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1.
Cell ; 153(2): 362-75, 2013 Apr 11.
Artigo em Inglês | MEDLINE | ID: mdl-23582326

RESUMO

The functions of Nr4a1-dependent Ly6C(low) monocytes remain enigmatic. We show that they are enriched within capillaries and scavenge microparticles from their lumenal side in a steady state. In the kidney cortex, perturbation of homeostasis by a TLR7-dependent nucleic acid "danger" signal, which may signify viral infection or local cell death, triggers Gαi-dependent intravascular retention of Ly6C(low) monocytes by the endothelium. Then, monocytes recruit neutrophils in a TLR7-dependent manner to mediate focal necrosis of endothelial cells, whereas the monocytes remove cellular debris. Prevention of Ly6C(low) monocyte development, crawling, or retention in Nr4a1(-/-), Itgal(-/-), and Tlr7(host-/-BM+/+) and Cx3cr1(-/-) mice, respectively, abolished neutrophil recruitment and endothelial killing. Prevention of neutrophil recruitment in Tlr7(host+/+BM-/-) mice or by neutrophil depletion also abolished endothelial cell necrosis. Therefore, Ly6C(low) monocytes are intravascular housekeepers that orchestrate the necrosis by neutrophils of endothelial cells that signal a local threat sensed via TLR7 followed by the in situ phagocytosis of cellular debris.


Assuntos
Células Endoteliais/metabolismo , Monitorização Imunológica , Monócitos/imunologia , Animais , Moléculas de Adesão Celular/metabolismo , Micropartículas Derivadas de Células , Endotélio Vascular/citologia , Endotélio Vascular/imunologia , Humanos , Inflamação , Molécula 1 de Adesão Intercelular/metabolismo , Rim/irrigação sanguínea , Rim/metabolismo , Receptores de Lipopolissacarídeos/metabolismo , Antígeno-1 Associado à Função Linfocitária/metabolismo , Camundongos , Monócitos/metabolismo , Neutrófilos/imunologia , Membro 1 do Grupo A da Subfamília 4 de Receptores Nucleares/metabolismo , Receptores de Quimiocinas/metabolismo
2.
J Exp Bot ; 73(16): 5543-5558, 2022 09 12.
Artigo em Inglês | MEDLINE | ID: mdl-35617147

RESUMO

Pollen development is dependent on the tapetum, a sporophytic anther cell layer surrounding the microspores that functions in pollen wall formation but is also essential for meiosis-associated development. There is clear evidence of crosstalk and co-regulation between the tapetum and microspores, but how this is achieved is currently not characterized. ABORTED MICROSPORES (AMS), a tapetum transcription factor, is important for pollen wall formation, but also has an undefined role in early pollen development. We conducted a detailed investigation of chromosome behaviour, cytokinesis, radial microtubule array (RMA) organization, and callose formation in the ams mutant. Early meiosis initiates normally in ams, shows delayed progression after the pachytene stage, and then fails during late meiosis, with disorganized RMA, defective cytokinesis, abnormal callose formation, and microspore degeneration, alongside abnormal tapetum development. Here, we show that selected meiosis-associated genes are directly repressed by AMS, and that AMS is essential for late meiosis progression. Our findings indicate that AMS has a dual function in tapetum-meiocyte crosstalk by playing an important regulatory role during late meiosis, in addition to its previously characterized role in pollen wall formation. AMS is critical for RMA organization, callose deposition, and therefore cytokinesis, and is involved in the crosstalk between the gametophyte and sporophytic tissues, which enables synchronous development of tapetum and microspores.


Assuntos
Regulação da Expressão Gênica de Plantas , Pólen , Células Germinativas Vegetais , Meiose , Pólen/metabolismo , Fatores de Transcrição/metabolismo
3.
Hum Mol Genet ; 27(3): 463-474, 2018 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-29194538

RESUMO

FUS (fused in sarcoma) mislocalization and cytoplasmic aggregation are hallmark pathologies in FUS-related amyotrophic lateral sclerosis and frontotemporal dementia. Many of the mechanistic hypotheses have focused on a loss of nuclear function in the FUS-opathies, implicating dysregulated RNA transcription and splicing in driving neurodegeneration. Recent studies describe an additional somato-dendritic localization for FUS in the cerebral cortex implying a regulatory role in mRNA transport and local translation at the synapse. Here, we report that FUS is also abundant at the pre-synaptic terminal of the neuromuscular junction (NMJ), suggesting an important function for this protein at peripheral synapses. We have previously reported dose and age-dependent motor neuron degeneration in transgenic mice overexpressing human wild-type FUS, resulting in a motor phenotype detected by ∼28 days and death by ∼100 days. Now, we report the earliest structural events using electron microscopy and quantitative immunohistochemistry. Mitochondrial abnormalities in the pre-synaptic motor nerve terminals are detected at postnatal day 6, which are more pronounced at P15 and accompanied by a loss of synaptic vesicles and synaptophysin protein coupled with NMJs of a smaller size at a time when there is no detectable motor neuron loss. These changes occur in the presence of abundant FUS and support a peripheral toxic gain of function. This appearance is typical of a 'dying-back' axonopathy, with the earliest manifestation being mitochondrial disruption. These findings support our hypothesis that FUS has an important function at the NMJ, and challenge the 'loss of nuclear function' hypothesis for disease pathogenesis in the FUS-opathies.


Assuntos
Junção Neuromuscular/metabolismo , Proteína FUS de Ligação a RNA/metabolismo , Esclerose Lateral Amiotrófica/metabolismo , Animais , Córtex Cerebral/metabolismo , Modelos Animais de Doenças , Demência Frontotemporal/metabolismo , Humanos , Camundongos , Camundongos Transgênicos , Neurônios Motores , Proteína FUS de Ligação a RNA/genética , Sinapses/metabolismo , Sinaptofisina/metabolismo
4.
Chemphyschem ; 21(8): 702-706, 2020 04 20.
Artigo em Inglês | MEDLINE | ID: mdl-32065707

RESUMO

Ion pairing between the major phospholipids of the Staphylococcus aureus plasma membrane (phosphatidylglycerol - PG and lysyl-phosphatidylglycerol - LPG) confers resistance to antimicrobial peptides and other antibiotics. We developed 3adLPG, a stable synthetic analogue which can substitute for the highy-labile native LPG, in biophysical experiments examining the membrane-protecting role of lipid ion pairing, in S. aureus and other important bacteria. Here we examine the surface charge and lipid packing characteristics of synthetic biomimetic mixtures of DPPG and DP3adLPG in Langmuir monolayers, using a combination of complementary surface-probing techniques such as infrared reflection-absorption spectroscopy and grazing-incidence x-ray diffraction. The resultant phase diagram for the ion paired lipids sheds light on the mixing behavior of lipids in monolayer models of resistant phenotype bacterial membranes, and provides a platform for future biophysical studies.


Assuntos
Materiais Biomiméticos/química , Bicamadas Lipídicas/química , Lisina/química , Lipídeos de Membrana/química , Membranas Artificiais , Modelos Biológicos , Fosfatidilgliceróis/química , Staphylococcus aureus/química , Antibacterianos/farmacologia , Fenômenos Biofísicos , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/metabolismo , Propriedades de Superfície
5.
Proc Natl Acad Sci U S A ; 114(10): E1986-E1995, 2017 03 07.
Artigo em Inglês | MEDLINE | ID: mdl-28209776

RESUMO

Neurons receive a multitude of synaptic inputs along their dendritic arbor, but how this highly heterogeneous population of synaptic compartments is spatially organized remains unclear. By measuring N-methyl-d-aspartic acid receptor (NMDAR)-driven calcium responses in single spines, we provide a spatial map of synaptic calcium signals along dendritic arbors of hippocampal neurons and relate this to measures of synapse structure. We find that quantal NMDAR calcium signals increase in amplitude as they approach a thinning dendritic tip end. Based on a compartmental model of spine calcium dynamics, we propose that this biased distribution in calcium signals is governed by a gradual, distance-dependent decline in spine size, which we visualized using serial block-face scanning electron microscopy. Our data describe a cell-autonomous feature of principal neurons, where tapering dendrites show an inverse distribution of spine size and NMDAR-driven calcium signals along dendritic trees, with important implications for synaptic plasticity rules and spine function.


Assuntos
Cálcio/metabolismo , Espinhas Dendríticas/metabolismo , Hipocampo/metabolismo , Células Piramidais/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Animais , Sinalização do Cálcio , Espinhas Dendríticas/ultraestrutura , Embrião de Mamíferos , Feminino , Expressão Gênica , Hipocampo/citologia , Camundongos , Camundongos Endogâmicos C57BL , Microscopia Eletrônica , Microtomia , N-Metilaspartato/metabolismo , Plasticidade Neuronal , Gravidez , Cultura Primária de Células , Células Piramidais/ultraestrutura , Ratos , Ratos Sprague-Dawley , Receptores de N-Metil-D-Aspartato/genética , Sinapses/fisiologia
6.
Proc Natl Acad Sci U S A ; 114(13): 3439-3444, 2017 03 28.
Artigo em Inglês | MEDLINE | ID: mdl-28292906

RESUMO

In the asexual blood stages of malarial infection, merozoites invade erythrocytes and replicate within a parasitophorous vacuole to form daughter cells that eventually exit (egress) by sequential rupture of the vacuole and erythrocyte membranes. The current model is that PKG, a malarial cGMP-dependent protein kinase, triggers egress, activating malarial proteases and other effectors. Using selective inhibitors of either PKG or cysteine proteases to separately inhibit the sequential steps in membrane perforation, combined with video microscopy, electron tomography, electron energy loss spectroscopy, and soft X-ray tomography of mature intracellular Plasmodium falciparum parasites, we resolve intermediate steps in egress. We show that the parasitophorous vacuole membrane (PVM) is permeabilized 10-30 min before its PKG-triggered breakdown into multilayered vesicles. Just before PVM breakdown, the host red cell undergoes an abrupt, dramatic shape change due to the sudden breakdown of the erythrocyte cytoskeleton, before permeabilization and eventual rupture of the erythrocyte membrane to release the parasites. In contrast to the previous view of PKG-triggered initiation of egress and a gradual dismantling of the host erythrocyte cytoskeleton over the course of schizont development, our findings identify an initial step in egress and show that host cell cytoskeleton breakdown is restricted to a narrow time window within the final stages of egress.


Assuntos
Citoesqueleto/metabolismo , Membrana Eritrocítica/parasitologia , Eritrócitos/parasitologia , Malária Falciparum/parasitologia , Plasmodium falciparum/fisiologia , Proteínas Quinases Dependentes de GMP Cíclico/genética , Proteínas Quinases Dependentes de GMP Cíclico/metabolismo , Citoesqueleto/genética , Membrana Eritrocítica/metabolismo , Eritrócitos/metabolismo , Humanos , Malária Falciparum/genética , Malária Falciparum/metabolismo , Plasmodium falciparum/enzimologia , Plasmodium falciparum/genética , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo
7.
Proc Natl Acad Sci U S A ; 113(9): 2484-9, 2016 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-26884193

RESUMO

Hepatitis C virus (HCV) is a unique enveloped virus that assembles as a hybrid lipoviral particle by tightly interacting with host lipoproteins. As a result, HCV virions display a characteristic low buoyant density and a deceiving coat, with host-derived apolipoproteins masking viral epitopes. We previously described methods to produce high-titer preparations of HCV particles with tagged envelope glycoproteins that enabled ultrastructural analysis of affinity-purified virions. Here, we performed proteomics studies of HCV isolated from culture media of infected hepatoma cells to define viral and host-encoded proteins associated with mature virions. Using two different affinity purification protocols, we detected four viral and 46 human cellular proteins specifically copurifying with extracellular HCV virions. We determined the C terminus of the mature capsid protein and reproducibly detected low levels of the viral nonstructural protein, NS3. Functional characterization of virion-associated host factors by RNAi identified cellular proteins with either proviral or antiviral roles. In particular, we discovered a novel interaction between HCV capsid protein and the nucleoporin Nup98 at cytosolic lipid droplets that is important for HCV propagation. These results provide the first comprehensive view to our knowledge of the protein composition of HCV and new insights into the complex virus-host interactions underlying HCV infection.


Assuntos
Hepacivirus/fisiologia , Complexo de Proteínas Formadoras de Poros Nucleares/fisiologia , Proteômica , Proteínas Virais/metabolismo , Vírion/metabolismo , Sequência de Aminoácidos , Linhagem Celular , Hepacivirus/metabolismo , Humanos , Espectrometria de Massas , Dados de Sequência Molecular , Morfogênese , Proteínas Virais/química
8.
EMBO Rep ; 17(9): 1326-42, 2016 09.
Artigo em Inglês | MEDLINE | ID: mdl-27418313

RESUMO

Defective FUS metabolism is strongly associated with amyotrophic lateral sclerosis and frontotemporal dementia (ALS/FTD), but the mechanisms linking FUS to disease are not properly understood. However, many of the functions disrupted in ALS/FTD are regulated by signalling between the endoplasmic reticulum (ER) and mitochondria. This signalling is facilitated by close physical associations between the two organelles that are mediated by binding of the integral ER protein VAPB to the outer mitochondrial membrane protein PTPIP51, which act as molecular scaffolds to tether the two organelles. Here, we show that FUS disrupts the VAPB-PTPIP51 interaction and ER-mitochondria associations. These disruptions are accompanied by perturbation of Ca(2+) uptake by mitochondria following its release from ER stores, which is a physiological read-out of ER-mitochondria contacts. We also demonstrate that mitochondrial ATP production is impaired in FUS-expressing cells; mitochondrial ATP production is linked to Ca(2+) levels. Finally, we demonstrate that the FUS-induced reductions to ER-mitochondria associations and are linked to activation of glycogen synthase kinase-3ß (GSK-3ß), a kinase already strongly associated with ALS/FTD.


Assuntos
Esclerose Lateral Amiotrófica/metabolismo , Retículo Endoplasmático/metabolismo , Demência Frontotemporal/metabolismo , Glicogênio Sintase Quinase 3 beta/metabolismo , Mitocôndrias/metabolismo , Proteínas Mitocondriais/metabolismo , Proteínas Tirosina Fosfatases/metabolismo , Proteína FUS de Ligação a RNA/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Cálcio/metabolismo , Modelos Animais de Doenças , Retículo Endoplasmático/ultraestrutura , Ativação Enzimática , Expressão Gênica , Humanos , Camundongos , Camundongos Transgênicos , Mitocôndrias/ultraestrutura , Mutação , Ligação Proteica , Proteína FUS de Ligação a RNA/genética
9.
EMBO J ; 32(20): 2722-34, 2013 Oct 16.
Artigo em Inglês | MEDLINE | ID: mdl-24076656

RESUMO

The epidermal growth factor receptor (EGFR) plays an essential role during development and diseases including cancer. Lamellipodin (Lpd) is known to control lamellipodia protrusion by regulating actin filament elongation via Ena/VASP proteins. However, it is unknown whether this mechanism supports endocytosis of the EGFR. Here, we have identified a novel role for Lpd and Mena in clathrin-mediated endocytosis (CME) of the EGFR. We have discovered that endogenous Lpd is in a complex with the EGFR and Lpd and Mena knockdown impairs EGFR endocytosis. Conversely, overexpressing Lpd substantially increases the EGFR uptake in an F-actin-dependent manner, suggesting that F-actin polymerization is limiting for EGFR uptake. Furthermore, we found that Lpd directly interacts with endophilin, a BAR domain containing protein implicated in vesicle fission. We identified a role for endophilin in EGFR endocytosis, which is mediated by Lpd. Consistently, Lpd localizes to clathrin-coated pits (CCPs) just before vesicle scission and regulates vesicle scission. Our findings suggest a novel mechanism in which Lpd mediates EGFR endocytosis via Mena downstream of endophilin.


Assuntos
Actinas/fisiologia , Aciltransferases/fisiologia , Proteínas de Transporte/fisiologia , Endocitose/genética , Receptores ErbB/metabolismo , Proteínas de Membrana/fisiologia , Proteínas dos Microfilamentos/fisiologia , Citoesqueleto de Actina/metabolismo , Citoesqueleto de Actina/fisiologia , Actinas/genética , Actinas/metabolismo , Aciltransferases/genética , Aciltransferases/metabolismo , Animais , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Células Cultivadas , Clatrina/metabolismo , Invaginações Revestidas da Membrana Celular/metabolismo , Invaginações Revestidas da Membrana Celular/fisiologia , Células HEK293 , Células HeLa , Humanos , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Proteínas dos Microfilamentos/genética , Proteínas dos Microfilamentos/metabolismo , Células NIH 3T3 , Transdução de Sinais/genética , Transdução de Sinais/fisiologia
10.
Plant Cell ; 26(4): 1544-1556, 2014 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-24781116

RESUMO

Mature pollen is covered by durable cell walls, principally composed of sporopollenin, an evolutionary conserved, highly resilient, but not fully characterized, biopolymer of aliphatic and aromatic components. Here, we report that ABORTED MICROSPORES (AMS) acts as a master regulator coordinating pollen wall development and sporopollenin biosynthesis in Arabidopsis thaliana. Genome-wide coexpression analysis revealed 98 candidate genes with specific expression in the anther and 70 that showed reduced expression in ams. Among these 70 members, we showed that AMS can directly regulate 23 genes implicated in callose dissociation, fatty acids elongation, formation of phenolic compounds, and lipidic transport putatively involved in sporopollenin precursor synthesis. Consistently, ams mutants showed defective microspore release, a lack of sporopollenin deposition, and a dramatic reduction in total phenolic compounds and cutin monomers. The functional importance of the AMS pathway was further demonstrated by the observation of impaired pollen wall architecture in plant lines with reduced expression of several AMS targets: the abundant pollen coat protein extracellular lipases (EXL5 and EXL6), and CYP98A8 and CYP98A9, which are enzymes required for the production of phenolic precursors. These findings demonstrate the central role of AMS in coordinating sporopollenin biosynthesis and the secretion of materials for pollen wall patterning.

11.
J Pathol ; 239(3): 374-83, 2016 07.
Artigo em Inglês | MEDLINE | ID: mdl-27126290

RESUMO

The epidermis is the outermost layer of skin that acts as a barrier to protect the body from the external environment and to control water and heat loss. This barrier function is established through the multistage differentiation of keratinocytes and the presence of bioactive sphingolipids such as ceramides, the levels of which are tightly regulated by a balance of ceramide synthase and ceramidase activities. Here we reveal the essential role of alkaline ceramidase 1 (Acer1) in the skin. Acer1-deficient (Acer1(-/-) ) mice showed elevated levels of ceramide in the skin, aberrant hair shaft cuticle formation and cyclic alopecia. We demonstrate that Acer1 is specifically expressed in differentiated interfollicular epidermis, infundibulum and sebaceous glands and consequently Acer1(-/-) mice have significant alterations in infundibulum and sebaceous gland architecture. Acer1(-/-) skin also shows perturbed hair follicle stem cell compartments. These alterations result in Acer1(-/-) mice showing increased transepidermal water loss and a hypermetabolism phenotype with associated reduction of fat content with age. We conclude that Acer1 is indispensable for mammalian skin homeostasis and whole-body energy homeostasis. © 2016 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.


Assuntos
Ceramidase Alcalina/metabolismo , Alopecia/enzimologia , Ceramidas/metabolismo , Metabolismo Energético , Homeostase , Ceramidase Alcalina/genética , Alopecia/fisiopatologia , Animais , Diferenciação Celular , Epiderme/anormalidades , Epiderme/enzimologia , Feminino , Folículo Piloso/anormalidades , Folículo Piloso/enzimologia , Humanos , Queratinócitos/enzimologia , Queratinócitos/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Hipófise/anormalidades , Hipófise/enzimologia , Glândulas Sebáceas/anormalidades , Glândulas Sebáceas/enzimologia , Pele/enzimologia , Anormalidades da Pele , Esfingolipídeos/metabolismo
12.
Am J Dermatopathol ; 38(3): 239-43, 2016 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-26894777

RESUMO

Woolly hair nevus is a mosaic disorder characterized by unruly, tightly curled hair in a circumscribed area of the scalp. This condition may be associated with epidermal nevi. We describe an 11-year-old boy who initially presented with multiple patches of woolly hair and with epidermal nevi on his left cheek and back. He had no nail, teeth, eye, or cardiac abnormalities. Analysis of plucked hairs from patches of woolly hair showed twisting of the hair shaft and an abnormal hair cuticle. Histopathology of a woolly hair patch showed diffuse hair follicle miniaturization with increased vellus hairs.


Assuntos
Doenças do Cabelo/patologia , Folículo Piloso/fisiologia , Folículo Piloso/ultraestrutura , Criança , Humanos , Masculino , Microscopia Eletrônica de Varredura , Mosaicismo , Nevo/patologia
13.
J Biol Chem ; 288(2): 859-72, 2013 Jan 11.
Artigo em Inglês | MEDLINE | ID: mdl-23184933

RESUMO

Sustained activation of X-box-binding protein 1 (XBP1) results in endothelial cell (EC) apoptosis and atherosclerosis development. The present study provides evidence that XBP1 mRNA splicing triggered an autophagic response in ECs by inducing autophagic vesicle formation and markers of autophagy BECLIN-1 and microtubule-associated protein 1 light chain 3ß (LC3-ßII). Endostatin activated autophagic gene expression through XBP1 mRNA splicing in an inositol-requiring enzyme 1α (IRE1α)-dependent manner. Knockdown of XBP1 or IRE1α by shRNA in ECs ablated endostatin-induced autophagosome formation. Importantly, data from arterial vessels from XBP1 EC conditional knock-out (XBP1eko) mice demonstrated that XBP1 deficiency in ECs reduced the basal level of LC3ß expression and ablated response to endostatin. Chromatin immunoprecipitation assays further revealed that the spliced XBP1 isoform bound directly to the BECLIN-1 promoter at the region from nt -537 to -755. BECLIN-1 deficiency in ECs abolished the XBP1-induced autophagy response, whereas spliced XBP1 did not induce transcriptional activation of a truncated BECLIN-1 promoter. These results suggest that XBP1 mRNA splicing triggers an autophagic signal pathway through transcriptional regulation of BECLIN-1.


Assuntos
Proteínas Reguladoras de Apoptose/genética , Autofagia/genética , Proteínas de Ligação a DNA/genética , Endotélio Vascular/metabolismo , Proteínas de Membrana/genética , Splicing de RNA , RNA Mensageiro/genética , Fatores de Transcrição/genética , Ativação Transcricional/genética , Animais , Sequência de Bases , Proteína Beclina-1 , Células Cultivadas , Imunoprecipitação da Cromatina , Primers do DNA , Endotélio Vascular/citologia , Técnica Indireta de Fluorescência para Anticorpo , Humanos , Camundongos , Camundongos Knockout , Microscopia Eletrônica de Transmissão , Reação em Cadeia da Polimerase em Tempo Real , Fatores de Transcrição de Fator Regulador X , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteína 1 de Ligação a X-Box
14.
New Phytol ; 202(4): 1212-1222, 2014 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-24641449

RESUMO

Root elongation and bending require the coordinated expansion of multiple cells of different types. These processes are regulated by the action of hormones that can target distinct cell layers. We use a mathematical model to characterise the influence of the biomechanical properties of individual cell walls on the properties of the whole tissue. Taking a simple constitutive model at the cell scale which characterises cell walls via yield and extensibility parameters, we derive the analogous tissue-level model to describe elongation and bending. To accurately parameterise the model, we take detailed measurements of cell turgor, cell geometries and wall thicknesses. The model demonstrates how cell properties and shapes contribute to tissue-level extensibility and yield. Exploiting the highly organised structure of the elongation zone (EZ) of the Arabidopsis root, we quantify the contributions of different cell layers, using the measured parameters. We show how distributions of material and geometric properties across the root cross-section contribute to the generation of curvature, and relate the angle of a gravitropic bend to the magnitude and duration of asymmetric wall softening. We quantify the geometric factors which lead to the predominant contribution of the outer cell files in driving root elongation and bending.


Assuntos
Arabidopsis/fisiologia , Gravitropismo , Raízes de Plantas/fisiologia , Arabidopsis/citologia , Arabidopsis/crescimento & desenvolvimento , Parede Celular/metabolismo , Fenômenos Mecânicos , Microscopia Eletrônica de Transmissão , Modelos Teóricos , Especificidade de Órgãos , Raízes de Plantas/citologia , Raízes de Plantas/crescimento & desenvolvimento
15.
J Magn Reson Imaging ; 39(3): 598-608, 2014 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-24006053

RESUMO

PURPOSE: To investigate a very small iron-oxide particle (VSOP) in a mouse model of acute ischemia-reperfusion to access the mechanism of such particles in areas of myocardial inflammation. MATERIALS AND METHODS: Animals were injected with VSOP at several time points, in a mouse model of acute myocardial infarction (MI), before and after MI. MRI was used to localize areas of VSOP enhancement, evaluate VSOP areas extension, and determine the related T2* values. Histology, electron microscopy, macrophage counting, and Evan's Blue staining were also performed. RESULTS: We found that areas of VSOP uptake decreased from 1 to 8 days post-MI while the related T2* values increased. T2* and VSOP areas, defined from MRI data, correlated well between 1 and 3 days post-MI but not at 7 days after injection. Histological analysis and electron microscopy showed colocalization of macrophages with areas of VSOP staining. However, there was no correlation between number of macrophages and the extension of the VSOP areas achieved by MR. We found that only areas of increased permeability (assessed by Evan's Blue staining) showed colocalization of macrophages and VSOP uptake. CONCLUSION: This study demonstrates that VSOP allows the assessment of myocardial inflammation associated with increased permeability during infarct healing in a mouse model of ischemia-reperfusion.


Assuntos
Compostos Férricos/farmacologia , Imageamento por Ressonância Magnética/métodos , Infarto do Miocárdio/diagnóstico , Reperfusão Miocárdica/métodos , Animais , Meios de Contraste , Modelos Animais de Doenças , Feminino , Inflamação/patologia , Injeções Intravenosas , Camundongos , Camundongos Endogâmicos C57BL , Infarto do Miocárdio/patologia , Infarto do Miocárdio/cirurgia , Distribuição Aleatória , Valores de Referência , Sensibilidade e Especificidade
16.
Chem Res Toxicol ; 27(4): 558-67, 2014 Apr 21.
Artigo em Inglês | MEDLINE | ID: mdl-24575710

RESUMO

ZnO nanoparticles (NPs) are prone to dissolution, and uncertainty remains whether biological/cellular responses to ZnO NPs are solely due to the release of Zn(2+) or whether the NPs themselves have additional toxic effects. We address this by establishing ZnO NP solubility in dispersion media (Dulbecco's modified Eagle's medium, DMEM) held under conditions identical to those employed for cell culture (37 °C, 5% CO2, and pH 7.68) and by systematic comparison of cell-NP interaction for three different ZnO NP preparations. For NPs at concentrations up to 5.5 µg ZnO/mL, dissolution is complete (with the majority of the soluble zinc complexed to dissolved ligands in the medium), taking ca. 1 h for uncoated and ca. 6 h for polymer coated ones. Above 5.5 µg/mL, the results are consistent with the formation of zinc carbonate, keeping the solubilized zinc fixed to 67 µM of which only 0.45 µM is as free Zn(2+), i.e., not complexed to dissolved ligands. At these relatively high concentrations, NPs with an aliphatic polyether-coating show slower dissolution (i.e., slower free Zn(2+) release) and reprecipitation kinetics compared to those of uncoated NPs, requiring more than 48 h to reach thermodynamic equilibrium. Cytotoxicity (MTT) and DNA damage (Comet) assay dose-response curves for three epithelial cell lines suggest that dissolution and reprecipitation dominate for uncoated ZnO NPs. Transmission electron microscopy combined with the monitoring of intracellular Zn(2+) concentrations and ZnO-NP interactions with model lipid membranes indicate that an aliphatic polyether coat on ZnO NPs increases cellular uptake, enhancing toxicity by enabling intracellular dissolution and release of Zn(2+). Similarly, we demonstrate that needle-like NP morphologies enhance toxicity by apparently frustrating cellular uptake. To limit toxicity, ZnO NPs with nonacicular morphologies and coatings that only weakly interact with cellular membranes are recommended.


Assuntos
Nanopartículas Metálicas/toxicidade , Óxido de Zinco/toxicidade , Linhagem Celular , Linhagem Celular Tumoral , Humanos , Cinética , Nanopartículas Metálicas/química , Microscopia Eletrônica de Transmissão , Solubilidade , Óxido de Zinco/química
17.
PLoS One ; 18(10): e0286278, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-37874822

RESUMO

Blood-brain barrier (BBB) dysfunction may be involved in the increased sensitivity of Alzheimer's disease (AD) patients to antipsychotics, including amisulpride. Studies indicate that antipsychotics interact with facilitated glucose transporters (GLUT), including GLUT1, and that GLUT1 BBB expression decreases in AD. We tested the hypotheses that amisulpride (charge: +1) interacts with GLUT1, and that BBB transport of amisulpride is compromised in AD. GLUT1 substrates, GLUT1 inhibitors and GLUT-interacting antipsychotics were identified by literature review and their physicochemical characteristics summarised. Interactions between amisulpride and GLUT1 were studied using in silico approaches and the human cerebral endothelial cell line, hCMEC/D3. Brain distribution of [3H]amisulpride was determined using in situ perfusion in wild type (WT) and 5xFamilial AD (5xFAD) mice. With transmission electron microscopy (TEM) we investigated brain capillary degeneration in WT mice, 5xFAD mice and human samples. Western blots determined BBB transporter expression in mouse and human. Literature review revealed that, although D-glucose has no charge, charged molecules can interact with GLUT1. GLUT1 substrates are smaller (184.95±6.45g/mol) than inhibitors (325.50±14.40g/mol) and GLUT-interacting antipsychotics (369.38±16.04). Molecular docking showed beta-D-glucose (free energy binding: -15.39kcal/mol) and amisulpride (-29.04kcal/mol) interact with GLUT1. Amisulpride did not affect [14C]D-glucose hCMEC/D3 accumulation. [3H]amisulpride uptake into the brain (except supernatant) of 5xFAD mice compared to WT remained unchanged. TEM revealed brain capillary degeneration in human AD. There was no difference in GLUT1 or P-glycoprotein BBB expression between WT and 5xFAD mice. In contrast, caudate P-glycoprotein, but not GLUT1, expression was decreased in human AD capillaries versus controls. This study provides new details about the BBB transport of amisulpride, evidence that amisulpride interacts with GLUT1 and that BBB transporter expression is altered in AD. This suggests that antipsychotics could potentially exacerbate the cerebral hypometabolism in AD. Further research into the mechanism of amisulpride transport by GLUT1 is important for improving antipsychotics safety.


Assuntos
Doença de Alzheimer , Antipsicóticos , Humanos , Camundongos , Animais , Barreira Hematoencefálica/metabolismo , Amissulprida , Doença de Alzheimer/metabolismo , Transportador de Glucose Tipo 1/metabolismo , Simulação de Acoplamento Molecular , Encéfalo/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Antipsicóticos/farmacologia , Antipsicóticos/metabolismo , Glucose/metabolismo , Proteínas Facilitadoras de Transporte de Glucose/metabolismo , Subfamília B de Transportador de Cassetes de Ligação de ATP/metabolismo
18.
Nanoscale ; 15(4): 1763-1774, 2023 Jan 27.
Artigo em Inglês | MEDLINE | ID: mdl-36601869

RESUMO

Fluorescent InP-based quantum dots have emerged as valuable nanomaterials for display technologies, biological imaging, and optoelectronic applications. The inclusion of zinc can enhance both their emissive and structural properties and reduce interfacial defects with ZnS or CdS shells. However, the sub-particle distribution of zinc and the role this element plays often remains unclear, and it has previously proved challenging to synthesise Zn-alloyed InP-based nanoparticles using aminophosphine precursors. In this report, we describe the synthesis of alloyed InZnP using zinc carboxylates, achieving colour-tuneable fluorescence from the unshelled core materials, followed by a one-pot ZnS or CdS deposition using diethyldithiocarbamate precursors. Structural analysis revealed that the "core/shell" particles synthesised here were more accurately described as homogeneous extended alloys with the constituent shell elements diffusing through the entire core, including full-depth inclusion of zinc.

19.
Dis Model Mech ; 16(2)2023 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-36715290

RESUMO

Phenotypic cell-based screens are critical tools for discovering candidate drugs for development, yet identification of the cellular target and mode of action of a candidate drug is often lacking. Using an imaging-based screen, we recently discovered an N-[(4-hydroxychroman-4-yl)methyl]-sulphonamide (N-4HCS) compound, DDD01035881, that blocks male gamete formation in the malaria parasite life cycle and subsequent transmission of the parasite to the mosquito with nanomolar activity. To identify the target(s) of DDD01035881, and of the N-4HCS class of compounds more broadly, we synthesised a photoactivatable derivative, probe 2. Photoaffinity labelling of probe 2 coupled with mass spectrometry identified the 16 kDa Plasmodium falciparum parasitophorous vacuole membrane protein Pfs16 as a potential parasite target. Complementary methods including cellular thermal shift assays confirmed that the parent molecule DDD01035881 stabilised Pfs16 in lysates from activated mature gametocytes. Combined with high-resolution, fluorescence and electron microscopy data, which demonstrated that parasites inhibited with N-4HCS compounds phenocopy the targeted deletion of Pfs16 in gametocytes, these data implicate Pfs16 as a likely target of DDD01035881. This finding establishes N-4HCS compounds as being flexible and effective starting candidates from which transmission-blocking antimalarials can be developed in the future.


Assuntos
Malária , Plasmodium , Animais , Masculino , Proteínas de Membrana/metabolismo , Vacúolos/metabolismo , Sulfonamidas/farmacologia , Sulfonamidas/uso terapêutico , Sulfonamidas/metabolismo
20.
Cell Rep ; 42(5): 112397, 2023 05 30.
Artigo em Inglês | MEDLINE | ID: mdl-37074915

RESUMO

Excitatory synapses are typically described as single synaptic boutons (SSBs), where one presynaptic bouton contacts a single postsynaptic spine. Using serial section block-face scanning electron microscopy, we found that this textbook definition of the synapse does not fully apply to the CA1 region of the hippocampus. Roughly half of all excitatory synapses in the stratum oriens involved multi-synaptic boutons (MSBs), where a single presynaptic bouton containing multiple active zones contacted many postsynaptic spines (from 2 to 7) on the basal dendrites of different cells. The fraction of MSBs increased during development (from postnatal day 22 [P22] to P100) and decreased with distance from the soma. Curiously, synaptic properties such as active zone (AZ) or postsynaptic density (PSD) size exhibited less within-MSB variation when compared with neighboring SSBs, features that were confirmed by super-resolution light microscopy. Computer simulations suggest that these properties favor synchronous activity in CA1 networks.


Assuntos
Hipocampo , Terminações Pré-Sinápticas , Sinapses , Neurônios , Dendritos
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