RESUMO
PIEZOs are mechanosensitive ion channels that convert force into chemoelectric signals1,2 and have essential roles in diverse physiological settings3. In vitro studies have proposed that PIEZO channels transduce mechanical force through the deformation of extensive blades of transmembrane domains emanating from a central ion-conducting pore4-8. However, little is known about how these channels interact with their native environment and which molecular movements underlie activation. Here we directly observe the conformational dynamics of the blades of individual PIEZO1 molecules in a cell using nanoscopic fluorescence imaging. Compared with previous structural models of PIEZO1, we show that the blades are significantly expanded at rest by the bending stress exerted by the plasma membrane. The degree of expansion varies dramatically along the length of the blade, where decreased binding strength between subdomains can explain increased flexibility of the distal blade. Using chemical and mechanical modulators of PIEZO1, we show that blade expansion and channel activation are correlated. Our findings begin to uncover how PIEZO1 is activated in a native environment. More generally, as we reliably detect conformational shifts of single nanometres from populations of channels, we expect that this approach will serve as a framework for the structural analysis of membrane proteins through nanoscopic imaging.
Assuntos
Canais Iônicos , Membrana Celular/metabolismo , Fluorescência , Canais Iônicos/química , Canais Iônicos/metabolismo , Modelos Moleculares , Movimento , Conformação Proteica , Análise de Célula ÚnicaRESUMO
It has been hypothesized that mitochondria evolved from a bacterial ancestor that initially became established in an archaeal host cell as an endosymbiont. Here we model this first stage of mitochondrial evolution by engineering endosymbiosis between Escherichia coli and Saccharomyces cerevisiae An ADP/ATP translocase-expressing E. coli provided ATP to a respiration-deficient cox2 yeast mutant and enabled growth of a yeast-E. coli chimera on a nonfermentable carbon source. In a reciprocal fashion, yeast provided thiamin to an endosymbiotic E. coli thiamin auxotroph. Expression of several SNARE-like proteins in E. coli was also required, likely to block lysosomal degradation of intracellular bacteria. This chimeric system was stable for more than 40 doublings, and GFP-expressing E. coli endosymbionts could be observed in the yeast by fluorescence microscopy and X-ray tomography. This readily manipulated system should allow experimental delineation of host-endosymbiont adaptations that occurred during evolution of the current, highly reduced mitochondrial genome.
Assuntos
Bioengenharia/métodos , Mitocôndrias/genética , Simbiose/genética , Trifosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Evolução Biológica , Escherichia coli/genética , Escherichia coli/metabolismo , Mitocôndrias/metabolismo , Modelos Biológicos , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Tiamina/metabolismoRESUMO
The Tie family of endothelial-specific receptor tyrosine kinases is essential for cell proliferation, migration, and survival during angiogenesis. Despite considerable similarity, experiments with Tie1- or Tie2-deficient mice highlight distinct functions for these receptors in vivo. The Tie2 receptor is further unique with respect to its structurally homologous ligands. Angiopoietin-2 and -3 can function as agonists or antagonists; angiopoietin-1 and -4 are constitutive agonists. To address the role of Tie1 in angiopoietin-mediated Tie2 signaling and determine the basis for the behavior of the individual angiopoietins, we used an in vivo FRET-based proximity assay to monitor Tie1 and -2 localization and association. We provide evidence for Tie1-Tie2 complex formation on the cell surface and identify molecular surface areas essential for receptor-receptor recognition. We further demonstrate that the Tie1-Tie2 interactions are dynamic, inhibitory, and differentially modulated by angiopoietin-1 and -2. Based on the available data, we propose a unified model for angiopoietin-induced Tie2 signaling.
Assuntos
Angiopoietina-1/metabolismo , Angiopoietina-2/metabolismo , Células Endoteliais/enzimologia , Receptor de TIE-1/metabolismo , Receptor TIE-2/metabolismo , Transdução de Sinais , Linhagem Celular , Membrana Celular/enzimologia , Transferência Ressonante de Energia de Fluorescência , Humanos , Ligantes , Modelos Moleculares , Mutação , Conformação Proteica , Multimerização Proteica , Estrutura Terciária de Proteína , Interferência de RNA , Receptor Cross-Talk , Receptor de TIE-1/química , Receptor de TIE-1/genética , Receptor TIE-2/química , Receptor TIE-2/genética , Proteínas Recombinantes de Fusão/metabolismo , Relação Estrutura-Atividade , Fatores de Tempo , TransfecçãoRESUMO
Statins, 3-hydroxyl-3-methylglutaryl coenzyme A reductase inhibitors, are the first-line medications prescribed for the prevention and treatment of coronary artery diseases. The efficacy of statins has been attributed not only to their systemic cholesterol-lowering actions but also to their pleiotropic effects that are unrelated to cholesterol reduction. These pleiotropic effects have been increasingly recognized as essential in statins therapy. This study was designed to investigate the pleiotropic actions of simvastatin, one of the most commonly prescribed statins, on macrophage cholesterol homeostasis with a focus on lysosomal free cholesterol egression. With simultaneous nile red and filipin staining, analysis of confocal/multi-photon imaging demonstrated that simvastatin markedly attenuated unesterified (free) cholesterol buildup in macrophages loaded with oxidized low-density lipoprotein but had little effect in reducing the sizes of cholesteryl ester-containing lipid droplets; the reduction in free cholesterol was mainly attributed to decreases in lysosome-compartmentalized cholesterol. Functionally, the egression of free cholesterol from lysosomes attenuated pro-inflammatory cytokine secretion. It was determined that the reduction of lysosomal free cholesterol buildup by simvastatin was due to the up-regulation of Niemann-Pick C1 (NPC1), a lysosomal residing cholesterol transporter. Moreover, the enhanced enzymatic production of 7-hydroxycholesterol by cytochrome P450 7A1 and the subsequent activation of liver X receptor α underscored the up-regulation of NPC1. These findings reveal a novel pleiotropic effect of simvastatin in affecting lysosomal cholesterol efflux in macrophages and the associated significance in the treatment of atherosclerosis.
Assuntos
Colesterol 7-alfa-Hidroxilase/metabolismo , Colesterol/metabolismo , Lipoproteínas LDL/farmacologia , Receptores X do Fígado/metabolismo , Lisossomos/metabolismo , Macrófagos/metabolismo , Proteínas/metabolismo , Sinvastatina/farmacologia , Animais , Transporte Biológico/efeitos dos fármacos , Citocinas/metabolismo , Mediadores da Inflamação/metabolismo , Interleucina-1beta/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular , Lisossomos/efeitos dos fármacos , Macrófagos/efeitos dos fármacos , Camundongos Endogâmicos C57BL , Proteína C1 de Niemann-Pick , RNA Interferente Pequeno/metabolismo , Transdução de Sinais/efeitos dos fármacos , Regulação para Cima/efeitos dos fármacosRESUMO
Our goals are to simultaneously determine the three-dimensional distribution patterns of KCNQ1 and KCNE1 in cardiac myocytes and to study the mechanism and functional implications for variations in KCNQ1/KCNE1 colocalization in myocytes. We monitored the distribution patterns of KCNQ1, KCNE1, and markers for subcellular compartments/organelles using immunofluorescence/confocal microscopy and confirmed the findings in ventricular myocytes by directly observing fluorescently tagged KCNQ1-GFP and KCNE1-dsRed expressed in these cells. We also monitored the effects of stress on KCNQ1-GFP and endoplasmic reticulum (ER) remodeling during live cell imaging. The data showed that 1) KCNE1 maintained a stable cell surface localization, whereas KCNQ1 exhibited variations in the cytosolic compartment (striations versus vesicles) and the degree of presence on the cell surface; 2) the degree of cell surface KCNQ1/KCNE1 colocalization was positively correlated with slow delayed rectifier (IKs) current density; 3) KCNQ1 and calnexin (an ER marker) shared a cytosolic compartment; and 4) in response to stress ([Ca(2+)]i elevation, oxidative overload, or AT1R stimulation), KCNQ1 exited the cytosolic compartment and trafficked to the cell periphery in vesicles. This was accompanied by partial ER fragmentation. We conclude that the cellular milieu regulates KCNQ1 distribution in cardiac myocytes and that stressful conditions can increase IKs by inducing KCNQ1 movement to the cell surface. This represents a hitherto unrecognized mechanism by which IKs fulfills its function as a repolarization reserve in ventricular myocytes.
Assuntos
Cálcio/metabolismo , Canais de Potássio Ativados por Cálcio de Condutância Intermediária/metabolismo , Canal de Potássio KCNQ1/metabolismo , Miócitos Cardíacos/metabolismo , Canais de Potássio de Abertura Dependente da Tensão da Membrana/metabolismo , Animais , Células COS , Calnexina/metabolismo , Membrana Celular/metabolismo , Chlorocebus aethiops , Citosol/metabolismo , Transferência Ressonante de Energia de Fluorescência , Cobaias , Átrios do Coração/citologia , Átrios do Coração/metabolismo , Ventrículos do Coração/citologia , Ventrículos do Coração/metabolismo , Humanos , Canal de Potássio KCNQ1/genética , Estresse Oxidativo , Canais de Potássio de Abertura Dependente da Tensão da Membrana/genética , Transporte Proteico , Ratos , Receptor Tipo 1 de Angiotensina/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismoRESUMO
Natural Killer (NK) cells utilize effector functions, including antibody-dependent cellular cytotoxicity (ADCC), for the clearance of viral infection and cellular malignancies. NK cell ADCC is mediated by Fc γ RIIIa (CD16a) binding to the fragment crystallizable (Fc) region of immunoglobulin G (IgG) within immune complexes on a target cell surface. While antibody-induced clustering of CD16a is thought to drive ADCC, the molecular basis for this activity has not been fully described. Here we use MINFLUX nanoscopy to map the spatial distribution of stoichiometrically labeled CD16a across the NK cell membrane, revealing the presence of pairs of CD16a molecules with intra-doublet distance of approximately 17 nm. NK cells activated on supported lipid bilayers by Trastuzumab results in an increase of synaptic regions with greater CD16a density. Our results provide the highest spatial resolution yet described for CD16a imaging, offering new insight into how CD16a organization within the immune synapse could influence ADCC activity. MINFLUX holds great promise to further unravel the molecular details driving CD16a-based activation of NK cells.
RESUMO
Primary ciliary dyskinesia (PCD), resulting from defects in cilia assembly or motility, is caused by mutations in a number of genes encoding axonemal proteins. PCD phenotypes are variable, and include recurrent respiratory tract infections, bronchiectasis, hydrocephaly, situs inversus, and male infertility. We generated knockout mice for the sperm-associated antigen-17 (Spag17) gene, which encodes a central pair (CP) protein present in the axonemes of cells with "9 + 2" motile cilia or flagella. The targeting of Spag17 resulted in a severe phenotype characterized by immotile nasal and tracheal cilia, reduced clearance of nasal mucus, profound respiratory distress associated with lung fluid accumulation and disruption of the alveolar epithelium, cerebral ventricular expansion consistent with emerging hydrocephalus, failure to suckle, and neonatal demise within 12 hours of birth. Ultrastructural analysis revealed the loss of one CP microtubule in approximately one quarter of tracheal cilia axonemes, an absence of a C1 microtubule projection, and other less frequent CP structural abnormalities. SPAG6 and SPAG16 (CP proteins that interact with SPAG17) were increased in tracheal tissue from SPAG17-deficient mice. We conclude that Spag17 plays a critical role in the function and structure of motile cilia, and that neonatal lethality is likely explained by impaired airway mucociliary clearance.
Assuntos
Movimento Celular , Cílios/metabolismo , Proteínas dos Microtúbulos/metabolismo , Sequência de Aminoácidos , Animais , Animais Recém-Nascidos , Axonema/metabolismo , Axonema/ultraestrutura , Cílios/ultraestrutura , Feminino , Síndrome de Kartagener/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microscopia Eletrônica de Transmissão , Proteínas dos Microtúbulos/genética , Mutação , Mucosa Nasal/metabolismo , Fenótipo , Análise de Sobrevida , Fatores de Tempo , Traqueia/anatomia & histologia , Traqueia/metabolismo , Traqueia/patologiaRESUMO
Fluorescence labeled oligonucleotides have a long history of being used to monitor nucleic acid transport and uptake. However, it is not known if the fluorescent moiety itself physically limits the number of pathways that can be used by the cell due to steric, hydrophobic, or other chemical characteristics. Here, we report a method for comparing the uptake kinetics of oligonucleotides labeled either with the fluorescent pteridine, 3-methyl-8-(2-deoxy-ß-D-ribofuranosyl) isoxanthopterin (3MI), or the common fluorophore 5-carboxyfluorescein (5-FAM). We use a multiphoton microscopic technique to monitor nucleic acid uptake LLC-PK1, a pig renal tubular cell line that is known to have multiple uptake pathways. We find that the two fluorophores enter the cells at different rates, suggesting that choice of fluorescent moiety influences the uptake pathway used by a cell. Finally, we reconstituted an LLC-PK1 membrane channel that is selective for nucleic acids in planar lipid bilayers, and tested the ability of the labeled nucleic acids to permeate the channel. We find that 3MI, and not 5-FAM labeled oligonucleotides can traverse the plasma membrane through the channel. These results have implications for future studies aimed at delivering pteridine moieties to cells and for tracking nucleic acid transport into tissues.
RESUMO
A fundamental feature of the mammalian visual system is the presence of separate channels that work in parallel to efficiently extract and analyze specific elements of a visual scene. Despite the extensive use of the mouse as a model system, it is not clear whether such parallel organization extends beyond the retina to subcortical structures, such as the dorsal lateral geniculate (dLGN) of thalamus. To begin to address this, we examined the morphology of biocytin-filled relay cells recorded in dLGN of mice. Based on a quantitative assessment of their dendritic architecture, we found that even at early postnatal ages relay cells could be readily classified as X-like (biconical), Y-like (symmetrical), or W-like (hemispheric) and that each cell type was regionally specified in dLGN. X-like cells were confined primarily to the monocular ventral region of dLGN. Y-like cells occupied a central core that also contained ipsilateral eye projections, whereas W-like cells were found along the perimeter of dLGN. Similar to cat, Y-like cells were more prevalent than X- and W-like cells, and X-like cells tended to be smaller than other cell types. However, the dendritic fields of X- and W-like cells did not exhibit an orientation bias with respect to optic tract or boundaries of dLGN. Although we found clear morphological differences among relay cells, an analysis of their electrophysiological properties did not reveal any additional distinguishing characteristics. Overall, these data coupled with recent observations in the retina suggest that the mouse has many of the hallmark features of a system-wide parallel organization.
Assuntos
Dendritos/fisiologia , Corpos Geniculados/citologia , Neurônios/citologia , Animais , Forma Celular/fisiologia , Tamanho Celular , Corpos Geniculados/fisiologia , Camundongos , Neurônios/fisiologiaRESUMO
Exposure of MCF-7 breast tumor cells or HCT-116 colon carcinoma cells to clinically relevant concentrations of doxorubicin (Adriamycin; Farmitalia Research Laboratories, Milan, Italy) or camptothecin results in both autophagy and senescence. To determine whether autophagy is required for chemotherapy-induced senescence, reactive oxygen generation induced by Adriamycin was suppressed by N-acetyl cysteine and glutathione, and the induction of ataxia telangiectasia mutated, p53, and p21 was modulated pharmacologically and/or genetically. In all cases, autophagy and senescence were collaterally suppressed. The close association between autophagy and senescence indicated by these experiments reflects their collateral regulation via common signaling pathways. The potential relationship between autophagy and senescence was further examined through pharmacologic inhibition of autophagy with chloroquine and 3-methyl-adenine and genetic ablation of the autophagy-related genes ATG5 and ATG7. However, inhibition of autophagy by pharmacological and genetic approaches could not entirely abrogate the senescence response, which was only reduced and/or delayed. Taken together, our findings suggest that autophagy and senescence tend to occur in parallel, and furthermore that autophagy accelerates the development of the senescent phenotype. However, these responses are not inexorably linked or interdependent, as senescence can occur when autophagy is abrogated.
Assuntos
Antineoplásicos/farmacologia , Autofagia/efeitos dos fármacos , Camptotecina/farmacologia , Senescência Celular/efeitos dos fármacos , Dano ao DNA , Doxorrubicina/farmacologia , Autofagia/genética , Western Blotting , Técnicas de Cultura de Células , Senescência Celular/genética , Citometria de Fluxo , Células HCT116 , Humanos , Células MCF-7 , Microscopia Eletrônica de Transmissão , Microscopia de Fluorescência , Espécies Reativas de Oxigênio/metabolismoRESUMO
Dysregulated secretion in neutrophil leukocytes associates with human inflammatory disease. The exocytosis response to triggering stimuli is sequential; gelatinase granules modulate the initiation of the innate immune response, followed by the release of pro-inflammatory azurophilic granules, requiring stronger stimulation. Exocytosis requires actin depolymerization which is actively counteracted under non-stimulatory conditions. Here we show that the actin nucleator, WASH, is necessary to maintain azurophilic granules in their refractory state by granule actin entrapment and interference with the Rab27a-JFC1 exocytic machinery. On the contrary, gelatinase granules of WASH-deficient neutrophil leukocytes are characterized by decreased Rac1, shortened granule-associated actin comets and impaired exocytosis. Rac1 activation restores exocytosis of these granules. In vivo, WASH deficiency induces exacerbated azurophilic granule exocytosis, inflammation, and decreased survival. WASH deficiency thus differentially impacts neutrophil granule subtypes, impairing exocytosis of granules that mediate the initiation of the neutrophil innate response while exacerbating pro-inflammatory granule secretion.
Assuntos
Actinas , Neutrófilos , Grânulos Citoplasmáticos , Exocitose , Gelatinases , Humanos , Inflamação , Proteínas dos MicrofilamentosRESUMO
Single-walled carbon nanohorns (SWNHs) are new carbonaceous materials. In this paper, we report the first successful preparation of SWNHs encapsulating trimetallic nitride template endohedral metallofullerenes (TNT-EMFs). The resultant materials were functionalized by a high-speed vibration milling method and conjugated with CdSe/ZnS quantum dots (QDs). The successful encapsulation of TNT-EMFs and external functionalization with QDs provide a dual diagnostic platform for in vitro and in vivo biomedical applications of these new carbonaceous materials.
Assuntos
Fulerenos/química , Metais/química , Nanotubos de Carbono , Pontos Quânticos , Técnicas In VitroRESUMO
HIV-1 Gag and Gag-Pol are responsible for viral assembly and maturation and represent a major paradigm for enveloped virus assembly. Numerous intracellular Gag-containing complexes (GCCs) have been identified in cellular lysates using sucrose gradient ultracentrifugation. While these complexes are universally present in Gag-expressing cells, their roles in virus assembly are not well understood. Here we demonstrate that most GCC species are predominantly comprised of monomeric or dimeric Gag molecules bound to ribosomal complexes, and as such, are not on-pathway intermediates in HIV assembly. Rather, these GCCs represent a population of Gag that is not yet functionally committed for incorporation into a viable virion precursor. We hypothesize that these complexes act as a reservoir of monomeric Gag that can incorporate into assembling viruses, and serve to mitigate non-specific intracellular Gag oligomerization. We have identified a subset of large GCC complexes, comprising more than 20 Gag molecules, that may be equivalent to membrane-associated puncta previously shown to be bona fide assembling-virus intermediates. This work provides a clear rationale for the existence of diverse GCCs, and serves as the foundation for characterizing on-pathway intermediates early in virus assembly.
Assuntos
HIV-1/metabolismo , Montagem de Vírus/fisiologia , Produtos do Gene gag do Vírus da Imunodeficiência Humana/química , Produtos do Gene gag do Vírus da Imunodeficiência Humana/metabolismo , Linhagem Celular , Genoma Viral , Células HEK293 , Humanos , Marcação por Isótopo , Vírion/metabolismo , Produtos do Gene gag do Vírus da Imunodeficiência Humana/genéticaRESUMO
BACKGROUND: The most important determinant of longevity in pulmonary arterial hypertension is right ventricular (RV) function, but in contrast to experimental work elucidating the pathobiology of left ventricular failure, there is a paucity of data on the cellular and molecular mechanisms of RV failure. METHODS AND RESULTS: A mechanical animal model of chronic progressive RV pressure overload (pulmonary artery banding, not associated with structural alterations of the lung circulation) was compared with an established model of angioproliferative pulmonary hypertension associated with fatal RV failure. Isolated RV pressure overload induced RV hypertrophy without failure, whereas in the context of angioproliferative pulmonary hypertension, RV failure developed that was associated with myocardial apoptosis, fibrosis, a decreased RV capillary density, and a decreased vascular endothelial growth factor mRNA and protein expression despite increased nuclear stabilization of hypoxia-induced factor-1alpha. Induction of myocardial nuclear factor E2-related factor 2 and heme-oxygenase 1 with a dietary supplement (Protandim) prevented fibrosis and capillary loss and preserved RV function despite continuing pressure overload. CONCLUSIONS: These data brought into question the commonly held concept that RV failure associated with pulmonary hypertension is due strictly to the increased RV afterload.
Assuntos
Pressão Sanguínea , Insuficiência Cardíaca/metabolismo , Hipertensão Pulmonar/metabolismo , Hipertrofia Ventricular Direita/metabolismo , Artéria Pulmonar/metabolismo , Animais , Apoptose , Modelos Animais de Doenças , Fibrose , Regulação da Expressão Gênica , Insuficiência Cardíaca/patologia , Insuficiência Cardíaca/fisiopatologia , Heme Oxigenase (Desciclizante)/biossíntese , Hipertensão Pulmonar/patologia , Hipertensão Pulmonar/fisiopatologia , Hipertrofia Ventricular Direita/patologia , Hipertrofia Ventricular Direita/fisiopatologia , Subunidade alfa do Fator 1 Induzível por Hipóxia/biossíntese , Masculino , Artéria Pulmonar/patologia , Artéria Pulmonar/fisiopatologia , RNA Mensageiro/biossíntese , Ratos , Ratos Sprague-Dawley , Fator A de Crescimento do Endotélio Vascular/biossíntese , Função Ventricular DireitaRESUMO
Multisensory convergence is the first, requisite step in the process that generates neural responses to events involving more than one sensory modality. Although anatomical studies have documented the merging of afferents from different sensory modalities within a given area, they do not provide insight into the architecture of connectivity at the neuronal level that underlies multisensory processing. In fact, few anatomical studies of multisensory convergence at the neuronal level have been conducted. The present study used a combination of tract-tracing, immunocytochemistry, and confocal microscopic techniques to examine the connections related to crossmodal auditory cortical inputs to somatosensory area SIV. Axons labeled from auditory cortex were found in contact with immunolabeled interneurons in SIV, some of which also colocalized vesicular glutamate transporter 1, indicating the presence of an active, glutamatergic synapse. No specific subtype of inhibitory interneuron appeared to be targeted by the crossmodal contacts. These results provide insight into the structural basis for multisensory processing at the neuronal level and offer anatomical evidence for the direct involvement of inhibitory interneurons in multisensory processing.
Assuntos
Córtex Auditivo/citologia , Percepção Auditiva/fisiologia , Interneurônios/fisiologia , Vias Neurais/citologia , Córtex Somatossensorial/citologia , Percepção do Tato/fisiologia , Animais , Córtex Auditivo/fisiologia , Axônios/fisiologia , Axônios/ultraestrutura , Gatos , Citometria por Imagem , Interneurônios/citologia , Microscopia Confocal , Vias Neurais/fisiologia , Técnicas de Rastreamento Neuroanatômico , Córtex Somatossensorial/fisiologia , Sinapses/fisiologia , Sinapses/ultraestrutura , Transmissão Sináptica/fisiologiaRESUMO
Interstitial cells of Cajal (ICCs) have been identified as pacemaker cells in the upper urinary tract and urethra, but the role of ICCs in the bladder remains to be determined. We tested the hypotheses that ICCs express cyclooxygenase (COX), and that COX products (prostaglandins), are the cause of spontaneous rhythmic contraction (SRC) of isolated strips of rabbit bladder free of urothelium. SRC was abolished by 10 microM indomethacin and ibuprofen (non-selective COX inhibitors). SRC was concentration-dependently inhibited by selective COX-1 (SC-560 and FR-122047) and COX-2 inhibitors (NS-398 and LM-1685), and by SC-51089, a selective antagonist for the PGE-2 receptor (EP) and ICI-192,605 and SQ-29,548, selective antagonists for thromboxane receptors (TP). The partial agonist/antagonist of the PGF-2alpha receptor (FP), AL-8810, inhibited SRC by approximately 50%. Maximum inhibition was approximately 90% by SC-51089, approximately 80-85% by the COX inhibitors and approximately 70% by TP receptor antagonists. In the presence of ibuprofen to abolish SRC, PGE-2, sulprostone, misoprostol, PGF-2alpha and U-46619 (thromboxane mimetic) caused rhythmic contractions that mimicked SRC. Fluorescence immunohistochemistry coupled with confocal laser scanning microscopy revealed that c-Kit and vimentin co-localized to interstitial cells surrounding detrusor smooth muscle bundles, indicating the presence of extensive ICCs in rabbit bladder. Co-localization of COX-1 and vimentin, and COX-2 and vimentin by ICCs supports the hypothesis that ICCs were the predominant cell type in rabbit bladder expressing both COX isoforms. These data together suggest that ICCs appear to be an important source of prostaglandins that likely play a role in regulation of SRC. Additional studies on prostaglandin-dependent SRC may generate opportunities for the application of novel treatments for disorders leading to overactive bladder.
Assuntos
Ciclo-Oxigenase 1/metabolismo , Ciclo-Oxigenase 2/metabolismo , Contração Muscular , Músculo Liso/metabolismo , Bexiga Urinária/metabolismo , Animais , Inibidores de Ciclo-Oxigenase/farmacologia , Feminino , Hidrazinas/farmacologia , Ibuprofeno/farmacologia , Indometacina/farmacologia , Células Intersticiais de Cajal , Oxazepinas/farmacologia , Prostaglandinas/metabolismo , Coelhos , Vimentina/metabolismoRESUMO
H460 non-small cell lung, HCT116 colon and 4T1 breast tumor cell lines induced into senescence by exposure to either etoposide or doxorubicin were able to recover proliferative capacity both in mass culture and when enriched for the senescence-like phenotype by flow cytometry (based on ß-galactosidase staining and cell size, and a senescence-associated reporter, BTG1-RFP). Recovery was further established using both real-time microscopy and High-Speed Live-Cell Interferometry (HSLCI) and was shown to be accompanied by the attenuation of the Senescence-Associated Secretory Phenotype (SASP). Cells enriched for the senescence-like phenotype were also capable of forming tumors when implanted in both immunodeficient and immunocompetent mice. As chemotherapy-induced senescence has been identified in patient tumors, our results suggest that certain senescence-like phenotypes may not reflect a terminal state of growth arrest, as cells that recover with self-renewal capacity may ultimately contribute to disease recurrence.
Assuntos
Antineoplásicos/farmacologia , Proliferação de Células/efeitos dos fármacos , Senescência Celular/efeitos dos fármacos , Carga Tumoral/efeitos dos fármacos , Animais , Linhagem Celular Tumoral , Proliferação de Células/fisiologia , Senescência Celular/fisiologia , Células HCT116 , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos SCID , Carga Tumoral/fisiologia , Ensaios Antitumorais Modelo de Xenoenxerto/métodosRESUMO
PURPOSE: Previous studies have documented a synaptic translocation of calcineurin (CaN) and increased CaN activity following status epilepticus (SE); however, the cellular effect of these changes in CaN in the pathology of SE remains to be elucidated. This study examined a CaN-dependent modification of the dendritic cytoskeleton. CaN has been shown to induce dephosphorylation of cofilin, an actin depolymerization factor. The ensuing actin depolymerization can lead to a number of physiological changes that are of interest in SE. METHODS: SE was induced by pilocarpine injection, and seizure activity was monitored by video-EEG. Subcellular fractions were isolated by differential centrifugation. CaN activity was assayed using a paranitrophenol phosphate (pNPP) assay protocol. Cofilin phosphorylation was assessed using phosphocofilin-specific antibodies. Cofilin-actin binding was determined by coimmunoprecipitation, and actin polymerization was measured using a triton-solubilization protocol. Spines were visualized using a single-section rapid Golgi impregnation procedure. RESULTS: The immunoreactivity of phosphocofilin decreased significantly in hippocampal and cortical synaptosomal samples after SE. SE-induced cofilin dephosphorylation could be partially blocked by the preinjection of CaN inhibitors. Cofilin activation could be further demonstrated by increased actin-cofilin binding and a significant depolymerization of neuronal actin, both of which were also blocked by CaN inhibitors. Finally, we demonstrated a CaN-dependent loss of dendritic spines histologically. DISCUSSION: The data demonstrate a CaN-dependent, cellular mechanism through which prolonged seizure activity results in loss of dendritic spines via cofilin activation. Further research into this area may provide useful insights into the pathology of SE and epileptogenic mechanisms.
Assuntos
Encéfalo/ultraestrutura , Espinhas Dendríticas/patologia , Estado Epiléptico/patologia , Actinas/metabolismo , Análise de Variância , Animais , Encéfalo/patologia , Calcineurina/metabolismo , Dendritos/efeitos dos fármacos , Dendritos/patologia , Espinhas Dendríticas/efeitos dos fármacos , Modelos Animais de Doenças , Fosfoproteína 32 Regulada por cAMP e Dopamina/metabolismo , Imunoprecipitação/métodos , Fosforilação , Pilocarpina , Ratos , Ratos Sprague-Dawley , Coloração pela Prata/métodos , Estado Epiléptico/induzido quimicamente , Frações Subcelulares/ultraestrutura , Fatores de TempoRESUMO
Photoconversion, the method by which a fluorescent dye is transformed into a stable, osmiophilic product that can be visualized by transmission electron microscopy, is the most widely used method to enable the ultrastructural analysis of fluorescently labeled cellular structures. Nevertheless, the conventional method of photoconversion using widefield fluorescence microscopy requires long reaction times and results in low resolution cell targeting which limit its utility. Accordingly, we developed a photoconversion method that ameliorates these limitations by adapting confocal laser scanning microscopy to the procedure. We confirmed that photoconversion times were dramatically reduced when using a confocal laser scanning microscope in the photoconversion process. We also demonstrated that the region of interest scanning capabilities of a confocal laser scanning microscope equipped with an acousto-optical tunable filter represented a unique tool to facilitate the targeting of the photoconversion process to individual cellular or subcellular elements within a fluorescent field. Moreover, region of interest scanning greatly reduced the area of the cell exposed to light energy, ameliorating the ultrastructural damage common to this process when widefield microscopes are employed. The potential of this new methodology extends beyond the neurosciences to any scientific modality which requires ultrastructural analysis of fluorescently labeled specimens, especially those where discrete photoconversion on a cellular or subcellular basis could be beneficial.
Assuntos
Astrócitos/ultraestrutura , Corantes Fluorescentes/efeitos da radiação , Microscopia Confocal/métodos , Neurônios/ultraestrutura , Raios Ultravioleta , Animais , Animais Recém-Nascidos , Astrócitos/metabolismo , Encéfalo/citologia , Técnicas de Cocultura/métodos , Embrião de Mamíferos , Gânglios Espinais/citologia , Proteína Glial Fibrilar Ácida/metabolismo , Microscopia Eletrônica de Transmissão , Proteínas do Tecido Nervoso/metabolismo , Neurônios/metabolismo , Ratos , Ratos Sprague-DawleyRESUMO
We describe the use of the fast Fourier transform (FFT) in the measurement of anisotropy in electrospun scaffolds of gelatin as a function of the starting conditions. In electrospinning, fiber alignment and overall scaffold anisotropy can be manipulated by controlling the motion of the collecting mandrel with respect to the source electrospinning solution. By using FFT to assign relative alignment values to an electrospun matrix it is possible to systematically evaluate how different processing variables impact the structure and material properties of a scaffold. Gelatin was suspended at varying concentrations (80, 100, 130, 150 mg/ml) and electrospun from 2,2,2 trifluoroethanol onto rotating mandrels (200-7000 RPM). At each starting concentration, fiber diameter remained constant over a wide range of mandrel RPM. Scaffold anisotropy developed as a function of fiber diameter and mandrel RPM. The induction of varying degrees of anisotropy imparted distinctive material properties to the electrospun scaffolds. The FFT is a rapid method for evaluating fiber alignment in tissue-engineering materials.