Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 104
Filtrar
Mais filtros










Base de dados
Intervalo de ano de publicação
3.
Int J Food Microbiol ; 339: 108987, 2021 Feb 02.
Artigo em Inglês | MEDLINE | ID: mdl-33321431

RESUMO

Bacterial and fungal population dynamics in cider for distillation have so far been explored by culture-dependant methods. Cider for distillation can be produced by the spontaneous fermentation of apples that do not undergo any intervention during the process. In this study, cider microbiomes extracted from six tanks containing ciders for distillation from four producers in Normandy were characterized at three main stages of the fermentation process: fermentation Initiation (I), end of the alcoholic Fermentation (F) and end of the Maturation period (M). Cider samples were subjected to Illumina MiSeq sequencing (rRNA 16S V1-V3 and ITS1 region targeting) to determine bacterial and fungal communities. Yeasts (YGC), Zymomonas (mZPP) and lactic acid bacteria selective media (mMRS, mMLO, mPSM) were also used to collect 807 isolates. Alcoholic levels, glycerol, sugar content (glucose, fructose and sucrose), pH, total and volatile acidity, nitrogen, malic and lactic acid contents were determined at all sampling points. Alpha diversity indexes show significant differences (p < 0.05) in microbial populations between I, F and M. Fungal communities were characterized by microorganisms from the environment and phytopathogens at I followed by the association of yearsts with alcoholic fermentation like Saccharomyces and non-Saccharomyces yeasts (Hanseniaspora, Candida). A maturation period for cider leads to an increase of the Dekkera/Brettanomyces population, which is responsible for off-flavors in cider for all producers. Among bacterial communities, the genera community associated to malolactic fermentation (Lactobacillus sp., Leuconostoc sp., Oenococcus sp.) was the most abundant at F and M. Acetic acid bacteria such as Acetobacter sp., Komagataeibacter sp. and Gluconobacter sp. were also detected during the process. Significant differences (p < 0.05) were found in fungal and bacterial populations between the four producers and during the fermentation process. The development of microorganisms associated with cider spoilage such as Zymomonas mobilis, Lactobacillus collinoides or Brettanomyces/Dekkera sp. was anticipated by a metagenomic approach. The monitoring of microbial diversity via high throughput sequencing combined with physical-chemical analysis is an interesting approach to improve the fermentation performance of cider for distillation and therefore, the quality of Calvados.


Assuntos
Bebidas Alcoólicas/microbiologia , Fenômenos Fisiológicos Bacterianos , Biodiversidade , Destilação , Fungos/fisiologia , Bactérias/genética , DNA Espaçador Ribossômico/genética , Fermentação , Malus , RNA Ribossômico 16S/genética
4.
Eur J Neurol ; 26(10): 1274-1280, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31021023

RESUMO

BACKGROUND AND PURPOSE: Neurosarcoidosis is a rare inflammatory disorder of unknown cause. The aim of this study was to evaluate the value of T/B lymphocyte population counts and the concentrations of the cytokines interleukin (IL) 6 and IL-10 in the cerebrospinal fluid (CSF) of neurosarcoidosis patients. METHODS: A retrospective study CSF biomarkers was conducted in patients with neurosarcoidosis who underwent CSF analysis between 2012 and 2017 as well as various control populations. RESULTS: Forty-three patients with neurosarcoidosis, 14 with multiple sclerosis (MS) and 48 with other inflammatory disorders were analyzed. The CSF IL-6 levels were higher in sarcoidosis patients than in MS patients (median 8 vs. 3 pg/ml, P = 0.006). The CSF CD4/CD8 ratio was higher in sarcoidosis patients than in MS patients and in patients with other inflammatory disorders (median 3.18 vs. 2.36 and 2.10, respectively, P = 0.008). The CSF IL-6 level was higher in patients with active neurosarcoidosis than in non-active neurosarcoidosis patients (median 13 vs. 3 pg/ml, P = 0.0005). In patients with neurosarcoidosis, a CSF IL-6 concentration >50 pg/ml was associated with a higher risk of relapse or progression-free survival (hazard ratio 3.60; 95% confidence interval 1.78-23.14). A refractory neurosarcoidosis patient was treated with an anti-IL-6 monoclonal antibody that produced a complete neurological response. CONCLUSIONS: The CSF CD4/CD8 ratio and IL-6 concentration are increased in neurosarcoidosis compared to MS and other inflammatory disorders. A CSF IL-6 concentration >50 pg/ml is associated with relapse or progression of neurosarcoidosis. IL-10 levels may be elevated in neurosarcoidosis.


Assuntos
Relação CD4-CD8 , Doenças do Sistema Nervoso Central/líquido cefalorraquidiano , Líquido Cefalorraquidiano/citologia , Interleucina-10/líquido cefalorraquidiano , Interleucina-6/líquido cefalorraquidiano , Sarcoidose/líquido cefalorraquidiano , Adulto , Biomarcadores/líquido cefalorraquidiano , Doenças do Sistema Nervoso Central/imunologia , Feminino , Humanos , Inflamação/líquido cefalorraquidiano , Masculino , Pessoa de Meia-Idade , Esclerose Múltipla/líquido cefalorraquidiano , Intervalo Livre de Progressão , Recidiva , Estudos Retrospectivos , Sarcoidose/imunologia , Resultado do Tratamento , Adulto Jovem
5.
Ecology ; 99(11): 2592-2604, 2018 11.
Artigo em Inglês | MEDLINE | ID: mdl-30198573

RESUMO

Effects of species diversity on population and community stability (or more precisely, the effects of species richness on temporal variability) have been studied for several decades, but there have been no large-scale tests in natural communities of predictions from theory. We used 91 data sets including plants, fish, small mammals, zooplankton, birds, and insects, to examine the relationship between species richness and temporal variability in populations and communities. Seventy-eight of 91 data sets showed a negative relationship between species richness and population variability; 46 of these relationships were statistically significant. Only five of the 13 positive richness-population variability relationships were statistically significant. Similarly, 51 of 91 data sets showed a negative relationship between species richness and community variability; of these, 26 were statistically significant. Seven of the 40 positive richness-community-variability relationships were statistically significant. We were able to test transferability (i.e., the predictive ability of models for sites that are spatially distinct from sites that were used to build the models) for 69 of 91 data sets; 35 and 31 data sets were transferable at the population and community levels, respectively. Only four were positive at the population level, and two at the community level. We conclude that there is compelling evidence of a negative relationship between species richness and temporal variability for about one-half of the ecological communities we examined. However, species richness explained relatively little of the variability in population or community abundances and resulted in small improvements in predictive ability.


Assuntos
Biodiversidade , Ecossistema , Animais , Plantas , Dinâmica Populacional , Zooplâncton
6.
Mol Psychiatry ; 21(7): 936-45, 2016 07.
Artigo em Inglês | MEDLINE | ID: mdl-26370147

RESUMO

Autism spectrum disorder (ASD) is a common neurodevelopmental condition characterized by marked genetic heterogeneity. Recent studies of rare structural and sequence variants have identified hundreds of loci involved in ASD, but our knowledge of the overall genetic architecture and the underlying pathophysiological mechanisms remains incomplete. Glycine receptors (GlyRs) are ligand-gated chloride channels that mediate inhibitory neurotransmission in the adult nervous system but exert an excitatory action in immature neurons. GlyRs containing the α2 subunit are highly expressed in the embryonic brain, where they promote cortical interneuron migration and the generation of excitatory projection neurons. We previously identified a rare microdeletion of the X-linked gene GLRA2, encoding the GlyR α2 subunit, in a boy with autism. The microdeletion removes the terminal exons of the gene (GLRA2(Δex8-9)). Here, we sequenced 400 males with ASD and identified one de novo missense mutation, p.R153Q, absent from controls. In vitro functional analysis demonstrated that the GLRA2(Δex8)(-)(9) protein failed to localize to the cell membrane, while the R153Q mutation impaired surface expression and markedly reduced sensitivity to glycine. Very recently, an additional de novo missense mutation (p.N136S) was reported in a boy with ASD, and we show that this mutation also reduced cell-surface expression and glycine sensitivity. Targeted glra2 knockdown in zebrafish induced severe axon-branching defects, rescued by injection of wild type but not GLRA2(Δex8-9) or R153Q transcripts, providing further evidence for their loss-of-function effect. Glra2 knockout mice exhibited deficits in object recognition memory and impaired long-term potentiation in the prefrontal cortex. Taken together, these results implicate GLRA2 in non-syndromic ASD, unveil a novel role for GLRA2 in synaptic plasticity and learning and memory, and link altered glycinergic signaling to social and cognitive impairments.


Assuntos
Glicina/metabolismo , Receptores de Glicina/genética , Receptores de Glicina/metabolismo , Adolescente , Adulto , Animais , Transtorno do Espectro Autista/metabolismo , Transtorno Autístico/metabolismo , Criança , Pré-Escolar , Glicina/genética , Humanos , Potenciação de Longa Duração/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neurogênese/efeitos dos fármacos , Plasticidade Neuronal/efeitos dos fármacos , Neurônios/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transmissão Sináptica/fisiologia , Peixe-Zebra
7.
J Thromb Haemost ; 13(5): 815-20, 2015 May.
Artigo em Inglês | MEDLINE | ID: mdl-25728415

RESUMO

BACKGROUND: von Willebrand factor (VWF) is cleared in a shear stress- and macrophage-dependent manner by LRP1. von Willebrand disease (VWD)-type 2B mutants are endocytosed more efficiently than wild-type (wt)-VWF by macrophages. OBJECTIVE: To investigate if VWD-type 2B mutations in the VWF A1-domain affect LRP1 binding and LRP1-dependent clearance. METHODS: Recombinant Fc-tagged A1 domain (A1-Fc, A2-Fc, A3-Fc) and full-length VWF (wt or mutants thereof) were tested for binding to LRP1 or a recombinant fragment thereof in a static immunosorbent assay. Mutant and wt-VWF were also compared for clearance in mice lacking macrophage LRP1 (macLRP1(-) ) and control mice (macLRP1(+) ). RESULTS: We found that A1-Fc but not A2-Fc or A3-Fc binds dose-dependently to LRP1. Binding of A1-Fc to LRP1 was markedly enhanced by the VWD-type 2B mutation p.V1316M. As expected, full-length wt-VWF was unable to bind LRP1 under static conditions unless ristocetin was added. In contrast, the presence of the p.V1316M or p.R1306Q mutation induced spontaneous binding to LRP1 without the need for ristocetin or shear stress. Both mutants were cleared more rapidly than wt-VWF in control macLRP1(+) mice. Surprisingly, deletion of macrophage LRP1 abrogated the increased clearance of the VWF/p.R1306Q and VWF/p.V1316M mutant. CONCLUSION: The VWF A1-domain contains a binding site for LRP1. Certain VWD-type 2B mutations relieve the need for shear stress to induce LRP1 binding. Enhanced LRP1 binding coincides with a reduced survival of VWF/p.R1306Q and VWF/p.V1316M. Our data provide a rationale for reduced VWF levels in at least some VWD-type 2B patients.


Assuntos
Proteína-1 Relacionada a Receptor de Lipoproteína de Baixa Densidade/metabolismo , Mutação , Resistência ao Cisalhamento , Estresse Mecânico , Fator de von Willebrand/metabolismo , Animais , Feminino , Masculino , Camundongos , Ligação Proteica , Fator de von Willebrand/genética
8.
Neuroscience ; 242: 110-27, 2013 Jul 09.
Artigo em Inglês | MEDLINE | ID: mdl-23506738

RESUMO

Glycine acts as a neuromodulator to regions rich in glutamatergic synapses, such as the forebrain. However, recent evidences for synaptic release of glycine in hippocampal cultured neurons and synaptosomes argue for the existence of functional glycinergic synapses in the hippocampus. It is well established that GABA and glycine act in concert at inhibitory synapses, while the existence of synapses which utilize both glutamate and glycine is less common. The purpose of the present study was to investigate the distribution of glycine and its role in hippocampal neurotransmission. Using immunohistochemistry, we demonstrate that vesicular glycine is preferentially stored in glutamatergic, rather than GABAergic presynaptic terminals. Using the sniffer patch technique, we found that glycine could be released upon presynaptic activity. Furthermore, using whole-cell patch-clamp recordings, we show for the first time the presence of a postsynaptic strychnine-sensitive chloride current in response to presynaptic stimulation. The small amplitude of this current is likely due to the paucity of postsynaptic glycine receptors rather than a low level of glycine release. Taken together, our results suggest that glycine is stored in glutamatergic presynaptic terminals. It is likely that the major role of glycine that is released from presynaptic terminals is to modulate N-methyl-d-aspartate receptor function but may also play a role in decreasing neuronal excitability by opposing glutamatergic neurotransmission in pathological states such as epilepsy or ischemia.


Assuntos
Ácido Glutâmico/metabolismo , Glicina/metabolismo , Hipocampo/metabolismo , Terminações Pré-Sinápticas/metabolismo , Vesículas Sinápticas/metabolismo , Animais , Tronco Encefálico/metabolismo , Células CHO , Cricetulus , Glutamato Descarboxilase/metabolismo , Glicina/fisiologia , Hipocampo/fisiologia , Potenciais da Membrana/fisiologia , Camundongos , Neurônios/metabolismo , Neurônios/fisiologia , Terminações Pré-Sinápticas/fisiologia , Receptores de Glicina/metabolismo , Receptores de Glicina/fisiologia , Receptores de N-Metil-D-Aspartato/metabolismo , Transmissão Sináptica/fisiologia , Ácido gama-Aminobutírico/metabolismo
9.
Glia ; 59(4): 675-95, 2011 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-21305616

RESUMO

Microglial cells invade the central nervous system during embryonic development, but their developmental functional roles in vivo remain largely unknown. Accordingly, their invasion pattern during early embryonic development is still poorly understood. To address this issue, we analyzed the initial developmental pattern of microglial cell invasion in the spinal cord of CX3CR1-eGFP mouse embryos using immunohistochemistry. Microglial cells began to invade the mouse embryonic spinal cord at a developmental period corresponding to the onset of spontaneous electrical activity and of synaptogenesis. Microglial cells reached the spinal cord through the peripheral vasculature and began to invade the parenchyma at 11.5 days of embryonic age (E11.5). Remarkably, at E12.5, activated microglial cells aggregated in the dorsolateral region close to terminals of dying dorsal root ganglia neurons. At E13.5, microglial cells in the ventral marginal zone interacted with radial glial cells, whereas ramified microglial cells within the parenchyma interacted with growing capillaries. At this age, activated microglial cells (Mac-2 staining) also accumulated within the lateral motor columns at the onset of the developmental cell death of motoneurons. This cell aggregation was still observed at E14.5, but microglial cells no longer expressed Mac-2. At E15.5, microglial cells were randomly distributed within the parenchyma. Our results provide the essential basis for further studies on the role of microglial cells in the early development of spinal cord neuronal networks in vivo.


Assuntos
Microglia/fisiologia , Rede Nervosa/embriologia , Neurônios/fisiologia , Medula Espinal/embriologia , Animais , Imuno-Histoquímica , Camundongos , Camundongos Transgênicos , Microglia/citologia , Microscopia Confocal , Rede Nervosa/citologia , Rede Nervosa/metabolismo , Neurônios/citologia , Medula Espinal/citologia , Medula Espinal/metabolismo
10.
Ecology ; 91(11): 3343-53, 2010 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-21141195

RESUMO

Spatially organized distribution patterns of species and communities are shaped by both autogenic processes (neutral mechanism theory) and exogenous processes (niche theory). In the latter, environmental variables that are themselves spatially organized induce spatial structure in the response variables. The relative importance of these processes has not yet been investigated in urban habitats. We compared the variance explained by purely spatial, spatially structured environmental, and purely environmental components for the community composition of spiders (Araneae), bees (Apidae), and birds (Aves) at 96 locations in three Swiss cities. Environmental variables (topography, climate, land cover, urban green management) were measured on four different radii around sampling points (< 10 m, 50 m, 250 m, 1000 m), while Moran's eigenvector maps (MEMs) acted as spatial variables. All three taxonomic groups showed weak spatial structure. Spider communities reacted to very fine-scaled environmental changes of lawn and meadow management and climate. Bird community composition was determined by woody plants as well as solar radiation at all radii, the scale of the influence varying among species. Bee communities were weakly explained by isolated variables only. Our results suggest that the anthropogenic structuring of urban areas has disrupted the spatial organization of environmental variables and inhibited the development of biotic spatial processes. The near absence of spatial structure may therefore be a feature typical of urban species assemblages, resulting in urban community composition mainly influenced by local environmental variables. Urban environments represent a close-knit mosaic of habitats that are regularly disturbed. Species communities in urban areas are far from equilibrium. Our analysis also suggests that urban communities need to be considered as being in constant change to adapt to disturbances and changes imposed by human activities.


Assuntos
Abelhas/fisiologia , Aves/fisiologia , Ecossistema , Aranhas/fisiologia , Animais , Cidades , Modelos Biológicos , Dinâmica Populacional , Processos Estocásticos , Suíça
11.
Ecol Appl ; 20(6): 1704-20, 2010 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-20945769

RESUMO

Clearing vegetation for traditional agriculture diminishes native habitat and reduces plant transpiration, leading to increased groundwater recharge and onset of dryland salinization due to rising groundwater and mobilization of salt stores in the soil profile. This change in hydrology and salinity can also negatively affect biodiversity in many semiarid regions. Alternating native perennial tree belts with mono-species agriculture within the tree belt alleys is one possible system that can provide recharge control and recover some of the ecosystem services of degraded agricultural landscapes. To assess the effect of this agroforestry technique on groundwater levels, an alley farming trial was established in 1995, incorporating different combinations of belt width, alley width, and revegetation density. Transects of piezometers within each design have been monitored from October 1995 to January 2008. The data set consisted of 70 piezometers monitored on 39 dates. Two trends were observed within the raw data: An increase in water table depth with time and an increase in the range of depths monitored at the site were clearly discernible. However, simple hydrograph analysis of the data has proved unsuccessful at distinguishing the effect of the tree belts on the water table morphology. The statistical techniques employed in this paper to show the effect of the experiment on the water table were variation partitioning, principal coordinates of neighbor matrices (PCNM), and canonical redundancy analysis (RDA). The environmental variables (alley farming design, distance of piezometer from the tree belt, and percentage vegetation cover including edge effect) explained 20-30% of the variation of the transformed and detrended data for the entire site. The spatial PCNM variables explained a further 20-30% of the variation. Partitioning of the site into a northern and southern block increased the proportion of explained variation for the plots in the northern block. The spatial PCNM variables and vegetation cover remained the most significant variables. The PCNM analysis revealed no spatial pattern that could be attributed to the trial. The high proportion of unexplained variation may be due to site variables that have not been considered in this study.


Assuntos
Agricultura/métodos , Produtos Agrícolas/fisiologia , Árvores/fisiologia , Água , Fenômenos Geológicos , Raízes de Plantas/crescimento & desenvolvimento , Fatores de Tempo , Austrália Ocidental
13.
Mol Ecol ; 19(17): 3824-35, 2010 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-20723057

RESUMO

A major challenges facing landscape geneticists studying adaptive variation is to include all the environmental variables that might be correlated with allele frequencies across the genome. One way of identifying loci that are possibly under selection is to see which ones are associated with environmental gradient or heterogeneity. Since it is difficult to measure all environmental variables, one may take advantage of the spatial nature of environmental filters to incorporate the effect of unaccounted environmental variables in the analysis. Assuming that the spatial signature of these variables is broad-scaled, broad-scale Moran's eigenvector maps (MEM) can be included as explanatory variables in the analysis as proxies for unmeasured environmental variables. We applied this approach to two data sets of the alpine plant Arabis alpina. The first consisted of 140 AFLP loci sampled at 130 sites across the European Alps (large scale). The second one consisted of 712 AFLP loci sampled at 93 sites (regional scale) in three mountain massifs (local scale) of the French Alps. For each scale, we regressed the frequencies of each AFLP allele on a set of eco-climatic and MEM variables as predictors. Twelve (large scale) and 11% (regional scale) of all loci were detected as significantly correlated to at least one of the predictors ( > 0.5), and, except for one massif, 17% at the local scale. After accounting for spatial effects, temperature and precipitation were the two major determinants of allele distributions. Our study shows how MEM models can account for unmeasured environmental variation in landscape genetics models.


Assuntos
Adaptação Fisiológica/genética , Arabis/genética , Variação Genética , Análise do Polimorfismo de Comprimento de Fragmentos Amplificados , Ecologia/métodos , Meio Ambiente , Europa (Continente) , Frequência do Gene , Genótipo , Modelos Estatísticos , Análise de Componente Principal
14.
Proc Natl Acad Sci U S A ; 104(9): 3273-7, 2007 Feb 27.
Artigo em Inglês | MEDLINE | ID: mdl-17360637

RESUMO

In population ecology, there has been a fundamental controversy about the relative importance of competition-driven (density-dependent) population regulation vs. abiotic influences such as temperature and precipitation. The same issue arises at the community level; are population sizes driven primarily by changes in the abundances of cooccurring competitors (i.e., compensatory dynamics), or do most species have a common response to environmental factors? Competitive interactions have had a central place in ecological theory, dating back to Gleason, Volterra, Hutchison and MacArthur, and, more recently, Hubbell's influential unified neutral theory of biodiversity and biogeography. If competitive interactions are important in driving year-to-year fluctuations in abundance, then changes in the abundance of one species should generally be accompanied by compensatory changes in the abundances of others. Thus, one necessary consequence of strong compensatory forces is that, on average, species within communities will covary negatively. Here we use measures of community covariance to assess the prevalence of negative covariance in 41 natural communities comprising different taxa at a range of spatial scales. We found that species in natural communities tended to covary positively rather than negatively, the opposite of what would be expected if compensatory dynamics were important. These findings suggest that abiotic factors such as temperature and precipitation are more important than competitive interactions in driving year-to-year fluctuations in species abundance within communities.


Assuntos
Comportamento Competitivo , Ecossistema , Meio Ambiente , Modelos Teóricos , Dinâmica Populacional , Análise de Variância , Animais
15.
Oncogene ; 26(19): 2695-706, 2007 Apr 26.
Artigo em Inglês | MEDLINE | ID: mdl-17057735

RESUMO

An instability of the mature cell phenotype is thought to participate to the formation of gliomas, primary brain tumors deriving from astrocytes and/or neural stem cells. Transforming growth factor alpha (TGFalpha) is an erbB1 ligand overexpressed in the earliest stages of gliomas, and exerts trophic effects on gliomal cells and astrocytes. Here, we questioned whether prolonged TGFalpha exposure affects the stability of the normal mature astrocyte phenotype. We first developed astrocyte cultures devoid of residual neural stem cells or progenitors. We demonstrate that days of TGFalpha treatment result in the functional conversion of a population of mature astrocytes into radial glial cells, a population of neural progenitors. TGFalpha-generated radial glial cells support embryonic neurons migration, and give birth to cells of the neuronal lineage, expressing neuronal markers and the electrophysiological properties of neuroblasts. Lengthening TGFalpha treatment to months results in the delayed appearance of cells with neural stem cells properties: they form floating cellular spheres that are self-renewing, can be clonally derived from a single cell and differentiated into cells of the neuronal lineage. This study uncovers a novel population of mature astrocytes capable, in response to a single epigenetic factor, to regress progressively into a neural stem-like cell stage via an intermediate progenitor stage.


Assuntos
Astrócitos/citologia , Diferenciação Celular/efeitos dos fármacos , Neurônios/metabolismo , Células-Tronco/citologia , Fator de Crescimento Transformador alfa/farmacologia , Animais , Astrócitos/metabolismo , Linhagem da Célula , Movimento Celular , Células Cultivadas , Eletrofisiologia , Embrião de Mamíferos/citologia , Embrião de Mamíferos/metabolismo , Receptores ErbB/metabolismo , Feminino , Feto/citologia , Feto/metabolismo , Humanos , Immunoblotting , Imunoprecipitação , Camundongos , Camundongos Endogâmicos C57BL , Neuroglia/citologia , Neurônios/citologia , Receptor ErbB-2/metabolismo , Proteínas Recombinantes/farmacologia , Células-Tronco/metabolismo
16.
J Thromb Haemost ; 5(2): 321-8, 2007 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-17087728

RESUMO

BACKGROUND: von Willebrand disease (VWD) type 2 is associated with mutations in von Willebrand factor (VWF) that affect its secretion, multimeric pattern, affinity for platelet receptors and clearance of the protein. While increased proteolysis by a disintegrin-like and metalloprotease with thrombospondin type 1 motifs-13 (ADAMTS-13) has been clearly established for VWF type 2A, only little is known about VWF types 2B and 2M in this regard. OBJECTIVES: Sensitivity of wild-type (WT) and mutated recombinant (r) VWF to proteolysis by ADAMTS-13 was investigated to better understand the role of this process in the pathophysiology of VWD. METHODS: We used human rADAMTS-13-WT to digest 11 full-length recombinant forms of VWF carrying molecular abnormalities identified in patients with VWD type 2A (E1638K and P1648S), type 2B (InsM1303, R1306W, R1308P and V1314F) and type 2M (G1324A, E1359K, K1362T, R1374H and I1425F). RESULTS: Using low ionic strength conditions, all mutations induced increased proteolysis of rVWF by rADAMTS-13 as compared with rVWF-WT. The susceptibility of mutants decreased in the following order: type 2A > type 2B > type 2M > rVWF-WT. At physiological salt concentration (150 mm NaCl) the sensitivity of all rVWF to rADAMTS-13 was significantly decreased. However, type 2A and type 2B mutants still exhibited a significantly higher susceptibility to rADAMTS-13 than rVWF-WT, whereas type 2M mutants normalized. CONCLUSIONS: Type 2M mutants and rVWF-WT exhibit a similar sensitivity to rADAMTS-13-mediated proteolysis, in agreement with the normal multimeric pattern in vivo. In VWD type 2B, the spontaneous binding to platelets and excessive degradation by ADAMTS-13 of VWF high-molecular-weight multimers may account for their clearance from plasma.


Assuntos
Proteínas ADAM/metabolismo , Doenças de von Willebrand/genética , Fator de von Willebrand/metabolismo , Proteínas ADAM/genética , Proteína ADAMTS13 , Dimerização , Predisposição Genética para Doença , Humanos , Mutação de Sentido Incorreto , Concentração Osmolar , Doenças de von Willebrand/classificação , Doenças de von Willebrand/etiologia
17.
Neuroscience ; 140(2): 389-402, 2006 Jun 30.
Artigo em Inglês | MEDLINE | ID: mdl-16564635

RESUMO

In vertebrates, most glycinergic inhibitory neurons discharge phasically at a relatively low frequency. Such a pattern of glycine liberation from presynaptic terminals may affect the kinetics of post-synaptic glycine receptors. To examine this influence, we have analyzed the behavior of glycine receptors in response to repetitive stimulation at frequencies at which consecutive outside-out currents did not superimpose (0.5-4 Hz). Neurotransmitter release was mimicked on outside-out patches from zebrafish hindbrain Mauthner cells using fast flow application techniques. The amplitude of outside-out currents evoked by short (1 ms) repetitive applications of a saturating concentration (3 mM) of glycine remained unchanged for application frequencies1 Hz. Glycine-evoked current simulations using a simple Markov model describing zebrafish glycine receptor kinetic behavior, indicates that this down-regulation of glycine receptor efficacy is due to a progressive accumulation of the receptors in a long lasting desensitization state. Our simulations suggest that this down-regulation can occur even when spontaneous inhibitory currents were generated randomly at a frequency>1 Hz.


Assuntos
Glicina/metabolismo , Inibição Neural/fisiologia , Neurônios/metabolismo , Receptores de Glicina/metabolismo , Rombencéfalo/metabolismo , Transmissão Sináptica/fisiologia , Animais , Relação Dose-Resposta a Droga , Regulação para Baixo/efeitos dos fármacos , Regulação para Baixo/fisiologia , Vias Eferentes/citologia , Vias Eferentes/efeitos dos fármacos , Vias Eferentes/metabolismo , Estimulação Elétrica , Glicina/farmacologia , Cadeias de Markov , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/fisiologia , Modelos Neurológicos , Inibição Neural/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Técnicas de Patch-Clamp , Receptores de Glicina/efeitos dos fármacos , Formação Reticular/citologia , Formação Reticular/efeitos dos fármacos , Formação Reticular/metabolismo , Rombencéfalo/citologia , Rombencéfalo/efeitos dos fármacos , Transmissão Sináptica/efeitos dos fármacos
18.
J Thromb Haemost ; 4(1): 236-46, 2006 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-16409474

RESUMO

BACKGROUND: Alpha(IIb)beta3-mediated platelet adhesive interactions in the vasculature, which are dependent on the functional state of this receptor, may be sensitive to shear forces. OBJECTIVES: To evaluate the influence of the alpha(IIb)beta3 affinity state on cell attachment under flow, we compared Chinese hamster ovary cells expressing the low affinity alpha(IIb)beta3 wild-type (wt) receptor to those expressing the high affinity alpha(IIb)beta3 T562N receptor. MATERIALS AND METHODS: We designed a real-time videomicroscopy adhesion assay for von Willebrand factor (VWF) or fibrinogen under flow conditions. RESULTS: At 50 s(-1), alpha(IIb)beta3 T562N supported higher cell adhesion to fibrinogen (63.3 +/- 2.9 cells/field) than alpha(IIb)beta3 wt (38.7 +/- 2.4 cells/field, P < 0.0001). At 100 s(-1), alpha(IIb)beta3 T562N mediated cell adhesion (40.5 +/- 3.8 cells/field), while alpha(IIb)beta3 wt did not (5.3 +/- 1.4 cells/field, P < 0.001), allowing to discriminate the efficiency of each receptor. Similar findings were observed for adhesion to VWF. Complete inhibition of cell adhesion to fibrinogen was achieved with 800 microM fibrinogen gamma-chain dodecapeptide [HHLGGAKQAGDV (H12)], while Arg-Gly-Asp-Ser (RGDS) peptide (10-1000 microM) induced a dose-dependent cell detachment. These results suggest that the H12 motif allows initial attachment, in contrast to the RGDS site, which strengthens the stability of adhesion. Interestingly, compared with wt, a 10-fold lower concentration of RGDS was required to reach a similar reduction of cell adhesion mediated by alpha(IIb)beta3 T562N. CONCLUSIONS: Our data show that alpha(IIb)beta3 activation is associated with a stabilization of integrin binding to fibrinogen or VWF under shear.


Assuntos
Adesão Celular/genética , Mutação de Sentido Incorreto , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/genética , Animais , Células CHO , Cricetinae , Fibrinogênio/metabolismo , Fibrinogênio/farmacologia , Microscopia de Vídeo , Oligopeptídeos/farmacologia , Fragmentos de Peptídeos/farmacologia , Ligação Proteica/genética , Estresse Mecânico , Fator de von Willebrand/metabolismo
19.
J Physiol ; 553(Pt 2): 369-86, 2003 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-12972628

RESUMO

Ionotropic glycine receptors (GlyRs) are present in the central nervous system well before the establishment of synaptic contacts. Immature nerve cells are known, at least in the spinal cord, to express alpha2 homomeric GlyRs, the properties of which are relatively unknown compared to those of the adult synaptic form of the GlyR (mainly alpha1/beta heteromeres). Here, the kinetics properties of GlyRs at the single-channel level have been recorded in real-time by means of the patch-clamp technique in the outside-out configuration coupled with an ultra-fast flow application system (< 100 micros). Recordings were performed on chinese hamster ovary (CHO) cells stably transfected with the alpha2 GlyR subunit. We show that the onset, the relaxation and the desensitisation of alpha2 homomeric GlyR-mediated currents are slower by one or two orders of magnitude compared to synaptic mature GlyRs and to other ligand-gated ionotropic channels involved in fast synaptic transmission. First latency analysis performed on single GlyR channels revealed that their slow activation time course was due to delayed openings. When synaptic release of glycine was mimicked (1 mM glycine; 1 ms pulse duration), the opening probability of alpha2 homomeric GlyRs was low (P(o) approximately = 0.1) when compared to mature synaptic GlyRs (Po = 0.9). This low Po is likely to be a direct consequence of the relatively slow activation kinetics of alpha2 homomeric GlyRs when compared to the activation kinetics of mature alpha1/beta GlyRs. Such slow kinetics suggest that embryonic alpha2 homomeric GlyRs cannot be activated by fast neurotransmitter release at mature synapses but rather could be suited for a non-synaptic paracrine-like release of agonist, which is known to occur in the embryo.


Assuntos
Receptores de Glicina/fisiologia , Sinapses/fisiologia , Algoritmos , Animais , Células CHO , Simulação por Computador , Cricetinae , Cricetulus , Dimerização , Relação Dose-Resposta a Droga , Processamento Eletrônico de Dados , Glicina/farmacologia , Cinética , Potenciais da Membrana/efeitos dos fármacos , Modelos Biológicos , Comunicação Parácrina/fisiologia , Técnicas de Patch-Clamp , Receptores de Glicina/química , Receptores de Glicina/genética , Transmissão Sináptica/efeitos dos fármacos , Transmissão Sináptica/fisiologia , Transfecção
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...