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The French Armed Forces Biomedical Research Institute (IRBA) deeply involved in research on SARS-COV-2, participated in the creation of the Obépine sentinel network in charge of detecting, qualifying and quantifying the virus genome in wastewater in France. During this pandemic, wastewater-based epidemiology has proven to be a first class public health tool for assessing viral dynamics in populations and environment. Obépine has also conducted research demonstrating the low infectivity of faeces and wastewater and allowed for early detection of epidemic waves linked to new variants. The IRBA has adapted this powerful tool to the monitoring of viral infections on board the aircraft carrier Charles-de-Gaulle in order to get an operational system for anticipation after the first local outbreak in 2020. The presence of this surveillance and anticipation tool has allowed a better management of SARS-CoV-2 contingent introductions on board during stopovers or crewmembers entries. The combination of a mandatory vaccination protocol and the surveillance of viral circulation in black waters has made it possible to identify and locate cases, and thus to continue the operational mission in the COVID-19 environment while limiting the spread and preserving the health of the crew. This innovative tool can easily be redirected to the search for any other pathogens in blackwater or even, in the long term, to ensure health surveillance of any military establishment, at sea or on land, in France or on overseas bases.
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IntroductionSevere acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the etiological agent of coronavirus disease (COVID-19). People infected with SARS-CoV-2 may exhibit no or mild non-specific symptoms; thus, they may contribute to silent circulation of the virus among humans. Since SARS-CoV-2 RNA can be detected in stool samples, monitoring SARS-CoV-2 RNA in waste water (WW) has been proposed as a complementary tool to investigate virus circulation in human populations.AimTo test if the quantification of SARS-CoV-2 genomes in WW correlates with the number of symptomatic or non-symptomatic carriers.MethodWe performed a time-course quantitative analysis of SARS-CoV-2 by RT-qPCR in raw WW samples collected from several major WW treatment plants in Greater Paris. The study period was 5 March to 23 April 2020, including the lockdown period in France (from 17 March).ResultsWe showed that the increase of genome units in raw WW accurately followed the increase of human COVID-19 cases observed at the regional level. Of note, the viral genome could be detected before the epidemic grew massively (around 8 March). Equally importantly, a marked decrease in the quantities of genome units was observed concomitantly with the reduction in the number of new COVID-19 cases, 29 days following the lockdown.ConclusionThis work suggests that a quantitative monitoring of SARS-CoV-2 genomes in WW could generate important additional information for improved monitoring of SARS-CoV-2 circulation at local or regional levels and emphasises the role of WW-based epidemiology.
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COVID-19/epidemiologia , Controle de Doenças Transmissíveis/métodos , Genoma Viral , Distanciamento Físico , Quarentena , RNA Viral/análise , SARS-CoV-2/isolamento & purificação , Eliminação de Partículas Virais , Águas Residuárias/virologia , COVID-19/transmissão , Controle de Doenças Transmissíveis/estatística & dados numéricos , França , Humanos , Paris/epidemiologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Carga ViralRESUMO
Wastewater-based epidemiology is experiencing exponential development. Despite undeniable advantages compared to patient-centered approaches (cost, anonymity, survey of large populations without bias, detection of asymptomatic infected peoples ), major technical limitations persist. Among them is the low sensitivity of the current methods used for quantifying and sequencing viral genomes from wastewater. In situations of low viral circulation, during initial stages of viral emergences, or in areas experiencing heavy rains, the extremely low concentrations of viruses in wastewater may fall below the limit of detection of the current methods. The availability during crisis and the cost of the commercial kits, as well as the requirement of expensive materials such as high-speed centrifuge, can also present major blocks to the development of wastewater-based epidemiological survey, specifically in low-income countries. Thereby, highly sensitive, low cost and standardized methods are still needed, to increase the predictability of the viral emergences, to survey low-circulating viruses and to make the results from different labs comparable. Here, we outline and characterize new protocols for concentrating and quantifying SARS-CoV-2 from large volumes (500 mL-1 L) of untreated wastewater. In addition, we report that the methods are applicable for monitoring and sequencing. Our nucleic acid extraction technique (the routine C: 5 mL method) does not require sophisticated equipment such as automatons and is not reliant on commercial kits, making it readily available to a broader range of laboratories for routine epidemiological survey. Furthermore, we demonstrate the efficiency, the repeatability, and the high sensitivity of a new membrane-based concentration method (MBC: 500 mL method) for enveloped (SARS-CoV-2) and non-enveloped (F-specific RNA phages of genogroup II / FRNAPH GGII) viruses. We show that the MBC method allows the quantification and the monitoring of viruses in wastewater with a significantly improved sensitivity compared to the routine C method. In contexts of low viral circulation, we report quantifications of SARS-CoV-2 in wastewater at concentrations as low as 40 genome copies per liter. In highly diluted samples collected in wastewater treatment plants of French Guiana, we confirmed the accuracy of the MBC method compared to the estimations done with the routine C method. Finally, we demonstrate that both the routine C method processing 5 mL and the MBC method processing 500 mL of untreated wastewater are both compatible with SARS-CoV-2 sequencing. We show that the quality of the sequence is correlated with the concentration of the extracted viral genome. Of note, the quality of the sequences obtained with some MBC processed wastewater was improved by dilutions or enzyme substitutions suggesting the presence of specific enzyme inhibitors in some wastewater. To the best of our knowledge, our MBC method is one of the first efficient, sensitive, and repeatable method characterized for SARS-CoV-2 quantification and sequencing from large volumes of wastewater.
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Ácidos Nucleicos , Águas Residuárias , Humanos , Genoma Viral , Genótipo , Laboratórios , RNA ViralRESUMO
SARS-CoV-2 is a coronavirus causing a globalized outbreak called COVID-19. SARS-CoV-2 transmission is associated with inhalation of contaminated respiratory droplets and could causes severe complications. Until today several "waves" of infections have been observed despite implementation of strict health policies. Decisions for such sanitary measures are based on population health monitoring. Unfortunately, for COVID-19, a significant proportion of individuals are asymptomatic but play a role in the virus transmission. To overcome these limitations, several strategies were developed including genome quantification in wastewater that could allow monitoring of the health status of population, since shedding of SARS-CoV-2 in patient stool is frequent. Wastewater-based epidemiology (WBE) was established and several countries implemented this approach to allow COVID-19 outbreak monitoring. In France, the OBEPINE project performed a quantitative analysis of SARS-CoV-2 in raw wastewater samples collected from major wastewater treatment plants (WWTP) since March 2020. In the greater Paris area 1101 samples (507 for five WWTP and 594 for sewer) were collected. This 16 months monitoring allows us to observe the outbreak dynamics. Comparison of WBE indicators with health data lead to several important observation; the good level of correlation with incidence rates, the average 3 days lead time, and the sensitivity (WBE change when incidence is > to 7/100000 inhabitants). We also compared the local monitoring (city level) with the regional monitoring, to help cluster identification. Moreover, variants of concern (VOC) emerged due to the selection pressure. We developed a specific RT-qPCR method targeting the deletion H69-V70 in the spike protein, using this deletion as a proxy of the B.1.1.7 presence in the wastewater. With this data we demonstrate the predominant role played by this strain in the third wave. All these results allow a better description and understanding of the pandemic and highlight the role of such WBE indicators.
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COVID-19 , SARS-CoV-2 , Surtos de Doenças , Humanos , Aerossóis e Gotículas Respiratórios , Águas ResiduáriasRESUMO
Like other pathogenic viruses, HIV-1 down-modulates surface expression of major histocompatibility complex class I (MHC-I) molecules in infected cells, thus impairing lysis by cytotoxic T lymphocytes. We have observed that this phenomenon depends on the expression of Nef. nef is an early gene of primate lentiviruses, which is necessary for maintaining high virus loads and inducing AIDS. Nef is not necessary for viral replication in vitro and stimulates the endocytosis of CD4. We show that the expression of MHC-I at the surface of lymphoid, monocytic and epithelial cells was reduced in the presence of Nef protein from various HIV-1 strains. Whereas MHC-I protein synthesis and transport through the endoplasmic reticulum and cis Golgi apparatus occurred normally in Nef(+) cells, surface MHC-I molecules were rapidly internalized, accumulated in endosomal vesicles and were degraded. The stimulation of MHC-I endocytosis by Nef represents a previously undocumented viral mechanism for evading the immune response.
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Endocitose/imunologia , Produtos do Gene nef/fisiologia , HIV-1/imunologia , Antígenos de Histocompatibilidade Classe I/metabolismo , Linhagem Celular , Regulação para Baixo , Produtos do Gene nef/genética , Produtos do Gene nef/imunologia , Genes nef , Infecções por HIV/imunologia , Infecções por HIV/virologia , HIV-1/genética , HIV-1/fisiologia , Humanos , Produtos do Gene nef do Vírus da Imunodeficiência HumanaRESUMO
INTRODUCTION: Stress cardiopathy, also called "Tako Tsubo" is a cardiac pathology linked to an acute coronary syndrome with electrocardiographic signs and an increase in the level of cardiac enzymes, without any abnormality on coronarography. This syndrome is secondary to great physical or mental stress. Mortality and the risk of recurrence are low. However, there is no consensus for treatment or prevention. CASE REPORT: We report the case of 75-year-old woman presenting recurrent cardiogenic shocks. A symptomatic sinusal dysfunction motivated the introduction of a pacemaker in March 2008. One month later, she was hospitalized for a new cardiogenic shock with left ventricular dysfunction, a 40% ejection fraction, and a third degree mitral insufficiency. Cardiac enzymes were initially elevated. Electrocardiogram showed an ST elevation. The transthoracic echocardiogram revealed a left anterolateral ventriculogram dysfunction, and cardiac catheterization showed healthy coronary arteries. The cardiologist wondered about the existence of an anxiodepressive syndrome. No personal psychiatric background was known. The patient was widowed 3 years earlier. She described herself as a naturally anxious person. She hadn't experienced any recent stressing event. She was not depressed and wasn't taking any psychotropic drug. Her family was caring for her. The next day, the patient had another cardiogenic shock and died a few hours later. Maybe the introduction of the pacemaker occasioned one more stress for this patient DISCUSSION: We know that people with a stressing job have probably more chance to suffer a myocardial infarction (the risks are 1.5 or two times greater for them). The prevalence of cardiomyopathy syndrome is 4.9% for women. These women have gone through the menopause, with a history of hypertension and anxiodepressive symptoms. However, we do not find any similar description (behavioural scheme type A) as is shown by the psychosomatic school in cases of patients who have gone through myocardial infarction. We also can question ourselves about the fact that some people can be predisposed to suffer from "Tako Tsubo" cardiomyopathy and about the existence of personality disorders. What then is the role of the psychiatrist with these patients?
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Síndrome Coronariana Aguda/psicologia , Síndrome Coronariana Aguda/terapia , Psiquiatria , Encaminhamento e Consulta , Cardiomiopatia de Takotsubo/psicologia , Cardiomiopatia de Takotsubo/terapia , Síndrome Coronariana Aguda/diagnóstico , Síndrome Coronariana Aguda/mortalidade , Idoso , Comportamento Cooperativo , Angiografia Coronária , Diagnóstico Diferencial , Ecocardiografia , Evolução Fatal , Feminino , Humanos , Comunicação Interdisciplinar , Marca-Passo Artificial/psicologia , Recidiva , Fatores de Risco , Choque Cardiogênico/diagnóstico , Choque Cardiogênico/psicologia , Choque Cardiogênico/terapia , Estresse Psicológico/complicações , Cardiomiopatia de Takotsubo/diagnóstico , Cardiomiopatia de Takotsubo/mortalidadeRESUMO
The ongoing global pandemic of coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been a public health emergency of international concern. Although SARS-CoV-2 is considered to be mainly transmitted by inhalation of contaminated droplets and aerosols, SARS-CoV-2 is also detected in human feces and to a less extent in urine, and in raw wastewaters (to date viral RNA only) suggesting that other routes of infection may exist. Monitoring SARS-CoV-2 genomes in wastewaters has been proposed as a complementary approach for tracing the dynamics of virus transmission within human population connected to wastewater network. The understanding on SARS-CoV-2 transmission through wastewater surveillance, the development of epidemic modeling and the evaluation of SARS-CoV-2 transmission from contaminated wastewater are largely limited by our knowledge on viral RNA genome persistence and virus infectivity preservation in such an environment. Using an integrity based RT-qPCR assay this study led to the discovery that SARS-CoV-2 RNA can persist under several forms in wastewaters, which provides important information on the presence of SARS-CoV-2 in raw wastewaters and associated risk assessment.
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COVID-19 , Vigilância Epidemiológica Baseada em Águas Residuárias , Humanos , RNA Viral , Medição de Risco , SARS-CoV-2 , Águas ResiduáriasRESUMO
The MDM2 oncoprotein is a cellular inhibitor of the p53 tumor suppressor in that it can bind the transactivation domain of p53 and downregulate its ability to activate transcription. In certain cancers, MDM2 amplification is a common event and contributes to the inactivation of p53. The crystal structure of the 109-residue amino-terminal domain of MDM2 bound to a 15-residue transactivation domain peptide of p53 revealed that MDM2 has a deep hydrophobic cleft on which the p53 peptide binds as an amphipathic alpha helix. The interface relies on the steric complementarity between the MDM2 cleft and the hydrophobic face of the p53 alpha helix and, in particular, on a triad of p53 amino acids-Phe19, Trp23, and Leu26-which insert deep into the MDM2 cleft. These same p53 residues are also involved in transactivation, supporting the hypothesis that MDM2 inactivates p53 by concealing its transactivation domain. The structure also suggests that the amphipathic alpha helix may be a common structural motif in the binding of a diverse family of transactivation factors to the TATA-binding protein-associated factors.
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Proteínas Nucleares , Conformação Proteica , Proteínas Proto-Oncogênicas/química , Ativação Transcricional , Proteína Supressora de Tumor p53/química , Sítios de Ligação , Cristalização , Cristalografia por Raios X , Ligação de Hidrogênio , Modelos Moleculares , Ligação Proteica , Dobramento de Proteína , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-mdm2 , Fatores de Transcrição/química , Fatores de Transcrição/metabolismo , Proteína Supressora de Tumor p53/metabolismoRESUMO
In the 20th century dengue fever became one of the leading causes of morbidity and mortality throughout the tropics. The dengue virus is an arbovirus transmitted by Aedes mosquitoes. There are four distinct serotypes of dengue arbovirus (DENV-1, 2, 3, 4). According to the World Health Organization, a person infected by one of the dengue viruses can develop symptoms ranging from the classical self-limiting form characterized by high temperature, headache, myalgia, and arthralgia to the severe, potentially fatal, form known as dengue shock syndrome. For over 40 years the main explanation for the pathogenesis of dengue has been based on the "antibody-dependent enhancement" (ADE) concept stating that enhancing antibodies acquired during a primary infection increase the number of infected cells, and thus the level of viremia, during secondary infection. However the severity of dengue is not limited to dengue shock syndrome and there are many cases that do not conform to the ADE concept. A meta-analysis could provide crucial information for resolving this controversy and open the way for development of a monovalent vaccine against the dengue virus as for the closely related yellow fever virus.
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Dengue/fisiopatologia , Anticorpos Facilitadores , Dengue/imunologia , Vacinas contra Dengue , Vírus da Dengue/imunologia , Vírus da Dengue/patogenicidade , Humanos , Fatores de RiscoRESUMO
The 90-kDa cellular protein encoded by the mouse mdm-2 oncogene binds to the p53 protein in vivo and inhibits its transactivation function (J. Momand, G. P. Zambetti, D. C. Olson, D. George, and A. J. Levine, Cell 69:1237-1245, 1992). cDNA clones encoding the human homolog of the mdm-2 protein (also called hdm-2) were isolated from a HeLa cell cDNA library. A series of monoclonal antibodies have been generated against human mdm-2 protein, and the epitopes recognized by these antibodies have been mapped. By construction of a series of deletion mutants, the region of the mdm-2 protein that is critical for complex formation with the p53 protein has been mapped to the N-terminal portion of the human mdm-2 protein. Interestingly, a monoclonal antibody with an epitope located in this same region failed to immunoprecipitate the mdm-2-p53 complex and appeared to recognize only free mdm-2 protein. The domain of the p53 protein that is sufficient for interaction with human mdm-2 protein has been mapped to the N-terminal 52 amino acid residues of the p53 protein. This region contains the transactivation domain of p53, suggesting that mdm-2 may inhibit p53 function by disrupting its interaction with the general transcription machinery.
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Proteínas de Neoplasias/metabolismo , Proteínas Nucleares , Proteínas Proto-Oncogênicas , Proteína Supressora de Tumor p53/metabolismo , Animais , Anticorpos Monoclonais , Sequência de Bases , Sítios de Ligação , DNA/isolamento & purificação , Eletroforese em Gel de Poliacrilamida , Epitopos , Células HeLa , Humanos , Camundongos , Dados de Sequência Molecular , Proteínas de Neoplasias/genética , Oncogenes , Proteínas Proto-Oncogênicas c-mdm2 , Proteína Supressora de Tumor p53/genéticaRESUMO
Throughout the purification of the mdm-2 or mdm-2-p53 protein complexes, a protein with a molecular weight of 34,000 was observed to copurify with these proteins. Several monoclonal antibodies directed against distinct epitopes in the mdm-2 or p53 protein coimmunoprecipitated this 34,000-molecular-weight protein, which did not react to p53 or mdm-2 polyclonal antisera in a Western immunoblot. The N-terminal amino acid sequence of this 34,000-molecular-weight protein demonstrated that the first 40 amino acids were identical to the ribosomal L5 protein, found in the large rRNA subunit and bound to 5S RNA. Partial peptide maps of the authentic L5 protein and the 34,000-molecular-weight protein were identical. mdm-2-L5 and mdm-2-L5-p53 complexes were shown to bind 5S RNA specifically, presumably through the known specificity of L5 protein for 5S RNA. In 5S RNA-L5-mdm-2-p53 ribonucleoprotein complexes, it was also possible to detect the 5.8S RNA which has been suggested to be covalently linked to a percentage of the p53 protein in a cell. These experiments have identified a unique ribonucleoprotein complex composed of 5S RNA, L5 protein, mdm-2 proteins, p53 protein, and possibly the 5.8S RNA. While the function of such a ribonucleoprotein complex is not yet clear, the identity of its component parts suggests a role for these proteins and RNA species in ribosomal biogenesis, ribosomal transport from the nucleus to the cytoplasm, or translational regulation in the cell.
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Proteínas de Neoplasias/metabolismo , Proteínas Nucleares , Proteínas Proto-Oncogênicas , Proteínas Ribossômicas/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Primers do DNA/genética , Sondas de DNA/genética , Substâncias Macromoleculares , Camundongos , Dados de Sequência Molecular , Peso Molecular , Mapeamento de Peptídeos , Proteínas Proto-Oncogênicas c-mdm2 , RNA Ribossômico 5S/genética , RNA Ribossômico 5S/metabolismo , Proteínas Ribossômicas/química , Proteínas Ribossômicas/genéticaRESUMO
Genetic alterations in the p53 and mdm2 genes have been reported to occur in soft tissue sarcomas. This study was designed to determine the prevalence and potential clinical value of detected molecular abnormalities and altered patterns of expression of mdm2 and p53 genes in adult soft tissue sarcomas. A cohort of 211 soft tissue sarcomas from adults that were both clinically and pathologically well characterized was analyzed. Monoclonal antibodies directed against mdm2 and p53 proteins were used to measure overexpression of these proteins in the nuclei of cells from sections of these tumors. Seventy-six of 207 tumors had abnormally high levels of mdm2 proteins and 56 of 211 tumors overexpressed p53 protein. Twenty-two cases had abnormally high levels of both mdm2 and p53 proteins based upon immunoreactivity with these antibodies. There was a striking statistically significant correlation between the overexpression of p53 and mdm2 proteins in the same tumor and poor survival (P < 0.05) of the patients. A group of 73 soft tissue sarcomas was chosen for analysis using Southern blots, single strand conformation polymorphisms, and direct DNA sequencing to confirm mdm2 gene amplifications and p53 mutations and correlate these with the results of the immunoreactivities. The overexpression of p53 and mdm2 proteins in the nuclei of tumor cells did not always correlate well with gene amplification at the mdm2 locus or mutation at the p53 gene. The possible reasons for these discrepancies are discussed.
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Genes p53/genética , Proteínas de Neoplasias/genética , Proteínas Nucleares , Proteínas Proto-Oncogênicas , Sarcoma/genética , Neoplasias de Tecidos Moles/genética , Adulto , Análise de Variância , Núcleo Celular/fisiologia , Estudos de Coortes , DNA de Neoplasias/análise , Amplificação de Genes/genética , Deleção de Genes , Expressão Gênica/genética , Genótipo , Humanos , Imuno-Histoquímica , Fenótipo , Mutação Puntual/genética , Proteínas Proto-Oncogênicas c-mdm2RESUMO
The Mdm2 gene is overexpressed in several human tumors. The oncogenic potential of Mdm2 is partially explained by the inhibition of the activity of the tumor suppressor protein p53. Determination of the three-dimensional structure of complexes between Mdm2 and the N-terminal p53 peptide provided a molecular basis for the inhibition of the transcriptional function of p53 by Mdm2. More dramatically, p53 is targeted by Mdm2 for rapid degradation. The Mdm2 gene itself is activated by p53, which gives the opportunity for feed-back control of p53 activity. Keeping p53 under control is most likely the major task of Mdm2 during early development. Recently, evidence was provided for an alternative, p53-independent function of Mdm2.
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Proteínas de Neoplasias/fisiologia , Proteínas Nucleares , Proteínas Proto-Oncogênicas/fisiologia , Proteína Supressora de Tumor p53/fisiologia , Sequência de Aminoácidos , Animais , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Dados de Sequência Molecular , Proteínas de Neoplasias/química , Proteínas de Neoplasias/genética , Proteínas Proto-Oncogênicas/química , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas c-mdm2 , Relação Estrutura-Atividade , Proteína Supressora de Tumor p53/química , Proteína Supressora de Tumor p53/genéticaRESUMO
A protein product of the mdm-2 oncogene (p90) has been recently shown to associate with the protein encoded by the tumor-suppressor gene p53. The mdm-2 gene was originally identified as a gene amplified in a spontaneously transformed Balb/c 3T3 cell line (3T3DM). This report describes the characterization of mdm-2 gene products and their interactions with the p53 protein. Polyclonal and monoclonal antibodies were generated against murine and human mdm-2 protein. These antibodies detected the mdm-2 p90 protein and at least four additional polypeptides (p85, p76, p74, p58-p57) in cultured cells. These additional proteins may arise from different spliced mRNA forms of the mdm-2 gene or post-translational modifications of the mdm-2 protein. The monoclonal antibodies distinguished at least three sets of mdm-2 proteins with distinct combinations of epitopes (p90 and p85; p76 and p74; p58-57). One or two of these proteins forms a complex with the p53 protein (p90, p58). These mdm-2 proteins were found to be overexpressed in 3T3DM cells and a subset of these proteins were complexed with p53. In 3T3DM cells, p90, like p53, had a short half-life of approximately 20 min and was localized to the cell nucleus. In resting cells stimulated with serum p90 levels and p90/p53 complex levels increased in the late G1 phase of the cell cycle. The p90 mdm-2 protein could regulate p53 activity in the late G1 phase of the cell cycle.
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Ciclo Celular , Proteínas de Neoplasias/metabolismo , Proteínas Nucleares/metabolismo , Fosfoproteínas/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Animais , Anticorpos Monoclonais/imunologia , Clonagem Molecular , Epitopos , Humanos , Substâncias Macromoleculares , Camundongos , Fosfoproteínas/imunologia , Ligação Proteica , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/imunologia , Proteínas Proto-Oncogênicas c-mdm2 , Fase S , Dedos de ZincoRESUMO
MDM-2 is one of the target genes of the p53 tumor suppressor protein. Its best characterized function is found in the inhibition of p53's ability to modulate transcription. Deregulated expression of MDM-2 could thus at least partially substitute for p53 mutation in the process of tumorigenesis. We show here that MDM-2 is highly expressed in biopsies of normal human skin or in vitro reconstituted human skin. The protein is detected in the nucleus of keratinocytes throughout the different layers of the epidermis and in reconstituted skin as early as the two to three cell layer stage. The 90 kiloDalton (kD) protein is one of the major forms detected in Western blot experiments. MDM-2 is detected in skin reconstituted from keratinocytes in which p53 is inactivated by mutation or degradation by E6 protein, providing evidence that MDM-2 expression in the skin can occur in the absence of wild type p53. Moreover, we found no correlation between the p53 status and MDM-2 expression levels in a series of basal and squamous cell carcinomas or Bowen diseases. Our data provide first evidence for the expression of MDM-2 in a differentiated adult tissue.
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Proteínas Nucleares , Proteínas Proto-Oncogênicas/metabolismo , Pele/metabolismo , Western Blotting , Doença de Bowen/metabolismo , Carcinoma Basocelular/metabolismo , Carcinoma de Células Escamosas/metabolismo , Epiderme/metabolismo , Técnica Indireta de Fluorescência para Anticorpo , Humanos , Hibridização In Situ , Queratinócitos/metabolismo , Proteínas Proto-Oncogênicas c-mdm2 , RNA Mensageiro/análise , Células Tumorais Cultivadas , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismoRESUMO
The Mdm2 gene is the best known cellular regulator of p53 tumor suppressor activity. We report here the cloning and characterization of Xdm2, its homolog in Xenopus laevis. Human, mouse and Xenopus MDM2 proteins are more than 65% identical in several regions which are likely to be important for the biological activities of MDM2. Region I is sufficient for binding p53 and inhibiting its G1 arrest and apoptosis functions. Region II contains most of a central acidic region required for interaction with the L5 ribosomal protein and a putative C4 zinc finger. Region III is nearly identical from Xenopus to human and comprises the RING finger domain. We show that this structural conservation is associated with the conservation of three biochemical activities of MDM2; binding to the p53 and L5 proteins and specifically to RNA. Lastly, Xdm2 expression during early development is mainly restricted from the oocyte stage I/II to the blastula stage and is possibly independent of transcriptional activation by p53. These data as well as the utilization of Xenopus laevis to investigate the roles of MDM2 and p53 during early embryogenesis are discussed.
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Proteínas Nucleares , Proteínas Proto-Oncogênicas/química , Xenopus laevis/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Camundongos , Dados de Sequência Molecular , Ligação Proteica , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-mdm2 , Proteínas Ribossômicas/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Proteína Supressora de Tumor p53/metabolismo , Proteínas de Xenopus , Xenopus laevis/embriologiaRESUMO
Lysosomal enzymes secreted or externally supplied into the extracellular medium can be internalized by cells and targeted to lysosomes after binding to specific membrane receptors. This process allows for the replacement of the missing enzyme activity in deficient cells. Using a retroviral vector, we have introduced the human beta-glucuronidase cDNA into primary mouse skin fibroblasts. The genetically modified cells were then engrafted into neo-organs that had been previously implanted into the peritoneal cavity of syngeneic recipient mice. The hypervascularized structures, made of collagen and basic fibroblast growth factor-coated synthetic fibers embedded into extracellular matrix gel, allowed in vivo survival of engrafted fibroblasts that expressed the human beta-glucuronidase cDNA for at least 3 months. The human enzyme was detected in the liver, lung, and spleen of experimental animals, but became undetectable after removal of the neo-organ. This observation indicated that the human enzyme was secreted into the serum and then captured by distant organs. The use of genetically modified fibroblasts implanted into neo-organs may, therefore, represent a convenient approach to enzyme replacement therapy in lysosomal storage diseases.
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Fibroblastos/fisiologia , Glucuronidase/genética , Glucuronidase/metabolismo , Lisossomos/enzimologia , Transfecção/métodos , Células 3T3 , Animais , Órgãos Artificiais , Sequência de Bases , DNA/genética , Matriz Extracelular/fisiologia , Fator 2 de Crescimento de Fibroblastos , Fibroblastos/citologia , Fibroblastos/metabolismo , Humanos , Camundongos , Camundongos Endogâmicos C3H , Dados de Sequência Molecular , Cavidade Peritoneal/fisiologia , Politetrafluoretileno , Próteses e Implantes , Pele/citologia , Transplante de Pele , Transfecção/genéticaRESUMO
We have developed a quantitative RT-PCR method that can be used to determine the amount of enterovirus RNA in urban sludge samples. This method combines Taq-Man technology with the ABI Prism 7700 real-time sequence detection system. We optimized a one-step RT-PCR that uses a dual-labeled fluorogenic probe to quantify the 5' noncoding region of enteroviruses. For accurate quantification of the number of copies, a Mahoney type 1 poliovirus RNA standard was designed and produced using genetic engineering. This fragment, quantified using the Ribogreen method, was used in serial dilutions as an external standard. The method had a 7-log dynamic range (5 to 2 x 10(7)). PCR inhibitors were removed by extracting viral RNA (after virus concentration) using the RNeasy mini kit with added polyvinylpyrrolidone (PVP) and running the amplification reaction with a mixture containing PVP and T4 gene 32 protein. This real-time quantification of enterovirus RNA allows large numbers of samples to be screened. Its sensitivity, simplicity and reproducibility render it suitable as a screening method with which to characterize enteroviruses, the presence of infectious particles being subsequently confirmed by cell culture.
Assuntos
Enterovirus/genética , RNA Viral/análise , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Corantes Fluorescentes , Esgotos/virologiaRESUMO
To study the prevalence of p53 inactivation and MDM2/p21(WAFI/CIP1) expression in severe combined immunodeficient (SCID) mice Epstein-Barr virus (EBV)-induced lymphoproliferation, 19 samples obtained after ip injection of peripheral blood mononuclear cells (PBMCs) from EBV-seropositive donors or lymphoblastoid cell lines (LCL) were analyzed. In all samples tested, overexpression of Ki-67 antigen was shown by immunohistochemistry, indicating a high proliferative index of SCID mice EBV-induced lymphoproliferation. P53 mutations were screened by functional assay in yeast in 14 samples. With this test, a p53-inactivating mutation was found in only one case; the remaining cases exhibited a wild-type p53 pattern. However, an accumulation of p53 protein was detected by immunohistochemistry in six of 19 samples. P21 expression was found in seven of 19 samples but was not correlated with the rate of p53 protein in tumors. In contrast, high levels of nuclear accumulation of MDM2 were found in all samples by immunohistochemistry. These results suggest that a high Ki-67 proliferative index in SCID mice EBV-induced lymphoproliferation is not due to the inactivation of p53 by mutation, but could be associated with an overexpression of MDM2, which would act by a p53-independent mechanism.(J Histochem Cytochem 47:1315-1321, 1999)
Assuntos
Ciclinas/biossíntese , Transtornos Linfoproliferativos/metabolismo , Proteínas Nucleares , Proteínas Proto-Oncogênicas/biossíntese , Animais , Divisão Celular , Linhagem Celular , Inibidor de Quinase Dependente de Ciclina p21 , Inibidores Enzimáticos/metabolismo , Infecções por Vírus Epstein-Barr/metabolismo , Feminino , Humanos , Imuno-Histoquímica , Antígeno Ki-67/metabolismo , Leucócitos Mononucleares/citologia , Leucócitos Mononucleares/metabolismo , Leucócitos Mononucleares/transplante , Transtornos Linfoproliferativos/patologia , Transtornos Linfoproliferativos/virologia , Camundongos , Camundongos SCID , Proteínas de Neoplasias/biossíntese , Proteínas Proto-Oncogênicas c-mdm2 , Células-Tronco/citologia , Células-Tronco/metabolismo , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismoRESUMO
We report here the use of a simple, rapid, reproducible, and quantitative assay to detect the putative activity of anti-HIV agents. It can be employed with different T cell cultures, such as peripheral blood lymphocytes or CEM-C113 cells, a sensitive clone of CEM cells. It can be used alone as a rapid screening test in order to measure both cytotoxicity and the potential antiviral activity of large numbers of compounds. It can also be combined for thorough studies with other classic tests that detect virus production. Among these tests, a micro-method to measure reverse transcriptase activity is shown to be very useful. To illustrate the practicability of the assay, 3'-azido-2',3'-dideoxythymidine (AZT) and ribavirin are studied.