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1.
Sensors (Basel) ; 23(12)2023 Jun 20.
Artigo em Inglês | MEDLINE | ID: mdl-37420929

RESUMO

The number of vehicles equipped with radars on the road has been increasing for years and is expected to reach 50% of cars by 2030. This rapid rise in radars will likely increase the risk of harmful interference, especially since radar specifications from standardization bodies (e.g., ETSI) provide requirements in terms of maximum transmit power but do no mandate specific radar waveform parameters nor channel access scheme policies. Techniques for interference mitigation are thus becoming very important to ensure the long-term correct operation of radars and upper-layer ADAS systems that depend on them in this complex environment. In our previous work, we have shown that organizing the radar band into time-frequency resources that do not interfere with each other vastly reduces the amount of interference by facilitating band sharing. In this paper, a metaheuristic is presented to find the optimal resource sharing between radars, knowing their relative positions and thereby the line-of-sight and non-line-of-sight interference risks during a realistic scenario. The metaheuristic aims at optimally minimizing interference while minimizing the number of resource changes that radars have to make. It is a centralized approach where everything about the system is known (e.g., the past and future positions of the vehicles). This and the high computational load induce that this algorithm is not meant to be used in real-time. However, the metaheuristic approach can be extremely useful for finding near optimal solutions in simulations, allowing for the extraction of efficient patterns, or as data generation for machine learning.


Assuntos
Algoritmos , Radar , Automóveis
2.
IEEE Trans Pattern Anal Mach Intell ; 43(1): 77-88, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-31295104

RESUMO

Barcode encoding schemes impose symbolic constraints which fix certain segments of the image. We present, implement, and assess a method for blind deblurring and denoising based entirely on Kullback-Leibler divergence. The method is designed to incorporate and exploit the full strength of barcode symbologies. Via both standard barcode reading software and smartphone apps, we demonstrate the remarkable ability of our method to blindly recover simulated images of highly blurred and noisy barcodes. As proof of concept, we present one application on a real-life out of focus camera image.

3.
Food Res Int ; 123: 364-372, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31284988

RESUMO

The development of new fermented foods and beverages requires more and more the use of new dehydrated yeasts species. In this context, the non-Saccharomyces (NS) yeasts Torulaspora delbrueckii, Metschnikowia pulcherrima and Lachancea thermotolerans are developed especially in winemaking as co-culture in the fermentation of the must or for the must bioprotection. However, during formulation-dehydration the yeast cells are exposed to several stresses that reduce cellular activity. Among these, the oxidative stress induced by the stabilization processes has been described as one of the main causes of cell death. In this study, we analyzed the effects of growth medium associated with two dehydration kinetics on the accumulation of reactive oxygen species (ROS) and lipid peroxidation levels. The cultivability of tested yeast strains was dependent on growth and dehydration conditions. The L. thermotolerans strain was the most sensible to dehydration when growing in nutrient-poor media, which was characterized by high levels of ROS, lipid peroxidation and reduced cultivability. In contrast, this yeast was able to restore its cultivability when growing in nutrient-rich medium before dehydration. The other NS yeast strains acquired resistance to oxidative stress similarly but in a growth-dehydration condition less dependent manner. These results showed that modulation of growing medium composition is a simple way to improve resistance to oxidative attack imposed by dehydration in NS yeasts. This was the first time that multiple quantitative and qualitative indicators of oxidative stress response in these three NS yeast strains were explored.


Assuntos
Desidratação , Estresse Oxidativo , Saccharomyces/metabolismo , Vinho/microbiologia , Fermento Seco/metabolismo , Estudos de Avaliação como Assunto , Fermentação , Peroxidação de Lipídeos , Malondialdeído/metabolismo , Metschnikowia/metabolismo , Viabilidade Microbiana , Espécies Reativas de Oxigênio/metabolismo , Saccharomycetales/metabolismo , Torulaspora/metabolismo , Vinho/análise
4.
J Acoust Soc Am ; 145(6): 3727, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31255114

RESUMO

The effective dynamic bulk modulus and density are presented for random media consisting of particles in a viscous host fluid, using a core-shell, self-consistent effective medium model, under the large compressional wavelength assumption. These properties are relevant to acoustic or dynamic processes in nano- and micro-particle fluids including particle density determination, resonant acoustic mixing, and acoustic characterisation. Analytical expressions are obtained for the effective bulk modulus and mass density, incorporating the viscous nature of the fluid host into the core-shell model through wave mode conversion phenomena. The effective density is derived in terms of particle concentration, particle and host densities, particle size, and the acoustic and shear wavenumbers of the liquid host. The analytical expressions obtained agree with prior known results in the limit of both static and inviscid cases; the ratio of the effective bulk modulus to that of the fluid is found to be quasi-static. Numerical calculations demonstrate the dependence of the effective mass density on frequency, particle size (from nano- to micro-regime), and concentration. Herein it is demonstrated both theoretically and numerically that the viscosity, often neglected in the literature, indeed plays a significant role in the effective properties of nanofluids.

5.
Food Microbiol ; 79: 137-146, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30621869

RESUMO

In food industry and winemaking, the use of active dehydrated yeast (ADY) Saccharomyces cerevisiae is a frequent practice because of the long-term stability and high efficiency of ADY. Nowadays, there is an increasing interest for new yeasts strains, such as Torulaspora delbrueckii (Td), Metschnikowia pulcherrima (Mp) and Lachancea thermotolerans (Lt). However, the yeasts transformation processes into the solidified form generate several stresses that reduce the cell viability. In this case, understanding the phenomena of yeast cell resistance before, during and after dehydration is of great importance. In this study we analyzed two compounds associated with resistance to stress and produced by cells, glutathione (total, oxidized and reduced) and trehalose, at different stages of the process. The impact of growing and dehydration conditions on cell viability was analyzed by flow cytometry and two-photon laser scanning microscopy. The results showed that cells naturally enriched in glutathione or trehalose acquired resistance to dehydration, preventing the oxidation of glutathione in a growth/dehydration condition dependent manner. This is the first time that simultaneous metabolic and dehydration responses were observed in three non-Saccharomyces strains. These findings represent an opportunity to better understand the yeast's dehydration resistance phenomena and thus to promote the efficient industrial production of new dried yeasts.


Assuntos
Desidratação , Glutationa/metabolismo , Saccharomycetales/fisiologia , Trealose/metabolismo , Adaptação Fisiológica , Membrana Celular/metabolismo , Cinética , Viabilidade Microbiana , Oxirredução , Saccharomycetales/crescimento & desenvolvimento , Saccharomycetales/metabolismo , Saccharomycetales/ultraestrutura , Vinho/microbiologia
6.
Neuropharmacology ; 121: 247-260, 2017 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-28456688

RESUMO

In cerebellar cortex, mGlu4 receptors located on parallel fibers play an essential role in normal motor function, but the molecular mechanisms involved are not yet completely understood. Using a strategy combining biochemical and electrophysiological approaches in the rodent cerebellum, we demonstrate that presynaptic mGlu4 receptors control synaptic transmission through an atypical activation of Gαq proteins. First, the Gαq subunit, PLC and PKC signaling proteins present in cerebellar extracts are retained on affinity chromatography columns grafted with different sequences of the cytoplasmic domain of mGlu4 receptor. The i2 loop and the C terminal domain were used as baits, two domains that are known to play a pivotal role in coupling selectivity and efficacy. Second, in situ proximity ligation assays show that native mGlu4 receptors and Gαq subunits are in close physical proximity in cerebellar cortical slices. Finally, electrophysiological experiments demonstrate that the molecular mechanisms underlying mGlu4 receptor-mediated inhibition of transmitter release at cerebellar Parallel Fiber (PF) - Molecular Layer Interneuron (MLI) synapses involves the Gαq-PLC signaling pathway. Taken together, our results provide compelling evidence that, in the rodent cerebellar cortex, mGlu4 receptors act by coupling to the Gαq protein and PLC effector system to reduce glutamate synaptic transmission.


Assuntos
Córtex Cerebelar/citologia , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/metabolismo , Receptores de Glutamato Metabotrópico/metabolismo , Transdução de Sinais/fisiologia , Transmissão Sináptica/fisiologia , Animais , Animais Recém-Nascidos , Benzopiranos/farmacologia , Citoplasma/metabolismo , Ativação Enzimática/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Fármacos Atuantes sobre Aminoácidos Excitatórios/farmacologia , Potenciais Pós-Sinápticos Excitadores/efeitos dos fármacos , Potenciais Pós-Sinápticos Excitadores/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Modelos Biológicos , Rede Nervosa/efeitos dos fármacos , Propionatos/farmacologia , Ratos , Ratos Sprague-Dawley , Receptores de Glutamato Metabotrópico/genética , Transdução de Sinais/efeitos dos fármacos , Transmissão Sináptica/efeitos dos fármacos , Transmissão Sináptica/genética
7.
J Biol Chem ; 292(19): 7954-7970, 2017 05 12.
Artigo em Inglês | MEDLINE | ID: mdl-28302728

RESUMO

P4-ATPases, also known as phospholipid flippases, are responsible for creating and maintaining transbilayer lipid asymmetry in eukaryotic cell membranes. Here, we use limited proteolysis to investigate the role of the N and C termini in ATP hydrolysis and auto-inhibition of the yeast flippase Drs2p-Cdc50p. We show that limited proteolysis of the detergent-solubilized and purified yeast flippase may result in more than 1 order of magnitude increase of its ATPase activity, which remains dependent on phosphatidylinositol 4-phosphate (PI4P), a regulator of this lipid flippase, and specific to a phosphatidylserine substrate. Using thrombin as the protease, Cdc50p remains intact and in complex with Drs2p, which is cleaved at two positions, namely after Arg104 and after Arg 1290, resulting in a homogeneous sample lacking 104 and 65 residues from its N and C termini, respectively. Removal of the 1291-1302-amino acid region of the C-terminal extension is critical for relieving the auto-inhibition of full-length Drs2p, whereas the 1-104 N-terminal residues have an additional but more modest significance for activity. The present results therefore reveal that trimming off appropriate regions of the terminal extensions of Drs2p can greatly increase its ATPase activity in the presence of PI4P and demonstrate that relief of such auto-inhibition remains compatible with subsequent regulation by PI4P. These experiments suggest that activation of the Drs2p-Cdc50p flippase follows a multistep mechanism, with preliminary release of a number of constraints, possibly through the binding of regulatory proteins in the trans-Golgi network, followed by full activation by PI4P.


Assuntos
ATPases Transportadoras de Cálcio/metabolismo , Fosfatos de Fosfatidilinositol/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Trifosfato de Adenosina/química , Arginina/química , Hidrólise , Mutação , Proteínas de Transferência de Fosfolipídeos/química , Fosfolipídeos/química , Fosforilação , Ligação Proteica , Domínios Proteicos , Proteólise , Trombina/química
8.
J Acoust Soc Am ; 142(6): 3691, 2017 12.
Artigo em Inglês | MEDLINE | ID: mdl-29289091

RESUMO

After identifying the parameters of the piezoelectric transducer, the mechanical impedance of the front medium is deduced. More particularly, the wave propagation velocity and attenuation are deduced in the inspected plate. By fitting the electrical impedance measurement results, the aging of composite materials is quantified, showing the effectiveness of this means of nondestructive evaluation. A specific measurement tool and protocol are proposed. Several estimators are identified on the basis of Gaussian fits of the resonances observed on the electrical impedance measurements. Those estimators are identified and the obtained results show that both the frequencies and widths of the resonances peaks vary according to the health of the plate, or aging duration. This method is applied successfully on non-perfect sandwich plates, porous, and with non-ideally flat surface. The sensitivity and limits of the most relevant estimators are discussed for the two studied plate families.

9.
Ultrasonics ; 62: 103-11, 2015 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-26138595

RESUMO

Defects may appear in composite structures during their life cycle. A 10MHz 128 elements phased array transducer was investigated to characterize join bonds and defects in sandwich honeycomb composite structures. An adequate focal law throughout the composite skin gives the ultrasonic dispersive properties of the composite skin and glue layer behind. The resulting B-scan cartographies allow characterizing locally the honeycomb adhesion. Experimental measurements are compared in good agreement with the Debye Series Method (DSM). In the processed C-scan image, flaws are detectable and measurable, localized both in the scanning plane and in the thickness of the composite skin.

10.
Ultrasonics ; 56: 308-17, 2015 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-25238692

RESUMO

The perturbation factors involved in ultrasonic broadband characterization of viscous fluids are analyzed. Precisely, the normal incidence error and the thermal sensitivity of the properties have been identified as dominant parameters. Thus, the sensitivity of the ultrasonic parameters of attenuation and phase velocity were measured at room temperature in the MHz frequency range for two reference silicone oils, namely 47V50 and 47V350 (Rhodorsil). Several methods of characterization were carried out: time of flight, cross-correlation and spectral method. These ultrasonic parameters are measured at room temperature. For this family of silicone oil, the dispersion of the attenuation spectrum is modeled by a power law. The velocity dispersion is modeled by two dispersion models: the quasi-local and the temporal causal. The impact of the experimental reproducibility of the phase velocity and acoustic attenuation was measured in the MHz frequency range, using a set of ultrasonic transducers with different center frequencies. These measurements are used to identify the dispersion of the ultrasonic parameters as a function of the frequency. A first experimental and descriptive approach is developed to assess the reproducibility of the normal incidence between the acoustic beam and the viscoelastic material. Thus, the relative error on the measurements of velocity and attenuation are directly related to the angular deviation of the ultrasonic wave, as well as the sampling and signal-to-noise ratio. A second experimental and phenomenological approach deals with the effect of a temperature change, typical of a polymerization reaction. As a result, the sensitivity of the phase velocity of silicone oil 47V50 was evaluated around -2 ms(-1) K(-1).

11.
J Proteome Res ; 14(1): 59-71, 2015 Jan 02.
Artigo em Inglês | MEDLINE | ID: mdl-25208982

RESUMO

Glutathionylation, the reversible post-translational formation of a mixed disulfide between a cysteine residue and glutathione (GSH), is a crucial mechanism for signal transduction and regulation of protein function. Until now this reversible redox modification was studied mainly in eukaryotic cells. Here we report a large-scale proteomic analysis of glutathionylation in a photosynthetic prokaryote, the model cyanobacterium Synechocystis sp. PCC6803. Treatment of acellular extracts with N,N-biotinyl glutathione disulfide (BioGSSG) induced glutathionylation of numerous proteins, which were subsequently isolated by affinity chromatography on streptavidin columns and identified by nano LC-MS/MS analysis. Potential sites of glutathionylation were also determined for 125 proteins following tryptic cleavage, streptavidin-affinity purification, and mass spectrometry analysis. Taken together the two approaches allowed the identification of 383 glutathionylatable proteins that participate in a wide range of cellular processes and metabolic pathways such as carbon and nitrogen metabolisms, cell division, stress responses, and H2 production. In addition, the glutathionylation of two putative targets, namely, peroxiredoxin (Sll1621) involved in oxidative stress tolerance and 3-phosphoglycerate dehydrogenase (Sll1908) acting on amino acids metabolism, was confirmed by biochemical studies on the purified recombinant proteins. These results suggest that glutathionylation constitutes a major mechanism of global regulation of the cyanobacterial metabolism under oxidative stress conditions.


Assuntos
Proteínas de Bactérias/metabolismo , Dissulfeto de Glutationa/metabolismo , Proteoma/metabolismo , Synechocystis/metabolismo , Sequência de Aminoácidos , Dados de Sequência Molecular , Fragmentos de Peptídeos/química , Processamento de Proteína Pós-Traducional , Proteômica
12.
J Biol Chem ; 289(49): 33850-61, 2014 Dec 05.
Artigo em Inglês | MEDLINE | ID: mdl-25301946

RESUMO

Sarcolipin (SLN) is a regulatory peptide present in sarcoplasmic reticulum (SR) from skeletal muscle of animals. We find that native rabbit SLN is modified by a fatty acid anchor on Cys-9 with a palmitic acid in about 60% and, surprisingly, an oleic acid in the remaining 40%. SLN used for co-crystallization with SERCA1a (Winther, A. M., Bublitz, M., Karlsen, J. L., Moller, J. V., Hansen, J. B., Nissen, P., and Buch-Pedersen, M. J. (2013) Nature 495, 265-2691; Ref. 1) is also palmitoylated/oleoylated, but is not visible in crystal structures, probably due to disorder. Treatment with 1 m hydroxylamine for 1 h removes the fatty acids from a majority of the SLN pool. This treatment did not modify the SERCA1a affinity for Ca(2+) but increased the Ca(2+)-dependent ATPase activity of SR membranes indicating that the S-acylation of SLN or of other proteins is required for this effect on SERCA1a. Pig SLN is also fully palmitoylated/oleoylated on its Cys-9 residue, but in a reverse ratio of about 40/60. An alignment of 67 SLN sequences from the protein databases shows that 19 of them contain a cysteine and the rest a phenylalanine at position 9. Based on a cladogram, we postulate that the mutation from phenylalanine to cysteine in some species is the result of an evolutionary convergence. We suggest that, besides phosphorylation, S-acylation/deacylation also regulates SLN activity.


Assuntos
Cisteína/química , Proteínas Musculares/química , Músculo Esquelético/metabolismo , Ácido Oleico/química , Ácido Palmítico/química , Fenilalanina/química , Processamento de Proteína Pós-Traducional , Proteolipídeos/química , Sequência de Aminoácidos , Animais , Evolução Biológica , Cristalografia por Raios X , Cisteína/metabolismo , Expressão Gênica , Hidroxilamina/química , Cinética , Lipoilação , Modelos Moleculares , Dados de Sequência Molecular , Proteínas Musculares/classificação , Proteínas Musculares/genética , Proteínas Musculares/metabolismo , Músculo Esquelético/química , Ácido Oleico/metabolismo , Ácido Palmítico/metabolismo , Fenilalanina/metabolismo , Filogenia , Proteolipídeos/classificação , Proteolipídeos/genética , Proteolipídeos/metabolismo , Coelhos , Retículo Sarcoplasmático , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/química , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/genética , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismo , Alinhamento de Sequência , Especificidade da Espécie , Suínos , Termodinâmica
13.
Appl Environ Microbiol ; 79(19): 5907-17, 2013 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-23872561

RESUMO

Streptomyces lividans TK24 is a strain that naturally produces antibiotics at low levels, but dramatic overproduction of antibiotics occurs upon interruption of the ppk gene. However, the role of the Ppk enzyme in relation to the regulation of antibiotic biosynthesis remains poorly understood. In order to gain a better understanding of the phenotype of the ppk mutant, the proteomes of the wild-type (wt) and ppk mutant strains, grown for 96 h on R2YE medium limited in phosphate, were analyzed. Intracellular proteins were separated on two-dimensional (2D) gels, spots were quantified, and those showing a 3-fold variation or more were identified by mass spectrometry. The expression of 12 proteins increased and that of 29 decreased in the ppk mutant strain. Our results suggested that storage lipid degradation rather than hexose catabolism was taking place in the mutant. In order to validate this hypothesis, the triacylglycerol contents of the wt and ppk mutant strains of S. lividans as well as that of Streptomyces coelicolor M145, a strain that produces antibiotics at high levels and is closely related to S. lividans, were assessed using electron microscopy and thin-layer chromatography. These studies highlighted the large difference in triacylglycerol contents of the three strains and confirmed the hypothetical link between storage lipid metabolism and antibiotic biosynthesis in Streptomyces.


Assuntos
Antibacterianos/biossíntese , Proteínas de Bactérias/análise , Metabolismo dos Lipídeos , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Proteoma/análise , Streptomyces lividans/enzimologia , Streptomyces lividans/metabolismo , Eletroforese em Gel Bidimensional , Deleção de Genes , Espectrometria de Massas , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Streptomyces lividans/genética
14.
Ultrasonics ; 53(3): 642-7, 2013 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-23259980

RESUMO

The paper deals with the study of the resonances of 1D periodic media composed of N elementary cells formed with two perfectly bonded layers which exhibit a high acoustic impedance contrast. In the case of a periodic bilayer structure constituted of a fluid layer and an elastic plate, it was shown in previous theoretical works that additional modes appear compared to those of a single plate. These are called structure modes. At low frequency, the so-called vertical modes are found. Approximate expressions of their cut-off frequencies are given and their numerical values match with the exact ones. At high frequency, the Lamb type modes are degenerated and modes in the fluid layers are also observed. Preliminary experimental results have already proved the existence of such phenomena for one and two periods. In our work, an experimental validation has been performed in the case of N periods made with a glass isotropic elastic plate and a water fluid layer, where the number N ranges from two to five. A good agreement is shown compared to theoretical works.

15.
J Biol Chem ; 287(24): 20176-86, 2012 Jun 08.
Artigo em Inglês | MEDLINE | ID: mdl-22528491

RESUMO

The eight pre- or/and post-synaptic metabotropic glutamatergic receptors (mGluRs) modulate rapid excitatory transmission sustained by ionotropic receptors. They are classified in three families according to their percentage of sequence identity and their pharmacological properties. mGluR4 belongs to group III and is mainly localized presynaptically. Activation of group III mGluRs leads to depression of excitatory transmission, a process that is exclusively provided by mGluR4 at parallel fiber-Purkinje cell synapse in rodent cerebellum. This function relies at least partly on an inhibition of presynaptic calcium influx, which controls glutamate release. To improve the understanding of molecular mechanisms of the mGluR4 depressant effect, we decided to identify the proteins interacting with this receptor. Immunoprecipitations using anti-mGluR4 antibodies were performed with cerebellar extracts. 183 putative partners that co-immunoprecipitated with anti-mGluR4 antibodies were identified and classified according to their cellular functions. It appears that native mGluR4 interacts with several exocytosis proteins such as Munc18-1, synapsins, and syntaxin. In addition, native mGluR4 was retained on a Sepharose column covalently grafted with recombinant Munc18-1, and immunohistochemistry experiments showed that Munc18-1 and mGluR4 colocalized at plasma membrane in HEK293 cells, observations in favor of an interaction between the two proteins. Finally, affinity chromatography experiments using peptides corresponding to the cytoplasmic domains of mGluR4 confirmed the interaction observed between mGluR4 and a selection of exocytosis proteins, including Munc18-1. These results could give indications to explain how mGluR4 can modulate glutamate release at parallel fiber-Purkinje cell synapses in the cerebellum in addition to the inhibition of presynaptic calcium influx.


Assuntos
Cálcio/metabolismo , Exocitose/fisiologia , Células de Purkinje/metabolismo , Receptores de Glutamato Metabotrópico/metabolismo , Sinapses/metabolismo , Animais , Células HEK293 , Humanos , Proteínas Munc18/genética , Proteínas Munc18/metabolismo , Células de Purkinje/citologia , Ramos Subendocárdicos/citologia , Ramos Subendocárdicos/metabolismo , Proteínas Qa-SNARE/genética , Proteínas Qa-SNARE/metabolismo , Ratos , Sinapses/genética , Sinapsinas/genética , Sinapsinas/metabolismo
16.
J Bacteriol ; 194(3): 587-97, 2012 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-22123248

RESUMO

Corynebacterineae is a specific suborder of Gram-positive bacteria that includes Mycobacterium tuberculosis and Corynebacterium glutamicum. The cell wall of these bacteria is composed of a heteropolymer of peptidoglycan (PG) linked to arabinogalactan (AG), which in turn is covalently associated with an atypical outer membrane, here called mycomembrane (M). The latter structure has been visualized by cryo-electron microscopy of vitreous sections, but its biochemical composition is still poorly defined, thereby hampering the elucidation of its physiological function. In this report, we show for the first time that the mycomembrane-linked heteropolymer of PG and AG (M-AG-PG) of C. glutamicum can be physically separated from the inner membrane on a flotation density gradient. Analysis of purified M-AG-PG showed that the lipids that composed the mycomembrane consisted almost exclusively of mycolic acid derivatives, with only a tiny amount, if any, of phospholipids and lipomannans, which were found with the characteristic lipoarabinomannans in the plasma membrane. Proteins associated with or inserted in the mycomembrane were extracted from M-AG-PG with lauryl-dimethylamine-oxide (LDAO), loaded on an SDS-PAGE gel, and analyzed by tandem mass spectrometry or by Western blotting. Sixty-eight different proteins were identified, 19 of which were also found in mycomembrane fragments released by the terminal-arabinosyl-transferase-defective ΔAftB strain. Almost all of them are predicted to contain a signal sequence and to adopt the characteristic ß-barrel structure of Gram-negative outer membrane proteins. These presumed mycomembrane proteins include the already-known pore-forming proteins (PorA and PorB), 5 mycoloyltransferases (cMytA, cMytB, cMytC, cMytD, and cMytF), several lipoproteins, and unknown proteins typified by a putative C-terminal hydrophobic anchor.


Assuntos
Membrana Celular/química , Membrana Celular/metabolismo , Corynebacterium glutamicum/metabolismo , Ácidos Micólicos/metabolismo , Proteínas da Membrana Bacteriana Externa/química , Proteínas da Membrana Bacteriana Externa/genética , Proteínas da Membrana Bacteriana Externa/metabolismo , Membrana Celular/genética , Corynebacterium glutamicum/química , Corynebacterium glutamicum/genética , Espectrometria de Massas , Ácidos Micólicos/análise
17.
Appl Microbiol Biotechnol ; 93(5): 2125-34, 2012 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-21863313

RESUMO

Based on the observation that shocks provoked by heat or amphiphilic compounds present some similarities, this work aims at studying whether cells grown on oleate (amphiphilic pre-stress) acquire a tolerance to heat shock. In rich media, changing glucose for oleate significantly enhanced the cell resistance to the shock, however, cells grown on a minimal oleate medium lost their ability to grow on agar with the same kinetic than glucose-grown cells (more than 7-log decrease in 18 min compared with 3-log for oleate-grown cells). Despite this difference in kinetics, the sequence of events was similar for oleate-grown cells maintained at 50°C with a (1) loss of ability to form colonies at 27°C, (2) loss of membrane integrity and (3) lysis (observed only for some minimal-oleate-grown cells). Glucose-grown cells underwent different changes. Their membranes, which were less fluid, lost their integrity as well and cells were rapidly inactivated. But, surprisingly, their nuclear DNA was not stained by propidium iodide and other cationic fluorescent DNA-specific probes but became stainable by hydrophobic ones. Moreover, they underwent a dramatic increase in membrane viscosity. The evolution of lipid bodies during the heat shock depended also on the growth medium. In glucose-grown cells, they seemed to coalesce with the nuclear membrane whereas for oleate-grown cells, they coalesced together forming big droplets which could be released in the medium. In some rare cases of oleate-grown cells, lipid bodies were fragmented and occupied all the cell volume. These results show that heat triggers programmed cell death with uncommon hallmarks for glucose-grown cells and necrosis for methyl-oleate-grown cells.


Assuntos
Glucose/metabolismo , Viabilidade Microbiana/efeitos da radiação , Ácido Oleico/metabolismo , Yarrowia/metabolismo , Yarrowia/efeitos da radiação , Membrana Celular/fisiologia , Membrana Celular/efeitos da radiação , Meios de Cultura/química , Temperatura Alta , Estresse Fisiológico , Yarrowia/crescimento & desenvolvimento , Yarrowia/fisiologia
18.
Ultrasonics ; 52(3): 427-34, 2012 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-22071268

RESUMO

A measurement method is proposed for the ultrasonic characterization of a fluid layer, corresponding to the resin transfer molding (RTM) manufacturing process. The ultrasonic velocity and attenuation of the silicone oil are measured in three samples having different viscosities. The measurement method is established on the basis of the attenuation of ultrasonic waves in fluids. A correction of the beam diffraction is implemented to improve measurement precision. A single element transducer with central frequency of 15 MHz is used. The tested fluids simulate the industrial resin used to manufacture composite materials. When injecting this resin, its viscosity increases until it reaches a critical state of polymerization. In this paper we focus on ultrasonic characterization of three fluids representing three intermediate cases of fluid resin during its injection before reaching the polymerization state.


Assuntos
Transdutores , Ultrassom , Polímeros , Óleos de Silicone , Viscosidade
19.
Proteomics ; 10(13): 2418-28, 2010 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-20405473

RESUMO

The importance of redox-regulation in Arabidopsis thaliana roots has been investigated through the identification of the proteins interacting with thioredoxin (TRX), an ubiquitous thiol-disulfide reductase. We have applied a proteomic approach based on affinity chromatography on a monocysteinic mutant of plastidial y-type TRX used as a bait to trap putative partners in a crude extract of root proteins. Seventy-two proteins have been identified, functioning mainly in metabolism, detoxification and response to stress, protein processing and signal transduction. This study allowed us to isolate 24 putative new targets and to propose the mevalonic acid-dependent biosynthesis of isoprenoids as a new redox-mediated process. The redox-regulation of phenylpropanoid biosynthesis is also suggested, three enzymes of this pathway being retained on the column. We also provided experimental evidence that phenylammonia-lyase was enzymatically more active when reduced by TRXy in root crude extract. Among the high number of partners involved in defense against stress we isolated from the column, we focused on plastidial monodehydroascorbate reductase and showed that its activity was dramatically increased in vitro in the presence of DTT-reduced TRXy1 in root crude extracts. Our data strongly suggest that TRXy1 could be the physiological regulator of monodehydroascorbate reductase in root plastids.


Assuntos
Arabidopsis/química , Raízes de Plantas/química , Tiorredoxinas/metabolismo , Arabidopsis/metabolismo , Ácidos Graxos/biossíntese , Oxirredução , Extratos Vegetais/química , Raízes de Plantas/metabolismo , Propanóis/química , Propanóis/metabolismo , Transdução de Sinais , Terpenos/metabolismo
20.
Appl Microbiol Biotechnol ; 87(3): 1089-99, 2010 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-20422183

RESUMO

In hydrophobic compounds biotechnology, medium-chain-length metabolites often perturb cell activity. Their effect is usually studied in model conditions of growth in glucose media. Here, we study whether culture on lipids has an impact on the resistance of Yarrowia lipolytica to such compounds: Cells were cultured on glucose or oleate and submitted to gamma-dodecalactone. After a 60-min exposure to 3 g L(-1), about 80% of the glucose-grown cells (yeast extract peptone dextrose (YPD) cells) had lost their cultivability, 38% their membrane integrity, and 31% their reducing capacity as shown with propidium iodide and methylene blue, respectively. For oleate-grown cells, treatment at 6 g L(-1) did not alter cultivability despite some transient loss of membrane integrity from 3 g L(-1). It was shown with diphenylhexatriene and 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene that oleate-grown cells had membranes more fluid and less sensitive to the lactone-induced fluidization. Analyses revealed also higher contents of ergosterol but, for YPD- and minimum-oleate-grown cells (YNBO cells), the addition of lactone provoked a decrease in the concentration of ergosterol in a way similar to the depletion by methyl-beta-cyclodextrin and an important membrane fluidization. Ergosterol depletion or incorporation increased or decreased, respectively, cell sensitivity to lactone. This study shows that the embedment of oleate moieties into membranes as well as higher concentrations of sterol play a role in the higher resistance to lactone of oleate-grown cells (YPO cells). Similar oleate-induced increase in resistance was also observed for Rhodotorula and Candida strains able to grow on oleate as the sole carbon source whereas Saccharomyces and Sporidiobolus cells were more sensitive after induction.


Assuntos
Membrana Celular/química , Meios de Cultura/química , Lactonas/química , Yarrowia/metabolismo , Membrana Celular/metabolismo , Meios de Cultura/metabolismo , Glucose/metabolismo , Lactonas/metabolismo , Viabilidade Microbiana , Ácido Oleico/metabolismo , Yarrowia/química , Yarrowia/crescimento & desenvolvimento
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