcription characteristics. We describe in Trypanosoma brucei a complex homologous to TFIIH, a multisubunit transcription factor involved in the control of transcription by RNA Pol I and RNA Pol II, but also in DNA repair and cell cycle control. Bioinformatics analyses allowed the detection of five genes encoding four putative core TFIIH subunits (TbXPD, TbXPB, Tbp44, Tbp52), including a novel XPB variant, TbXPBz. In all cases sequences known to be important for TFIIH functions were conserved. We performed a molecular analysis of this core complex, focusing on the two subunits endowed with a known enzymatic (helicase) activity, XPD and XPB. The involvement of these T. brucei proteins in a bona fide TFIIH core complex was supported by (i) colocalization by immunofluorescence in the nucleus, (ii) direct physical interaction of TbXPD and its interacting regulatory subunit Tbp44 as determined by double-hybrid assay and tandem affinity purification of the core TFIIH, (iii) involvement of the core proteins in a high molecular weight complex and (iv) occurrence of transcription, cell cycle and DNA repair phenotypes upon either RNAi knock-down or overexpression of essential subunits.
Assuntos
Fator de Transcrição TFIIH/química , Fator de Transcrição TFIIH/metabolismo , Trypanosoma brucei brucei/genética , Sequência de Aminoácidos , Animais , Animais Geneticamente Modificados , Sítios de Ligação , Núcleo Celular/metabolismo , Sequência Conservada , DNA Helicases , Reparo do DNA , Dados de Sequência Molecular , Ligação Proteica , Estrutura Terciária de Proteína , Subunidades Proteicas/química , Subunidades Proteicas/genética , Interferência de RNA , Homologia de Sequência de Aminoácidos , Fator de Transcrição TFIIH/genética , Transcrição Gênica , Técnicas do Sistema de Duplo-HíbridoRESUMO
Two protein extraction procedures were tested in order to remove interfering compounds prior to 2-DE of potato tubers. These methods using SDS lysis buffer and phenol-phase extraction were compared regarding the quality of the resulting 2-D gel. While the resolution of SDS extracts on semipreparative gels seems better, both methods lead to similar extraction yields and total number of spots. The procedures are complementary regarding the Mr range of preferentially extracted proteins.
Assuntos
Eletroforese em Gel Bidimensional/métodos , Proteínas de Plantas/isolamento & purificação , Proteômica/métodos , Solanum tuberosum/química , Concentração de Íons de Hidrogênio , Fenol , Proteínas de Plantas/química , Tubérculos/química , Dodecilsulfato de SódioRESUMO
The Trypanosoma brucei homolog of the RNA polymerase II (RNA Pol II) subunit RPB9 was cloned and characterized. Contrary to what occurs in Saccharomyces cerevisiae, in T. brucei this protein was found to be essential since the knock down of its expression by RNAi led to lethality in both bloodstream and procyclic forms of the parasite. As expected, TbRPB9 knock down specifically inhibited transcription by RNA Pol II, but not by RNA Pol I and III. TbRPB9 was used as bait to isolate the RNA Pol II core complex by tandem affinity purification. Nine subunits homologous to the other eukaryotic RNA Pol II, namely RPB1, RPB2, RPB3, RPB4, RPB5, RPB6, RPB7, RPB8 and RPB11, were identified in the purified complex. Interestingly, the RPB5 homolog associated with RNA Pol II was different from the one previously found in RNA Pol I. Analysis of the genome database revealed the presence of genes for all purified subunits plus RPB10. As in the case of TbRPB5, two genes coding for different isoforms of TbRPB6 were identified, suggesting the existence of polymerase-specific isoforms for both TbRPB5 and TbRPB6.
Assuntos
Subunidades Proteicas/genética , Proteínas de Protozoários/genética , RNA Polimerase II/genética , Transcrição Gênica , Trypanosoma brucei brucei/enzimologia , Sequência de Aminoácidos , Animais , Clonagem Molecular , Genes de Protozoários , Dados de Sequência Molecular , Alinhamento de Sequência , Trypanosoma brucei brucei/genéticaRESUMO
Using a proteomic approach, we characterized different protein expression profiles in anterior gills of the Chinese mitten crab, Eriocheir sinensis, after cadmium (Cd) exposure. Two experimental conditions were tested: (i) an acute exposure (i.e. 500 microg Cd l(-1) for 3 days) for which physiological, biochemical and ultrastructural damage have been observed previously; (ii) a chronic exposure (i.e. 50 microg Cd l(-1) for 30 days) resulting in physiological acclimation, i.e. increased resistance to a subsequent acute exposure. Two-dimensional gel electrophoresis (2-DE) revealed six protein spots differentially expressed after acute, and 31 after chronic Cd exposure. From these spots, 15 protein species were identified using MS/MS micro-sequencing and MS BLAST database searches. Alpha tubulin, glutathione S-transferase and crustacean calcium-binding protein 23 were down-regulated after an acute exposure, whereas another glutathione S-transferase isoform was up-regulated. Furthermore, analyses revealed the over-expression of protein disulfide isomerase, thioredoxin peroxidase, glutathione S-transferase, a proteasome subunit and cathepsin D after chronic exposure. Under the same condition, ATP synthase beta, alpha tubulin, arginine kinase, glyceraldehyde-3-phosphate dehydrogenase and malate dehydrogenase were down-regulated. These results demonstrate that acute and chronic exposure to waterborne Cd induced different responses at the protein expression level. Protein identification supports the idea that Cd mainly exerts its toxicity through oxidative stress induction and sulfhydryl-group binding. As a result, analyses showed the up-regulation of several antioxidant enzymes and chaperonins during acclimation process. The gill proteolytic capacity seems also to be increased. On the other hand, the clearly decreased abundance of several enzymes involved in energy transfer suggests that chronic metal exposure induced an important metabolic reshuffling.
Assuntos
Aclimatação/fisiologia , Braquiúros/efeitos dos fármacos , Braquiúros/fisiologia , Cádmio/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Animais , Regulação para Baixo/fisiologia , Exposição Ambiental , Perfilação da Expressão Gênica , Brânquias/efeitos dos fármacos , Brânquias/fisiologia , Espectrometria de Massas/veterinária , Estresse Oxidativo/fisiologia , Consumo de Oxigênio/efeitos dos fármacos , Proteômica/métodos , Regulação para Cima/fisiologia , Poluentes Químicos da Água/farmacologiaRESUMO
(R)-Roscovitine (CYC202) is often referred to as a "selective inhibitor of cyclin-dependent kinases." Besides its use as a biological tool in cell cycle, neuronal functions, and apoptosis studies, it is currently evaluated as a potential drug to treat cancers, neurodegenerative diseases, viral infections, and glomerulonephritis. We have investigated the selectivity of (R)-roscovitine using three different methods: 1) testing on a wide panel of purified kinases that, along with previously published data, now reaches 151 kinases; 2) identifying roscovitine-binding proteins from various tissue and cell types following their affinity chromatography purification on immobilized roscovitine; 3) investigating the effects of roscovitine on cells deprived of one of its targets, CDK2. Altogether, the results show that (R)-roscovitine is rather selective for CDKs, in fact most kinases are not affected. However, it binds an unexpected, non-protein kinase target, pyridoxal kinase, the enzyme responsible for phosphorylation and activation of vitamin B6. These results could help in interpreting the cellular actions of (R)-roscovitine but also in guiding the synthesis of more selective roscovitine analogs.
Assuntos
Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/metabolismo , Proteínas Quinases/metabolismo , Purinas/química , Purinas/metabolismo , Piridoxal Quinase/metabolismo , Trifosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Animais , Ciclo Celular/fisiologia , Sobrevivência Celular , Células Cultivadas , Cromatografia de Afinidade , Fibroblastos/citologia , Fibroblastos/fisiologia , Humanos , Camundongos , Camundongos Knockout , Modelos Moleculares , Conformação Molecular , Dados de Sequência Molecular , Estrutura Molecular , Estrutura Terciária de Proteína , Piridoxal/metabolismo , Piridoxal Quinase/antagonistas & inibidores , Piridoxal Quinase/genética , Fosfato de Piridoxal/metabolismo , Ratos , Roscovitina , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Distribuição TecidualRESUMO
The Trypanosoma brucei homologue of the RNA polymerase I (RNA Pol I) subunit Rpa12p of Saccharomyces cerevisiae was cloned and characterized. This protein did not appear to be essential for growth in either bloodstream or procyclic forms of the parasite. Trypanosomes expressing a C-terminal tagged version of TbRPA12 were generated in order to purify RNA Pol I from both developmental stages. Tandem affinity purification (TAP) revealed a number of proteins associating with TbRPA12, some of which appeared to be stage-specific. Mass spectrometry allowed the identification of four subunits in addition to TbRPA12, namely TbRPA1, TbRPA2, TbRPC40 and one isoform of TbRPB5 (Tb1RPB5), as well as an unknown 30kDa protein and histones H2A and H3. Whereas these studies demonstrated that TbRPA1 was phosphorylated, no evidence for phosphorylation of TbRPA2 was found.
Assuntos
Subunidades Proteicas , RNA Polimerase I , Trypanosoma brucei brucei/enzimologia , Sequência de Aminoácidos , Animais , Variação Antigênica , Regulação da Expressão Gênica no Desenvolvimento , Estágios do Ciclo de Vida , Dados de Sequência Molecular , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , RNA Polimerase I/química , RNA Polimerase I/genética , RNA Polimerase I/metabolismo , Proteínas Recombinantes , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Alinhamento de Sequência , Trypanosoma brucei brucei/genética , Trypanosoma brucei brucei/crescimento & desenvolvimentoRESUMO
The O-antigen of the gram negative bacteria Brucella is composed of an homopolymer of 4,6-dideoxy-4-formamido-alpha-D-mannopyranosyl (or perosamine). Several mAb interact specifically with only the O-antigen of certain Brucella species. Although, many studies show that this specific recognition results mainly from the ratios of alpha 1-2 and alpha 1-3 link between the different Brucella strain perosamine residues, little is known about the mAb recognising this O-antigen. In this paper, we describe the binding profile of five anti-Brucella O-antigen mAb to the LPS of two Brucella strains and a bacteria possessing a nearly identical O-antigen: Yersinia enterocolitica 0:9. We show that the specificity of these five mAb can be correlated to their germ line gene usage. Besides, their relative affinity to the different LPS is correlated to their ability to protect against Brucella infection by passive transfer in a mouse model. The analysis of their 3D structure gives new hypothesis of the epitopes recognised.
Assuntos
Anticorpos Antibacterianos/imunologia , Anticorpos Monoclonais/imunologia , Brucella/imunologia , Lipopolissacarídeos/imunologia , Antígenos O/imunologia , Sequência de Aminoácidos , Animais , Anticorpos Antibacterianos/química , Anticorpos Antibacterianos/genética , Anticorpos Monoclonais/química , Anticorpos Monoclonais/genética , Feminino , Camundongos , Dados de Sequência MolecularRESUMO
In this work, stable overexpression of peroxiredoxin VI was generated in WI-38 human diploid fibroblasts using a retrovirus-mediated transfection system. Estimation of cell survival showed that peroxiredoxin VI provides a significant protection against tert-butylhydroperoxide- or UVB-caused cytotoxicity. No protection was found against ethanol- or H(2)O(2)-caused cytotoxicity. These effects are correlated with the known functions of Prx VI.
Assuntos
Fibroblastos/citologia , Fibroblastos/fisiologia , Peroxidases/genética , Retroviridae/genética , terc-Butil Hidroperóxido/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos da radiação , Células Cultivadas , Senescência Celular/efeitos dos fármacos , Diploide , Etanol/farmacologia , Regulação Enzimológica da Expressão Gênica , Regulação Viral da Expressão Gênica , Humanos , Peróxido de Hidrogênio/farmacologia , Oxidantes/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Peroxirredoxina VI , Peroxirredoxinas , Solventes/farmacologia , Transfecção , Raios UltravioletaRESUMO
Exposure of human proliferative cells to subcytotoxic stress triggers stress-induced premature senescence (SIPS) which is characterized by many biomarkers of replicative senescence. Proteomic comparison of replicative senescence and stress-induced premature senescence indicates that, at the level of protein expression, stress-induced premature senescence and replicative senescence are different phenotypes sharing however similarities. In this study, we identified 30 proteins showing changes of expression level specific or common to replicative senescence and/or stress-induced premature senescence. These changes affect different cell functions, including energy metabolism, defense systems, maintenance of the redox potential, cell morphology and transduction pathways.
Assuntos
Senescência Celular/fisiologia , Proteínas/química , Sequência de Bases , Western Blotting , Linhagem Celular , Primers do DNA , Eletroforese em Gel Bidimensional , Proteínas/fisiologia , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
In this paper, we illustrate how a proteomic analysis can be useful to approach complex biological problems, in this case the concept of stress-induced premature senescence (SIPS). According to the stochastic theories of ageing, damage that accumulate with time in the cellular components are responsible for cellular ageing. As a corollary, some sort of premature senescence should appear if the damage level is artificially increased due to the presence of stressing agents at subcytotoxic level. It has been shown, in several different models, that at a long-term after subcytotoxic stresses of many different natures, human diploid fibroblasts (HDFs) display biomarkers of replicative senescence (RS), which led to the concept of SIPS as compared to telomere-dependent RS. We compared RS and SIPS of HDFs by proteome analysis. SIPS was induced by two very different stressors: tert-butyhydroperoxide or ethanol. First, only a part of the protein expression changes observed in RS were also observed in SIPS. Second, HDFs in SIPS show changes specific either to the long-term effects of t-BHP or ethanol or independent of the nature of the stress. These changes have been termed "molecular scars" of subcytotoxic stresses. This work is also an excellent opportunity to discuss on important methodological issue in proteomics: the absolute requirement to start from reliable and reproducible models, which was the case in this study. We also focus on the data handling and statistical analysis allowing to use two-dimensional gel electrophoresis patterns in a semi-quantitative analysis.
Assuntos
Senilidade Prematura/metabolismo , Senescência Celular/fisiologia , Proteoma/análise , Estresse Fisiológico/metabolismo , Eletroforese em Gel Bidimensional/métodos , Humanos , FenótipoRESUMO
The Hayflick limit-senescence of proliferative cell types-is a fundamental feature of proliferative cells in vitro. Various human proliferative cell types exposed in vitro to many types of subcytotoxic stresses undergo stress-induced premature senescence (SIPS) (also called stress-induced premature senescence-like phenotype, according to the definition of senescence). The known mechanisms of appearance the main features of SIPS are reviewed: senescent-like morphology, growth arrest, senescence-related changes in gene expression, telomere shortening. Long before telomere-shortening induces senescence, other factors such as culture conditions or lack of 'feeder cells' can trigger either SIPS or prolonged reversible G(0) phase of the cell cycle. In vivo, 'proliferative' cell types of aged individuals are likely to compose a mosaic made of cells irreversibly growth arrested or not. The higher level of stress to which these cells have been exposed throughout their life span, the higher proportion of the cells of this mosaic will be in SIPS rather than in telomere-shortening dependent senescence. All cell types undergoing SIPS in vivo, most notably the ones in stressful conditions, are likely to participate in the tissular changes observed along ageing. For instance, human diploid fibroblasts (HDFs) exposed in vivo and in vitro to pro-inflammatory cytokines display biomarkers of senescence and might participate in the degradation of the extracellular matrix observed in ageing.