RESUMO
Cancer cells are able to reach distant tissues by migration and invasion processes. Enhanced ability to cope with physical stresses leading to cell membrane damages may offer to cancer cells high survival rate during metastasis. Consequently, down-regulation of the membrane repair machinery may lead to metastasis inhibition. We show that migration of MDA-MB-231 cells on collagen I fibrils induces disruptions of plasma membrane and pullout of membrane fragments in the wake of cells. These cells are able to reseal membrane damages thanks to annexins (Anx) that are highly expressed in invasive cancer cells. In vitro membrane repair assays reveal that MDA-MB-231 cells respond heterogeneously to membrane injury and some of them possess a very efficient repair machinery. Finally, we show that silencing of AnxA5 and AnxA6 leads to the death of migrating MDA-MB-231 cells due to major defect of the membrane repair machinery. Disturbance of the membrane repair process may therefore provide a new avenue for inhibiting cancer metastasis.
Assuntos
Anexina A5/metabolismo , Membrana Celular/metabolismo , Proteínas de Neoplasias/metabolismo , Neoplasias/metabolismo , Linhagem Celular Tumoral , Membrana Celular/patologia , Sobrevivência Celular , Humanos , Invasividade Neoplásica , Metástase Neoplásica , Neoplasias/patologiaRESUMO
The motion of ships can cause discomfort and stress in humans, but little is known about the impact on sheep welfare, despite many sheep traveling long distances by ship during live export. We tested whether exposing sheep to roll (side to side movement), heave (up and down movement), and pitch (front to back movement) with similar amplitude and period conditions to a commercial livestock transport vessel would affect their behavior and physiology. Specifically, we tested the effects of these motions and a control treatment on behavior, heart rate variability, rumination, body posture, and balance of sheep. Four sheep (37 ± 0.1 kg) were restrained in pairs in a crate, which was placed on a moveable and programmable platform that generated roll and pitch motions. An electric forklift was used to produce heave motion. The treatments were applied for 30 min each time in a changeover design with 1 repetition over 8 consecutive days. Sheep behavior was recorded continuously from video records, and heart rate monitors were attached to determine heart rate and its variability. Heave reduced the time that sheep spent ruminating, compared with the other 3 treatments ( < 0.001). The 2 sheep spent more time during heave with their heads 1 above the head of the other ( < 0.001) and looking toward their companion ( = 0.02), indicating greater affiliative behavior. Sheep spent more time during heave standing with their back supported on the crate ( = 0.006) and less time lying down ( = 0.01). Roll caused more stepping motions than pitch and control, indicating loss of balance ( < 0.001). Sheep experiencing heave and roll had increased heart rates and reduced interbeat intervals (IBI) compared to the control ( < 0.001). The IBI of sheep in the heave treatment had an increased ratio of low to high frequency duration ( = 0.01), indicating reduced parasympathetic control of stress responses. Therefore, there was both behavioral and physiological evidence that heave and roll caused stress, with sheep experiencing roll apparently coping better by regular posture changes and heave causing the sheep to seek the close presence of their companion.
Assuntos
Comportamento Animal/fisiologia , Movimento (Física) , Ovinos/fisiologia , Navios , Bem-Estar do Animal , Animais , Frequência Cardíaca , Masculino , Estresse FisiológicoRESUMO
Development of antibodies (Abs) against factor VIII (FVIII) is a severe complication of haemophilia A treatment. Recent publications suggest that domain specificity of anti-FVIII antibodies, particularly during immune tolerance induction (ITI), might be related to the outcome of the treatment. Obtaining suitable tools for a fine mapping of discontinuous epitopes could thus be helpful. The aim of this study was to map discontinuous epitopes on FVIII A2 domain using a new epitope prediction functionality of the PEPOP bioinformatics tool and a peptide inhibition assay based on the Luminex technology. We predicted, selected and synthesized 40 peptides mimicking discontinuous epitopes on the A2 domain of FVIII. A new inhibition assays using Luminex technology was performed to identify peptides able to inhibit the binding of anti-A2 Abs to A2 domain. We identified two peptides (IFKKLYHVWTKEVG and LYSRRLPKGVKHFD) able to block the binding of anti-A2 allo-antibodies to this domain. The three-dimensional representation of these two peptides on the A2 domain revealed that they are localized on a limited region of A2. We also confirmed that residues 484-508 of the A2 domain define an antigenic site. We suggest that dissection of the antibody response during ITI using synthetic peptide epitopes could provide important information for the management of patients with inhibitors.
Assuntos
Simulação por Computador , Mapeamento de Epitopos , Epitopos/química , Fator VIII/química , Modelos Moleculares , Peptídeos/química , Domínios e Motivos de Interação entre Proteínas , Algoritmos , Sequência de Aminoácidos , Inibidores dos Fatores de Coagulação Sanguínea/imunologia , Inibidores dos Fatores de Coagulação Sanguínea/metabolismo , Epitopos/imunologia , Epitopos/metabolismo , Fator VIII/imunologia , Fator VIII/metabolismo , Hemofilia A/tratamento farmacológico , Hemofilia A/imunologia , Humanos , Isoanticorpos/imunologia , Isoanticorpos/metabolismo , Peptídeos/síntese química , Peptídeos/imunologia , Peptídeos/metabolismo , Ligação Proteica , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas/imunologiaRESUMO
AIM: The need for developing efficient and safe alternatives to parabens has been growing in the cosmetic and pharmaceutical industries. To this end, the antimicrobial activities and safety of methyl-ß-d-maltoside, methyl-ß-d-maltotrioside and their dodecyl homologues were investigated. METHODS AND RESULTS: Antimicrobial activities of methyl- and dodecyl-ß-d-oligomaltoside have been tested on European pharmacopoeia reference strains. When effective, minimal inhibitory concentrations ranged from 32 to 1024 mg l(-1) . Methyl derivatives exhibited greater antibacterial properties, while their dodecyl homologues were more active on fungal strains. To elucidate the mechanism of action of methyl compounds, enzymatic inhibition assays of key maltose metabolism enzymes have been conducted. Methyl-ß-maltotrioside (MeG3) was found to be effective in inhibiting microbial glucoamylase and α-amylase. MeG3 and dodecyl-ß-maltotrioside cytotoxicity were also checked on HepG2 cells and were found to be low, compared with benzalkonium chloride or parabens. CONCLUSION: Methyl- and dodecyl-ß-maltoside or maltotrioside were found to be active against reference strains used for preservatives efficacy testing. Methyl derivatives could act through an interesting inhibition of the microbial enzymatic metabolism. SIGNIFICANCE AND IMPACT OF THE STUDY: Given their very simple chemical structure, low toxicity and good antimicrobial activity, methyl-ß-d-oligomaltosides could represent a valuable alternative to currently used preservatives such as parabens.
Assuntos
Anti-Infecciosos/farmacologia , Glucosídeos/farmacologia , Conservantes Farmacêuticos/farmacologia , Anti-Infecciosos/toxicidade , Cosméticos , Inibidores Enzimáticos/farmacologia , Glucosídeos/toxicidade , Testes de Sensibilidade Microbiana , Parabenos/farmacologia , Conservantes Farmacêuticos/toxicidade , alfa-Amilases/antagonistas & inibidoresRESUMO
The venom of Loxosceles intermedia (Li) spiders is responsible for cutaneous lesions and other clinical manifestations. We previously reported that the monoclonal antibody LimAb7 can neutralize the dermonecrotic activity of crude Li venom. In this study, we observed that this antibody recognizes several proteins from the venom dermonecrotic fraction (DNF), including LiD1. Identifying the epitope of such a neutralizing antibody could help designing immunogens for producing therapeutic sera or vaccination approaches. To this aim, two sets of 25- and 15-mer overlapping peptides that cover the complete amino acid sequence of LiD1 were synthesized using the SPOT technique. None of them was recognized by LimAb7, suggesting that the epitope is discontinuous. Then, the screening of four peptide phage-display libraries yielded four possible epitope mimics that, however, did not show any obvious similarity with the LiD1 sequence. These mimotopes, together with a 3D model of LiD1, were used to predict with the MIMOP bioinformatic tool the putative epitope region (residues C197, Y224, W225, T226, D228, K229, R230, T232 and Y248 of LiD1) recognized by LimAb7. This analysis and the results of alanine-scanning experiments highlighted a few residues (such as W225 and D228) that are found in the active site of different SMases D and that may be important for LiD1 enzymatic activity. Finally, the only mimotope NCNKNDHLFACW that interacts with LimAb7 by SPOT and its analog NSNKNDHLFASW were used as immunogens in rabbits. The resulting antibodies could neutralize some of the biological effects induced by crude Li venom, demonstrating a mimotope-induced protection against L. intermedia venom.
Assuntos
Anticorpos Neutralizantes/sangue , Antitoxinas/sangue , Aracnídeos , Epitopos/imunologia , Venenos de Aranha/antagonistas & inibidores , Vacinas de Subunidades Antigênicas/imunologia , Animais , Mapeamento de Epitopos , Feminino , Biblioteca de Peptídeos , Perciformes , Coelhos , Venenos de Aranha/toxicidadeRESUMO
The Amm VIII protein was previously isolated from the venom of the scorpion Androctonus mauretanicus mauretanicus. Despite 87% identity with AaH II, the most toxic alpha-type scorpion toxin, Amm VIII is not toxic to mice. However, antisera against Amm VIII protect mice from AaH II lethal action. Here, we report that the Amm VIII protein elicits antibodies that only recognize discontinuous-type epitopes since we could not observe any antibody binding to overlapping 12-mer peptides covering the whole Amm VIII sequence. By using a new bioinformatic tool, 24 peptides mimicking discontinuous regions of Amm VIII were designed in silico, then prepared by Spot synthesis. Seven of these discontinuous-continuous peptides were recognized by anti-Amm VIII antibodies. Analysis of the 3D location of the segments that compose the antigenically reactive discontinuous-continuous peptides, allowed us to group those antigenic segments into three regions of Amm VIII, putatively corresponding to discontinuous antigenic regions of alpha-type scorpion toxins. Anti-Amm VIII antibodies were also found to cross-react towards several of the discontinuous-continuous peptides designed from the AaH II structure, pointing to a possible involvement of the corresponding discontinuous epitopes in the capacity displayed by anti-Amm VIII antibodies to neutralize AaH II. Altogether, our results show that it is possible to design antibody-reactive peptides from discontinuous parts of scorpion toxins. The position of the reactive segments in the structural context of scorpion toxins highlights the antigenic properties of the Amm VIII anatoxin and concurs to explain the capacity of anti-Amm VIII antibodies to neutralize the potent AaH II toxin.
Assuntos
Antitoxinas/imunologia , Mapeamento de Epitopos , Epitopos/imunologia , Dados de Sequência Molecular , Venenos de Escorpião/imunologia , Escorpiões/imunologia , Sequência de Aminoácidos , Animais , Anticorpos Neutralizantes/imunologia , Reações Cruzadas , Epitopos/genética , Camundongos , Camundongos Endogâmicos C57BL , Modelos Moleculares , Testes de Neutralização , Estrutura Terciária de Proteína , Coelhos , Venenos de Escorpião/genética , Escorpiões/genéticaRESUMO
Linear and non-linear thermo-optical dynamical regimes were investigated in a photonic crystal cavity. First, we have measured the thermal relaxation time in an InP-based nano-cavity with quantum dots in the presence of optical pumping. The experimental method presented here allows one to obtain the dynamics of temperature in a nanocavity based on reflectivity measurements of a cw probe beam coupled through an adiabatically tapered fiber. Characteristic times of 1.0+/-0.2 micros and 0.9+/-0.2 micros for the heating and the cooling processes were obtained. Finally, thermal dynamics were also investigated in a thermo-optical bistable regime. Switch-on/off times of 2 micros and 4 micros respectively were measured, which could be explained in terms of a simple non-linear dynamical representation.
RESUMO
Antibodies (Abs) that block factor VIII (FVIII) activity occur in hemophilia A patients treated with FVIII replacement therapy and severely impair treatment. In this work, we designed and synthesized ten peptides whose sequences are found in putative epitopes at the surface of a1 and C2 domains of the FVIII molecule. These peptides were screened for their ability to inhibit the binding of anti-FVIII Abs from plasmas of hemophilia A patients to FVIII. All peptides were efficient in inhibiting anti-FVIII Abs in plasma from patients with inhibitors, with however different efficiencies. It was found that each tested patient's plasma had a different profile of reactivity with peptides, consistent with an individual anti-FVIII Ab specificity. The profile of recognized peptides was also changing during the treatment of the patients. Three peptides were used in an affinity chromatography assay to attempt to remove anti-FVIII Abs from patients' plasma. Anti-FVIII IgGs were significantly captured by the peptide-Sepharose affinity matrixes as assessed by enzyme-linked immunosorbent assay. However, due to the low level of Abs in the plasma samples, other methods (Chromogenic and Bethesda assays) were not sensitive enough to properly detect the reduction of inhibitors.
Assuntos
Autoanticorpos/metabolismo , Epitopos/metabolismo , Fator VIII/imunologia , Hemofilia A/imunologia , Fragmentos de Peptídeos/metabolismo , Reações Antígeno-Anticorpo , Ligação Competitiva , Cromatografia de Afinidade/métodos , Ensaio de Imunoadsorção Enzimática/métodos , Humanos , Epitopos Imunodominantes/metabolismo , Masculino , Fragmentos de Peptídeos/imunologiaRESUMO
Polymyxo graminis, a ubiquitous plasmodiophorid obligate root endoparasite, is recognized as the vector of about 15 viruses on cereals and groundnut in temperate and tropical areas. Within the species, five special forms have been distinguished on the basis of specific ribotypes. Three of them occur in tropical areas: P. graminis f.sp. colombiana on rice, P. graminis f.sp. subtropicalis on cereals cropped in the tropics such as maize, pearl millet and sorghum but also on barley and/or wheat, and P. graminis f.sp. tropicalis mainly on maize, pearl millet and sorghum. Their particular host ranges distinguish them significantly from P. graminis f.sp. temperata and P. graminis f.sp. tepida found in temperate areas on barley and wheat. In order to assess whether these special forms commonly infect these cereals, barley and wheat plants were grown under controlled conditions on two soils from Belgium and France and both infested by P. graminis f.sp. temperata and P. graminis f.sp. tepida. The infection of each cereal species by each form was quantified by real-time quantitative PCR with specific primers and Taqman probes. The infection of P. graminis f.sp. temperata was significantly higher on barley than on wheat, whereas the quantities of P. graminis f.sp. tepida on wheat were higher than on barley. These results show that the distinction between these special forms, based on the ribotype, reflects differences in ecological features.
Assuntos
Clima , Grão Comestível/parasitologia , Mixomicetos/classificação , Mixomicetos/patogenicidade , Reação em Cadeia da Polimerase/métodos , Animais , Sequência de Bases , DNA de Protozoário/química , DNA de Protozoário/genética , Grão Comestível/classificação , Hordeum/parasitologia , Dados de Sequência Molecular , Mixomicetos/crescimento & desenvolvimento , Mixomicetos/isolamento & purificação , Doenças das Plantas/parasitologia , Raízes de Plantas/parasitologia , Ribotipagem , Especificidade da Espécie , Triticum/parasitologiaRESUMO
Barley yellow mosaic virus (BaYMV) is the causal agent of a soil-borne systemic mosaic disease on barley. It has been reported in Belgium since the 1980s. The control of this disease is managed almost exclusively through the use of resistant varieties. The resistance of most commercial barley cultivars grown in Europe is conferred mainly by a single recessive gene, rym4. This monogenic resistance provides immunity against BaYMV pathotype 1 and has been mapped on barley chromosome 3HL and shown to be caused by mutations in the translation initiation factor eIF4E. Another pathotype, BaYMV pathotype 2, which appeared in the late 1980s (in Belgium, in the early 1990s), is able to overcome the rym4-controlled resistance. Until recently, this pathotype remained confined to specific locations. During a systematic survey in 2003, mosaic symptoms were observed only on susceptible barley cultivars collected in Belgian fields. BaYMV was detected by ELISA and RT-PCR on the susceptible cultivars and only by RT-PCR on the resistant cultivars. In 2004, mosaic symptoms were observed on susceptible and resistant cultivars. BaYMV was detected by ELISA and RT-PCR on both cultivars. In addition to developing RT-PCR methods for detecting and identifying BaYMV and Barley mild mosaic virus (BaMMV), an RT-PCR targeting the VPg/NIa viral protein part of the genome, known to discriminate the two BaYMV pathotypes, was set up to accurately identify the pathotype(s) now present in Belgium. The sequences from the generated amplicons revealed the single nucleotide substitution resulting in an amino acid change from lysine to asparagine specific to BaYMV pathotype 2. The possible reasons for the change in the BaYMV pathotype situation in Belgium, such as climatic change or a progressive build-up of soil inoculum potential, will be discussed, as well as the use of eIF4E-based resistance.
Assuntos
Farmacorresistência Viral/genética , Hordeum/genética , Hordeum/virologia , Vírus do Mosaico/patogenicidade , Doenças das Plantas/genética , Sequência de Aminoácidos , Substituição de Aminoácidos , Bélgica , Ensaio de Imunoadsorção Enzimática , Genes Recessivos , Dados de Sequência Molecular , Doenças das Plantas/virologia , Proteínas de Plantas/genética , Plantas Geneticamente Modificadas , RNA Viral/química , Reação em Cadeia da Polimerase Via Transcriptase Reversa , VirulênciaRESUMO
Polymyxa graminis f. sp. temperata and P. graminis f. sp. tepida are distinguished on the basis of their specific ribosomal DNA sequences. In order to evaluate whether or not host specialization is associated with the special form, the occurrence of infection of both forms on barley and wheat was studied. P. graminis inocula were obtained from soils collected in Belgium and France. Their ribotypes were characterized using molecular tools specific to P. graminis f. sp. temperata or P. graminis f. sp. tepida such as restriction fragment length polymorphism (RFLP) analysis of polymerase chain reaction (PCR)-amplified rDNA, nested and multiplex PCR. Both special forms were found in each country and coexisted in some soils. The host specificity of P. graminis special forms for barley and wheat was studied from two soils collected at Gembloux (Belgium) and Chambon-sur-Cisse (France), each infested by bymo- and furoviruses. P. graminis f. sp. temperata is more frequent on barley and P. graminis f. sp. tepida on wheat. Furthermore, the quantification of each form on barley and wheat by two separated real-time quantitative PCR assays confirms the observations on the vector specialization. These results suggest a certain but not exclusive host specificity of P. graminis special forms.
RESUMO
We report on quantum cascade lasers employing waveguides based on a predominant air confinement mechanism in which the active region is located immediately at the device top surface. The lasers employ ridge-waveguide resonators with narrow lateral electrical contacts only, with a large, central top region not covered by metallization layers. Devices based on this principle have been reported in the past; however, they employed a thick, doped top-cladding layer in order to allow for uniform current injection. We find that the in-plane conductivity of the active region - when the material used is of high quality - provides adequate electrical injection. As a consequence, the devices demonstrated in this work are thinner, and most importantly they can simultaneously support air-guided and surface-plasmon waveguide modes. When the lateral contacts are narrow, the optical mode is mostly located below the air-semiconductor interface. The mode is predominantly air-guided and it leaks from the top surface into the surrounding environment, suggesting that these lasers could be employed for surface-sensing applications. These laser modes are found to operate up to room temperature under pulsed injection, with an emission spectrum centered around l (1/4) 7:66 mum.
RESUMO
Reported myocardial pathology resulting from increased levels of catecholamines in vivo has led us to investigate the effect of adrenalin on the gap junction proteins connexin 40 (Cx40) and Cx43 and the possible relationship to vascular toxicity. Adrenalin and its known metabolites, adrenochrome and adrenolutin, were used in this study. Utilizing the A7r5 rat aortic cell line, we evaluated the effects of adrenalin, adrenochrome, and adrenolutin on the expression and function of connexin 40 and 43 that are present in both cardiac and vascular tissues.
RESUMO
In order to assess the occurrence of Wheat spindle streak mosaic virus (WSSMV) in Belgium, a reverse-transcription polymerase chain reaction (RT-PCR) was developed, targeting WSSMV isolates from Canada, France, Germany, Italy, and the United States. The primers also were designed for virus quantification by real-time RT-PCR with SYBR-Green. No cross-reaction with soilborne cereal viruses such as Barley mild mosaic virus, Barley yellow mosaic virus, Soilborne cereal mosaic virus, and Soil-borne wheat mosaic virus was observed. The RT-PCR and real-time quantitative RT-PCR allowed a more sensitive detection of WSSMV than enzymelinked immunosorbent assay. The incidence of WSSMV in Belgium was evaluated using a bioassay with wheat cvs. Cezanne and Savannah and rye cv. Halo, grown in 104 Belgian soils. The presence of WSSMV was detected from plants grown in 32% of the soils. The RT-PCR methods developed here, combined with large sampling, allowed WSSMV to be detected for the first time in Belgium. The real-time quantitative RT-PCR was developed as a tool for evaluating the resistance to WSSMV by quantifying the virus concentration in wheat cultivars.
RESUMO
UNLABELLED: The objective was to design and validate a method for tele-operating (from an expert site) an echographic examination in an isolated site. METHOD: The isolated places, defined as areas with reduced medical facilities, could be secondary hospitals 20 to 50 km from the university hospital, or dispensaries in Africa or Amazonia, or a moving structure like a rescue vehicle or the International Space Station (ISS). At the expert center, the ultrasound medical expert moves a fictive probe, connected to a computer (n degrees 1) which sends, the coordinate changes of this probe via an ISDN or satellite line to a second computer (n degrees 2), located at the isolated site, which applies them to the robotic arm holding the real echographic probe. RESULTS: The system was tested at Tours Hospital on 105 patients. A complete investigation (visualization) of all the organs requested for different clinical cases was obtained in 76% of the cases with the robot, and 87% at the reference echography: In 11% of the cases, at least one of the organ visualized at reference echo could not be investigated by the robot, thus the diagnostic was not done. The number of repositioning was higher for the robot (6.5 +/- 2) than for the reference echo (5.1 +/- 2 = or > 24% more with robot). The duration of the examination was higher with the robot (16 +/- 10 min) than for the reference echography (11 +/- 4 min = or > +43% with the robot compare to reference echography. The system was also tested successfully using satellite links in a limited number of cases (approx 30).
Assuntos
Medicina Aeroespacial/instrumentação , Robótica , Voo Espacial/instrumentação , Astronave/instrumentação , Telemedicina/instrumentação , Ultrassonografia/métodos , Desenho de Equipamento , Humanos , Consulta Remota , Sensibilidade e Especificidade , Telemetria/instrumentação , Telemetria/métodos , Ultrassonografia/instrumentaçãoRESUMO
OBJECTIVE: This phase II study was performed to evaluate the activity and toxicity of gemcitabine plus cisplatin as first-line treatment of advanced epithelial ovarian cancer. METHODS: Chemonaive patients with histologically or cytologically confirmed FIGO stage III or IV epithelial ovarian carcinoma were enrolled. Patients received cisplatin 75 mg/m(2) on Day 1 and gemcitabine 1250 mg/m(2) on Days 1 (after cisplatin) and 8 of a 21-day cycle. RESULTS: Of the 42 female patients (median age 60 years) enrolled, 81% had a Zubrod performance status of 0 or 1. Among the 37 response-evaluable patients, there were 5 (13.5%) pathological complete responses (CRs), 16 (43.2%) pathological partial responses (PRs), and 3 (8.1%) clinical PRs, for an overall response rate of 64.9% (95% CI: 47.4-79.8%) and a pathological response rate of 56.8%. Per an intent-to-treat analysis, the overall response rate was 57.1% (95% CI: 41.0-72.3%). After a median follow-up time of 15.8 months, the median survival was 24.0 months and median progression-free survival was 13.4 months. Grade 3/4 neutropenia and thrombocytopenia occurred in 69.0 and 33.3% of patients, respectively, with no febrile neutropenia or hemorrhage. Grade 3/4 nausea and vomiting occurred in 35.7% and grade 3 alopecia in 21.4% of the patients. One patient died due to a toxicity-related death (dyspnea). CONCLUSIONS: Gemcitabine plus cisplatin is active and feasible as first-line treatment of advanced epithelial ovarian cancer. Further clinical trials adding gemcitabine to first-line treatment seem warranted.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Desoxicitidina/análogos & derivados , Neoplasias Ovarianas/tratamento farmacológico , Adulto , Idoso , Protocolos de Quimioterapia Combinada Antineoplásica/efeitos adversos , Cisplatino/administração & dosagem , Cisplatino/efeitos adversos , Desoxicitidina/administração & dosagem , Desoxicitidina/efeitos adversos , Intervalo Livre de Doença , Esquema de Medicação , Feminino , Humanos , Infusões Intravenosas , Pessoa de Meia-Idade , Taxa de Sobrevida , GencitabinaRESUMO
Due to the increasing complexity of food production systems and the concerns that these systems raise, there has been increasing demand from the general public for more State control of these processes. In France, it is the official Veterinary Services who are responsible for food safety and who must respond to these demands. The Veterinary Service is formulating a quality assurance procedure in accordance with standard EN 45004-ISO 17020, which determines the requirements that inspection bodies must follow to be recognised, at national, European and international level, as competent and reliable. As part of this procedure, the Veterinary Service will review requirements in terms of organisation, functions, qualifications and resources. The progress of inspection service orders, from their conception by the Central Administration, to their implementation by decentralised services, must be carefully managed. It is essential that service orders be implemented effectively and systematically by using recognised methods and issuing adequate inspection reports. The training and qualifications of inspectors are very important: their skills must remain up-to-date so that there is always a network of qualified staff, that is, staff who have an understanding of production processes and who have recognised competences in terms of initial training, continuous professional development and adequate experience. The quality systems implemented will only meet expectations if they are continuously monitored by means of regular evaluations. For this reason, both internal and external audits are performed. These new practices contribute to establishing a basis for the improvement of internal evaluation. In order to facilitate the implementation of a quality assurance procedure for inspection services, several tools, that are linked with the information system of the government department responsible for food, are, or will be, at the disposal of the decentralised Veterinary Services, i.e. a national database, mail and service order processing software, and inspection procedures.
Assuntos
Inspeção de Alimentos/normas , Garantia da Qualidade dos Cuidados de Saúde , Medicina Veterinária/organização & administração , Medicina Veterinária/normas , Animais , Qualidade de Produtos para o Consumidor , Comportamento Cooperativo , França , Humanos , Desenvolvimento de Programas , Avaliação de Programas e Projetos de Saúde , Garantia da Qualidade dos Cuidados de Saúde/organização & administração , Garantia da Qualidade dos Cuidados de Saúde/normas , Controle de QualidadeRESUMO
Nuclear magnetic resonance is an important tool for high-resolution structural studies of proteins. It demands high protein concentration and high purity; however, the expression of proteins at high levels often leads to protein aggregation and the protein purification step can correspond to a high percentage of the overall time in the structural determination process. In the present article we show that the step of sample optimization can be simplified by selective labeling the heterologous protein expressed in Escherichia coli by the use of rifampicin. Yeast thioredoxin and a coix transcription factor Opaque 2 leucine zipper (LZ) were used to show the effectiveness of the protocol. The (1)H/(15)N heteronuclear correlation two-dimensional NMR spectrum (HMQC) of the selective (15)N-labeled thioredoxin without any purification is remarkably similar to the spectrum of the purified protein. The method has high yields and a good (1)H/(15)N HMQC spectrum can be obtained with 50 ml of M9 growth medium. Opaque 2 LZ, a difficult protein due to the lower expression level and high hydrophobicity, was also probed. The (15)N-edited spectrum of Opaque 2 LZ showed only the resonances of the protein of heterologous expression (Opaque 2 LZ) while the (1)H spectrum shows several other resonances from other proteins of the cell lysate. The demand for a fast methodology for structural determination is increasing with the advent of genome/proteome projects. Selective labeling the heterologous protein can speed up NMR structural studies as well as NMR-based drug screening. This methodology is especially effective for difficult proteins such as hydrophobic transcription factors, membrane proteins, and others.
Assuntos
Proteínas de Ligação a DNA/química , Escherichia coli/metabolismo , Proteínas Fúngicas/química , Proteínas de Plantas/química , Tiorredoxinas/química , Fatores de Transcrição/química , Proteínas de Ligação a DNA/biossíntese , Proteínas Fúngicas/biossíntese , Zíper de Leucina , Ressonância Magnética Nuclear Biomolecular , Proteínas de Plantas/biossíntese , Rifampina/química , Rifampina/farmacologia , Tiorredoxinas/biossíntese , Fatores de Transcrição/biossínteseRESUMO
We examined the role of the microtubule cytoskeleton during vaccinia virus infection. We found that newly assembled virus particles accumulate in the vicinity of the microtubule-organizing centre in a microtubule- and dynein-dynactin complex-dependent fashion. Microtubules are required for efficient intracellular mature virus (IMV) formation and are essential for intracellular enveloped virus (IEV) assembly. As infection proceeds, the microtubule cytoskeleton becomes dramatically reorganized in a fashion reminiscent of overexpression of microtubule-associated proteins (MAPs). Consistent with this, we report that the vaccinia proteins A10L and L4R have MAP-like properties and mediate direct binding of viral cores to microtubules in vitro. In addition, vaccinia infection also results in severe reduction of proteins at the centrosome and loss of centrosomal microtubule nucleation efficiency. This represents the first example of viral-induced disruption of centrosome function. Further studies with vaccinia will provide insights into the role of microtubules during viral pathogenesis and regulation of centrosome function.
Assuntos
Centrossomo/fisiologia , Proteínas Associadas aos Microtúbulos/metabolismo , Microtúbulos/fisiologia , Vaccinia virus/fisiologia , Proteínas Virais/metabolismo , Citoesqueleto/efeitos dos fármacos , Complexo Dinactina , Dineínas/metabolismo , Imunofluorescência , Genoma Viral , Nocodazol/farmacologia , Proteínas Virais/isolamento & purificação , Montagem de VírusRESUMO
Wiskott-Aldrich syndrome protein (WASP) and N-WASP have emerged as key proteins connecting signalling cascades to actin polymerization. Here we show that the amino-terminal WH1 domain, and not the polyproline-rich region, of N-WASP is responsible for its recruitment to sites of actin polymerization during Cdc42-independent, actin-based motility of vaccinia virus. Recruitment of N-WASP to vaccinia is mediated by WASP-interacting protein (WIP), whereas in Shigella WIP is recruited by N-WASP. Our observations show that vaccinia and Shigella activate the Arp2/3 complex to achieve actin-based motility, by mimicking either the SH2/SH3-containing adaptor or Cdc42 signalling pathways to recruit the N-WASP-WIP complex. We propose that the N-WASP-WIP complex has a pivotal function in integrating signalling cascades that lead to actin polymerization.