RESUMO
1. This study assessed the impact of dietary dihydroquercetin (DHQ) in wheat-based diets on egg production, composition and quality when fed to laying hens. A total of 80 Hy-Line Brown hens were allocated to 20 enriched layer cages, over two tiers, in groups of four birds.2. Two wheat-based diets were used in the study. A basal diet, meeting the nutrient requirement of the hens, containing 11.56 MJ/kg AME and 172 g/kg crude protein, was mixed and split into two parts. One part was fed as prepared to the control group of birds. The second diet was made by adding 1.5 g DHQ per kg basal diet and fed to the treatment group of birds. This level was relatively high and extended the data on levels normally fed. The diets were fed in a meal form and did not contain any coccidiostat, antimicrobial growth promoters or other similar additives. Each diet was fed to hens in 10 replicate cages for 4 weeks, from 22 to 26 weeks of age, following randomisation.3. Subsequently, eggs were investigated to determine the impact of dietary DHQ on the quality variables of fresh and 28-d stored eggs.4. Overall, feeding 1.5 g/kg dietary DHQ for 4 weeks did not affect (P > 0.05) egg production or the quality of fresh and stored eggs. Any observed egg quality changes (P < 0.05) confirmed the expected effects of egg storage.
Assuntos
Galinhas , Triticum , Animais , Feminino , Ração Animal/análise , Dieta/veterinária , Suplementos Nutricionais , Ovos , ÓvuloRESUMO
1. In this study, 240 ISA Brown hens were fed diets containing different levels of hybrid rye, and the influence of xylanase addition on laying performance and egg quality was evaluated. 2. Birds were allocated to 10 treatment groups with 12 replicates (cages) of two hens and were fed, from week 26 to 50, isocaloric and isonitrogenous experimental diets. A 5 × 2 experimental arrangement was applied, using diets with increasing level of rye (0%, 10%, 15%, 20% or 25%) with or without xylanase supplementation (200 mg/kg of feed; Ronozyme WX (CT) with minimum xylanase activity of 1,000 FXU/g). 3. Increasing dietary level of rye did not affect daily mass of eggs, mean egg weight or feed conversion ratio (P > 0.05). Laying rate decreased in all groups fed with rye. Egg and eggshell quality indices were unaffected by dietary rye grain (P > 0.05); however, rye inclusion significantly decreased yolk colour on the DSM scale (P < 0.05). In comparison with the control group, high dietary levels of rye (25%) significantly increased viscosity of small intestine content (P < 0.05). Diet supplementation with xylanase had no significant effect on egg production indices and egg quality (except for yolk colour) but decreased the viscosity of intestinal content in laying hens fed high levels of rye (P < 0.05). 4. The results of this experiment suggest that rye may be incorporated to a level of 25% in the diet of laying hens without any strong negative effect on egg performance, while xylanase added to high-rye grain reduced the viscosity of intestinal content; however, it did not positively affect the laying performance or egg quality.
Assuntos
Galinhas/fisiologia , Óvulo/efeitos dos fármacos , Reprodução/efeitos dos fármacos , Secale/química , Xilano Endo-1,3-beta-Xilosidase/metabolismo , Ração Animal/análise , Fenômenos Fisiológicos da Nutrição Animal/efeitos dos fármacos , Animais , Dieta/veterinária , Suplementos Nutricionais/análise , Casca de Ovo/efeitos dos fármacos , Casca de Ovo/fisiologia , Feminino , Óvulo/fisiologia , Reprodução/fisiologia , Xilano Endo-1,3-beta-Xilosidase/administração & dosagemRESUMO
Vascular integrity is primarily determined by endothelial cell (EC) cytoskeletal structure that is differentially regulated by various stimuli. In this study, atomic force microscopy (AFM) was used to characterize structural and mechanical properties in the cytoskeleton of cultured human pulmonary artery EC (HPAEC) and human lung microvascular EC (HLMVEC) by determining elastic properties (Young's modulus) in response to endogenous barrier protective agents sphingosine 1-phosphate (S1P) and hepatocyte growth factor (HGF), or the barrier disruptive molecule thrombin. Initial studies in unstimulated cells indicate higher baseline peripheral elastic modulus values in HPAEC (mean 2.9 KPa) than in HLMVEC (1.8 KPa). After 30 min of stimulation, S1P induced the highest Young's modulus increase (6.1 KPa) compared to the other barrier enhancing stimuli, HGF (5.8 KPa) and the pharmaceutical agent and S1P analog FTY720 (4.1 KPa). In contrast, the barrier disruptive agent thrombin decreased values from 2.5 KPa to 0.7 KPa depending on the cell type and treatment time. AFM topographical imaging supports these quantitative biophysical data regarding differential peripheral elastic properties in EC. Overall, these AFM studies provide novel insights into the biomechanical properties of human lung EC that regulate vascular barrier function and have potential applicability to pathophysiologic vascular leak syndromes such as acute lung injury.
Assuntos
Módulo de Elasticidade , Endotélio Vascular/citologia , Endotélio Vascular/metabolismo , Pulmão/irrigação sanguínea , Fenômenos Biomecânicos , Linhagem Celular , Citoesqueleto/metabolismo , Fator de Crescimento de Hepatócito/metabolismo , Humanos , Lisofosfolipídeos/metabolismo , Microscopia de Força Atômica , Artéria Pulmonar/citologia , Esfingosina/análogos & derivados , Esfingosina/metabolismo , Trombina/metabolismoRESUMO
The vasopressin 1b receptor (Avpr1b) is critical for social memory and social aggression in rodents, yet little is known about its specific roles in these behaviors. Some clues to Avpr1b function can be gained from its profile of expression in the brain, which is largely limited to the pyramidal neurons of the CA2 region of the hippocampus, and from experiments showing that inactivation of the gene or antagonism of the receptor leads to a reduction in social aggression. Here we show that partial replacement of the Avpr1b through lentiviral delivery into the dorsal CA2 region restored the probability of socially motivated attack behavior in total Avpr1b knockout mice, without altering anxiety-like behaviors. To further explore the role of the Avpr1b in this hippocampal region, we examined the effects of Avpr1b agonists on pyramidal neurons in mouse and rat hippocampal slices. We found that selective Avpr1b agonists induced significant potentiation of excitatory synaptic responses in CA2, but not in CA1 or in slices from Avpr1b knockout mice. In a way that is mechanistically very similar to synaptic potentiation induced by oxytocin, Avpr1b agonist-induced potentiation of CA2 synapses relies on NMDA (N-methyl-D-aspartic acid) receptor activation, calcium and calcium/calmodulin-dependent protein kinase II activity, but not on cAMP-dependent protein kinase activity or presynaptic mechanisms. Our data indicate that the hippocampal CA2 is important for attacking in response to a male intruder and that the Avpr1b, likely through its role in regulating CA2 synaptic plasticity, is a necessary mediator.
Assuntos
Agressão/fisiologia , Região CA2 Hipocampal/citologia , Plasticidade Neuronal/genética , Receptores de Vasopressinas/metabolismo , Sinapses/genética , Animais , Antagonistas dos Receptores de Hormônios Antidiuréticos/farmacologia , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Contagem de Células , Comportamento Exploratório/fisiologia , Feminino , Lentivirus/genética , Masculino , Aprendizagem em Labirinto/fisiologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Ratos , Ratos Sprague-Dawley , Receptores de N-Metil-D-Aspartato/metabolismo , Receptores de Ocitocina/genética , Receptores de Ocitocina/metabolismo , Receptores de Vasopressinas/agonistas , Receptores de Vasopressinas/genética , Transdução GenéticaRESUMO
Endothelial dysfunction underlies the pathophysiology of vascular disorders such as acute lung injury (ALI) syndromes. Recent work has identified the Abl family kinases (c-Abl and Arg) as important regulators of endothelial cell (EC) barrier function and suggests that their inhibition by currently available pharmaceutical agents such as imatinib may be EC protective. Here we describe novel and differential effects of imatinib in regulating lung pathophysiology in two clinically relevant experimental models of ALI. Imatinib attenuates endotoxin (LPS)-induced vascular leak and lung inflammation in mice but exacerbates these features in a mouse model of ventilator-induced lung injury (VILI). We next explored these discrepant observations in vitro through investigation of the roles for Abl kinases in cultured lung EC. Imatinib attenuates LPS-induced lung EC permeability, restores VE-cadherin junctions, and reduces inflammation by suppressing VCAM-1 expression and inflammatory cytokine (IL-8 and IL-6) secretion. Conversely, in EC exposed to pathological 18% cyclic stretch (CS) (in vitro model of VILI), imatinib decreases VE-cadherin expression, disrupts cell-cell junctions, and increases IL-8 levels. Downregulation of c-Abl expression with siRNA attenuates LPS-induced VCAM-1 expression, whereas specific reduction of Arg reduces VE-cadherin expression in 18% CS-challenged ECs to mimic the imatinib effects. In summary, imatinib exhibits pulmonary barrier-protective and anti-inflammatory effects in LPS-injured mice and lung EC; however, imatinib exacerbates VILI as well as dysfunction in 18% CS-EC. These findings identify the Abl family kinases as important modulators of EC function and potential therapeutic targets in lung injury syndromes.
Assuntos
Anti-Inflamatórios/farmacologia , Benzamidas/farmacologia , Piperazinas/farmacologia , Pirimidinas/farmacologia , Lesão Pulmonar Induzida por Ventilação Mecânica/tratamento farmacológico , Animais , Anti-Inflamatórios/uso terapêutico , Benzamidas/uso terapêutico , Fenômenos Biomecânicos , Permeabilidade Capilar/efeitos dos fármacos , Células Cultivadas , Citocinas/biossíntese , Avaliação Pré-Clínica de Medicamentos , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/imunologia , Endotélio Vascular/fisiopatologia , Humanos , Mesilato de Imatinib , Lipopolissacarídeos/farmacologia , Pulmão/irrigação sanguínea , Pulmão/efeitos dos fármacos , Pulmão/imunologia , Masculino , Camundongos Endogâmicos C57BL , Piperazinas/uso terapêutico , Proteínas Proto-Oncogênicas c-abl/metabolismo , Pirimidinas/uso terapêutico , Estresse Fisiológico , Lesão Pulmonar Induzida por Ventilação Mecânica/imunologia , alfa-Fetoproteínas/metabolismoRESUMO
The field of phenomics has been investigating network structure among large arrays of phenotypes, and genome-wide association studies (GWAS) have been used to investigate the relationship between genetic variation and single diseases/outcomes. A novel approach has emerged combining both the exploration of phenotypic structure and genotypic variation, known as the phenome-wide association study (PheWAS). The Population Architecture using Genomics and Epidemiology (PAGE) network is a National Human Genome Research Institute (NHGRI)-supported collaboration of four groups accessing eight extensively characterized epidemiologic studies. The primary focus of PAGE is deep characterization of well-replicated GWAS variants and their relationships to various phenotypes and traits in diverse epidemiologic studies that include European Americans, African Americans, Mexican Americans/Hispanics, Asians/Pacific Islanders, and Native Americans. The rich phenotypic resources of PAGE studies provide a unique opportunity for PheWAS as each genotyped variant can be tested for an association with the wide array of phenotypic measurements available within the studies of PAGE, including prevalent and incident status for multiple common clinical conditions and risk factors, as well as clinical parameters and intermediate biomarkers. The results of PheWAS can be used to discover novel relationships between SNPs, phenotypes, and networks of interrelated phenotypes; identify pleiotropy; provide novel mechanistic insights; and foster hypothesis generation. The PAGE network has developed infrastructure to support and perform PheWAS in a high-throughput manner. As implementing the PheWAS approach has presented several challenges, the infrastructure and methodology, as well as insights gained in this project, are presented herein to benefit the larger scientific community.
Assuntos
Estudos de Associação Genética/estatística & dados numéricos , Bases de Dados Genéticas , Etnicidade/genética , Variação Genética , Estudo de Associação Genômica Ampla/estatística & dados numéricos , Humanos , Modelos Genéticos , Modelos Estatísticos , Fenótipo , Polimorfismo de Nucleotídeo Único , Grupos Raciais/genéticaRESUMO
Infection with Borrelia spirochaetes leads to the launch of specific and non-specific immunological response in humans. Activation of the complement system is one of the first defence mechanisms against penetrating pathogens. The aim of this study was to select genes related to the alternative pathway of the complement system [including complement factor H (CFH)], differentiating the type of infection in the system model, that is, a culture of normal human diploid fibroblasts infected with three different spirochaete genospecies: Borrelia afzelii, Borrelia garinii and Borrelia burgdorferii sensu stricto, by comparing the infected fibroblast culture with the control fibroblast one. With the use of oligonucleotide microarrays HGU-133A, the differences in the expression of genes selected on the basis of a scientific database Affymetrix were studied by comparing transcriptomes from the four cultures of fibroblasts. In the result of infection of fibroblast cultivation with a specific Borrelia genospecies, a variable expression of certain CFH and complement system-associated genes, specific for one genospecies only, B. afzelii- C1QBP, CD59, C2, CD46 and FHL1; B. garinii - C1S and CLU; Borrelia burgeforii- CFB, A2M and VSIG4, was observed. CFH differentiates infections induced by B. afzelii and B. garinii from infections induced by B. burgdorferii sensu stricto.
Assuntos
Grupo Borrelia Burgdorferi/imunologia , Via Alternativa do Complemento/genética , Fibroblastos/imunologia , Doença de Lyme/genética , Células Cultivadas , Fator H do Complemento/genética , Fator H do Complemento/imunologia , Via Alternativa do Complemento/imunologia , Fibroblastos/citologia , Fibroblastos/metabolismo , Expressão Gênica/imunologia , Perfilação da Expressão Gênica , Interações Hospedeiro-Patógeno , Humanos , Doença de Lyme/imunologia , Doença de Lyme/microbiologia , Análise de Sequência com Séries de Oligonucleotídeos , Especificidade da EspécieRESUMO
Gene-gene interactions are proposed as an important component of the genetic architecture of complex diseases, and are just beginning to be evaluated in the context of genome-wide association studies (GWAS). In addition to detecting epistasis, a benefit to interaction analysis is that it also increases power to detect weak main effects. We conducted a knowledge-driven interaction analysis of a GWAS of 931 multiple sclerosis (MS) trios to discover gene-gene interactions within established biological contexts. We identify heterogeneous signals, including a gene-gene interaction between CHRM3 (muscarinic cholinergic receptor 3) and MYLK (myosin light-chain kinase) (joint P=0.0002), an interaction between two phospholipase C-ß isoforms, PLCß1 and PLCß4 (joint P=0.0098), and a modest interaction between ACTN1 (actinin alpha 1) and MYH9 (myosin heavy chain 9) (joint P=0.0326), all localized to calcium-signaled cytoskeletal regulation. Furthermore, we discover a main effect (joint P=5.2E-5) previously unidentified by single-locus analysis within another related gene, SCIN (scinderin), a calcium-binding cytoskeleton regulatory protein. This work illustrates that knowledge-driven interaction analysis of GWAS data is a feasible approach to identify new genetic effects. The results of this study are among the first gene-gene interactions and non-immune susceptibility loci for MS. Further, the implicated genes cluster within inter-related biological mechanisms that suggest a neurodegenerative component to MS.
Assuntos
Esclerose Múltipla/genética , Cálcio/metabolismo , Citoesqueleto/genética , Citoesqueleto/metabolismo , Suscetibilidade a Doenças , Epistasia Genética , Loci Gênicos , Estudo de Associação Genômica Ampla , Humanos , Polimorfismo de Nucleotídeo Único/genética , Transdução de Sinais/genéticaRESUMO
Strategies to improve pulmonary endothelial barrier function are needed to reverse the devastating effects of vascular leak in acute respiratory distress syndrome. FTY720 is a pharmaceutical analogue of the potent barrier-enhancing phospholipid sphingosine 1-phosphate (S1P). FTY720 decreases vascular permeability by an incompletely characterised mechanism that differs from S1P. Here, we describe its barrier-promoting effects on intracellular signalling and junctional assembly formation in human pulmonary endothelium. Permeability of cultured human pulmonary endothelial cells was assessed using transendothelial electrical resistance and dextran transwell assays. Junctional complex formation was assessed using membrane fractionation and immunofluorescence. Pharmacological inhibitors and small interfering (si)RNA were utilised to determine the effects of individual components on permeability. Unlike S1P, FTY720 failed to induce membrane translocation of adherens junction or tight junction proteins. ß-catenin, occludin, claudin-5 or zona occludens protein (ZO)-1/ZO-2 siRNAs did not alter FTY720-induced barrier enhancement. FTY720 induced focal adhesion kinase (FAK) phosphorylation and focal adhesion formation, with FAK siRNA partially attenuating the prolonged phase of barrier enhancement. Inhibition of Src, protein kinase (PK)A, PKG, PKC or protein phosphatase 2A failed to alter FTY720-induced barrier enhancement. FTY720 increased c-Abl tyrosine kinase activity and c-Abl siRNA attenuated peak barrier enhancement after FTY720. FTY720 enhances endothelial barrier function by a novel pathway involving c-Abl signalling.
Assuntos
Células Endoteliais/citologia , Regulação da Expressão Gênica , Pulmão/efeitos dos fármacos , Propilenoglicóis/farmacologia , Proteínas Proto-Oncogênicas c-abl/metabolismo , Esfingosina/análogos & derivados , Junções Aderentes/patologia , Células Cultivadas , Cloridrato de Fingolimode , Humanos , Inflamação , Lisofosfolipídeos/metabolismo , Permeabilidade , Fosforilação , Artéria Pulmonar/citologia , RNA Interferente Pequeno/metabolismo , Transdução de Sinais , Esfingosina/metabolismo , Esfingosina/farmacologia , Frações Subcelulares/metabolismo , Junções Íntimas/patologiaRESUMO
Novel therapies are needed to address the vascular endothelial cell (EC) barrier disruption that occurs in inflammatory diseases such as acute lung injury (ALI). We previously demonstrated the potent barrier-enhancing effects of both sphingosine 1-phosphate (S1P) and the structurally similar compound FTY720 [2-amino-2-(2-[4-octylphenyl]ethyl)-1,3-propanediol] in inflammatory lung injury. In this study, we examined the therapeutic potential of several novel FTY720 analogs to reduce vascular leak. Similar to S1P and FTY720, the (R)- and (S)-enantiomers of FTY720 phosphonate and enephosphonate analogs produce sustained EC barrier enhancement in vitro, as seen by increases in transendothelial electrical resistance (TER). In contrast, the (R)- and (S)-enantiomers of FTY720-regioisomeric analogs disrupt EC barrier integrity in a dose-dependent manner. Barrier-enhancing FTY720 analogs demonstrate a wider protective concentration range in vitro (1-50 microM) and greater potency than either S1P or FTY720. In contrast to FTY720-induced EC barrier enhancement, S1P and the FTY720 analogs dramatically increase TER within minutes in association with cortical actin ring formation. Unlike S1P, these FTY720 analogs exhibit differential phosphorylation effects without altering the intracellular calcium level. Inhibitor studies indicate that barrier enhancement by these analogs involves signaling via G(i)-coupled receptors, tyrosine kinases, and lipid rafts. Consistent with these in vitro responses, the (S)-phosphonate analog of FTY720 significantly reduces multiple indices of alveolar and vascular permeability in a lipopolysaccharide-mediated murine model of ALI (without significant alterations in leukocyte counts). These results demonstrate the capacity for FTY720 analogs to significantly decrease pulmonary vascular leakage and inflammation in vitro and in vivo.
Assuntos
Permeabilidade Capilar/efeitos dos fármacos , Permeabilidade Capilar/fisiologia , Cloridrato de Fingolimode/análogos & derivados , Mediadores da Inflamação/síntese química , Mediadores da Inflamação/farmacologia , Organofosfonatos/síntese química , Organofosfonatos/farmacologia , Propilenoglicóis/síntese química , Propilenoglicóis/farmacologia , Artéria Pulmonar/efeitos dos fármacos , Esfingosina/análogos & derivados , Animais , Linhagem Celular , Cloridrato de Fingolimode/síntese química , Cloridrato de Fingolimode/farmacologia , Humanos , Pulmão/irrigação sanguínea , Pulmão/efeitos dos fármacos , Pulmão/patologia , Camundongos , Camundongos Endogâmicos C57BL , Artéria Pulmonar/patologia , Esfingosina/síntese química , Esfingosina/farmacologiaRESUMO
Stimulation of phosphoinositide hydrolysis by excitatory amino acids was studied in synaptoneurosomes of kitten striate cortex at several postnatal ages. Ibotenate and glutamate stimulated phosphoinositide turnover during the second and third postnatal months; N-methyl-D-aspartate and DL-alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) were without effect. The developmental profile of ibotenate-stimulated phosphoinositide turnover parallels the postnatal changes in cortical susceptibility to visual deprivation. The transient increase in ibotenate-stimulated phosphoinositide turnover does not occur in visual cortex of kittens reared in complete darkness.
Assuntos
Fosfatidilinositóis/metabolismo , Sinapses/fisiologia , Córtex Visual/crescimento & desenvolvimento , Envelhecimento , Animais , Ácido Aspártico/farmacologia , Carbacol/farmacologia , Bovinos , Glutamatos/farmacologia , Ácido Glutâmico , Ácido Ibotênico/farmacologia , N-Metilaspartato , Fenômenos Fisiológicos Oculares , Sinapses/efeitos dos fármacos , Sinapses/metabolismo , Visão Ocular , Córtex Visual/metabolismo , Córtex Visual/fisiologiaRESUMO
Activity-dependent synaptic plasticity in the superficial layers of juvenile cat and adult rat visual neocortex was compared with that in adult rat hippocampal field CA1. Stimulation of neocortical layer IV reliably induced synaptic long-term potentiation (LTP) and long-term depression (LTD) in layer III with precisely the same types of stimulation protocols that were effective in CA1. Neocortical LTP and LTD were specific to the conditioned pathway and, as in the hippocampus, were dependent on activation of N-methyl-D-aspartate receptors. These results provide strong support for the view that common principles may govern experience-dependent synaptic plasticity in CA1 and throughout the superficial layers of the mammalian neocortex.
Assuntos
Córtex Cerebral/fisiologia , Hipocampo/fisiologia , Plasticidade Neuronal/fisiologia , Sinapses/fisiologia , Potenciais de Ação , Envelhecimento/fisiologia , Animais , Gatos , Estimulação Elétrica , Técnicas In Vitro , Vias Neurais/fisiologia , Ratos , Receptores de N-Metil-D-Aspartato/fisiologiaRESUMO
We measured rate constants of thermal, interfacial electron transfer through oligophenylenevinylene bridges between a gold electrode and a tethered redox species in contact with an aqueous electrolyte using the indirect laser-induced temperature jump technique. Analysis of the distance dependence indicates that, unlike other bridges studied to date, the rate constants are not limited by electronic coupling for bridges up to 28 angstroms long. The energy levels of the bridges relative to those of the redox species rule out hopping through the bridge. We conclude that, out to 28 angstroms, the transfer is limited by structural reorganization and that electron tunneling occurs in less than 20 picoseconds, suggesting that oligophenylenevinylene bridges could be useful for wiring molecular electronic elements.
Assuntos
Elétrons , Compostos Ferrosos/química , Estilbenos/química , Eletroquímica , Eletrodos , Eletrólitos , Eletrônica , Ouro , Metalocenos , Oxirredução , TemperaturaRESUMO
For in vitro LTD to remain viable as a model for synaptic weakening in visual cortical plasticity, it is crucial that it display a critical period for its induction within layer IV. A complicating factor, however, is that LTD in layer IV is modulated by inhibitory postsynaptic potentials (IPSPs); postsynaptic responses characterized as containing IPSPs do not depress in response to 1 Hz afferent stimulation. By blocking IPSPs intracellularly, we find that the ability to induce LTD in layer IV neurons is restored in juvenile, but not in mature animals. This developmental down-regulation of LTD induction is specific for layer IV when compared with LTD induction in layers II/III. These data are consistent with the hypothesis that an LTD-like phenomenon is involved in critical period plasticity and is apparently independent of developmental changes in inhibitory circuitry.
Assuntos
Regulação para Baixo , Leucotrieno D4/fisiologia , Córtex Visual/fisiologia , Vias Visuais/fisiologia , Animais , Gatos , Cobaias , Microscopia Eletrônica , Plasticidade Neuronal/fisiologia , Neurônios/fisiologia , Neurônios/ultraestruturaRESUMO
Novel therapeutic strategies are needed to reverse the loss of endothelial cell (EC) barrier integrity that occurs during inflammatory disease states such as acute lung injury. We previously demonstrated potent EC barrier augmentation in vivo and in vitro by the platelet-derived phospholipid, sphingosine 1-phosphate (S1P) via ligation of the S1P1 receptor. The S1P analogue, FTY720, similarly exerts barrier-protective vascular effects via presumed S1P1 receptor ligation. We examined the role of the S1P1 receptor in sphingolipid-mediated human lung EC barrier enhancement. Both S1P and FTY-induced sustained, dose-dependent barrier enhancement, reflected by increases in transendothelial electrical resistance (TER), which was abolished by pertussis toxin indicating Gi-coupled receptor activation. FTY-mediated increases in TER exhibited significantly delayed onset and intensity relative to the S1P response. Reduction of S1P1R expression (via siRNA) attenuated S1P-induced TER elevations whereas the TER response to FTY was unaffected. Both S1P and FTY rapidly (within 5 min) induced S1P1R accumulation in membrane lipid rafts, but only S1P stimulated S1P1R phosphorylation on threonine residues. Inhibition of PI3 kinase activity attenuated S1P-mediated TER increases but failed to alter FTY-induced TER elevation. Finally, S1P, but not FTY, induced significant myosin light chain phosphorylation and dramatic actin cytoskeletal rearrangement whereas reduced expression of the cytoskeletal effectors, Rac1 and cortactin (via siRNA), attenuated S1P-, but not FTY-induced TER elevations. These results mechanistically characterize pulmonary vascular barrier regulation by FTY720, suggesting a novel barrier-enhancing pathway for modulating vascular permeability.
Assuntos
Células Endoteliais/efeitos dos fármacos , Imunossupressores/farmacologia , Propilenoglicóis/farmacologia , Receptores de Lisoesfingolipídeo/metabolismo , Esfingosina/análogos & derivados , Adenoviridae/genética , Permeabilidade Capilar , Células Cultivadas , Citoesqueleto/metabolismo , Impedância Elétrica , Endotélio Vascular/citologia , Cloridrato de Fingolimode , Humanos , Pulmão/citologia , Modelos Biológicos , Fosforilação , Artéria Pulmonar/citologia , RNA Interferente Pequeno/metabolismo , Transdução de Sinais , Esfingosina/farmacologia , Treonina/metabolismo , Proteínas rac1 de Ligação ao GTP/metabolismoRESUMO
Exploration of human pulmonary artery endothelial cell (EC) as a prototypical biomechanical system has important pathophysiologic relevance because this cell type plays a key role in the development of a wide variety of clinical conditions. The complex hierarchical organization ranging from the molecular scale up to the cellular level has an intimate and intricate relationship to the barrier function between lung tissue and blood. To understand the innate molecule-cell-tissue relationship across varied length-scales, the functional role of c-Abl kinase in the cytoskeletal nano-biomechanics of ECs in response to barrier-altering agonists was investigated using atomic force microscopy. Concurrently, the spatially specific arrangement of cytoskeleton structure and dynamic distribution of critical proteins were examined using scanning electron microscopy and immunofluorescence. Reduction in c-Abl expression by siRNA attenuates both thrombin- and sphingosine 1-phosphate (S1P)-mediated structural changes in ECs, specifically spatially-defined changes in elastic modulus and distribution of critical proteins. These results indicate that c-Abl kinase is an important determinant of cortical actin-based cytoskeletal rearrangement. Our findings directly bridge the gap between kinase activity, structural complexity, and functional connectivity across varied length-scales, and suggest that manipulation of c-Abl kinase activity may be a potential target for the treatment of pulmonary barrier disorders.
Assuntos
Citoesqueleto de Actina/efeitos dos fármacos , Células Endoteliais/efeitos dos fármacos , Mecanotransdução Celular , Proteínas Proto-Oncogênicas c-abl/genética , Citoesqueleto de Actina/metabolismo , Citoesqueleto de Actina/ultraestrutura , Antígenos CD/genética , Antígenos CD/metabolismo , Fenômenos Biomecânicos , Caderinas/genética , Caderinas/metabolismo , Linhagem Celular , Cortactina/genética , Cortactina/metabolismo , Módulo de Elasticidade , Células Endoteliais/metabolismo , Células Endoteliais/ultraestrutura , Regulação da Expressão Gênica , Humanos , Lisofosfolipídeos/farmacologia , Microscopia de Força Atômica , Paxilina/genética , Paxilina/metabolismo , Proteínas Proto-Oncogênicas c-abl/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-abl/metabolismo , Artéria Pulmonar/citologia , Artéria Pulmonar/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Esfingosina/análogos & derivados , Esfingosina/farmacologia , Trombina/farmacologiaRESUMO
The endothelium serves as a size-selective barrier and tightly controls the fluid exchange from the circulation to the surrounding tissues. In this study, a multiplexed microscopy characterization is developed to study the spatio-temporal effects of Abl kinases on endothelial cytoskeletal structure using AFM, SEM, and immunofluorescence. Sphingosine 1-phosphate (S1P) produces significant endothelial barrier enhancement by means of peripheral actin rearrangement. However, Abl kinase inhibition by imatinib reduces rapid redistribution of the important cytoskeletal proteins to the periphery and their association with the cortical actin ring. Herein, it moderates the thickness of the cortical actin ring, and diminishes the increase in elastic modulus at the periphery and cytoplasm. These findings demonstrate that imatinib attenuates multiple cytoskeletal changes associated with S1P-mediated endothelial barrier enhancement and suggest a novel role for Abl kinases in mediating these S1P effects. These observations bridge the gap between molecule dynamics, structure complexity and function connectivity across varied length-scales to improve our understanding on human pulmonary endothelial barrier regulation. Moreover, our study suggests a framework for understanding form-function relationships in other biomechanical subsystems, wherein complex hierarchical organization programmed from the molecular scale to the cellular and tissue levels has an intimate relationship to the overall physiological function.
Assuntos
Citoesqueleto/efeitos dos fármacos , Mesilato de Imatinib/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Artéria Pulmonar/citologia , Antígenos CD/metabolismo , Caderinas/metabolismo , Células Cultivadas , Cortactina/metabolismo , Citoesqueleto/ultraestrutura , Endotélio Vascular/citologia , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/metabolismo , Imunofluorescência , Humanos , Lisofosfolipídeos/farmacologia , Microscopia de Força Atômica , Microscopia Eletrônica de Varredura , Paxilina/metabolismo , Proteínas Proto-Oncogênicas c-abl/agonistas , Proteínas Proto-Oncogênicas c-abl/antagonistas & inibidores , Artéria Pulmonar/efeitos dos fármacos , Esfingosina/análogos & derivados , Esfingosina/farmacologiaRESUMO
Mitogen-activated protein kinase (MAPK) has been identified as a potential element in regulating excitability, long-term potentiation (LTP), and gene expression in hippocampal neurons. The objective of the present study was to determine whether the pattern and intensity of synaptic activity could differentially regulate MAPK phosphorylation via selective activation of different modes of calcium influx into CA1 pyramidal neurons. An antibody specific for the phosphorylated (active) form of MAPK was used to stain sections from hippocampal slices, which were first stimulated in vitro. LTP-inducing stimulation [theta-burst (TBS) and 100 Hz] was effective in inducing intense staining in both dendritic and somatic compartments of CA1 neurons. Phosphorylation of MAPK was also induced, however, with stimulation frequencies (3-10 Hz) not typically effective in inducing LTP. Intensity and extent of staining was better correlated with the spread of population spikes across the CA1 subfield than with frequency (above 3 Hz). Experiments using inhibitors of NMDA receptors and voltage-sensitive calcium channels (VSCCs) revealed that, although MAPK is activated after both TBS and 5 Hz stimulation, the relative contribution of calcium through L-type calcium channels differs. Blockade of NMDA receptors alone was sufficient to prevent MAPK phosphorylation in response to 5 Hz stimulation, whereas inhibitors of both NMDA receptors and VSCCs were necessary for inhibition of the TBS-induced staining. We conclude that the intensity and frequency of synaptic input to CA1 hippocampal neurons are critically involved in determining the path by which second-messenger cascades are activated to activate MAPK.
Assuntos
Cálcio/metabolismo , Dendritos/enzimologia , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Células Piramidais/fisiologia , Potenciais de Ação/efeitos dos fármacos , Potenciais de Ação/fisiologia , Animais , Bloqueadores dos Canais de Cálcio/farmacologia , Canais de Cálcio/metabolismo , Compartimento Celular/fisiologia , Dendritos/efeitos dos fármacos , Estimulação Elétrica/métodos , Antagonistas de Aminoácidos Excitatórios/farmacologia , Hipocampo/citologia , Hipocampo/fisiologia , Técnicas In Vitro , Potenciação de Longa Duração/efeitos dos fármacos , Potenciação de Longa Duração/fisiologia , Camundongos , Fosforilação , Células Piramidais/efeitos dos fármacos , Células Piramidais/ultraestrutura , Ratos , Ratos Sprague-Dawley , Receptores de N-Metil-D-Aspartato/antagonistas & inibidoresRESUMO
One Hertz stimulation of afferents for 15 min with constant interstimulus intervals (regular stimulation) can induce long-term depression (LTD) of synaptic strength in the neocortex. However, it is unknown whether natural patterns of low-frequency afferent spike activity induce LTD. Although neurons in the neocortex can fire at overall rates as low as 1 Hz, the intervals between spikes are irregular. This irregular spike activity (and thus, presumably, irregular activation of the synapses of that neuron onto postsynaptic targets) can be approximated by stimulation with Poisson-distributed interstimulus intervals (Poisson stimulation). Therefore, if low-frequency presynaptic spike activity in the intact neocortex is sufficient to induce a generalized LTD of synaptic transmission, then Poisson stimulation, which mimics this spike activity, should induce LTD in slices. We tested this hypothesis by comparing changes in the strength of synapses onto layer 2/3 pyramidal cells induced by regular and Poisson stimulation in slices from adult visual cortex. We find that regular stimulation induces LTD of excitatory synaptic transmission as assessed by field potentials and intracellular postsynaptic potentials (PSPs) with inhibition absent. However, Poisson stimulation does not induce a net LTD of excitatory synaptic transmission. When the PSP contained an inhibitory component, neither Poisson nor regular stimulation induced LTD. We propose that the short bursts of synaptic activity that occur during a Poisson train have potentiating effects that offset the induction of LTD that is favored with regular stimulation. Thus, natural (i.e., irregular) low-frequency activity in the adult neocortex in vivo should not consistently induce LTD.
Assuntos
Potenciais de Ação/fisiologia , Neurônios/fisiologia , Córtex Visual/fisiologia , Animais , Estimulação Elétrica , Eletrofisiologia , Cobaias , Plasticidade Neuronal/fisiologia , Sinapses/fisiologiaRESUMO
We investigated levels of nucleotide polymorphism within and among populations of the highly self-fertilizing Brassicaceous species, Arabidopsis thaliana. Four-cutter RFLP data were collected at one mitochondrial and three nuclear loci from 115 isolines representing 11 worldwide population collections, as well as from seven commonly used ecotypes. The collections include multiple populations from North America and Eurasia, as well as two pairs of collections from locally proximate sites, and thus allow a hierarchical geographic analysis of polymorphism. We found no variation at the mitochondrial locus Nad5 and very low levels of intrapopulation nucleotide diversity at Adh, Dhs1, and Gpa1. Interpopulation nucleotide diversity was also consistently low among the loci, averaging 0.0014. gst, a measure of population differentiation, was estimated to be 0.643. Interestingly, we found no association between geographical distance between populations and genetic distance. Most haplotypes have a worldwide distribution, suggesting a recent expansion of the species or long-distance gene flow. The low level of polymorphism found in this study is consistent with theoretical models of neutral mutations and background selection in highly self-fertilizing species.