RESUMO
Nicotine, the addictive component of tobacco products, is an agonist at nicotinic acetylcholine receptors (nAChRs) in the brain. The subtypes of nAChR are defined by their α- and ß-subunit composition. The α6ß2ß3 nAChR subtype is expressed in terminals of dopaminergic neurons that project to the nucleus accumbens and striatum and modulate dopamine release in brain regions involved in nicotine addiction. Although subtype-dependent selectivity of nicotine is well documented, subtype-selective profiles of other tobacco product constituents are largely unknown and could be essential for understanding the addiction-related neurological effects of tobacco products. We describe the development and validation of a recombinant cell line expressing human α6/3ß2ß3V273S nAChR for screening and profiling assays in an automated patch clamp platform (IonWorks Barracuda). The cell line was pharmacologically characterized by subtype-selective and nonselective reference agonists, pore blockers, and competitive antagonists. Agonist and antagonist effects detected by the automated patch clamp approach were comparable to those obtained by conventional electrophysiological assays. A pilot screen of a library of Food and Drug Administration-approved drugs identified compounds, previously not known to modulate nAChRs, which selectively inhibited the α6/3ß2ß3V273S subtype. These assays provide new tools for screening and subtype-selective profiling of compounds that act at α6ß2ß3 nicotinic receptors.
Assuntos
Descoberta de Drogas , Ensaios de Triagem em Larga Escala , Técnicas de Patch-Clamp , Receptores Nicotínicos/metabolismo , Linhagem Celular , Clonagem Molecular , Descoberta de Drogas/métodos , Fenômenos Eletrofisiológicos/efeitos dos fármacos , Expressão Gênica , Vetores Genéticos/genética , Humanos , Ativação do Canal Iônico , Subunidades Proteicas/metabolismo , Receptores Nicotínicos/genética , Bibliotecas de Moléculas Pequenas , TransfecçãoRESUMO
The Family Smoking Prevention and Tobacco Control Act of 2009 (Public Law 111-31) gave the US Food and Drug Administration (FDA) the responsibility for regulating tobacco products. Nicotine is the primary addictive component of tobacco and its effects can be modulated by additional ingredients in manufactured products. Nicotine acts by mimicking the neurotransmitter acetylcholine on neuronal nicotinic acetylcholine receptors (nAChRs), which function as ion channels in cholinergic modulation of neurotransmission. Subtypes within the family of neuronal nAChRs are defined by their α- and ß-subunit composition. The subtype-selective profiles of tobacco constituents are largely unknown, but could be essential for understanding the physiological effects of tobacco products. In this report, we report the development and validation of electrophysiology-based high-throughput screens (e-HTS) for human nicotinic subtypes, α3ß4, α3ß4α5, α4ß2, and α7 stably expressed in Chinese Hamster Ovary cells. Assessment of agonist sensitivity and acute desensitization gave results comparable to those obtained by conventional manual patch clamp electrophysiology assays. The potency of reference antagonists for inhibition of the receptor channels and selectivity of positive allosteric modulators also were very similar between e-HTS and conventional manual patch voltage clamp data. Further validation was obtained in pilot screening of a library of FDA-approved drugs that identified α7 subtype-selective positive allosteric modulation by novel compounds. These assays provide new tools for profiling of nicotinic receptor selectivity.
Assuntos
Agonistas Nicotínicos/farmacologia , Antagonistas Nicotínicos/farmacologia , Subunidades Proteicas/fisiologia , Receptores Nicotínicos/fisiologia , Animais , Células CHO , Cricetinae , Cricetulus , Relação Dose-Resposta a Droga , Fenômenos Eletrofisiológicos/efeitos dos fármacos , Fenômenos Eletrofisiológicos/fisiologia , Humanos , Técnicas de Patch-Clamp/métodos , Subunidades Proteicas/agonistas , Subunidades Proteicas/antagonistas & inibidoresRESUMO
BACKGROUND: This study sought to quantify the total patient radiation exposure during fluoro-assisted direct anterior approach (DAA) total hip arthroplasty (THA). We hypothesized that the patient radiation exposure would fall within acceptable published limits for a 1-time patient exposure. METHODS: After institutional review board approval, we performed a retrospective chart review of consecutive unilateral primary DAA THAs at 2 institutions (N = 157) between 2012 and 2014 by a single fellowship-trained arthroplasty surgeon assisted by residents and fellows. Incomplete dose reporting information was the sole exclusion criterion. Patient electronic medical records were queried regarding exposure time (seconds), radiation emittance (mGy), and peak kilovoltage (kVp). Descriptive statistics were calculated. Pearson correlation coefficients were used to determine the correlation between variables. RESULTS: Mean radiation dose for patient exposure measured 2.97 ± 1.63 mGy (range: 0.29-9.83). Positive but weak linear relationship with radiation dose and body mass index (BMI; r = 0.34; P < .0002). Average exposure time per procedure was 23.74 s (range: 11.3-61.7). Average kVp per procedure was 75.38 (range: 65-86). Average BMI was 28.32 (range: 16.6-39.8). There was a significantly strong correlation between kVp and BMI (r = 0.75; P < .0001). CONCLUSIONS: Total patient radiation exposure was nearly identical with previously published values for a screening mammogram (3 mGy) and 4 times less than that of a standard chest computed tomography (13 mGy). Although it is difficult to ascertain the exact patient-absorbed radiation, our data suggest that a 1-time exposure during DAA THA is likely negligible and provides the surgeon with additional data for counseling patients preoperatively.
Assuntos
Artroplastia de Quadril/efeitos adversos , Artroplastia de Quadril/métodos , Quadril/diagnóstico por imagem , Doses de Radiação , Exposição à Radiação/prevenção & controle , Registros Eletrônicos de Saúde , Feminino , Fluoroscopia , Humanos , Consentimento Livre e Esclarecido , Masculino , Cuidados Pré-Operatórios , Radiação Ionizante , Estudos Retrospectivos , Fatores de Tempo , Tomografia Computadorizada por Raios XRESUMO
BACKGROUND: Highly porous metal acetabular components illustrate a decreased rate of aseptic loosening in short-term follow-up compared with previous registry data. This study compared the effect of component surface roughness at the bone-implant interface and the quality of the bone on initial pressfit stability. The null hypothesis is that a standard porous coated acetabular cup would show no difference in initial stability as compared with a highly porous acetabular cup when subjected to a bending moment. Second, would bone mineral density (BMD) be a significant variable under these test conditions. METHODS: In a cadaveric model, acetabular cup micromotion was measured during a 1-time cantilever bending moment applied to 2 generations of pressfit acetabular components. BMD data were also obtained from the femoral necks available for associated specimen. RESULTS: The mean bending moment at 150 µm was not found to be significantly different for Gription (24.6 ± 14.0 N m) cups vs Porocoat (25 ± 10.2 N m; P > .84). The peak bending moment tolerated by Gription cups (33.9 ± 20.3 N m) was not found to be significantly different from Porocoat (33.5 ± 12.2 N m; P > .92). No correlation between BMD and bending moment at 150 µm of displacement could be identified. CONCLUSION: The coefficient of friction provided by highly porous metal acetabular shells used in this study did not provide better resistance to migration under bending load when compared with a standard porous coated component.
Assuntos
Artroplastia de Quadril/instrumentação , Prótese de Quadril , Desenho de Prótese , Acetábulo , Feminino , Fricção , Humanos , Masculino , Metais , Porosidade , Falha de PróteseRESUMO
We conducted a study to evaluate biomechanical performance during destructive testing of several different suture materials in various arthroscopic knot configurations under both in vitro and in situ conditions. Surgeons of different levels of experience tied the knots. Three different arthroscopic knots (static surgeon's, Weston, Tennessee slider) with 3 reverse half-hitches on alternating posts were tested using Fiberwire, ForceFiber, Orthocord, and Ultrabraid suture materials under both in vitro and in situ (blood plasma at 37°C) conditions. Three surgeons of different experience levels tied the knots on a post 30 mm in circumference. A single load-to-failure test was performed. There were no significant in vitro-in situ differences for Ultrabraid in the different knot configurations or with the different experience levels. Surgeon B (intermediate experience) showed no significant differences between test conditions for any knot configuration or suture material. With Tennessee slider knots, surgeon C (least experience) showed significantly lower clinical failure load under both test conditions and had a higher percentage of complete knot slippage. Surgeon B had no knot slippage with use of Fiberwire. Both the aqueous environment and the surgeon's familiarity with certain knots have an effect on knot security.
Assuntos
Teste de Materiais , Técnicas de Sutura/normas , Suturas/normas , Artroscopia , Fenômenos BiomecânicosRESUMO
Metaxin, a mitochondrial outer membrane protein, is critical for TNF-induced cell death in L929 cells. Its deficiency, caused by retroviral insertion-mediated mutagenesis, renders L929 cells resistance to TNF killing. In this study, we further characterized metaxin deficiency-caused TNF resistance in parallel with Bcl-X(L) overexpression-mediated death resistance. We did not find obvious change in mitochondria membrane potential in metaxin-deficient (Met(mut)) and Bcl-X(L)-overexpressing cells, but we did find an increase in the release rate of the mitochondrial membrane potential probe rhodamine 123 (Rh123) that was preloaded into mitochondria. In addition, overexpression of a function-interfering mutant of metaxin (MetaΔTM/C) or Bcl-X(L) in MCF-7.3.28 cells also resulted in an acquired resistance to TNF killing and a faster rate of Rh123 release, indicating a close correlation between TNF resistance and higher rates of the dye release from the mitochondria. The release of Rh123 can be controlled by the mitochondrial membrane permeability transition (PT) pore, as targeting an inner membrane component of the PT pore by cyclosporin A (CsA) inhibited Rh123 release. However, metaxin deficiency and Bcl-X(L) overexpression apparently affect Rh123 release from a site(s) different from that of CsA, as CsA can overcome their effect. Though both metaxin and Bcl-X(L) appear to function on the outer mitochondrial membrane, they do not interact with each other. They may use different mechanisms to increase the permeability of Rh123, since previous studies have suggested that metaxin may influence certain outer membrane porins while Bcl-X(L) may form pores on the outer membrane. The alteration of the mitochondrial outer membrane properties by metaxin deficiency and Bcl-X(L) overexpression, as indicated by a quicker Rh123 release, may be helpful in maintaining mitochondrial integrity.
Assuntos
Apoptose , Permeabilidade da Membrana Celular , Membranas Mitocondriais/metabolismo , Proteínas/metabolismo , Fator de Necrose Tumoral alfa/fisiologia , Proteína bcl-X/metabolismo , Animais , Linhagem Celular Tumoral , Humanos , Potencial da Membrana Mitocondrial/fisiologia , Camundongos , Proteínas de Transporte da Membrana Mitocondrial/fisiologia , Poro de Transição de Permeabilidade Mitocondrial , Mutação , Necrose , Proteínas/genética , Espécies Reativas de Oxigênio/metabolismo , Rodamina 123/metabolismo , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
TSPs 1 and 2 function as endogenous inhibitors of angiogenesis. Although thrombospondins (TSPs) have been shown to induce apoptosis in HMVECs, we reasoned that a homeostatic mechanism would also be needed to inhibit EC growth without causing cell death, e.g., in the maintenance of a normal vascular endothelium. HMVECs, cultured in low serum, responded to VEGF with an increase in [(3)H]thymidine incorporation that was inhibited by TSPs and was accompanied by decreases in the phosphorylation of Akt and MAPK, without an increase in apoptosis. RAP, an inhibitor of the low-density lipoprotein (LDL) family of endocytic receptors, and blocking antibodies to VLDLR were as effective as TSPs in the inhibition of thymidine uptake in response to VEGF, and the effects of these agents were not additive. Supportive evidence for the role of the VLDLR in mediating this inhibition was provided by the demonstration of a high-affinity interaction between TSPs and the VLDLR. We propose that TSP1 and TSP2, together with the VLDLR, initiate a nonapoptotic pathway for maintenance of the normal adult vascular endothelium in a quiescent state, similar to that invoked for the regulation of mitogenesis by PDGF, but involving signaling via the VLDLR rather than LRP1.
Assuntos
Apoptose , Divisão Celular , Células Endoteliais/citologia , Endotélio Vascular/citologia , Receptores de LDL/metabolismo , Trombospondina 1/metabolismo , Trombospondinas/metabolismo , Animais , Apoptose/efeitos dos fármacos , Células CHO , Divisão Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Cricetinae , Cricetulus , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/enzimologia , Endotélio Vascular/enzimologia , Humanos , Imunoprecipitação , Camundongos , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Fosforilação/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/farmacologia , Ressonância de Plasmônio de Superfície , Trombospondinas/química , Trombospondinas/isolamento & purificação , Trombospondinas/farmacologia , Fator A de Crescimento do Endotélio Vascular/farmacologiaRESUMO
The gradual disorganization of collagen fibers in the stromal connective tissue of the uterine cervix is characteristic of progressive cervical softening during pregnancy. A lack of thrombospondin (TSP) 2 has been shown to be associated with altered collagen fibril morphology of connective-tissue-rich organs such as skin and tendon. The goal of this study was to determine the role of TSP2 in cervical softening by studying a TSP2-null mouse line. Creep testing showed that, in the nonpregnant animal and on Day 10 of pregnancy, there was no difference between the cervical extensibility of the wild-type and the TSP2-deficient mice. However, by Day 14 of pregnancy, the TSP2-null mice showed 4.5-fold increase in cervical extensibility, and by Day 18, a 6.1-fold increase, when compared with wild-type mice. A further indicator of compromised cervical integrity was that, on Days 14 and 18 of pregnancy, the cervix of TSP2-null mice broke rapidly under standard loading conditions that did not break the cervix of wild-type mice. Western blotting showed that TSP2 was expressed in the cervix of mice on Days 14 and 18 of pregnancy but not on Day 10 or in the nonpregnant animal. As determined by immunohistochemistry, the amount of matrix metalloproteinase 2 (MMP2) in the cervix of TSP2-null mice increased 11-fold on Day 14 of pregnancy and 19-fold on Day 18. Thus, TSP2-null mice provide an animal model to assist in the understanding of the molecular basis of spontaneous, premature softening of the uterine cervix.
Assuntos
Colo do Útero/metabolismo , Colo do Útero/fisiopatologia , Complicações na Gravidez/metabolismo , Complicações na Gravidez/fisiopatologia , Trombospondinas/genética , Animais , Colágeno/metabolismo , Modelos Animais de Doenças , Feminino , Expressão Gênica , Metaloproteinase 2 da Matriz/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Parto , Gravidez , Trombospondinas/deficiênciaRESUMO
Thrombospondins (TSPs) 1 and 2 are matricellular proteins with the well-characterized ability to inhibit angiogenesis in vivo, and the migration and proliferation of cultured microvascular endothelial cells (ECs). Angiogenesis in developing tumors and in various models of wound healing is diminished or delayed by the presence of TSP1 or 2. Sequences within the type I repeats of TSP1 and 2 have been demonstrated to mediate the anti-migratory effects of TSPs on microvascular EC, although, paradoxically, sequences in the N- and C-terminal domains have pro-angiogenic effects. A scavenger receptor, CD36, recognizes the active sequences in the type I repeats, and is required for the anti-angiogenic effects of TSP1 in the corneal neovascularization assay. However, interactions of TSPs with growth factors, proteases, histidine-rich glycoprotein, and other cell-surface receptors on EC have the potential to modulate CD36-mediated effects. Binding of TSP1 to CD36 has been shown to activate apoptosis by inducing p38 and Jun N-terminal kinase, members of the mitogen-activated protein kinase superfamily, and subsequently the cell-surface expression of FasL. Ligation of Fas by FasL then induces a caspase cascade and apoptotic cell death. However, we have recently shown that inhibition of proliferation of microvascular EC by TSPs can occur in the absence of cell death. This finding raises the possibility that TSPs can activate separate cell death and anti-proliferative pathways.
Assuntos
Inibidores da Angiogênese/metabolismo , Neovascularização Patológica , Trombospondina 1/metabolismo , Trombospondinas/metabolismo , Animais , Apoptose , Endotélio/citologia , Endotélio/metabolismo , Humanos , Trombospondina 1/química , Trombospondinas/químicaRESUMO
The matricellular protein thrombospondin 2 (TSP2) regulates a variety of cell-matrix interactions. A prominent feature of TSP2-null mice is increased microvascular density, particularly in connective tissues synthesized after injury. We investigated the cellular basis for the regulation of angiogenesis by TSP2 in cultures of murine and human fibroblasts and endothelial cells. Fibroblasts isolated from murine and human dermis synthesize TSP2 mRNA and secrete significant amounts of immunoreactive TSP2, whereas endothelial cells from mouse lung and human dermis did not synthesize TSP2 mRNA or protein. Recombinant mouse TSP2 inhibited growth of human microvascular endothelial cells (HMVECs) mediated by basic fibroblast growth factor, insulin-like growth factor-1, epidermal growth factor, and vascular endothelial growth factor (VEGF). HMVECs exposed to TSP2 in the presence of these growth factors had a decreased proportion of cells in S and G2/M phases. HMVECs cultured with a combination of basic fibroblast growth factor, insulin-like growth factor-1, and epidermal growth factor displayed an increased proportion of nonviable cells in the presence of TSP2, but the addition of VEGF blocked this TSP2-mediated impairment of cell viability. TSP2-mediated inhibition of DNA synthesis by HMVECs in the presence of VEGF was not affected by the broad-spectrum caspase inhibitor zVAD-fmk. Similar findings were obtained with TSP1. Taken together, these observations indicate that either TSP2 or TSP1 can inhibit HMVEC proliferation by inhibition of cell cycle progression and induction of cell death, but the mechanisms responsible for TSP2-mediated inhibition of cell cycle progression are independent from those leading to cell death.