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OBJECTIVES: To describe the characteristics and factors which may influence the maternal outcomes of maternal cardiac arrest (MCA). DESIGN: Retrospective analysis of cases. SETTING: China. POPULATION OR SAMPLE: A total of 61 MCA patients admitted or transferred to The Third Affiliated Hospital of Guangzhou Medical University from January 2000 to December 2019. METHODS: Clinical data for MCA were analysed retrospectively. The indicators included maternal age; BMI; gestational age; antenatal examination; income; MCA cause and place; cardiopulmonary resuscitation (CPR); mode of delivery; maternal prognosis; and neonatal outcome. MAIN OUTCOME MEASURES: The impact of case characteristics on maternal prognosis of MCA. RESULTS: The hospital received 61 patients with MCA, 36 of whom died (mortality 59.0%, 95% CI 46.3-71.7%). MCA was predominantly caused by treatable complications. Those who died were more likely to have collapsed in the ICU. CONCLUSIONS: Regular antenatal examination and early intervention can reduce the incidence of adverse pregnancy outcomes. The location of MCA occurred may be related to maternal prognosis. The leading causes of MCA were postpartum haemorrhage and amniotic fluid embolism. TWEETABLE ABSTRACT: A retrospective analysis describes the correlation between case characteristics of MCA and maternal outcomes.
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Paro Cardíaco/epidemiología , Complicaciones del Trabajo de Parto/epidemiología , Complicaciones Cardiovasculares del Embarazo/epidemiología , Adulto , Índice de Masa Corporal , Reanimación Cardiopulmonar , Cesárea/estadística & datos numéricos , China/epidemiología , Embolia de Líquido Amniótico/epidemiología , Femenino , Insuficiencia Cardíaca/epidemiología , Humanos , Unidades de Cuidados Intensivos , Admisión del Paciente , Hemorragia Posparto/epidemiología , Embarazo , Pronóstico , Insuficiencia Respiratoria/epidemiología , Estudios Retrospectivos , Choque Séptico/epidemiología , Accidente Cerebrovascular/epidemiologíaRESUMEN
Objective: To compare the mortality, survival rate and the therapeutic efficacy between mitral valve repair and replacement as treatment for severe ischemic mitral regurgitation (IMR), and explore the middle- and long-term outcomes. Methods: Between January 2000 and January 2016, 378 patients with severe IMR underwent coronary artery bypass grafting (CABG) combined with mitral valve repair (n=162) or mitral valve replacement (n=216) in the Department of Cardiovascular Surgery of Nanjing First Hospital. Clinical data, in-hospital morbidity and mortality of patients were retrospectively reviewed. The patients were followed up for the long term survival rate, heart function and re-admission. Results: No statistically significant differences of baseline data and operation details were found between the two groups except for left ventricular end-diastolic diameter[(61.3±10.2)mm in replacement group vs (56.2±9.0)mm in repair group, P<0.001]. Seven patients died during the perioperative period, with a total operation mortality of 1.9%.No significant difference of mortality was found between the two groups (5 cases in the replacement group and 2 cases in the repair group). The early outcome after the surgery showed that the rate of low cardiac output and ventricular arrhythmia of patients were significantly higher in the replacement group compared with the repair group (both P<0.05). The mortality of patients received mitral valve replacement was better than who received mitral valve repair when left ventricular end-diastolic diameter was over 65 mm (5.9% vs 10.0%, P=0.036). No significant differences were observed between the two groups in the middle- and -long term survival rate (87% for replacement group vs 85% for repair group, P=0.568). The follow-up time was 1-85 (52.8±21.5) months and the follow-up rate was 93%. The rate of valve-related complications was significantly higher in the repair group compared with the replacement group (8.82% vs 3.82%, P=0.003). Conclusions: We should choose the surgical methods carefully (replacement or repair) for severe IMR patients according to degree of left ventricular remodeling and pathological changes of mitral valve. Mitral valve replacement with preservation of the subvalvular apparatus is a safe and effective surgical alternative for mitral valve repair, especially for patients with complications or complex reflux.
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Implantación de Prótesis de Válvulas Cardíacas , Insuficiencia de la Válvula Mitral/cirugía , Puente de Arteria Coronaria , Humanos , Válvula Mitral/cirugía , Isquemia Miocárdica , Estudios Retrospectivos , Resultado del TratamientoRESUMEN
OBJECTIVE: Ovarian cancer is a gynecological malignancy with high mortality rates all over the world. Markers for diagnosis, prognosis and therapy are urgently required to improve the mortality rates. As a key proto-oncogene, CCND1 is known to be amplified in many different carcinomas, including breast cancer, esophageal cancer, bladder cancer, endometrial cancer and ovarian cancer, etc. CCND1 plays an important role in cancer development and progression. However, its function and mechanism have not been completely elucidated in ovarian cancer. MATERIALS AND METHODS: In the present study, we use cisplatin in vitro to inhibit the cell proliferation and promote cell apoptosis in epithelial ovarian cancer cell line SKOV-3. CCND1 expression, cell proliferation and cell cycle analysis were carried out by real-time PCR, CCK-8 and flow cytometry respectively. RESULTS: Our results demonstrated that cisplatin could inhibit the expression of CCND1 in human epithelial ovarian cancer cell line, which is related to the decreased cell proliferation and increased cell apoptosis. CONCLUSIONS: This study demonstrated that CCND1 is a potential therapeutic target for epithelial ovarian cancer treatment.
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Apoptosis , Línea Celular Tumoral , Proliferación Celular , Ciclina D1 , Neoplasias Glandulares y Epiteliales , Neoplasias Ováricas , Antineoplásicos/uso terapéutico , Carcinoma Epitelial de Ovario , Cisplatino/uso terapéutico , Femenino , Humanos , Proto-Oncogenes MasRESUMEN
OBJECTIVE: In this study, we sought to investigate the effects of 2-methoxyestradiol (2-ME) on cisplatin-induced apoptosis and growth inhibition in SKOV3 ovarian cancer cells. MATERIALS AND METHODS: Cells were treated with 2-ME, carboplatin, or both, the control group, and cell viability and growth inhibition assays were performed using the MTT method. Apoptosis was detected by flow cytometry analysis. Reverse transcription polymerase chain reaction and western blotting were used to monitor the mRNA and protein expression of the pro-apoptotic genes bax and caspase-3 and the anti-apoptotic gene bcl-2. The phosphorylation of Bcl-2 protein was monitored by western blotting. RESULTS: Cell viability was inhibited by all three treatments in a time-dependent manner. Importantly, the combination treatment resulted in significantly reduced cell growth compared with the other groups. The mRNA and protein expression of Bax and caspase-3 were increased in the combination treatment group, and the expression of Bcl-2 was decreased in the combination treatment group as compared with the other two groups. The ratio of bax to Bcl-2 mRNA in the combination treatment group was higher than that in the carboplatin-treated group. Finally, phosphorylation of Bcl-2 protein was increased stronger in the combination treatment group compared with the carboplatin-treated group. CONCLUSIONS: 2-ME promoted the growth inhibitory and apoptosis-inducing effects of platinum-based agents in SKOV3 ovarian cancer cells. The mechanism mediating this effect may be related to the phosphorylation of Bcl-2 protein, which reduces the formation of dimers and, thereby, increases apoptosis. Moreover, 2-ME promoted the mRNA and protein expression of Bax, thereby, increasing the Bax/Bcl-2 expression ratio and activating the mitochondrial apoptosis pathway.
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Proteínas Reguladoras de la Apoptosis/metabolismo , Apoptosis/efectos de los fármacos , Carboplatino/farmacología , Supervivencia Celular/efectos de los fármacos , Estradiol/análogos & derivados , Neoplasias Ováricas/metabolismo , 2-Metoxiestradiol , Secuencia de Bases , Caspasa 3/metabolismo , Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Cisplatino/farmacología , Estradiol/farmacología , Femenino , Humanos , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , ARN Mensajero/metabolismo , Proteína X Asociada a bcl-2/metabolismoRESUMEN
INTRODUCTION: The presence of subset of cancer stem cells (CSCs) called "Side population" (SP) cells has been identified in several solid tumors, responsible for treatment failure especially chemotherapy, and cancer relapse. The present study was aimed to isolate and characterize cancer stem like cells side population cells from high grade ovarian cancer. METHODS: The collected cancer samples were analyzed for presence of SP cells by FACS using Hoechst 33342 exclusion technique. Further the FACS sorted SP and non-SP cells were subjected to analysis of stem cell surface protein expression by western blot and immunocytochemistry, drug resistance and sphere formation assay. RESULTS: By FACS, we have identified 3.7% of cancer stem cell like side population cells in ovarian cancer whose prevalence was reduced to 0.5% upon treatment with verapamil an inhibitor of ABC transporter. Further, these sorted SP cells showed over expression of ABCG2 (ABC transporter), stem cell proteins such as CD144, CD44, EpCAM and antiapoptotic factor Bcl-2. Also the SP cells showed high resistance to chemotherapy drugs, have high survival rate and they are highly potential to form tumor spheres. CONCLUSION: Our data suggest that ovarian cancer contain small sub-population of side population cells which shares some characteristics of stem cells. The co-expression of ABC transporters and stem cells surface markers in SP cells may associate with resistance to chemotherapeutic agents, apoptosis and also supports a role for these cells in tumor recurrence, metastasis and invasion.
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Resistencia a Antineoplásicos/fisiología , Recurrencia Local de Neoplasia/patología , Neoplasias Ováricas/patología , Células de Población Lateral/patología , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 2 , Transportadoras de Casetes de Unión a ATP/metabolismo , Antígenos CD/metabolismo , Antígenos de Neoplasias/metabolismo , Cadherinas/metabolismo , Moléculas de Adhesión Celular/metabolismo , Línea Celular Tumoral , Molécula de Adhesión Celular Epitelial , Femenino , Humanos , Receptores de Hialuranos/metabolismo , Proteínas de Neoplasias/metabolismo , Recurrencia Local de Neoplasia/metabolismo , Células Madre Neoplásicas/efectos de los fármacos , Células Madre Neoplásicas/metabolismo , Células Madre Neoplásicas/patología , Neoplasias Ováricas/tratamiento farmacológico , Neoplasias Ováricas/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Células de Población Lateral/efectos de los fármacos , Células de Población Lateral/metabolismo , Verapamilo/farmacologíaRESUMEN
As an oncoprotein, Eps8 participates in v-Src-induced cellular transformation. To delineate the underlying mechanism, we conducted a yeast two-hybrid screening and identified IRSp53S, a protein critical in cell mobilization, as one of the Eps8-binding partners from a human brain cDNA library. The association was mediated by the multiple proline-rich regions of Eps8 and the C-terminal SH3-WWB containing domains of IRSp53S. In this study, we observed that Eps8 modulated the expression of IRSp53 in v-Src-transformed cells (IV5), raising the question of whether Eps8/IRSp53 interaction was crucial in carcinogenesis. To address this issue, we generated IV5-expressing irsp53 siRNA cells. Attenuation of IRSp53 reduced cell proliferation of IV5 in culture dish and tumor formation in mice, which could be partly rescued by ectopically expressed human IRSp53S. In addition, IRSp53 knockdown impaired activity of phosphatidylinositol 3-kinase (as reflected by Pi-Ser473 AKT) and Stat3 (as reflected by Pi-Tyr705 Stat3), and reduced cyclin D1 expression that culminated to impede G(1)-phase cell-cycle progression. Ectopically expressed human IRSp53S, but not its Eps8-binding defective mutants (that is, Delta363 and PPPDA), rescued these defects and partly restored cell proliferation. Remarkably, through activation of Src, EGF increased the formation of Eps8/IRSp53 complex and Stat3 activation in HeLa cells. With these results, we show for the first time that IRSp53, through its interaction with Eps8, not only affects cell migration but also dictates cellular growth in cancer cells.
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Transformación Celular Neoplásica , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Proteína Oncogénica pp60(v-src)/metabolismo , Proteínas Adaptadoras Transductoras de Señales , Animales , Ciclo Celular , Proliferación Celular , Ciclina D1/metabolismo , Activación Enzimática/efectos de los fármacos , Factor de Crecimiento Epidérmico/farmacología , Regulación Neoplásica de la Expresión Génica , Células HeLa , Humanos , Péptidos y Proteínas de Señalización Intracelular/química , Péptidos y Proteínas de Señalización Intracelular/deficiencia , Péptidos y Proteínas de Señalización Intracelular/genética , Ratones , Proteínas del Tejido Nervioso/química , Proteínas del Tejido Nervioso/deficiencia , Proteínas del Tejido Nervioso/genética , Unión Proteica/efectos de los fármacos , Estructura Terciaria de Proteína , Proteínas Proto-Oncogénicas c-akt/metabolismo , ARN Interferente Pequeño/genética , Factor de Transcripción STAT3/metabolismo , Eliminación de Secuencia , Técnicas del Sistema de Dos HíbridosRESUMEN
BACKGROUND: Paclitaxel has been reported to be a ligand to Toll like receptor 4 (TLR4). Myeloid differentiation factor 88(MyD88) was described as a myeloid differentiation primary response gene. TLR4 signalling owns two pathways: MyD88-dependent and MyD88-independent pathways. XIAP is a key member of the inhibitor of apoptosis protein family. Akt is a major downstream target of growth factor receptor tyrosine kinases, which negatively regulates apoptotic pathways through phosphorylation (pAkt). The aim of the present study is to investigate the role of TLR4 in paclitaxel resistance of ovarian cancer cells. MATERIALS AND METHODS: We reconstructed the RNA interference expression vector, pGenesil-1-U6 specifically targeting TLR4 mRNA, which was stable transfected into the human ovarian cancer cell line SKOV3 (MyD88-positive expression) and A2780 (MyD88-negative expression). Cell proliferation, cell cycle distribution and cell apoptosis were assessed in the cells transfected with scramble control shRNA (SKOV3/shControl, A2780/shControl) and TLR4 shRNA (SKOV3/shTLR4, A2780/shTLR4) to explore the possible functions of TLR4 in ovarian cancer cells growth. The expression of TLR4, MyD88, XIAP, Akt and pAkt was analysed by Western blot analysis. RESULTS: A knockdown of TLR4 levels down-regulated the expression of XIAP and pAkt. And it restored the inhibitory effect of paclitaxel on cell proliferation and impeding cell cycle progression in SKOV3 cells. CONCLUSIONS: It suggests that TLR4 negatively regulates paclitaxel chemotherapy and MyD88 is an essential downstream factor to TLR4 signalling for this resistance. Knockdown of TLR4 induces paclitaxel chemosensitivity which might depress the Akt pathway. The TLR4-MyD88 signalling represents an important source to promote tumour growth.
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Antineoplásicos Fitogénicos/farmacología , Factor 88 de Diferenciación Mieloide/metabolismo , Neoplasias Ováricas/tratamiento farmacológico , Paclitaxel/farmacología , Receptor Toll-Like 4/metabolismo , Apoptosis/efectos de los fármacos , Ciclo Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Femenino , Humanos , Proteínas Proto-Oncogénicas c-akt/metabolismo , Proteína Inhibidora de la Apoptosis Ligada a X/metabolismoRESUMEN
Some steroids rapidly alter neuronal excitability through interaction with neurotransmitter-gated ion channels in addition to their well-known genomic effects via intracellular steroid receptors. Such effects were found in GABA receptor, nicotinic receptors, yet not investigated in P2X purinoceptors. In this study, the effects of dehydroepiandrosterone sulfate on the P2 purinoceptor was investigated. Results show that dehydroepiandrosterone sulfate acutely inhibits P2X purinoceptor functions in PC12 cells. Dehydroepiandrosterone sulfate suppressed ATP-induced cytosolic free calcium concentration ([Ca(2+)](i)) rise, cytosolic free sodium concentration ([Na(+)](i)) rise, and dopamine secretion in the presence of external calcium, but had no effect on ATP-induced [Ca(2+)](i) rise in the absence of external calcium or on UTP-induced [Ca(2+)](i) rise in the absence or presence of external calcium. Our data show that dehydroepiandrosterone sulfate exerted its effect on P2X, but not on the P2Y purinoceptors found in PC12 cells. Estradiol and estrone have similar effects on P2X purinoceptor, but dehydroepiandrosterone and progesterone do not.
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Sulfato de Deshidroepiandrosterona/farmacología , Antagonistas del Receptor Purinérgico P2 , Adenosina Trifosfato/farmacología , Animales , Calcio/metabolismo , Calcio/farmacología , Citosol/metabolismo , Antagonistas de Dopamina/farmacología , Sinergismo Farmacológico , Concentración Osmolar , Células PC12/efectos de los fármacos , Ratas , Receptores Purinérgicos P2/fisiología , Receptores Purinérgicos P2X , Sodio/antagonistas & inhibidores , Suramina/farmacología , Uridina Trifosfato/farmacologíaRESUMEN
Cretinism is a disease characterized by neurological defects associated with severe iodine deficiency. In a rat model of severe iodine deficiency, we investigated the distribution pattern of trace elements (iodine [I], selenium [Se], and bromine [Br] in brain tissue samples; potassium [K], calcium [Ca], manganese [Mn], iron [Fe], copper [Cu], zinc [Zn], rubidium [Rb], and lead [Pb] in erythrocytes) after supplementing the rats with I and/or Se. Neutron activation analysis, proton induced x-ray emission and x-ray fluorescence were used. The serum levels of total and free thyroxine (T4, FT4), and of total, free, and reverse triiodothyronine (T3, FT3, rT3, respectively) were assessed by radioimmunoassay. The results were statistically evaluated by one-way analysis of variance and bivariate correlation. The study indicated that the levels of T4, FT4, and rT3 increased in the serum of iodine-deficient rats supplemented with I or I + Se. In the same animals, we documented alterations of the content of Br in the brain, and of Zn, Mn, Cu, and Rb in erythrocytes, whereas the brain content of I and Se was unchanged. Thus, I and I + Se supplementation improves thyroid hormone metabolism but affects the content of selected trace elements in erythrocytes and of Br in the brain. The data stimulate further clarification of the role of trace elements in the central nervous system.
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Encéfalo/metabolismo , Eritrocitos/metabolismo , Yodo/deficiencia , Yodo/farmacología , Selenio/farmacología , Hormonas Tiroideas/biosíntesis , Oligoelementos/sangre , Animales , Encéfalo/fisiopatología , Hipotiroidismo Congénito/metabolismo , Hipotiroidismo Congénito/fisiopatología , Modelos Animales de Enfermedad , Ratas , Ratas WistarRESUMEN
rBPI(23), a recombinant N-terminal fragment of human bactericidal/permeability-increasing protein (BPI), kills gram-negative bacteria and binds endotoxin. rBPI(21), a variant, in which cysteine 132 is changed to alanine, retains the activities of rBPI(23). Initial attempts using conventional ion-exchange chromatography to purify rBPI(23) from culture supernatants of transfected CHO-K1 cells resulted in lower than expected yields. Also, ELISA of supernatants from CHO-K1 transfectants expressing rBPI(23) or rBPI(21) yielded variable signals. Results from pulse-chase experiments using [(35)S]methionine had indicated that rBPI(23) could not be detected in the culture medium by 7 h of chase, suggesting that these proteins were degraded and/or bound to cells, media components, or vessel surfaces. To address these issues, we developed a novel process whereby sterile S-Sepharose beads were added directly to the cell culture medium. For attached cells, the beads were added to confluent cultures with serum-free medium for the expression phase, while for suspension-adapted cells, beads were added at the beginning of culture growth. The S-Sepharose was then separated from cells and media and washed, and BPI was eluted with high-salt buffer. This approach yielded up to a 50-fold improvement in recovery of rBPI(23) and rBPI(21) from roller bottles, shake flasks, and 2-liter fermenters. It also resulted in improved detection and quantitation of secreted rBPI(23) and rBPI(21) by ELISA. Results of competition binding studies with iodinated rBPI(21) in conjunction with unlabeled rBPI(21) and rBPI(23) or with heparin demonstrated that these proteins bound specifically and with high affinity to heparan-containing sites on the surface of the CHO-K1 cells. We conclude that the S-Sepharose included in the culture medium captures the BPI protein products as they are secreted and protects them from degradation and/or irreversible binding to cell surfaces. This method has been scaled up to a manufacturing process in large (2750 liter) fermenters for pharmaceutical production.
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Proteínas Sanguíneas/genética , Proteínas Sanguíneas/aislamiento & purificación , Proteínas de la Membrana , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/aislamiento & purificación , Animales , Péptidos Catiónicos Antimicrobianos , Unión Competitiva , Proteínas Sanguíneas/biosíntesis , Células CHO , Cricetinae , Medios de Cultivo , Fermentación , Humanos , Resinas de Intercambio Iónico , Fragmentos de Péptidos/biosíntesis , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Sefarosa , TransfecciónRESUMEN
Bactericidal/permeability-increasing protein (BPI) inhibited growth of cell wall-deficient Acholeplasma laidlawii and L forms of certain strains of Staphylococcus aureus and Streptococcus pyogenes. However, the same strains of S. aureus and S. pyogenes with intact cell walls were not susceptible to the growth-inhibitory effects of BPI.
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Acholeplasma laidlawii/efectos de los fármacos , Antibacterianos/farmacología , Proteínas Sanguíneas/farmacología , Proteínas de la Membrana , Staphylococcus aureus/efectos de los fármacos , Streptococcus pyogenes/efectos de los fármacos , Acholeplasma laidlawii/crecimiento & desarrollo , Péptidos Catiónicos Antimicrobianos , Staphylococcus aureus/clasificación , Staphylococcus aureus/crecimiento & desarrollo , Streptococcus pyogenes/clasificación , Streptococcus pyogenes/crecimiento & desarrolloRESUMEN
CrO3 was found to affect norepinephrine release in a biphasic manner: at concentrations above 100 microM, it inhibited, while at concentrations below 10 microM, it enhanced DMPP- and high K+-induced [3H]norepinephrine (NE) release from bovine adrenal medullary cells. Similar effects were found for K2Cr2O7. CrO3 inhibited the 45Ca2+ uptake induced by DMPP and high K+, suggesting that the voltage-gated Ca2+ channels are possible sites of the inhibitory action of CrO3. CrCl3, possessing a trivalent state in contrast to the hexavalent states of CrO3, K2Cr2O7, inhibited DMPP-induced [3H] release and inhibited, to a lesser extent, high K+-induced [3H]-NE release, suggesting that nicotinic receptors are also possible sites of Cr3+ action. In medullary cells permeabilized with digitonin, both CrO3 and CrCl3 induced [3H]-NE release from cells preloaded with [3H]-NE. In intact cells, CrO3 but not CrCl3 enhanced secretagogue-induced [3H]-NE release and entered into the cells as demonstrated by fluorescence quenching experiments. These results suggest that chromium compounds can induce catecholamine secretion after entering the cytoplasm. The enhancement of norepinephrine release induced by chromium ions appears to be due to interference with the intracellular functions of Ca2+ in the cytoplasm.
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Médula Suprarrenal/efectos de los fármacos , Médula Suprarrenal/metabolismo , Catecolaminas/metabolismo , Compuestos de Cromo/toxicidad , Médula Suprarrenal/citología , Animales , Calcio/metabolismo , Bovinos , Células Cultivadas , Cloruros/toxicidad , Norepinefrina/metabolismo , Dicromato de Potasio/toxicidad , Factores de TiempoRESUMEN
The effects of arginine on calcium mobilization in human SK-N-SH neuroblastoma cells were examined. It was found that arginine potentiated an increase in carbachol-induced Ca2+ from the external Ca2+ influx as opposed to an internal Ca2+ release from intracellular pools. The potentiation effect of arginine on carbachol-induced calcium mobilization was mimicked by either 8-bromo cyclic GMP or sodium nitroprusside. In addition, it was found that arginine induced NO production and an increase in cyclic GMP. Moreover, arginine-induced potentiation, NO production, and cyclic GMP increases were all suppressed after the preincubation of cells with N-methyl-L-arginine or N-nitro-L-arginine, nitric oxide synthase inhibitor. It is suggested that the NO production and subsequent cyclic GMP elevation induced by arginine are responsible for the potentiation of carbachol-induced Ca2+ increase. Our results show the existence of a NO/cyclic GMP pathway and an interconnection of NO and Ca2+ signaling pathways in human SK-N-SH neuroblastoma cells. We also observed that NO, which is produced by endothelial CPAE cells, has a modulating effect on cyclic GMP elevation in human SK-N-SH neuroblastoma cells. The intercellular communication role of NO and its cell-diffusing character may also affect the regulation of nonneuronal cells in their interactions with neuronal cells.
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Arginina/farmacología , Calcio/metabolismo , GMP Cíclico/metabolismo , Neuroblastoma/metabolismo , Óxido Nítrico/metabolismo , Receptores Muscarínicos/fisiología , Carbacol/farmacología , Difusión , Humanos , Membranas Intracelulares/metabolismo , Agonistas Muscarínicos/farmacología , Neuroblastoma/patología , Concentración Osmolar , Células Tumorales CultivadasRESUMEN
rBPI23 is a biologically active, recombinant N-terminal fragment of human bactericidal/permeability-increasing protein (BPI). While rBPI23 is readily purified from culture supernatants of Chinese hamster ovary (CHO)-K1 transfectants, it is heterogeneous, consisting of monomer and disulfide-linked dimer, characteristics due presumably to the presence of three cysteines within the molecule. We have examined the role of these cysteines in rBPI23 expression, function, and dimer formation by mutating their codons to alanine (C132A), serine (C135S), or alanine (C175A) and expressing analogues of N-terminal fragments ("variants") lacking one, two, or all three cysteines in permanently transfected CHO-K1 cells. We also expressed a variant in which serine 18 was changed to cysteine (S18C), as found in both bovine and rabbit BPI. The C132A variant was readily secreted and purified as a homogeneous, stable monomeric protein species. The C135S and S18C variants were produced as mixtures of monomer and dimer; the C135S variant was poorly secreted, difficult to purify, and unstable on storage. In contrast, the C175A variant and those lacking any two or all three cysteines were expressed but not secreted. Purified rBPI23 and the C132A and S18C variants had comparable bactericidal and lipopolysaccharide (LPS) binding activities and were similarly effective at neutralizing LPS-induced tumor necrosis factor synthesis by THP-1 cells; the purified C135S variant lacked all activities. From these studies with CHO-K1 transfectants, we conclude that (i) cysteines 135 and 175 are both necessary for efficient secretion of a biologically active N-terminal BPI fragment, presumably through the formation of a disulfide bond, (ii) cysteine 132 is responsible for dimer formation, and (iii) only the C132A modification yields a stable, biologically active, N-terminal BPI fragment (designated rBPI21) that is free of dimeric species.
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Proteínas Sanguíneas/genética , Cisteína/genética , Proteínas de Choque Térmico , Proteínas de la Membrana , Animales , Antiinfecciosos/farmacología , Péptidos Catiónicos Antimicrobianos , Unión Competitiva , Proteínas Sanguíneas/química , Proteínas Sanguíneas/aislamiento & purificación , Northern Blotting , Western Blotting , Células CHO , Proteínas Portadoras/metabolismo , Cricetinae , Cisteína/metabolismo , Disulfuros/metabolismo , Electroforesis en Gel de Poliacrilamida , Chaperón BiP del Retículo Endoplásmico , Humanos , Lipopolisacáridos/farmacología , Chaperonas Moleculares/metabolismo , Mutagénesis Sitio-Dirigida/genética , Conformación Proteica , Pliegue de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Compuestos de Sulfhidrilo/metabolismo , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Dehydroepiandrosterone sulfate (DHEAS) dose-dependently inhibited [3H]norepinephrine (NE) secretion and the corresponding [Ca2+]i rise induced by the nicotinic receptor agonist 1,1-dimethyl-4-phenylpoperazimium (DMPP) in bovine chromaffin cells. DHEAS at 10 microM, the physiological concentration in human serum, significantly inhibited both the release of [3H]NE and the rise of [Ca2+]i induced by DMPP in chromaffin cells. DHEAS also inhibited the [3H]NE release induced by the Na+ channel activator veratridine. However, DHEAS did not affect either the [3H]NE release, or the corresponding [Ca2+]i rise induced by high K+. Moreover, DHEAS suppressed the [Na+]i rise induced by either DMPP or high K+ as monitored by the fluorescence 340/380 ratio of SBFI loaded chromaffin cells. Our results suggest that the inhibitory effects of DHEAS on secretion mainly occur at nicotinic receptors as well as at the voltage-dependent Na+ channels.
Asunto(s)
Glándulas Suprarrenales/citología , Sistema Cromafín/citología , Sulfato de Deshidroepiandrosterona/farmacología , Norepinefrina/antagonistas & inhibidores , Glándulas Suprarrenales/metabolismo , Animales , Benzofuranos , Calcio/análisis , Catecolaminas/antagonistas & inhibidores , Catecolaminas/metabolismo , Bovinos , Células Cultivadas/efectos de los fármacos , Células Cultivadas/metabolismo , Sistema Cromafín/efectos de los fármacos , Sistema Cromafín/metabolismo , Citosol/química , Yoduro de Dimetilfenilpiperazina/farmacología , Éteres Cíclicos , Colorantes Fluorescentes , Fura-2 , Activación del Canal Iónico/fisiología , Agonistas Nicotínicos/farmacología , Norepinefrina/metabolismo , Sodio/análisis , Tritio/metabolismoRESUMEN
Previous studies have demonstrated that ATP activates calcium-dependent K+ channels thereby leading to plasma membrane hyperpolarization and chloride secretion in MDCK cells. However, the signaling pathway involved in regulating ATP-evoked Ca2+ mobilization in MDCK cells remains unclarified. The present study was performed to elucidate the mechanisms underlying ATP-evoked calcium mobilization and inositol phospholipid turnover in MDCK cells. Our results indicate that ATP exerted its effects by activating the P2u subtype purinoceptor because both ATP and UTP but not adenosine caused Ca2+ mobilization. ATP induced [Ca2+]i increase in a dose-dependent manner with a maximal and half-maximal effect at 100 microM and 5 microM, respectively. In the absence of external Ca2+, both UTP and ATP were capable of mobilizing Ca2+ from internal stores through an IP3-sensitive pathway and the response for the former was larger than the latter. While in the presence of external Ca2+, the magnitude of UTP-induced [Ca2+]i increase was similar to that of ATP. These observations suggest that other mechanisms involved in addition to P2u subtype receptors in regulating ATP-induced Ca2+ response. The Ca2+ transients observed in Ma(2+)-depleted media were greater than those seen in Mg(2+)-contained media. ATP-induced Na+ influx were evidenced by SBFI loaded MDCK cells in Mg(2+)-depleted media. These data support that non-selective ATP4-membrane pores were formed under the application of ATP. The ATP-induced Ca2+ rise was suppressed by either omega-conotoxin or La3+ but was unaffected by the L-type Ca2+ channel blockers, verapamil and nifedipine. The deprivation of external Na+ also enhanced the ATP-induced Ca2+ rise. These data indicate that a P2 receptor-mediated cation channel was also involved under the stimulation of ATP.
Asunto(s)
Adenosina Trifosfato/metabolismo , Calcio/metabolismo , Espacio Extracelular/metabolismo , Riñón/metabolismo , Adenosina Trifosfato/farmacología , Animales , Transporte Biológico/efectos de los fármacos , Bloqueadores de los Canales de Calcio/farmacología , Cationes/metabolismo , Línea Celular , Perros , Fosfatos de Inositol/biosíntesis , Riñón/citología , Deficiencia de Magnesio/metabolismoRESUMEN
The effects of caffeine on Ca2+ fluxes and catecholamine secretion in bovine adrenal chromaffin cells were examined. Caffeine inhibited secretion. 45Ca2+ uptake and cytosolic Ca2+ concentration ([Ca2+]i) rise induced by the nicotinic receptor agonist 1.1-dimethyl- 4-phenylpiperazinium (DMPP) and the Na+ channel activator veratridine. The inhibitory effect of caffeine on high K(+)-induced secretion was smaller than that on DMPP- and veratridine-induced responses. Caffeine only slightly inhibited high K(+)-induced 45Ca2+ uptake and did not affect [Ca2+]i rise. Caffeine also inhibited muscarinic receptor-mediated inositol phosphate generation. Our results suggest that the inhibitory effects of caffeine on bovine chromaffin cells mainly occur at both muscarinic and nicotinic receptors as well as at the voltage-dependent Na+ channels and to a smaller extent at site(s) distal to Ca2+ entry. The effects of caffeine on nicotinic receptors but not on muscarinic receptors can be explained by its ability to raise intracellular cAMP.
Asunto(s)
Médula Suprarrenal/efectos de los fármacos , Cafeína/farmacología , Calcio/metabolismo , Catecolaminas/metabolismo , Inhibidores de Fosfodiesterasa/farmacología , 1-Metil-3-Isobutilxantina/farmacología , Médula Suprarrenal/metabolismo , Animales , Bucladesina/farmacología , ATPasas Transportadoras de Calcio/antagonistas & inhibidores , Carbacol/antagonistas & inhibidores , Bovinos , Colforsina/farmacología , Yoduro de Dimetilfenilpiperazina/farmacología , Fosfatos de Inositol/metabolismo , Agonistas Nicotínicos/farmacología , Terpenos/farmacología , Tapsigargina , Veratridina/farmacologíaRESUMEN
We have used a simple and efficient procedure for the synthesis of N-5-carboxypentyl-1-deoxynojirimycin, an affinity ligand for alpha-glucosidase I (Bernotas, R. C. and Ganem, B., Biochem. J., 270, 539-540, 1990). The affinity gel was used to purify alpha-glucosidase I in one step from crude extract. In subsequent steps, partially purified alpha-glucosidase II was obtained. We have synthesized several analogues of castanospermine and studied their inhibition of alpha-glucosidase I in vitro using purified alpha-glucosidase I and in vivo in cultured B16F10 cells. Although the castanospermine analogues were significantly less active against the purified enzyme (IC50 approximately 1-23 micrograms/ml) as compared to castanospermine (IC50 = 0.02 microgram/ml), several compounds had up to 30-fold higher activity than castanospermine against alpha-glucosidase I in B16F10 cells, based on the accumulation of G3M7-9N2 oligosaccharide-containing glycoproteins. These results suggest that these analogues with lipophilic side chains cross the membrane barrier more efficiently than castanospermine. Once inside the cell, they may be converted to their active metabolite, castanospermine, by cellular esterases to give enzyme inhibition.
Asunto(s)
Glicoproteínas/biosíntesis , Inhibidores de Glicósido Hidrolasas , Indolizinas/farmacología , Riñón/enzimología , Animales , Secuencia de Carbohidratos , Línea Celular , Cromatografía de Afinidad , Glicopéptidos/química , Glicopéptidos/aislamiento & purificación , Glicosilación , Indolizinas/síntesis química , Datos de Secuencia Molecular , Estructura Molecular , Procesamiento Proteico-Postraduccional , Relación Estructura-Actividad , Porcinos , alfa-Glucosidasas/aislamiento & purificaciónRESUMEN
The lethality of organophosphorous compounds has been attributed to their inhibitory effect on acetylcholinesterase (AChE) in the nervous system. However, subacute exposure of humans to organophosphates induces cognitive and emotional defects which might not solely be attributable to AChE inhibition. Therefore we investigated the toxic effects of methyl parathion and malathion on bovine adrenal chromaffin cells. Catecholamine secretion and 45Ca2+ uptake evoked by either a nicotinic agonist (DMPP) or high [K+] were inhibited by methyl parathion and malathion. The [Ca2+]i rise induced by DMPP was inhibited by both compounds. We conclude that in addition to AChE, voltage-gated Ca2+ channels and nicotinic receptors are possible sites of action of organophosphates in mammalian systems.
Asunto(s)
Médula Suprarrenal/efectos de los fármacos , Calcio/metabolismo , Malatión/toxicidad , Metil Paratión/toxicidad , Norepinefrina/metabolismo , Médula Suprarrenal/citología , Médula Suprarrenal/metabolismo , Animales , Canales de Calcio/efectos de los fármacos , Bovinos , Células Cultivadas/efectos de los fármacos , Células Cultivadas/metabolismo , Gránulos Cromafines/efectos de los fármacos , Gránulos Cromafines/metabolismo , Activación del Canal Iónico/efectos de los fármacos , Receptores Nicotínicos/efectos de los fármacos , Receptores Nicotínicos/metabolismoRESUMEN
LPS-binding protein (LBP) mediates the pro-inflammatory effects of bacterial LPS by enhancing LPS-induced cytokine production by monocytic cells. LBP binds specifically to LPS to generate a complex that interacts with the CD14 receptor on the surface of responsive cells. To identify the biologically active regions of the protein responsible for mediating these activities, we cloned and expressed human rLBP (456 amino acids) as well as a truncated form encoding amino acids 1-197 (rLBP25). Both forms of LBP bound to LPS with the same affinity, and similarly inhibited LPS activity in the Limulus amebocyte lysate assay. These results demonstrate that the LPS-binding domain of LBP resides entirely within the N-terminal 197 amino acids of the protein. rLBP and rLBP25 were compared for their ability to mediate CD14-dependent LPS effects on cells. rLBP was effective in mediating uptake of LPS and stimulation of TNF production by human monocytic THP-1 cells, whereas rLBP25 had no significant activity in these assays. Similarly, rLBP was able to mediate LPS-induced TNF production by human PBMC whereas rLBP25 was essentially inactive. These results suggest that the structural features of LBP required for mediating LPS effects via CD14 are probably located in the C-terminal region of the protein. Thus, the LPS-binding activity of LBP can be separated from the CD14-stimulatory activity, suggesting these activities are mediated by structural elements residing in different regions of the protein.