RESUMEN
BACKGROUND: Bacterial pneumonia and sepsis are both common causes of end-organ dysfunction, especially in immunocompromised and critically ill patients. Pre-clinical data demonstrate that bacterial pneumonia and sepsis elicit the production of cytotoxic tau and amyloids from pulmonary endothelial cells, which cause lung and brain injury in naïve animal subjects, independent of the primary infection. The contribution of infection-elicited cytotoxic tau and amyloids to end-organ dysfunction has not been examined in the clinical setting. We hypothesized that cytotoxic tau and amyloids are present in the bronchoalveolar lavage fluid of critically ill patients with bacterial pneumonia and that these tau/amyloids are associated with end-organ dysfunction. METHODS: Bacterial culture-positive and culture-negative mechanically ventilated patients were recruited into a prospective, exploratory observational study. Levels of tau and Aß42 in, and cytotoxicity of, the bronchoalveolar lavage fluid were measured. Cytotoxic tau and amyloid concentrations were examined in comparison with patient clinical characteristics, including measures of end-organ dysfunction. RESULTS: Tau and Aß42 were increased in culture-positive patients (n = 49) compared to culture-negative patients (n = 50), independent of the causative bacterial organism. The mean age of patients was 52.1 ± 16.72 years old in the culture-positive group and 52.78 ± 18.18 years old in the culture-negative group. Males comprised 65.3% of the culture-positive group and 56% of the culture-negative group. Caucasian culture-positive patients had increased tau, boiled tau, and Aß42 compared to both Caucasian and minority culture-negative patients. The increase in cytotoxins was most evident in males of all ages, and their presence was associated with end-organ dysfunction. CONCLUSIONS: Bacterial infection promotes the generation of cytotoxic tau and Aß42 within the lung, and these cytotoxins contribute to end-organ dysfunction among critically ill patients. This work illuminates an unappreciated mechanism of injury in critical illness.
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Neumonía Bacteriana , Sepsis , Masculino , Animales , Humanos , Adulto , Persona de Mediana Edad , Anciano , Femenino , Estudios Prospectivos , Enfermedad Crítica , Células Endoteliales , Insuficiencia Multiorgánica , Irrigación Terapéutica , Líquido del Lavado Bronquioalveolar/microbiología , Neumonía Bacteriana/microbiología , Amiloide , Citotoxinas , Péptidos beta-Amiloides , Proteínas tauRESUMEN
AIM: Recent evidence has shown no difference in the risk of surgical site infection (SSI) with oral antibiotics alone (OA) and oral antibiotics in combination with mechanical bowel preparation (OA + MBP), suggesting that the use of MBP may be safely avoided. The aim of this work was to determine the absolute risk of SSI that patients would accept with OA relative to OA + MBP. METHOD: Standardized, in-person interviews were conducted using the threshold task with patients attending colorectal surgery clinics who had previously had MBP. Participants were asked which option they preferred when the absolute risk of SSI was 7% for both options. Next, their switch point was determined by increasing the risk of SSI with OA by 1% intervals until their preference changed from OA to OA + MBP. Median switch point scores were reported and represented the absolute increased risk of SSI that patients would accept with OA relative to OA + MBP. RESULTS: Fifty patients completed the interview. All participants chose OA over OA + MBP when the risk of SSI was 7% for both options. Switch points ranged from 8% to 25%, with a median of 10%, indicating that participants were willing to accept up to a 3% increase in absolute risk of developing a SSI with OA to avoid MBP. CONCLUSIONS: The results showed that patients are willing to accept an increased risk of up to 3% for SSI with OA relative to OA + MBP. Incorporating patient preferences into the planning of future trials has the potential to improve the uptake of trial results into clinical practice.
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Cirugía Colorrectal , Infección de la Herida Quirúrgica , Administración Oral , Antibacterianos/uso terapéutico , Profilaxis Antibiótica/métodos , Catárticos/uso terapéutico , Cirugía Colorrectal/efectos adversos , Cirugía Colorrectal/métodos , Procedimientos Quirúrgicos Electivos/métodos , Humanos , Cuidados Preoperatorios/métodos , Infección de la Herida Quirúrgica/etiología , Infección de la Herida Quirúrgica/prevención & controlAsunto(s)
Canal de Potasio KCNQ1/metabolismo , Neuralgia/metabolismo , Potenciales de Acción/efectos de los fármacos , Aminopiridinas/uso terapéutico , Analgésicos/uso terapéutico , Animales , Carbamatos/uso terapéutico , Humanos , Hiperalgesia/tratamiento farmacológico , Hiperalgesia/metabolismo , Canal de Potasio KCNQ1/antagonistas & inhibidores , Neuralgia/tratamiento farmacológico , Fenilendiaminas/uso terapéuticoRESUMEN
Merkel discs, located in skin touch domes and whisker hair follicles, are tactile end organs essential for environmental exploration, social interaction, and tactile discrimination. Recent studies from our group and two others have shown that mechanical stimulation excites Merkel cells via Piezo2 channel activation to subsequently activate sensory neural pathways. We have further shown that mechanical stimulation leads to the release of 5-HT from Merkel cells to synaptically transmit tactile signals to whisker afferent nerves. However, a more recent study using skin touch domes has raised the possibility that Merkel discs are adrenergic synapses. It was proposed that norepinephrine is released from Merkel cells upon mechanical stimulation to subsequently activate ß2 adrenergic receptors on Merkel disc nerve endings leading to nerve impulses. In the present study, we examined effects of norepinephrine and ß2 adrenergic receptor antagonist ICI 118,551 on Merkel disc mechanoreceptors in mouse whisker hair follicles. We show that norepinephrine did not directly induce impulses from Merkel disc mechanoreceptors. Furthermore, we found that ICI 118,551 at 50 µM inhibited voltage-gated Na+ channels and suppressed impulses of Merkel disc mechanoreceptors, but ICI 118,551 at 1 µM had no effects on the impulse. These findings challenge the hypothesis of Merkel discs being adrenergic synapses.
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Antagonistas Adrenérgicos beta/farmacología , Folículo Piloso/metabolismo , Mecanorreceptores/metabolismo , Células de Merkel/metabolismo , Norepinefrina/farmacología , Propanolaminas/farmacología , Sinapsis/metabolismo , Vibrisas/efectos de los fármacos , Vías Aferentes/efectos de los fármacos , Vías Aferentes/metabolismo , Animales , Folículo Piloso/efectos de los fármacos , Células de Merkel/efectos de los fármacos , Sinapsis/efectos de los fármacosRESUMEN
CONTEXT: Regulator of G-protein signaling-2 (RGS2) inhibits Gq-mediated regulation of Ca(2+) signalling in vascular smooth muscle cells (VSMC). OBJECTIVE: RGS2 knockout (RGS2KO) mice are hypertensive and show arteriolar remodeling. VSMC proliferation modulates intracellular Ca(2+) concentration [Ca(2+)]i. RGS2 involvement in VSMC proliferation had not been examined. METHODS: Thymidine incorporation and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) conversion assays measured cell proliferation. Fura-2 ratiometric imaging quantified [Ca(2+)]i before and after UTP and thapsigargin. [(3)H]-labeled inositol was used for phosphoinositide hydrolysis. Quantitative RT-PCR and confocal immunofluorescence of select Ca(2+) transporters was performed in primary aortic VSMC. RESULTS AND DISCUSSION: Platelet-derived growth factor (PDGF) increased S-phase entry and proliferation in VSMC from RGS2KO mice to a greater extent than in VSMC from wild-type (WT) controls. Consistent with differential PDGF-induced changes in Ca(2+) homeostasis, RGS2KO VSMC showed lower resting [Ca(2+)]i but higher thapsigargin-induced [Ca(2+)]i as compared with WT. RGS2KO VSMC expressed lower mRNA levels of plasma membrane Ca(2+) ATPase-4 (PMCA4) and Na(+) Ca(2+) Exchanger (NCX), but higher levels of sarco-endoplasmic reticulum Ca(2+) ATPase-2 (SERCA2). Western blot and immunofluorescence revealed similar differences in PMCA4 and SERCA2 protein, while levels of NCX protein were not reduced in RGS2KO VSMC. Consistent with decreased Ca(2+) efflux activity, (45)Ca-extrusion rates were lower in RGS2KO VSMC. These differences were reversed by the PMCA inhibitor La(3+), but not by replacing extracellular Na(+) with choline, implicating differences in the activity of PMCA and not NCX. CONCLUSION: RGS2-deficient VSMC exhibit higher rates of proliferation and coordinate plasticity of Ca(2+)-handling mechanisms in response to PDGF stimulation.
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Señalización del Calcio/fisiología , Calcio/metabolismo , Músculo Liso Vascular/citología , Músculo Liso Vascular/fisiología , Miocitos del Músculo Liso/citología , Miocitos del Músculo Liso/fisiología , Proteínas RGS/metabolismo , Animales , Señalización del Calcio/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Proliferación Celular/fisiología , Células Cultivadas , Ratones , Ratones Noqueados , Músculo Liso Vascular/efectos de los fármacos , Miocitos del Músculo Liso/efectos de los fármacos , Factor de Crecimiento Derivado de Plaquetas/farmacología , Proteínas RGS/genéticaRESUMEN
BACKGROUND: To overcome the limitations of conventional diffusion tensor magnetic resonance imaging resulting from the assumption of a Gaussian diffusion model for characterizing voxels containing multiple axonal orientations, Shannon's entropy was employed to evaluate white matter structure in human brain and in brain remodeling after traumatic brain injury (TBI) in a rat. METHODS: Thirteen healthy subjects were investigated using a Q-ball based DTI data sampling scheme. FA and entropy values were measured in white matter bundles, white matter fiber crossing areas, different gray matter (GM) regions and cerebrospinal fluid (CSF). Axonal densities' from the same regions of interest (ROIs) were evaluated in Bielschowsky and Luxol fast blue stained autopsy (n = 30) brain sections by light microscopy. As a case demonstration, a Wistar rat subjected to TBI and treated with bone marrow stromal cells (MSC) 1 week after TBI was employed to illustrate the superior ability of entropy over FA in detecting reorganized crossing axonal bundles as confirmed by histological analysis with Bielschowsky and Luxol fast blue staining. RESULTS: Unlike FA, entropy was less affected by axonal orientation and more affected by axonal density. A significant agreement (r = 0.91) was detected between entropy values from in vivo human brain and histologically measured axonal density from post mortum from the same brain structures. The MSC treated TBI rat demonstrated that the entropy approach is superior to FA in detecting axonal remodeling after injury. Compared with FA, entropy detected new axonal remodeling regions with crossing axons, confirmed with immunohistological staining. CONCLUSIONS: Entropy measurement is more effective in distinguishing axonal remodeling after injury, when compared with FA. Entropy is also more sensitive to axonal density than axonal orientation, and thus may provide a more accurate reflection of axonal changes that occur in neurological injury and disease.
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Lesiones Encefálicas/patología , Encéfalo/patología , Imagen de Difusión Tensora/métodos , Entropía , Adolescente , Adulto , Animales , Axones/patología , Lesiones Encefálicas/diagnóstico , Difusión , Femenino , Humanos , Masculino , Persona de Mediana Edad , Probabilidad , Ratas , Adulto JovenRESUMEN
Collagens in the atherosclerotic plaque signal regulation of cell behavior and provide tensile strength to the fibrous cap. Type VIII collagen, a short-chain collagen, is up-regulated in atherosclerosis; however, little is known about its functions in vivo. We studied the response to arterial injury and the development of atherosclerosis in type VIII collagen knockout mice (Col8(-/-) mice). After wire injury of the femoral artery, Col8(-/-) mice had decreased vessel wall thickening and outward remodeling when compared with Col8(+/+) mice. We discovered that apolipoprotein E (ApoE) is an endogenous repressor of the Col8a1 chain, and, therefore, in ApoE knockout mice, type VIII collagen was up-regulated. Deficiency of type VIII collagen in ApoE(-/-) mice (Col8(-/-);ApoE(-/-)) resulted in development of plaques with thin fibrous caps because of decreased smooth muscle cell migration and proliferation and reduced accumulation of fibrillar type I collagen. In contrast, macrophage accumulation was not affected, and the plaques had large lipid-rich necrotic cores. We conclude that in atherosclerosis, type VIII collagen is up-regulated in the absence of ApoE and functions to increase smooth muscle cell proliferation and migration. This is an important mechanism for formation of a thick fibrous cap to protect the atherosclerotic plaque from rupture.
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Aterosclerosis/patología , Colágeno Tipo VIII/fisiología , Placa Aterosclerótica/patología , Animales , Apolipoproteínas E/deficiencia , Apolipoproteínas E/fisiología , Aterosclerosis/metabolismo , Movimiento Celular/fisiología , Proliferación Celular , Células Cultivadas , Colágeno/metabolismo , Colágeno Tipo VIII/deficiencia , Colágeno Tipo VIII/genética , Elastina/metabolismo , Femenino , Arteria Femoral/lesiones , Gelatinasas/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Músculo Liso Vascular/patología , Necrosis , Placa Aterosclerótica/metabolismo , ARN Mensajero/genética , Transducción de Señal/fisiología , Regulación hacia Arriba/fisiologíaRESUMEN
Cells from multiple origins contribute to vascular smooth muscle cell (VSMC) development. Phenotypic heterogeneity of VSMCs is associated with their point of developmental origin; however, the mechanisms driving such differences are unknown. We here examined the mechanisms controlling vascular bed-specific differences in Rgs5 expression during development. Rgs5 levels were similar across different regions of the vasculature in neonatal animals but were >15-fold higher in descending aortas compared with carotid arteries of adult mice. Thus, vessel bed-specific changes in regulation of Rgs5 expression occurred during vessel maturation. Examination of adult Rgs5-LacZ reporter mice revealed lower Rgs5 expression in VSMCs originating from the third (carotid artery) branchial arch compared with those originating in the fourth and sixth (aortic B segment, right subclavian, and ductus arteriosus) branchial arches. Indeed, a mosaic Rgs5 expression pattern, with discreet LacZ boundaries between VSMCs derived from different developmental origins, was observed. Furthermore, Rgs5-LacZ expression was correlated with the site of VSMC origin (splanchic mesoderm ≈ local mesenchyme > somites > proepicardium > mesothelium). Surprisingly, Rgs5 reporter activity in cultured carotid artery- and descending aorta-derived cells did not recapitulate the differences observed in vivo. Consistent with a developmental origin-specific epigenetic mechanism driving the observed expression differences in vivo, the Rgs5 promoter showed increased methylation on CpG dinucleotides in carotid arteries compared with that in descending aortas in adult but not in neonatal mice. In vitro methylation of the Rgs5 promoter confirmed that its activity is sensitive to transcriptional down-regulation by CpG methylation. These data suggest that an origin-dependent epigenetic program regulates vascular bed- and maturation state-dependent regulation of VSMC-specific gene transcription.
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Aorta Torácica , Arterias Carótidas , Epigénesis Genética/fisiología , Neovascularización Fisiológica/genética , Proteínas RGS/genética , Proteínas RGS/metabolismo , Factores de Edad , Animales , Aorta Torácica/citología , Aorta Torácica/crecimiento & desarrollo , Aorta Torácica/fisiología , Arterias Carótidas/citología , Arterias Carótidas/crecimiento & desarrollo , Arterias Carótidas/fisiología , Diferenciación Celular/fisiología , Metilación de ADN/fisiología , Operón Lac/genética , Ratones , Ratones Endogámicos C57BL , Ratones Mutantes , Músculo Liso Vascular/citología , Músculo Liso Vascular/crecimiento & desarrollo , Músculo Liso Vascular/fisiología , Especificidad de Órganos , Fenotipo , Regiones Promotoras Genéticas/fisiología , ARN Mensajero/metabolismo , Transducción de Señal/fisiologíaRESUMEN
BACKGROUND AND PURPOSE: Sildenafil provides restorative therapeutic benefits in the treatment of experimental stroke. The majority of experimental studies on treatment of stroke have been performed in young animals; however, stroke is primarily a disease of the aged. Thus, using MRI, we evaluated the effects of sildenafil treatment of embolic stroke in aged animals. METHODS: Aged male Wistar rats (18 months) were subjected to embolic stroke and treated daily with saline (n=10) or with sildenafil (n=10) initiated at 24 hours and subsequently for 7 days after onset of ischemia. MRI measurements were performed at 24 hours and weekly to 6 weeks after embolization. RESULTS: MRI and histological measurements demonstrated that sildenafil treatment of aged rats significantly enhanced angiogenesis and axonal remodeling after stroke compared to saline-treated aged rats. Local cerebral blood flow in the angiogenic area was elevated and expansion of the ipsilateral ventricle and, consequently, brain atrophy was significantly reduced in the sildenafil-treated rats. CONCLUSIONS: Treatment of embolic stroke in aged rats with sildenafil significantly augments angiogenesis and axonal remodeling, which increased local blood flow and reduced expansion of the ipsilateral ventricle 6 weeks after stroke compared to control aged rats. MRI can be used to investigate brain repair after stroke in aged rats.
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Encéfalo/efectos de los fármacos , Embolia Intracraneal/tratamiento farmacológico , Imagen por Resonancia Magnética , Neovascularización Fisiológica/efectos de los fármacos , Piperazinas/uso terapéutico , Accidente Cerebrovascular/tratamiento farmacológico , Sulfonas/uso terapéutico , Vasodilatadores/uso terapéutico , Animales , Encéfalo/patología , Circulación Cerebrovascular/efectos de los fármacos , Embolia Intracraneal/patología , Estudios Longitudinales , Masculino , Piperazinas/farmacología , Purinas/farmacología , Purinas/uso terapéutico , Ratas , Ratas Wistar , Recuperación de la Función/efectos de los fármacos , Citrato de Sildenafil , Accidente Cerebrovascular/patología , Sulfonas/farmacología , Vasodilatadores/farmacologíaRESUMEN
IGF-I, a known secretory product of intestinal subepithelial myofibroblasts (ISEMFs), is essential for the intestinotropic effects of glucagon-like peptide-2 (GLP-2). Furthermore, GLP-2 increases IGF-I mRNA transcript levels in vitro in heterogeneous fetal rat intestinal cultures, as well as in vivo in the rodent small intestine. To determine the mechanism underlying the stimulatory effect of GLP-2 on intestinal IGF-I mRNA, murine ISEMF cells were placed into primary culture. Immunocytochemistry showed that the ISEMF cells appropriately expressed α-smooth muscle actin and vimentin but not desmin. The cells also expressed GLP-2 receptor and IGF-I mRNA transcripts. Treatment of ISEMF cells with (Gly2)GLP-2 induced IGF-I mRNA transcripts by up to 5-fold of basal levels after treatment with 10(-8) m GLP-2 for 2 h (P < 0.05) but did not increase transcript levels for other intestinal growth factors, such as ErbB family members. Immunoblot revealed a 1.6-fold increase in phospho (p)-Akt/total-(t)Akt with 10(-8) m GLP-2 treatment (P < 0.05) but no changes in cAMP, cAMP-dependent ß-galactosidase expression, pcAMP response element-binding protein/tcAMP response element-binding protein, pErk1/2/tErk1/2, or intracellular calcium. Furthermore, pretreatment of ISEMF cells with the phosphatidylinositol 3 kinase (PI3K) inhibitors, LY294002 and wortmannin, abrogated the IGF-I mRNA response to GLP-2, as did overexpression of kinase-dead Akt. The role of PI3K/Akt in GLP-2-induced IGF-I mRNA levels in the murine jejunum was also confirmed in vivo. These findings implicate the PI3K/Akt pathway in the stimulatory effects of GLP-2 to enhance intestinal IGF-I mRNA transcript levels and provide further evidence in support of a role for IGF-I produced by the ISEMF cells in the intestinotropic effects of GLP-2.
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Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Péptido 2 Similar al Glucagón/farmacología , Factor I del Crecimiento Similar a la Insulina/genética , Intestinos/citología , ARN Mensajero/genética , Androstadienos/farmacología , Animales , Cromonas/farmacología , AMP Cíclico/metabolismo , Femenino , Receptor del Péptido 2 Similar al Glucagón , Humanos , Inmunohistoquímica , Masculino , Ratones , Morfolinas/farmacología , Fosfatidilinositol 3-Quinasas/metabolismo , Inhibidores de las Quinasa Fosfoinosítidos-3 , Receptores de Glucagón/genética , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , WortmaninaRESUMEN
Understanding the mechanisms that underlie BP (blood pressure) variation in humans and animal models may provide important clues for reducing the burden of uncontrolled hypertension in industrialized societies. High BP is often associated with increased signalling via G-protein-coupled receptors. Three members of the RGS (regulator of G-protein signalling) superfamily RGS2, RGS4 and RGS5 have been implicated in the attenuation of G-protein signalling pathways in vascular and cardiac myocytes, as well as cells of the kidney and autonomic nervous system. In the present review, we discuss the current state of knowledge regarding their differential expression and function in cardiovascular tissues, and the likelihood that one or more of these alleles are candidate hypertension genes. Together, findings from the studies described herein suggest that development of methods to modulate the expression and function of RGS proteins may be a possible strategy for the treatment and prevention of hypertension and cardiovascular disease.
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Presión Sanguínea/fisiología , Sistema Cardiovascular/metabolismo , Proteínas RGS/fisiología , Animales , Proteínas de Unión al GTP/fisiología , Predisposición Genética a la Enfermedad , Humanos , Hipertensión/genética , Ratones , Miocitos Cardíacos/fisiología , Proteínas RGS/genética , Transducción de Señal/fisiologíaRESUMEN
Hypertension is a leading risk factor for the development of cardiovascular disease. Data from human and animal studies suggest that RGS2, a potent inhibitor of G(q) signaling, is important for blood pressure regulation. Several RGS2 mutations in the Japanese population have been found to be associated with hypertension. The product of one of these alleles, R44H, is mutated within the amino terminal amphipathic alpha-helix domain, the region responsible for plasma membrane-targeting. The functional consequence of this mutation and its potential link to the development of hypertension, however, are not known. In this study, we showed that R44H was a weaker inhibitor of receptor-mediated G(q) signaling than wild-type RGS2. Confocal microscopy revealed that YFP-tagged R44H bound to the plasma membrane less efficiently than wild-type RGS2. R44 is one of the basic residues positioned to stabilize lipid bilayer interaction of the RGS2 amphipathic helix domain. Tryptophan fluorescence and circular dichroism studies of this domain showed that the R44H mutation prevented proper entrenchment of hydrophobic residues into the lipid bilayer without disrupting helix-forming capacity. Together, these data suggest that decreasing the side-chain length and flexibility at R44 prevented proper lipid bilayer association and function of RGS2. Finally, the R44H protein did not behave as a dominant-negative interfering mutant. Thus, our data are consistent with the notion that a R44H missense mutation in human RGS2 produces a hypomorphic allele that may lead to altered receptor-mediated G(q) inhibition and contribute to the development of hypertension in affected subjects.
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Alelos , Membrana Celular/metabolismo , Subunidades alfa de la Proteína de Unión al GTP Gq-G11/antagonistas & inhibidores , Hipertensión/genética , Proteínas RGS/metabolismo , Secuencia de Aminoácidos , Línea Celular , Genes Dominantes , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Membrana Dobles de Lípidos , Liposomas , Modelos Biológicos , Datos de Secuencia Molecular , Proteínas Mutantes/química , Mutación Missense/genética , Fosfolípidos/metabolismo , Polimorfismo de Nucleótido Simple/genética , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Transporte de Proteínas , Proteínas RGS/química , Proteínas Recombinantes de Fusión/metabolismo , TransfecciónRESUMEN
The regulator of G-protein signaling (RGS2) contains a characteristic RGS domain flanked by short amino and carboxyl terminal sequences. The RGS domain mediates inhibition of Galpha(q) and Galpha(i) signaling, whereas the amino terminal domain (NTD) directs interaction with adenylyl cyclases, G-protein-coupled receptors, and other signaling partners. Here, we identify a set of novel RGS2 protein products that differ with respect to their amino terminal architecture and functional characteristics. An RGS2 expression reporter cassette revealed four distinct open reading frames (ORFs) that can be expressed from the RGS2 NTD. We hypothesized that alternative translation initiation from four AUG codons corresponding to amino acid positions 1, 5, 16, and 33 could produce the observed RGS2 expression profile. Selective disruption of each AUG confirmed that alternate sites of translation initiation accounted for each of the observed products. Proteins derived from ORFs 1 to 4 showed no difference in Galpha(q) inhibitory potential or recruitment from the nucleus in response to Galpha(q) signaling. By contrast, RGS2 products initiating from methionines at positions 16 (ORF3) and 33 (ORF4) were impaired as inhibitors of type V adenylyl cyclase (ACV) compared with full-length RGS2. We predicted that regulation of the RGS2 expression profile would allow cells to adapt to changing signaling conditions. Consistent with this model, activation of Galpha(s)/ACV but not Galpha(q) signaling increased the relative abundance of the full-length RGS2 protein, suggesting that alternative translation initiation of RGS2 is part of a novel negative feedback control pathway for adenylyl cyclase signaling.
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Biosíntesis de Proteínas , Proteínas RGS/genética , Secuencia de Bases , Línea Celular , Cartilla de ADN , HumanosRESUMEN
RGS2 and RGS5 are inhibitors of G-protein signaling belonging to the R4/B subfamily of RGS proteins. We here show that RGS2 is a much more potent attenuator of M1 muscarinic receptor signaling than RGS5. We hypothesize that this difference is mediated by variation in their ability to constitutively associate with the plasma membrane (PM). Compared with full-length RGS2, the RGS-box domains of RGS2 and RGS5 both show reduced PM association and activity. Prenylation of both RGS-box domains increases activity to RGS2 levels, demonstrating that lipid bilayer targeting increases RGS domain function. Amino-terminal domain swaps confirm that key determinants of localization and function are found within this important regulatory domain. An RGS2 amphipathic helix domain mutant deficient for phospholipid binding (L45D) shows reduced PM association and activity despite normal binding to the M1 muscarinic receptor third intracellular loop and activated Galpha(q). Replacement of a unique dileucine motif adjacent to the RGS2 helix with corresponding RGS5 residues disrupts both PM localization and function. These data suggest that RGS2 contains a hydrophobic extension of its helical domain that imparts high efficiency binding to the inner leaflet of the lipid bilayer. In support of this model, disruption of membrane phospholipid composition with N-ethylmaleimide reduces PM association of RGS2, without affecting localization of the M1 receptor or Galpha(q). Together, these data indicate that novel features within the RGS2 amphipathic alpha helix facilitate constitutive PM targeting and more efficient inhibition of M1 muscarinic receptor signaling than RGS5 and other members of the R4/B subfamily.
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Membrana Celular/metabolismo , Proteínas RGS/clasificación , Proteínas RGS/metabolismo , Secuencia de Aminoácidos , Animales , Calcio/metabolismo , Señalización del Calcio , Línea Celular , Secuencia Conservada , Secuencias Hélice-Asa-Hélice , Humanos , Hidrólisis , Interacciones Hidrofóbicas e Hidrofílicas , Leucina/genética , Leucina/metabolismo , Metabolismo de los Lípidos , Datos de Secuencia Molecular , Fosfatidilinositoles/metabolismo , Unión Proteica , Proteínas RGS/genética , Receptor Muscarínico M1/antagonistas & inhibidores , Receptor Muscarínico M1/metabolismo , Alineación de SecuenciaRESUMEN
Homeostatic plasticity is important to stabilize the activity level of neuronal circuits. Molecular mechanisms underlying neuronal homeostatic plasticity in response to activity deprivation are not completely understood. We found that prolonged alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor blockade by 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) resulted in larger, faster miniature excitatory postsynaptic current (mEPSC) events with enhanced frequency in cultured forebrain cortical neurons. Furthermore, GluR1 protein level and CREB-dependent transcription were up-regulated. Blockade of L-type Ca(2+) channels but not kainate receptors produced similar effects to the AMPA receptor blockade. Genetic deletion of AC1 (adenylyl cyclase 1), but not AC8, a key neuronal adenylyl cyclase, significantly reduced inactivity-induced GluR1 changes. Our results indicate the synthesis of homomeric GluR1 AMPA receptors and their possible insertion into synapses due to synaptic inactivity in the cortex. AC1 plays a subtype selective role in this process by coupling signals from L-type Ca(2+) channels to downstream signalling pathways.
Asunto(s)
Adenilil Ciclasas/genética , Adenilil Ciclasas/fisiología , Corteza Cerebral/enzimología , Corteza Cerebral/fisiología , Neuronas/enzimología , Neuronas/fisiología , Sinapsis/enzimología , Sinapsis/fisiología , 6-Ciano 7-nitroquinoxalina 2,3-diona/farmacología , Animales , Western Blotting , Canales de Calcio Tipo L/efectos de los fármacos , Células Cultivadas , Corteza Cerebral/citología , AMP Cíclico/fisiología , Interpretación Estadística de Datos , Electrofisiología , Antagonistas de Aminoácidos Excitadores/farmacología , Genes Reporteros/genética , Luciferasas/genética , Luciferasas/metabolismo , Ratones , Ratones Noqueados , Técnicas de Placa-Clamp , Prosencéfalo/enzimología , Prosencéfalo/fisiología , Receptores AMPA/efectos de los fármacos , Receptores de Ácido Kaínico/efectos de los fármacos , Receptores de N-Metil-D-Aspartato/efectos de los fármacos , Transducción de Señal/fisiologíaRESUMEN
PURPOSE: This study assessed the radiopacity of five luting cements, five dowels, and five core build-up materials using two target distances. MATERIALS AND METHODS: Materials were analyzed using a modified version of ISO protocol 4049. samples 1 mm thick were digitally radiographed alongside a stepwedge of aluminum alloy 1100 using a Trophy RVG-4 CCD sensor and 70 kVp X-ray generator. The gray-scale values of the stepwedge and sample were converted to X-ray absorbencies. The relationship between X-ray absorbance and aluminum thickness was linear for thicknesses less than 10 mm and followed a power-law relationship above 10 mm. These relations were used to convert the absorbencies of the samples into aluminum thicknesses. The radiopacity data was subjected to ANOVA/Student-Newman-Keuls testing. RESULTS: All materials were more radiopaque than equivalent thicknesses of aluminum. Each product category contained a wide range of radiopacities. Syringe-dispensed materials tended to be less radiopaque than materials dispensed by mechanically assisted syringe or mixed by hand (p < 0.01). Target distance did not affect the measured radiopacity so long as the exposure time was suitably adjusted (p= 0.86). CONCLUSIONS: All luting cements and core materials met or exceeded the ISO minimums. The tested metal-reinforced glass ionomer core build-up materials were extremely radiopaque. Some publications suggest that excessively radiopaque core materials can hinder a clinician's ability to spot voids or marginal defects.
Asunto(s)
Cementos Dentales/química , Técnica de Perno Muñón/instrumentación , Radiografía Dental Digital/métodos , Procesamiento de Imagen Asistido por Computador/métodosRESUMEN
OBJECTIVE: The IP3 receptor-1 (IP3R1) mediates Ca2+ signals critical to vascular smooth muscle cell (VSMC) proliferation. The cell cycle-associated transcription factor c-Myb increases Ca2+ at the G1/S transition. Here we show the mechanism through which c-Myb regulates expression of IP3R1. METHODS & RESULTS: Ribonuclease protection confirmed transcriptional start (TS), and qRT-PCR revealed a 6-fold increase in IP3R1 mRNA as immortalized VSMC progress from G0 to G1/S. A c-Myb neutralizing antibody decreased IP3R1 mRNA expression 3-fold, and abolished the 3.4-fold increase in IP3R1 protein observed at G1/S. Primary aortic VSMCs in culture and proliferating carotid VSMCs in vivo showed similar regulation of IP3R1 mRNA and protein. Sequence analysis of a 3.1-Kb mouse IP3R1 promoter revealed 17 putative c-Myb binding sites. Reporter assays demonstrated a 2-fold increase in promoter activity in G1/S- versus G0-synchronized VSMCs, which was abolished by functional c-Myb knockdown or deletion of promoter sequences upstream and downstream of TS. Point mutations in Myb sites-13 or -15 significantly blunted G1/S-specific promoter induction in both immortalized and primary VSMCs. Gel shift and ChIP confirmed binding of c-Myb to sites-13 and -15 in G1/S stage VSMCs. CONCLUSION: c-Myb regulates cell cycle-associated IP3R1 transcription in VSMCs via specific highly conserved Myb-binding sites in the IP3R1 promoter.
Asunto(s)
Canales de Calcio/metabolismo , Ciclo Celular/fisiología , Glicoproteínas de Membrana/metabolismo , Músculo Liso Vascular/metabolismo , Miocitos del Músculo Liso/metabolismo , Regiones Promotoras Genéticas , Proteínas Proto-Oncogénicas c-myb/metabolismo , Receptores Citoplasmáticos y Nucleares/metabolismo , Transcripción Genética , Animales , Calcio/metabolismo , Canales de Calcio/genética , Arterias Carótidas/cirugía , Enfermedades de las Arterias Carótidas/genética , Enfermedades de las Arterias Carótidas/metabolismo , Enfermedades de las Arterias Carótidas/fisiopatología , Línea Celular , Proliferación Celular , Inmunoprecipitación de Cromatina , Secuencia Conservada , ADN/metabolismo , Modelos Animales de Enfermedad , Ensayo de Cambio de Movilidad Electroforética , Genes Reporteros , Receptores de Inositol 1,4,5-Trifosfato , Luciferasas , Glicoproteínas de Membrana/genética , Ratones , Ratones Endogámicos C57BL , Músculo Liso Vascular/citología , Mutación , ARN Mensajero/metabolismo , Receptores Citoplasmáticos y Nucleares/genética , Análisis de Secuencia de ADN , Transfección , Regulación hacia ArribaRESUMEN
PURPOSE: This in vitro study determined the fracture strength of five core materials supported by two different endodontic dowels. Diametral tensile strength and microhardness of the three resin composite core materials used in this study were also tested. MATERIAL AND METHODS: The fracture strength study used one lanthanide-reinforced flowable resin composite (Ti-Core Auto E), one titanium- and lanthanide-reinforced composite (Ti-Core), one lanthanide-reinforced composite (Ti-Core Natural), and two metal-reinforced glass ionomer core materials (Ketac Silver and GC Miracle Mix). Two types of dowels were used: a multitiered, split-shank threaded dowel with a flange (#1 Flexi-Flange) and one without a flange design (#1 Flexi-Post). The specimens were divided into ten groups. Each tooth/dowel and core specimen was placed in a special jig at 45 degrees and subjected to a load by a universal testing machine. The diametral tensile strength and the microhardness of the three resin composite core materials were measured by a universal testing machine and Barcol hardness tester, respectively. All test groups contained ten specimens. RESULTS: The fracture strength value of the resin composite core materials was significantly larger ( p < 0.0001) than those for the metal-reinforced glass-ionomer core materials. Analysis of variance (ANOVA) also showed that the Flexi-Flange dowel interacted with Ti-Core and Ti-Core Auto E to significantly ( p < 0.0013) increase the fracture strength relative to the Flexi-Post. One-way ANOVA revealed that there were no significant differences between them in terms of diametral tensile strength. The Barcol hardness values of the composite core materials were statistically different ( p < 0.0001), with the Ti-Core the highest, followed by Ti-Core Natural, then Ti-Core Auto E. CONCLUSIONS: Resin composite core material performed better than glass ionomer material in this in vitro study. The flowable composite core material performed about the same in terms of fracture strength and diametral tensile strength compared with nonflowable composites. Combined with certain core materials, the flange design increased the fracture strength of the tooth/dowel and core combination.
Asunto(s)
Resinas Compuestas/química , Materiales Dentales/química , Técnica de Perno Muñón/instrumentación , Cementos Cermet/química , Aleaciones Dentales/química , Diseño de Prótesis Dental , Cementos de Ionómero Vítreo/química , Dureza , Humanos , Elementos de la Serie de los Lantanoides/química , Ensayo de Materiales , Plata/química , Estrés Mecánico , Resistencia a la Tracción , Titanio/química , ViscosidadRESUMEN
PURPOSE: This study evaluated the retention of dental post heads (No. 2 Flexi-Post) embedded in 5 core materials (1 automix resin composite, 2 hand-mixed resin composites, and 2 glass ionomers). MATERIALS AND METHODS: Samples were prepared by embedding post heads in 4.5-mm-thick disks of core material. RESULTS: The resin composite materials provided significantly more retention than the glass-ionomer-based materials. The post head retention of the automix resin composite was comparable to that of the hand-mixed resin composites. CONCLUSION: Unlike the resin composite samples, all the glass-ionomer samples fractured during testing. This is an unacceptable condition for a clinically successful restoration.
Asunto(s)
Resinas Compuestas , Retención de Prótesis Dentales , Cementos de Ionómero Vítreo , Técnica de Perno Muñón , Análisis de Varianza , Cementos Cermet , Análisis del Estrés Dental , Ensayo de MaterialesRESUMEN
OBJECTIVES: Radiopacity is a desirable property for most intra-oral materials. There are established ISO and ANSI/ADA protocols for determining radiopacity using film-based radiography. However, these methods are not always followed by researchers. This study aims to adapt those procedures by using digital radiography, a simplified stepwedge, and examine the effects of target distance and exposure time choice. METHODS: One millimetre thick samples of three dental materials were prepared by placing the materials into a 1.00 mm thick washer sandwiched between two glass slides. The samples were digitally radiographed alongside a stepwedge of aluminum alloy 1100 with an X-ray unit at 70 kVp using five different target distance/exposure time combinations. For each combination, the grey scale values of various thicknesses of the stepwedge were converted into absorbencies and plotted against their thickness. These plots were then linearly regressed in order to correlate absorbance with a thickness of aluminum for each target distance/exposure time combination. The absorbencies of each sample were then converted into radiopacities using these correlations. RESULTS: The correlations between the absorbance of the stepwedge and its thickness were highly linear. This linearity allows the correlation to be accurately deduced from fewer data points than required by the ISO and ANSI/ADA protocols. Varying exposure time did not significantly affect the mean radiopacity measured at a target distance of 30 cm. Varying the target distance did not significantly affect the measured radiopacity as long as the samples were properly exposed. SIGNIFICANCE: A simplified, consistent digital method for determining radiopacity is presented.