RESUMO
BACKGROUND: The aim of this study was to elucidate the morphological charac-teristics of the muscle bundles of the flexor digitorum superficialis (FDS) attached to the intermuscular aponeurosis (IMA) and any related structure that could potentially compress the ulnar nerve. MATERIALS AND METHODS: Fifty embalmed limbs of 34 adult cadavers were studied. RESULTS: The FDS arose as multiple separate bundles from the IMA of the lateral surface of the flexor carpi ulnaris in 76% of specimens. Below their origin, these separate bundles became attached continuously as a single mass to form the muscle belly. There were 1, 2, 3, 4 and 5 arising FDS muscle bundles in 28%, 30%, 4%, 10% and 4% of specimens, respectively. The muscle bundles were attached either only superficially (24% of cases) or across the entire width (20% of cases) of the IMA. In 32% of the specimens, bundles arose from the IMA in a combined fashion, being attached to the IMA superficially, deep and across the entire structure. The muscle bundles that arose from the deep part or entire width of the IMA were in contact with the ulnar nerve in 52% of specimens. In 11 (22%) specimens, the deep borders of the lowest muscle bundles close to the ulnar nerve were composed of tendinous fibres that divided from the IMA of the lateral surface of the flexor carpi ulnaris. The distance from the medial epicondyle to the lowest point of the FDS arising from the IMA was 62.0 ± 19.7 mm. CONCLUSIONS: The thick tendinous deep border of the lowest muscle bundle of the FDS where it attaches to the IMA is a potential cause of ulnar nerve compression.
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OBJECTIVE: Neointimal hyperplasia is a major complication of endovascular stent placement with consequent in-stent restenosis or occlusion. Improvements in the biocompatibility of stent designs could reduce stent-associated thrombosis and in-stent restenosis. We hypothesised that the use of a diamond-like carbon (DLC)-coated nitinol stent or a polyethylene glycol (PEG)-DLC-coated nitinol stent could reduce the formation of neointimal hyperplasia, thereby improving stent patency with improved biocompatibility. METHODS: A total of 24 stents were implanted, under general anaesthesia, into the iliac arteries of six dogs (four stents in each dog) using the carotid artery approach. The experimental study dogs were divided into three groups: the uncoated nitinol stent group (n = 8), the DLC-nitinol stent group (n = 8) and the PEG-DLC-nitinol stent group (n = 8). RESULTS: The mean percentage of neointimal hyperplasia was significantly less in the DLC-nitinol stent group (26.7±7.6%) than in the nitinol stent group (40.0±20.3%) (p = 0.021). However, the mean percentage of neointimal hyperplasia was significantly greater in the PEG-DLC-nitinol stent group (58.7±24.7%) than in the nitinol stent group (40.0±20.3%) (p = 0.01). CONCLUSION: Our findings indicate that DLC-coated nitinol stents might induce less neointimal hyperplasia than conventional nitinol stents following implantation in a canine iliac artery model; however, the DLC-coated nitinol stent surface when reformed with PEG induces more neointimal hyperplasia than either a conventional or DLC-coated nitinol stent.
Assuntos
Materiais Revestidos Biocompatíveis/uso terapêutico , Artéria Ilíaca/patologia , Neointima/prevenção & controle , Stents , Ligas/uso terapêutico , Animais , Implante de Prótese Vascular/instrumentação , Implante de Prótese Vascular/métodos , Cães , Hiperplasia/prevenção & controle , Neointima/patologia , Polietilenoglicóis/uso terapêutico , Stents/efeitos adversosRESUMO
The hemocompatibility of plasma-treated, silicon-incorporated, diamond-like carbon (Si-DLC) films was investigated. Si-DLC films with a Si concentration of 2at.% were prepared on Si (100) or Nitinol substrates using a capacitively coupled radiofrequency plasma-assisted chemical vapor deposition method using a mixed gas of benzene (C(6)H(6)) and diluted silane (SiH(4):H(2)=10:90). The Si-DLC films were then treated with O(2), CF(4) or N(2) glow discharge for surface modification. The plasma treatment revealed an intimate relationship between the polar component of the surface energy and its hemocompatibility. All in vitro characterizations, i.e. protein absorption behavior, activated partial thromboplastin time measurement and platelet adhesion behavior, showed improved hemocompatibility of the N(2-)- or O(2)-plasma-treated surfaces where the polar component of the surface energy was significantly increased. Si-O or Si-N surface bonds played an important role in improving hemocompatibility, as observed in a model experiment. These results support the importance of a negatively charged polar component of the surface in inhibiting fibrinogen adsorption and platelet adhesion.
Assuntos
Carbono/química , Diamante/química , Silício/química , Adsorção , Albuminas/química , Ligas/química , Humanos , Modelos Estatísticos , Nitrogênio/química , Oxigênio/química , Tempo de Tromboplastina Parcial , Adesividade Plaquetária , Propriedades de Superfície , Fatores de Tempo , ÁguaRESUMO
This paper aims to propose an emergency recognition system using multimodal information extracted by an image processing module, a voice processing module, and a gravity sensor processing module. Each processing module detects predefined events such as moving, stopping, fainting, and transfer them to the multimodal integration module. Multimodal integration module recognizes emergency situation by using the transferred events and rechecks it by asking the user some question and recognizing the answer. The experiment was conducted for a faint motion in the living room and bathroom. The results of the experiment show that the proposed system is robust than previous methods and effectively recognizes emergency situations at various situations.
Assuntos
Medicina de Emergência/métodos , Processamento de Sinais Assistido por Computador , Algoritmos , Processamento Eletrônico de Dados , Serviços Médicos de Emergência , Humanos , Processamento de Imagem Assistida por Computador/métodos , Sistemas de Informação , Modelos Neurológicos , Redes Neurais de Computação , Consulta Remota , Som , Análise de Sistemas , Transdutores , Interface Usuário-ComputadorRESUMO
Successful treatment of multiple cancer types requires early detection and identification of reliable biomarkers present in specific cancer tissues. To test the feasibility of identifying proteins from archival cancer tissues, we have developed a methodology, termed direct tissue proteomics (DTP), which can be used to identify proteins directly from formalin-fixed paraffin-embedded prostate cancer tissue samples. Using minute prostate biopsy sections, we demonstrate the identification of 428 prostate-expressed proteins using the shotgun method. Because the DTP method is not quantitative, we employed the absolute quantification method and demonstrate picogram level quantification of prostate-specific antigen. In depth bioinformatics analysis of these expressed proteins affords the categorization of metabolic pathways that may be important for distinct stages of prostate carcinogenesis. Furthermore, we validate Wnt-3 as an upregulated protein in cancerous prostate cells by immunohistochemistry. We propose that this general strategy provides a roadmap for successful identification of critical molecular targets of multiple cancer types.
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Biomarcadores Tumorais/metabolismo , Proteínas de Neoplasias/metabolismo , Proteômica , Sequência de Aminoácidos , Biologia Computacional , Formaldeído , Humanos , Imuno-Histoquímica , Masculino , Espectrometria de Massas , Dados de Sequência Molecular , Proteínas de Neoplasias/química , Inclusão em Parafina , Neoplasias da Próstata/metabolismoRESUMO
An approach to the systematic identification and quantification of the proteins contained in the microsomal fraction of cells is described. It consists of three steps: (1) preparation of microsomal fractions from cells or tissues representing different states; (2) covalent tagging of the proteins with isotope-coded affinity tag (ICAT) reagents followed by proteolysis of the combined labeled protein samples; and (3) isolation, identification, and quantification of the tagged peptides by multidimensional chromatography, automated tandem mass spectrometry, and computational analysis of the obtained data. The method was used to identify and determine the ratios of abundance of each of 491 proteins contained in the microsomal fractions of naïve and in vitro- differentiated human myeloid leukemia (HL-60) cells. The method and the new software tools to support it are well suited to the large-scale, quantitative analysis of membrane proteins and other classes of proteins that have been refractory to standard proteomics technology.
Assuntos
Microssomos/química , Proteínas/análise , Marcadores de Afinidade , Sequência de Aminoácidos , Diferenciação Celular , Células HL-60 , Humanos , Espectrometria de Massas , Dados de Sequência Molecular , Software , Acetato de Tetradecanoilforbol/farmacologiaRESUMO
Although Fas-mediated cell death may play a role in atherogenesis, causal data in support of this hypothesis are lacking. The present study investigated the possibility that endothelial cells are involved in vascular smooth muscle cell (VSMC) apoptosis via the Fas-FasL pathway, and hence in atherogenesis. FACS analysis detected FasL on the surface of human umbilical vein endothelial cells (HUVECs) and immunofluorescence staining of the HUVECs demonstrated high levels of FasL in the intracellular compartment. FasL was down-regulated 4 h after tumor necrosis factor (TNFalpha) treatment, coinciding with maximal surface expression of the adhesion molecules vascular cell adhesion molecule-1 and E-selectin. However, the down-regulation of FasL expression was transient, as surface expression returned within 24 h of TNFalpha treatment. When cocultured with VSMCs, the FasL-expressing EC could kill the VSMCs in a manner that could be blocked by recombinant Fas-Fc, deployed as a soluble receptor for Fas. Moreover, when human coronary arteries were studied with immunohistochemistry using G247-4 monoclonal antibody for the detection of FasL, few FasL positive EC were observed in diffuse intimal thickening. In contrast, endothelium overlying the plaque showed prominent and uniform expression of FasL. These findings suggest that the Fas/FasL pathway can be used by EC to induce VSMC apoptosis in the atherosclerotic lesion.
Assuntos
Apoptose/efeitos dos fármacos , Endotélio Vascular/química , Glicoproteínas de Membrana/farmacologia , Músculo Liso Vascular/efeitos dos fármacos , Adulto , Idoso , Técnicas de Cocultura , Doença da Artéria Coronariana/etiologia , Doença da Artéria Coronariana/patologia , Regulação para Baixo/efeitos dos fármacos , Endotélio Vascular/patologia , Proteína Ligante Fas , Feminino , Humanos , Imuno-Histoquímica , Masculino , Glicoproteínas de Membrana/metabolismo , Glicoproteínas de Membrana/fisiologia , Pessoa de Meia-Idade , Músculo Liso Vascular/citologia , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
A soluble, phosphatidic acid-preferring phospholipase A1, expressed in mature bovine testes but not in newborn calf testes, may contribute to the formation or function of sperm. Here we incubated a recombinant preparation of the phospholipase in vitro with several enzymes including protein kinase CK2 (CK2), extracellular signal-regulated kinase 2 (ERK2), and protein phosphatase 2A (PP2A) to identify effects that might be of regulatory importance in vivo. Major findings were that 1) CK2 phosphorylated the phospholipase on serines 93, 105, and 716; 2) ERK2 phosphorylated the enzyme on serine 730; 3) there was cross-antagonism between the reactions that phosphorylated serines 716 and 730; 4) PP2A selectively hydrolyzed phosphate groups that were esterified to serines 716 and 730; 5) CK2alpha formed a stable, MgATP/MgGTP-dependent complex with the phospholipase by a novel mechanism; and 6) the complex showed reduced phospholipase activity and resembled a complex identified in homogenates of macaque testis. These results provide the first available information about the effects of reactions of phosphorylation and dephosphorylation on the behavior of the phospholipase, shed light on properties of CK2alpha that may be required for the formation of complexes with its substrates, and raise the possibility that a complex containing CK2alpha and the phospholipase may play a special biological role in the testis.
Assuntos
Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Ácidos Fosfatídicos/metabolismo , Fosfolipases A/metabolismo , Fosfoproteínas Fosfatases/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Sequência de Aminoácidos , Animais , Caseína Quinase II , Cromatografia em Gel , Macaca , Masculino , Espectrometria de Massas , Dados de Sequência Molecular , Fosfolipases A/química , Fosfolipases A1 , Fosforilação , Testes de Precipitina , Ligação Proteica , Proteína Fosfatase 2 , Testículo/enzimologiaRESUMO
The isotope-coded affinity tag (ICAT) technology enables the concurrent identification and comparative quantitative analysis of proteins present in biological samples such as cell and tissue extracts and biological fluids by mass spectrometry. The initial implementation of this technology was based on microcapillary chromatography coupled on-line with electrospray ionization tandem mass spectrometry. This implementation lacked the ability to select proteins for identification based on their relative abundance and therefore to focus on differentially expressed proteins. In order to improve the sample throughput of this technology, we have developed a two-step approach that is focused on those proteins for which the abundance changes between samples: First, a new software program for the automated quantification of ICAT reagent labeled peptides analyzed by microcapillary electrospray ionization time-of-flight mass spectrometry determines those peptides that differ in their abundance and second, these peptides are identified by tandem mass spectrometry using an electrospray quadrupole time-of flight mass spectrometer and sequence database searching. Results from the application of this approach to the analysis of differentially expressed proteins secreted from nontumorigenic human prostate epithelial cells and metastatic cancerous human prostate epithelial cells are shown.
Assuntos
Proteínas/química , Proteoma/química , Autoanálise , Linhagem Celular , Células Epiteliais/química , Humanos , Indicadores e Reagentes , Masculino , Espectrometria de Massas , Peptídeos/química , Próstata/química , Próstata/citologia , Próstata/metabolismo , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Biossíntese de ProteínasRESUMO
A novel chemical modification of biological tissues was developed by the direct coupling heparin to bovine pericardium (BP). The heparinization involves pretreatment of BP using GA and followed by grafting heparin to BP by the reaction of residual aldehyde and amine group of heparin. BP was modified by direct coupling of heparin and the effect of heparin coupling on calcification was evaluated in vitro and in vivo. Heparinized BP was characterized by measuring shrinkage temperature, mechanical properties, digestion resistance to collagenase enzyme, in vitro cytotoxicity, and in vivo calcification. Thermal and mechanical properties showed that the durability of heparin-treated tissue increased as compared with fresh tissue and GA-treated tissue. Resistance to collagenase digestion revealed that heparin-treated tissue has greater resistance to enzyme digestion than did fresh tissue and GA-treated tissue. Heparinized tissue had shown to be non-cytotoxic, however, relatively high cytotoxicity was observed in the GA-treated tissues due to the release of GA. In vivo calcification study demonstrated much less calcium deposition on heparin-treated BP than GA-treated one. Obtained results attest to the usefulness of heparinized BP for cardiovascular bioprostheses.
Assuntos
Bioprótese , Prótese Vascular , Próteses Valvulares Cardíacas , Heparina , Pericárdio , Animais , Bovinos , Divisão Celular , Sobrevivência Celular , Células Cultivadas , Colagenases , Reagentes de Ligações Cruzadas , Glutaral , Heparina/farmacologia , Temperatura Alta , Masculino , Ratos , Ratos Sprague-Dawley , Resistência à TraçãoRESUMO
OBJECTIVE: c-FLIP is a natural homologue of caspase 8, and may antagonize activation of death pathways mediated by FADD. c-FLIP is highly expressed in the heart, and a recent report suggests that c-FLIP may protect against certain types of myocyte death. The present study was designed to define the expression patterns of c-FLIP in the heart. METHODS: The expression pattern of c-FLIP in end-stage human hearts, and rat cardiomyocyte grafting models was analyzed by in situ hybridization, immunohistochemistry and TUNEL assay. In addition, to determine whether Fas-dependent pathway is active in cardiomyocytes in vitro, we examined whether activated monocytes can kill neonatal cardiomyocytes in a co-culture system. RESULTS: c-FLIP mRNA and protein were abundantly expressed in normal cardiomyocytes from failing human heart. In animal models, c-FLIP protein was absent in TUNEL-positive grafted cardiomyocytes. Double staining demonstrated that c-FLIP-positive cells rarely had fragmented DNA, while TUNEL-positive cells rarely contained c-FLIP. Finally, activated monocytes induced death of neonatal rat cardiomyocytes via the Fas/FasL system. CONCLUSIONS: Loss of c-FLIP expression correlates with cardiomyocyte cell death. We hypothesize that diminished c-FLIP expression may predispose cardiomyocytes to apoptotic death.
Assuntos
Caspases/metabolismo , Insuficiência Cardíaca/metabolismo , Miocárdio/metabolismo , Adulto , Animais , Animais Recém-Nascidos , Apoptose , Western Blotting , Transplante de Células , Técnicas de Cocultura , Fragmentação do DNA , Cães , Inibidores Enzimáticos/metabolismo , Proteína Ligante Fas , Feminino , Expressão Gênica , Humanos , Imuno-Histoquímica , Hibridização In Situ , Marcação In Situ das Extremidades Cortadas , Leucócitos Mononucleares , Ativação Linfocitária , Masculino , Glicoproteínas de Membrana/metabolismo , Pessoa de Meia-Idade , Modelos Animais , RNA Mensageiro/análise , Ratos , Ratos Endogâmicos F344 , Receptor fas/metabolismoRESUMO
Using a heterologous yeast expression assay, we show that inhibitor of apoptosis proteins (IAPs) suppress caspase-3-mediated cytotoxicity in the order of XIAP>c-IAP2>c-IAP1>survivin. The same ordering of IAP activities was demonstrated in mammalian cells expressing an auto-activating caspase-3. The relative anti-apoptotic activities of each IAP depended on the particular death stimulus. For IAP-expressing cells treated with camptothecin, survival correlated with their intrinsic anti-caspase-3 activity. However, c-IAP1-transfected cells were disproportionately resistant to tumor necrosis factor-alpha, suggesting that its anti-apoptotic activities extend beyond caspase-3 or -7 inhibition. Yeast-based caspase assays provide rapid, reliable information on specificity and activity of the IAPs and aid in identifying critical targets in mammalian apoptotic pathways.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Caspases/metabolismo , Proteínas Associadas aos Microtúbulos , Proteínas/metabolismo , Saccharomyces cerevisiae/genética , Apoptose/efeitos dos fármacos , Western Blotting , Camptotecina/farmacologia , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Caspase 3 , Caspase 7 , Inibidores de Caspase , Caspases/genética , Linhagem Celular , Proteína de Domínio de Morte Associada a Fas , Humanos , Proteínas Inibidoras de Apoptose , Proteínas de Neoplasias , Fenótipo , Ligação Proteica , Proteínas/genética , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/metabolismo , Transdução de Sinais/efeitos dos fármacos , Survivina , Transfecção , Fator de Necrose Tumoral alfa/farmacologia , Ubiquitina-Proteína Ligases , Proteínas Virais/genética , Proteínas Virais/metabolismo , Proteína bcl-XRESUMO
Apoptosis of smooth muscle cells is a common feature of vascular lesions but its pathophysiological significance is not known. We demonstrate that signals initiated by regulated Fas-associated death domain protein overexpression in rat vascular smooth muscle cells in the carotid artery induce expression of monocyte-chemoattractant protein-1 and interleukin-8, and cause massive immigration of macrophages in vivo. These chemokines, and a specific set of other pro-inflammatory genes, are also upregulated in human vascular smooth muscle cells during Fas-induced apoptosis, in part through a process that requires interleukin-1alpha activation. Induction of a pro-inflammatory program by apoptotic vascular smooth muscle cells may thus contribute to the pathogenesis of vascular disease.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Apoptose , Proteínas de Transporte/metabolismo , Inflamação/genética , Músculo Liso Vascular/imunologia , Receptor fas/metabolismo , Animais , Artérias Carótidas/imunologia , Artérias Carótidas/patologia , Caspases/metabolismo , Quimiocina CCL2/biossíntese , Proteína de Domínio de Morte Associada a Fas , Regulação da Expressão Gênica , Humanos , Interleucina-1/metabolismo , Interleucina-8/biossíntese , Ratos , Ratos Endogâmicos F344 , Regulação para CimaRESUMO
We previously isolated MACH-related inducer of toxicity (MRIT), a homolog of caspase 8. MRIT, also known as c-FLICE-inhibitory protein (c-FLIP), is an enzymatically inactive homolog of caspase 8 with homology to viral FLIP (v-FLIP). Because of this homology and resemblance to dominant negative proteins, c-FLIP is widely believed to be an antagonist to the death receptor-initiated apoptotic pathways that use caspase 8. We generated a polyclonal antibody, MAG1, and show that this antibody specifically recognizes two splice forms, long form (c-FLIPL) and short form (c-FLIPS). By in situ hybridization and immunohistochemistry, we demonstrate that c-FLIP is expressed in endothelial cells, macrophages, and smooth muscle cells (SMCs) both in human coronary arteries and in cultured cells. In an uninjured rat carotid arteries, c-FLIP protein is abundant in the vascular media. After balloon angioplasty, c-FLIP protein is rapidly down-regulated in medial SMCs for 2 weeks and regains expression by 4 weeks. In contrast, the neointima is strongly immunoreactive to c-FLIP from day 7 after the initial injury and remains strongly immunoreactive until 4 to 6 weeks. Similarly there is strong c-FLIP immunoreactivity in SMCs from nonatherosclerotic diffuse intimal thickening and in the overlying endothelial cells. In contrast, c-FLIP immunoreactivity is uneven and often absent in SMCs within the atherosclerotic plaque. Double labeling with c-FLIP antibody and terminal deoxynucleotidyltransferase-mediated UDP end labeling (TUNEL) in the injured rat common carotid artery show that TUNEL-positive cells in the first 2 days after injury lack detectable c-FLIP, suggested a role for caspase 8 in this form of death. In contrast, there is no correlation of c-FLIP with the spontaneous elevation in death of intima seen at 7 days after injury. For human atherosclerotic plaques, the majority of TUNEL-positive cells lack detectable c-FLIP. The expression pattern of c-FLIP and the relation between c-FLIP and TUNEL suggest a role for c-FLIP- and caspase 8-driven death in control of viability of the cells of the atherosclerotic intima.
Assuntos
Lesões das Artérias Carótidas/metabolismo , Proteínas de Transporte/metabolismo , Vasos Coronários/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular , Adulto , Animais , Apoptose/fisiologia , Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD , Lesões das Artérias Carótidas/patologia , Células Cultivadas , Doença da Artéria Coronariana/metabolismo , Doença da Artéria Coronariana/patologia , Vasos Coronários/patologia , Vasos Coronários/fisiopatologia , Endotélio Vascular/metabolismo , Endotélio Vascular/patologia , Feminino , Humanos , Macrófagos/metabolismo , Masculino , Pessoa de Meia-Idade , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/patologia , Ratos , Ratos Sprague-Dawley , Valores de ReferênciaRESUMO
Antigen presenting cells (APCs) can take up exogenous antigenic peptides chaperoned by heat shock protein gp96 and re-present them through the endogenous pathway on their major histocompatibility class I molecules. The high efficiency of this process has been attributed previously to a receptor for gp96 on APCs. The CD91 molecule (also called alpha 2-macroglobulin receptor or the low density lipoprotein-related protein) is shown here to be a cell surface receptor for the heat shock protein gp96. CD91 binds gp96 directly, rather than through another ligand for CD91. The previously known CD91 ligand, alpha 2-macroglobulin, inhibits re-presentation of gp96-chaperoned antigenic peptides by macrophages, as do antibodies to CD91. As gp96 is exclusively intracellular and is released as a result of necrotic but not apoptotic cell death, we propose that CD91 acts as a sensor for necrotic cell death.
Assuntos
Antígenos de Neoplasias/metabolismo , Proteínas de Choque Térmico/metabolismo , Glicoproteínas de Membrana/metabolismo , Receptores Imunológicos/metabolismo , alfa-Macroglobulinas/metabolismo , Sequência de Aminoácidos , Animais , Apresentação de Antígeno/imunologia , Proteína-1 Relacionada a Receptor de Lipoproteína de Baixa Densidade , Dados de Sequência Molecular , Peptídeos/imunologia , Coelhos , Receptores Imunológicos/imunologiaRESUMO
Fas and its ligand, FasL, are a receptor-ligand pair identified as promoting cell death in several tissues. Vascular smooth muscle cells (VSMCs) are resistant to FasL or anti-Fas antibody (Ab) signal, and a number of in vitro studies show that VSMC death can only be induced by anti-Fas Ab or FasL in the presence of protein inhibitor or additional inflammatory mediators. It remains to be clarified whether known, constitutively expressed cytoprotective molecules are reduced by protein inhibitor, thereby accounting for sensitization to cell death by Fas/FasL signaling. We found that Fas mRNA and protein exist in several primary VSMCs, as previously reported. We also demonstrated (1) that critical death-signaling molecules, such as FADD, caspase-1/ICE, and caspase-3/YAMA, are present in these VSMCs, (2) that human VSMCs contain high concentrations of c-FLIP (3) and that following treatment with the protein inhibitor, CHX, cell extracts showed a decrease in c-FLIP protein that was dose- and time-dependent on the degree of apoptosis and inversely correlated with both caspase-8 and -3 activity. In contrast, there was neither a change nor an even modest upregulation of Bcl-2 family, even after 12 h of treatment with CHX. Taken together, these results may provide a novel insight into atherogenesis and suggest that c-FLIP may contribute to an apoptosis-resistant state of VSMC, and that a downregulation of c-FLIP may render VSMCs susceptible to apoptosis.
Assuntos
Apoptose , Proteínas de Arabidopsis , Proteínas de Transporte/fisiologia , Peptídeos e Proteínas de Sinalização Intracelular , Músculo Liso Vascular/efeitos dos fármacos , Receptor fas/fisiologia , Apoptose/efeitos dos fármacos , Arteriosclerose/fisiopatologia , Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD , Proteínas de Transporte/biossíntese , Proteínas de Transporte/genética , Caspase 1/biossíntese , Caspase 1/genética , Caspase 3 , Caspase 8 , Caspase 9 , Caspases/biossíntese , Caspases/genética , Células Cultivadas , Cicloeximida/farmacologia , Indução Enzimática , Ensaio de Imunoadsorção Enzimática , Proteína Ligante Fas , Ácidos Graxos Dessaturases/biossíntese , Ácidos Graxos Dessaturases/genética , Regulação da Expressão Gênica , Genes bcl-2 , Humanos , Marcação In Situ das Extremidades Cortadas , Glicoproteínas de Membrana/fisiologia , Músculo Liso Vascular/citologia , Inibidores da Síntese de Proteínas/farmacologia , Proteínas Proto-Oncogênicas c-bcl-2/biossíntese , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Recombinantes de Fusão/farmacologia , Transdução de Sinais , Proteína bcl-X , Receptor fas/biossíntese , Receptor fas/genéticaRESUMO
The post-genomic era is characterized by the deposition of sequence information for entire genomes in databases. Currently, besides the protein sequences for known human proteins, there are partial sequences from thousands more human proteins for which no biological function has been assigned. A powerful new tool for the unambiguous identification and characterization of gel-separated proteins is accomplished by the combination of mass spectrometry and sequence database searching. This combination provides the cancer biologist with the ability to (i) identify the potential protein:protein associations and (ii) fully characterize function-critical post-translational modifications, both directly from silver-stained polyacrylamide gels. In this report we describe the application of tandem mass spectrometry and database searching to two problems which are prototypical for cancer research and indeed for biomedical research in general. The first is the identification of gel-separated, low abundance proteins based on amino acid sequence composition following coimmunoprecipitation with the human apoptosis inhibitor protein BclX(L). The second is the determination of the precise sites of phosphorylation of the human regulatory protein 4E-BP1, which controls mRNA translation.
Assuntos
Bases de Dados Factuais , Espectrometria de Massas/métodos , Proteínas de Neoplasias/análise , Sequência de Aminoácidos , Sítios de Ligação , Linhagem Celular , Humanos , Dados de Sequência Molecular , Neoplasias , Fosfoproteínas , Fosforilação , Testes de Precipitina , PesquisaRESUMO
Caspase-3, a member of the caspase family of cell death proteases, cleaves cytoplasmic and nuclear substrates and promotes apoptotic cell death in mammalian cells. Although yeast homologs of apoptotic genes have not been identified, some components of apoptotic pathways retain function in yeast. Here we show that the expression of caspase-3 delays cell growth in Saccharomyces cerevesiae without causing cell death. Mutation of the caspase-3 QACRG active site abolished effects on yeast growth. Co-expression of caspase inhibitors alleviated growth inhibition in yeast as did the tripeptide caspase inhibitor ZVAD-fmk. These results suggest that substrates for caspase-3 are present in S. cerevesiae and may participate in the normal cell growth and division processes.
Assuntos
Apoptose/genética , Caspases/genética , Regulação Enzimológica da Expressão Gênica , Proteínas Proto-Oncogênicas c-bcl-2 , Saccharomyces cerevisiae/genética , Caspase 3 , Regulação Fúngica da Expressão Gênica , Proteínas Proto-Oncogênicas/genética , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/crescimento & desenvolvimento , Proteína X Associada a bcl-2RESUMO
To investigate the effect of the hydrophilic and hydrophobic microdomain structure on blood compatibility, a series of interpenetrating polymer networks (IPNs) composed of hydrophilic polyurethane (PU) and hydrophobic polystyrene (PS) was prepared. One series was prepared with varying cross-link densities of each network, the other with varying hydrophilicity of the PU component. All PU/PS IPNs exhibited microphase-separated structures that had dispersed PS domains in the continuous PU matrix. The domain size decreased with decreasing the hydrophilicity of the PU component and increasing the cross-link density of each network. As the cross-link density and hydrophobicity of the PU component was increased, an inward shift of Tgs was observed, which was due to the decrease in phase separation between the hydrophobic PS component and hydrophilic PU component. In the in vitro platelet adhesion test, as the microdomain size of PU/PS IPN surface decreased, the number of adhered platelets on the PU/PS IPN surface was reduced and deformation of the adhered platelets decreased. It could be concluded that blood compatibility of PU/PS IPN was mainly affected by the degree of mixing between PU and PS component, which was reflected by the domain size of PS rich phase.
Assuntos
Reagentes de Ligações Cruzadas/química , Adesividade Plaquetária/efeitos dos fármacos , Polímeros/síntese química , Poliestirenos/química , Poliuretanos/química , Temperatura Alta , Humanos , Microscopia Eletrônica de Varredura , Polímeros/farmacologia , Propriedades de SuperfícieRESUMO
Novel lactide-based poly(ethylene glycol) (PEG) polymer networks (GL9-PEGs) were prepared by UV copolymerization of a glycerol-lactide triacrylate (GL9-Ac) with PEG monoacrylate (PEG-Ac) to use as scaffolds in tissue engineering, and the surface properties and biocompatibility of these networks were investigated as a function of PEG molecular weight and content. Analysis by ATR-FTIR and ESCA revealed that PEG was incorporated well within the GL9-PEG polymer networks and was enriched at the surfaces. From the results of SEM, AFM, and contact angle analyses, GL9-PEG networks showed relatively rough and irregular surfaces compared to GL9 network, but the mobile PEG chains coupled at their termini were readily exposed toward the aqueous environment when contacting water such that the surfaces became smoother and more hydrophilic. This reorientation and increase in hydrophilicity were more extensive with increasing PEG molecular weight and content. As compared to GL9 network lacking PEG, protein adsorption as well as platelet and S. epidermidis adhesion to GL9-PEG networks were significantly reduced as the molecular weight and content of PEG was increased, indicating that GL9-PEG networks are more biocompatible than the GL9 network due to PEG's passivity. Based on the physical and biological characterization reported, the GL9-PEG materials would appear to be interesting candidates as matrices for tissue engineering.