RESUMO
In recent years, serum levels of the prohormone procalcitonin have been investigated in a number of studies in relation to postmortem sepsis diagnostics, as macroscopic and histomorphological findings are, as a rule, nonspecific. However, due to advanced haemolysis, it is often not possible to determine serum procalcitonin (PCT) levels in cases of sepsis-related death. Moreover, the impact of postmortem interval on PCT levels is largely unclarified. In view of this, the present pilot study investigated PCT levels in the serum, aqueous humour, and cerebrospinal fluid in a study population of 25 persons who died of sepsis and a control population of 25 deaths unrelated to sepsis. Using the Mann-Whitney U test, statistically significant differences in PCT levels were determined for all the analysed samples from the study and control populations. Logistic regression analysis was used to calculate cut-off values for sepsis diagnosis for all the sample types. Furthermore, the serum elimination rates published by Tsokos et al. (Int J Legal Med 114:237-243, 2001) were used to calculate the PCT levels at the time of death for the cases with a known postmortem interval. The results of our study demonstrate that, taking account of the postmortem elimination process, it is possible to infer the value at the time of death from the procalcitonin levels measured in all three sample types and to interpret this with the aid of a defined cut-off value. The findings need to be verified based on a larger study population.
Assuntos
Humor Aquoso/química , Calcitonina/análise , Precursores de Proteínas/análise , Sepse/diagnóstico , Idoso , Biomarcadores/análise , Peptídeo Relacionado com Gene de Calcitonina , Estudos de Casos e Controles , Feminino , Humanos , Modelos Logísticos , Masculino , Pessoa de Meia-Idade , Projetos Piloto , Mudanças Depois da Morte , Estudos Prospectivos , Sensibilidade e EspecificidadeRESUMO
The negative correlation between coronary heart disease and plasma levels of HDL has been attributed to the ability of HDL to take up cellular cholesterol. The HDL3-induced removal of cellular cholesterol was reported to be impaired in fibroblasts from patients with familial HDL deficiency (Tangier disease, TD). In addition, we have recently shown that HDL3 stimulates the hydrolysis of phosphatidylcholine (PC) in cholesterol-loaded fibroblasts. To investigate whether this cell signaling pathway is involved in cholesterol efflux mechanisms, we compared the HDL3-induced PC hydrolysis in normal fibroblasts and in fibroblasts from a TD kindred, in whom the HDL3- and apolipoprotein A-I (apo A-I)-induced mobilization of cellular cholesterol was found to be reduced by 50%. The HDL3-induced formation of phosphatidic acid (PA) via PC-specific phospholipase D (PC-PLD) was markedly reduced by 60-80% in these cells, whereas the formation of diacylglycerol (DG) via PC-specific phospholipase C (PC-PLC) was two- to threefold enhanced. Defective regulation of PC-PLC and PC-PLD was similarly observed in response to apo A-I and endothelin, but not in response to the receptor-independent stimulation of PC hydrolysis by PMA. A Tangier-like PA and DG formation pattern could be induced in normal cells after preincubation with pertussis toxin, suggesting the involvement of a G-protein. The impaired mobilization of radiolabeled cellular cholesterol in TD cells could completely be overcome by increasing the PA levels in the presence of the PA phosphohydrolase inhibitor propranolol. Conversely, the inhibition of PA formation in the presence of 0.3% butanol as well as the inhibition of DG formation in the presence of the PC-PLC inhibitor D 609 reduced the mobilization of cellular cholesterol both in normal and in TD cells. Our data indicate that the coordinate formation of PA and DG via PC-PLD and PC-PLC is essential for efficient cholesterol efflux. The molecular defect in this TD kindred appears to affect an upstream effector of protein kinase C responsible for the G-protein-dependent regulation of PC-specific phospholipases.
Assuntos
Fosfatidilcolinas/metabolismo , Fosfolipase D/genética , Fosfolipase D/fisiologia , Transdução de Sinais/genética , Transdução de Sinais/fisiologia , Doença de Tangier/genética , Doença de Tangier/metabolismo , Fosfolipases Tipo C/genética , Fosfolipases Tipo C/fisiologia , Apolipoproteína A-I/metabolismo , Apolipoproteína A-I/farmacologia , Hidrocarbonetos Aromáticos com Pontes/farmacologia , Butanóis/farmacologia , Células Cultivadas , Diglicerídeos/metabolismo , Endotelinas/farmacologia , Feminino , Fibroblastos/metabolismo , Proteínas de Ligação ao GTP/fisiologia , Humanos , Lipoproteínas HDL/análise , Lipoproteínas HDL/metabolismo , Masculino , Pessoa de Meia-Idade , Norbornanos , Linhagem , Ácidos Fosfatídicos/metabolismo , Inibidores de Fosfodiesterase/farmacologia , Fosfolipase D/metabolismo , Propranolol/farmacologia , Tiocarbamatos , Tionas/farmacologia , Fosfolipases Tipo C/metabolismoRESUMO
We isolated and identified nucleoside(5') oligophospho-(5') nucleosides containing adenosine and guanosine (ApnG; n = 3-6) as well as diguanosine polyphosphates (GpnG; n = 3-6) in human platelets. For identification, UV spectrometry, matrix-assisted laser desorption/ionization, postsource decay matrix-assisted laser desorption/ionization mass spectrometry, and enzymatic cleavage experiments were used. The adenosine(5') oligophospho-(5') guanosines act as vasoconstrictors and growth factors. The diguanosine polyphosphates are potent modulators of growth in vascular smooth muscle cells, but do not affect vascular tone.
Assuntos
Adenosina , Plaquetas/metabolismo , Fosfatos de Dinucleosídeos/metabolismo , Guanosina , Vasoconstritores/metabolismo , Fosfatos de Dinucleosídeos/isolamento & purificação , Espaço Extracelular/metabolismo , Humanos , Rim/metabolismo , Músculo Liso Vascular/enzimologia , Nucleotidases/metabolismo , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Vasoconstritores/isolamento & purificaçãoRESUMO
BACKGROUND: Previous studies demonstrated inactivation of vitamin B12 by nitrous oxide (N(2)O). The intraoperative exposure to N(2)O was shown to induce megaloblastic anaemia and myelopathy in subjects with subclinical vitamin B12 deficiency. In contrast, no data concerning the influence of occupational exposure to N(2)O on vitamin B12 metabolic status are available to date. In the present study, the vitamin B12 status in operating theatre personnel was assessed in relation to the extent of exposure. METHODS: Ninety-five operating theatre nurses with the history of exposure to N(2)O and 90 unexposed counterparts were examined. Vitamin B12 and folic acid were measured by immunoassay. Total homocysteine (tHcy), an indicator of impaired vitamin B12 metabolism, was determined by high performance liquid chromatography. N(2)O concentration was monitored by adsorption gas chromatography and mass spectrometry. RESULTS: No significant differences were found between both groups with respect to haematological parameters and folic acid. However, subjects exposed to N(2)O presented with lower vitamin B12 [372.8 (12.1) vs 436.8 (13.2) pmol litre(-1), P<0.001] and higher tHcy [11.2 (0.5) vs 8.9 (0.5) micromol litre(-1), P=0.006]. The changes in vitamin B12 status were aggravated in subjects exposed to N(2)O in concentrations substantially exceeding occupational exposure limit (180 mg m(-3)) [vitamin B12: 341.9 (17.7) vs 436.8 (13.2) pmol litre(-1), P=0.006; tHcy: 12.9 (0.7) vs 8.9 (0.5) micromol litre(-1), P=0.047]. CONCLUSIONS: Exposure to N(2)O in healthcare workers is associated with alterations of vitamin B12 metabolic status, the extent of which depends on the level of exposure.
Assuntos
Anestésicos Inalatórios/farmacologia , Óxido Nitroso/farmacologia , Exposição Ocupacional/análise , Salas Cirúrgicas , Vitamina B 12/sangue , Adulto , Anestésicos Inalatórios/análise , Coleta de Amostras Sanguíneas/métodos , Relação Dose-Resposta a Droga , Monitoramento Ambiental/métodos , Feminino , Ácido Fólico/sangue , Homocisteína/sangue , Humanos , Pessoa de Meia-Idade , Óxido Nitroso/análise , Enfermagem de Centro Cirúrgico , Ventilação/métodosRESUMO
Survival of human vascular endothelial cells depends on their ability to activate the transcription factor nuclear factor-kappaB (NF-kappaB), a regulator of antiapoptotic genes, such as the X chromosome-linked inhibitor of apoptosis protein (xIAP). In the present study, we demonstrated expression of xIAP in the endothelial lining of normal human arteries and veins and elevated levels in highly malignant human endothelial tumors. Using retroviral infection of human endothelial cells, we identified two novel survival mechanisms mediated by xIAP in endothelial cells. First, xIAP can activate the transcription factor NF-kappaB, a known survival factor for human endothelial cells. This positive feedback loop induced by xIAP is mediated via phosphorylation and sustained degradation of inhibitor (I) kappaBalpha. Second, xIAP can inhibit cell proliferation via downregulation of cyclins A and D1 and induction of the cyclin-dependent kinase inhibitors p21(Cip1/Waf1) and p27(Kip1). Cleavage of xIAP by caspases during endothelial cell apoptosis disables both of these biological functions of xIAP. Thus, caspase-mediated cleavage of xIAP interrupts a positive regulatory cytoprotective loop between NF-kappaB and xIAP and increases the vulnerability of the cell to apoptosis by releasing it from an xIAP-mediated quiescent state.
Assuntos
Ciclo Celular/fisiologia , NF-kappa B/metabolismo , Apoptose , Caspases/metabolismo , Divisão Celular , Sobrevivência Celular , Células Cultivadas , Endotélio Vascular/citologia , Endotélio Vascular/metabolismo , Regulação da Expressão Gênica , Humanos , Mutagênese Sítio-Dirigida , Mutação , Proteínas/genética , Proteínas/metabolismo , Especificidade por Substrato , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo XRESUMO
The effects of diadenosine pentaphosphate (AP5A), and diadenosine hexaphosphate (AP6A) on the cytosolic-free Ca2+ concentration ([Ca2+]i) were evaluated in cultured human fibroblast cells (HF cells) using the fluorescent dye technique. AP5A, and AP6A concentration-dependently increased [Ca2+]i in HF cells. The addition of 10 mumol/1 AP5A and AP6A significantly increased [Ca2+]i in HF cells from 71 +/- 3 nmol/1 (n = 184) to 241 +/- 39 nmol/1 (n = 11; P < 0.001 compared to resting value) and to 227 +/- 26 nmol/1 (n = 23; P < 0.001), respectively. The purinoceptor P2 blockers, suramin and pyridoxal-phosphate-6-azophenyl-2',4'-disulphonic acid (PPADS), inhibited the diadenosine polyphophate-induced [Ca2+]i increase, whereas the P2y purinoceptor blocker, reactive blue, had no effect. Adenosinetriphosphate (ATP) and the P2x agonist, alpha 1 beta-methylene-ATP also significantly increased [Ca2+]i in HF cells, whereas the P2y agonist methylthio-ATP showed only a small [Ca2+]i response. Diadenosine polyphosphates mainly induced transplasmamembrane Ca2+ influx as was confirmed by experiments in the absence of extracellular Ca2+ or by manganese quenching studies. Organic (verapamil) and inorganic Ca2+ channel blockers (NiCI2) significantly reduced the AP6A induced transplasmamembrane Ca2+ influx. The inhibitor of phosphatidylcholine-specific phospholipase C, D609, significantly reduced the effect of diadenosine polyphosphates on [Ca2+]i in HF cells. It is concluded that diadenosine polyphosphates regulate transplasmamembrane Ca2+ influx after occupation of P2x receptors via activation of phosphatidylcholine-specific phospholipase C and hence of voltage-operated Ca2+ channels.
Assuntos
Cálcio/metabolismo , Fosfatos de Dinucleosídeos/farmacologia , Receptores Purinérgicos/metabolismo , Fosfolipases Tipo C/metabolismo , Hidrocarbonetos Aromáticos com Pontes/farmacologia , Bloqueadores dos Canais de Cálcio/farmacologia , Células Cultivadas , Citosol/metabolismo , Fibroblastos , Fura-2/metabolismo , Humanos , Manganês/farmacologia , Níquel/farmacologia , Norbornanos , Inibidores de Fosfodiesterase/farmacologia , Antagonistas Purinérgicos , Suramina/farmacologia , Teobromina/análogos & derivados , Teobromina/farmacologia , Tiocarbamatos , Tionas/farmacologia , Fosfolipases Tipo C/antagonistas & inibidores , Vasoconstritores/farmacologia , Verapamil/farmacologia , Xantinas/farmacologiaRESUMO
Previously, we reported that the phosphatidylcholine-specific phospholipase C (PC-PLC) inhibitor tricyclodecan-9-yl xanthogenate (D609) potentiates thapsigargin-induced Ca(2+) influx in human lymphocytes. In the present study we examined the effect of D609 on the thapsigargin-induced Na(+) entry. We found that the early phase of the thapsigargin-induced increase in the intracellular Na(+) concentration (approx. 1-2 min after stimulation) was attenuated after preincubation of lymphocytes with D609. By contrast, thapsigargin-induced Na(+) influx was not affected in the presence butan-1-ol, which inhibits phosphatidylcholine-specific phospholipase D (PC-PLD). The thapsigargin-induced Na(+) influx could be mimicked by PC-PLC exogenously added to the lymphocyte suspension, whereas addition of PC-PLD had no effect. In addition, thapsigargin stimulated formation of the physiological PC-PLC products, diacylglycerol. Cell-permeable diacylglycerol analogue, dioctanoyl-glycerol (DOG), produced time- and concentration-dependent increase in the intracellular Na(+) concentration. Both thapsigargin- and DOG-induced Na(+) increases were not affected in the presence of Na(+)/H(+) antiport inhibitor, HOE609, or Na(+)/Ca(2+) antiport inhibitor, dimethylthiourea, as well as in the presence of Co(2+) and Ni(2+), which block store-operated Ca(2+) entry. By contrast, markedly reduced thapsigargin- and DOG-induced Na(+) influx were noted in the presence of flufenamic acid, which blocks the non-selective cation current (I(CRANC)). In conclusion, our results suggest that diacylglycerol released due to the PC-PLC activation contributes to the thapsigargin-induced Na(+) entry.
Assuntos
Hidrocarbonetos Aromáticos com Pontes/farmacologia , Inibidores Enzimáticos/farmacologia , Linfócitos/enzimologia , Inibidores de Fosfodiesterase/farmacologia , Sódio/metabolismo , Tapsigargina/farmacologia , Tionas/farmacologia , Fosfolipases Tipo C/antagonistas & inibidores , Transporte Biológico/efeitos dos fármacos , Transporte Biológico/fisiologia , Cálcio/metabolismo , Diglicerídeos/biossíntese , Diglicerídeos/farmacocinética , Citometria de Fluxo , Humanos , Linfócitos/efeitos dos fármacos , Norbornanos , Fosfolipase D/antagonistas & inibidores , Fosfolipase D/farmacologia , Proteína Quinase C/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Trocador de Sódio e Cálcio/metabolismo , Trocadores de Sódio-Hidrogênio/metabolismo , Tiocarbamatos , Fosfolipases Tipo C/farmacologiaRESUMO
High density lipoprotein (HDL) cholesterol is an important risk factor for coronary heart disease, and HDL exerts various potentially antiatherogenic properties, including the mediation of reverse transport of cholesterol from cells of the arterial wall to the liver and steroidogenic organs. Enhancement of cholesterol efflux and of reverse cholesterol transport (RCT) is considered an important target for antiatherosclerotic drug therapy. Levels and composition of HDL subclasses in plasma are regulated by many factors, including apolipoproteins, lipolytic enzymes, lipid transfer proteins, receptors, and cellular transporters. In vitro experiments as well as genetic family and population studies and investigation of transgenic animal models have revealed that HDL cholesterol plasma levels do not necessarily reflect the efficacy and antiatherogenicity of RCT. Instead, the concentration of HDL subclasses, the mobilization of cellular lipids for efflux, and the kinetics of HDL metabolism are important determinants of RCT and the risk of atherosclerosis.
Assuntos
Arteriosclerose/metabolismo , Colesterol/metabolismo , Lipoproteínas HDL/metabolismo , Animais , Transporte Biológico , HumanosRESUMO
Screening for apolipoprotein (apo) C-II variants in the plasma of 400 students, 600 patients of a cardiological rehabilitation center, and 1200 patients of an outpatient lipid clinic by isoelectric focusing and subsequent anti-apo C-II immunoblotting led to the identification of four individuals whose plasma samples contained an apo C-II isoform with an abnormal isoelectric point. In all cases direct sequencing of PCR-amplified DNA assessed a heterozygous A to C transversion in codon 19 of the apo C-II gene which leads to the replacement of lysine with threonine. Two of the four index patients presented with moderate hypertriglyceridemia; one suffered from severe hyperlipidemia, with triglyceride levels ranging between 180 and 1900 mg/dl, depending on dietary changes. Sequencing of this proband's lipoprotein lipase gene showed no alteration compared to the wild-type sequence. A study in his family revealed that heterozygosity for apo C-II(K19T) is not associated with differences in mean lipid and lipoprotein concentrations. In conclusion, apo C-II(K19T) occurs in Germany at a frequency of approximately 1 in 550. Although this variant is not sufficient to cause hypertriglyceridemia, it may be possible that apo C-II(K19T) cause hypertriglyceridemia in the presence of additional as yet unidentified environmental and/or genetic factors.
Assuntos
Apolipoproteínas C/sangue , Apolipoproteínas C/genética , Adulto , Sequência de Aminoácidos , Apolipoproteína C-II , Apolipoproteínas C/química , Sequência de Bases , Variação Genética , Heterozigoto , Humanos , Hipercolesterolemia/genética , Hipertrigliceridemia/genética , Focalização Isoelétrica , Masculino , Pessoa de Meia-Idade , Dados de Sequência MolecularRESUMO
In non-excitable cells, sustained intracellular Ca2+ increase critically depends on influx of extracellular Ca2+. Such Ca2+ influx is thought to occur by a 'store-operated' mechanism, i.e. the signal for Ca2+ entry is believed to result from the initial release of Ca2+ from inositol 1,4,5-trisphosphate-sensitive intracellular stores. Here we show that the depletion of cellular Ca2+ stores by thapsigargin or bradykinin is functionally linked to a phosphoinositide-specific phospholipase D (PLD) activity in cultured vascular smooth muscle cells (VSMC), and that phosphatidic acid formed via PLD enhances sustained calcium entry in this cell type. These results suggest a regulatory role for PLD in store-operated Ca2+ entry in VSMC.
Assuntos
Cálcio/metabolismo , Músculo Liso Vascular/metabolismo , Fosfolipase D/metabolismo , Animais , Bradicinina/farmacologia , Sinalização do Cálcio/efeitos dos fármacos , Sinalização do Cálcio/fisiologia , Células Cultivadas , Ativação Enzimática/efeitos dos fármacos , Líquido Intracelular/metabolismo , Transporte de Íons/efeitos dos fármacos , Músculo Liso Vascular/efeitos dos fármacos , Ácidos Fosfatídicos/metabolismo , Ratos , Tapsigargina/farmacologiaRESUMO
In the present study, we investigated the effect of high density lipoproteins 3 (HDL3) on Na+/H+ exchanger activity and cytosolic pH (pHi) in human platelets. HDL3 alone failed to affect pHi, but preincubation with HDL3 significantly enhanced the Na+/H+ antiport activation brought about by acidification with 100 mM sodium propionate or stimulation with 0.05 U/ml thrombin. the stimulatory effect of HDL3 was unaffected by indomethacin excluding a role for cyclooxygenase products. The HDL3 effect was not mediated by Ca2+/calmodulin-dependent protein kinase as HDL3 failed to increase cytosolic free calcium concentration. However, the potentiating effect of HDL3 was completely blocked in the presence of the protein kinase C inhibitor, bisindoylmaleimide and the phosphatidylcholine-specific phospholipase C inhibitor, D609. Furthermore, the effect of HDL3 was abolished after covalent modification of HDL3 with dimethylsuberimidate and was not observed in platelets from Glanzmann thrombasthenia type 1 which do not express GP IIb/IIIa, as well as in platelets preincubated with anti-GP IIb/IIIa polyclonal antibodies. We conclude that HDL3 enhances the sodium propionate- and thrombin-induced Na+/H+ antiport activity in human platelets via binding to GP IIb/IIIa and activation of protein kinase C and phosphatidylcholine-specific phospholipase C.
Assuntos
Plaquetas/efeitos dos fármacos , Lipoproteínas HDL/farmacologia , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/metabolismo , Trocadores de Sódio-Hidrogênio/efeitos dos fármacos , Plaquetas/metabolismo , Cálcio/sangue , Ativação Enzimática , Humanos , Fosfatidilcolinas/sangue , Fosfolipases/metabolismo , Propionatos/farmacologia , Ligação Proteica , Proteína Quinase C/metabolismo , Cloreto de Sódio/sangue , Trocadores de Sódio-Hidrogênio/sangue , Especificidade por Substrato , Trombina/farmacologiaRESUMO
Various studies have already shown that the fatty acid composition of dietary fat has different effects on hemostasis and platelet function. However, knowledge on this topic is incomplete. In the present study, fifty-eight healthy students received either a 4-week rapeseed oil [high content of monounsaturated fatty acids (MUFA) and high n-3/n-6 PUFA ratio], an olive oil (high content of MUFA, low n-3/n-6 PUFA ratio) or a sunflower oil (low content of MUFA, low n-3/n-6 PUFA ratio) diet. In each group, effects on hemostatic parameters were compared with a wash-in diet rich in saturated fatty acids with respect to intermediate-time effects on the hemostatic system and platelet function. With the olive oil diet, a reduction of coagulation factors VIIc, XIIc, XIIa, and Xc was found, whereas sunflower oil led to lower values of coagulation factors XIIc, XIIa, and IXc. In all study groups levels of plasmin-alpha2-antiplasmin were lower in week 4 than at baseline. Lower fibrinogen binding on platelets was found after the sunflower oil diet, whereas expression of CD62 and spontaneous platelet aggregation were slightly higher after the olive oil diet. However, given the major differences in the fatty acid compositions of the diets, the differences between the groups with respect to hemostasis tended to be small. Therefore, the clinical significance of the present findings remains to be evaluated.
Assuntos
Gorduras Insaturadas na Dieta/farmacologia , Hemostasia/efeitos dos fármacos , Óleos de Plantas/farmacologia , Adulto , Fator VII/efeitos dos fármacos , Fator XII/efeitos dos fármacos , Ácidos Graxos Monoinsaturados/farmacologia , Ácidos Graxos Ômega-3/farmacologia , Ácidos Graxos Ômega-6 , Ácidos Graxos Insaturados/farmacologia , Feminino , Humanos , Masculino , Azeite de Oliva , Testes de Função Plaquetária , Óleo de Brassica napus , Óleo de GirassolRESUMO
In this study we found that HDL acts as a potent and specific mitogen in vascular smooth muscle cells (VSMC) by stimulating entry into S-phase and DNA synthesis in a time- and concentration-dependent manner, induction of cyclins D1, E, and A, as well as activation of cyclin D-dependent kinases as inferred from phosphorylation of the retinoblastoma protein (pRb). Moreover, HDL induced activation of the mitogen-activated protein kinase pathway including Raf-, MEK-1, and ERK1/2, as well as the expression of proto-oncogen c-fos, which is controlled by ERK1/2. PD98059, an inhibitor of MEK-1 blocked the mitogenic activity of HDL and cyclin D1 expression. HDL-induced VSMC proliferation, cell cycle progression, cyclin D1 expression, and activation of the Raf-1/MEK-1/ERK1/2 cascade were blocked by preincubation of cells with pertussis toxin indicating involvement of trimeric G-protein. By contrast, none of these responses was inhibited by the protein kinase C inhibitor, GF109203X. The mitogenic effects of native HDL were not mimicked by apo A-I, reconstituted HDL containing apo A-I, or cholesterol-containing liposomes. In conclusion, HDL possesses an intrinsic property to induce G-protein- and MAP-kinase-dependent proliferation and cell cycle progression in VSMC. The strong and specific mitogenic effect of HDL should be taken into account, when therapeutic strategies to elevate the plasma level of these lipoproteins are developed.
Assuntos
Ciclo Celular/efeitos dos fármacos , Lipoproteínas HDL/farmacologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Mitógenos/farmacologia , Músculo Liso Vascular/efeitos dos fármacos , Animais , Western Blotting , Proteínas de Ciclo Celular/biossíntese , Proteínas de Ciclo Celular/genética , Células Cultivadas/efeitos dos fármacos , Replicação do DNA/efeitos dos fármacos , Ativação Enzimática/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Proteínas de Ligação ao GTP/efeitos dos fármacos , Proteínas de Ligação ao GTP/fisiologia , Regulação da Expressão Gênica/efeitos dos fármacos , Lipoproteínas LDL/farmacologia , Lipoproteínas VLDL/farmacologia , Proteínas Quinases Ativadas por Mitógeno/biossíntese , Proteínas Quinases Ativadas por Mitógeno/genética , Músculo Liso Vascular/patologia , Toxina Pertussis , Fosforilação/efeitos dos fármacos , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Ratos , Ratos Endogâmicos WKY , Proteína do Retinoblastoma/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fase S/efeitos dos fármacos , Fatores de Virulência de Bordetella/farmacologiaRESUMO
High levels of fibrinogen and low levels of high-density lipoprotein (HDL) cholesterol were reported to be risk factors for coronary heart disease. CD11b/CD18, a fibrinogen-binding protein, is expressed on the surface of monocytes, which play a crucial role in the formation of atherosclerotic lesions. In the present study, we investigate the effects of antibodies against CD11b and CD18, as well as HDL3 and low-density lipoprotein (LDL) cholesterol on fibrinogen binding on monocytes. We find that binding of fibrinogen on monocytes activated with adenosine diphosphate (ADP) was reduced to 66.0+/-8.3% (mean +/- SD) in the presence of anti-CD11b antibodies (12.5 microg/ml; P < or = 0.02) and to 54.5+/-4.9% in the presence of anti-CD18 antibodies (20 microg/ml; P < or = 0.01), respectively. Fibrinogen binding on Cytochalasin-B-activated monocytes was reduced to 79.8+/-6.0% in the presence of anti-CD18 (20 microg/ml; P < or = 0.05). Incubation of ADP-activated monocytes with HDL3 (0.5 g/l) led to a lowering of fibrinogen binding to 65.0+/-6.6% (P < or = 0.05). No effect of HDL3 on fibrinogen binding was seen on Cytochalasin-B-activated monocytes. A slight, non-significant stimulatory effect of LDL on fibrinogen binding on ADP-activated but not on Cytochalasin-B-activated monocytes was additionally observed. Neither incubation with HDL3 or with LDL had a significant influence on ADP-activated cellular binding of anti-CD11b or anti-CD18 antibodies. The inhibition of fibrinogen binding on monocytes in the presence of HDL3 is a major new finding of this study. Since inhibition of fibrinogen binding in the presence of HDL might impair both monocyte recruitment to the arterial wall and foam cells formation, our findings suggests a novel mechanism by which HDL may prevent development of arteriosclerosis.
Assuntos
Fibrinogênio/metabolismo , Lipoproteínas HDL/farmacologia , Monócitos/efeitos dos fármacos , Difosfato de Adenosina/farmacologia , Anticorpos , Antígenos CD18/imunologia , HDL-Colesterol/farmacologia , LDL-Colesterol/farmacologia , Relação Dose-Resposta a Droga , Fibrinogênio/efeitos dos fármacos , Humanos , Monócitos/metabolismo , Ligação Proteica/efeitos dos fármacosRESUMO
The secondary oxidation of biologically modified low-density lipoproteins (LDL) was demonstrated to contribute to the cytotoxicity and thereby to the atherogenicity of modified lipoproteins. Previously we have shown that chemical modification of LDL by carbon disulfide (CS(2)) mimicked the naturally occurring process of LDL modification. In the present study the cytotoxicity of CS(2)-modified LDL and their susceptibility to the secondary oxidative modification was investigated. The cytotoxicity of CS(2)-modified LDL did not significantly exceed that of native LDL. However, the Cu(2+) -oxidized form of CS(2)-modified LDL revealed to be more cytotoxic than oxidized native LDL. Oxidized CS(2)-modified LDL presented with altered physicochemical properties including derivatization of protein amino and - SH groups, increased negative charge, and electrophoretic mobility which exceeded that of oxidized native LDL. The secondary oxidative modification of CS(2)-modified LDL involved the process of Cu(2+) binding to CS(2)-derived dithiocarbamate -SH groups followed by covalent modification of - SH groups by products of lipid peroxidation. Taken together, these finding suggest that secondary oxidation of CS(2)-modified LDL may contribute to the atherogenic effect of the chronic occupational exposure to CS(2).
RESUMO
BACKGROUND: Protein disulfide isomerase (PDI) controls platelet integrin function, tissue-factor (TF) activation, and concentrates at fibrin and thrombus formation sites of vascular injury. OBJECTIVE: To investigate the involvement of surface thiol isomerases and especially PDI, in thrombin-mediated thrombin amplification on human platelets. METHODS/RESULTS: Using a newly developed thrombin-dependent platelet thrombin generation assay, we observed that the feedback activation of thrombin generation on the platelet surface does not depend on TF, as anti-TF antibodies inhibiting TF-induced thrombin formation in platelet-depleted plasma had no effect compared with vehicle-treated controls. Feedback activation of thrombin generation in the presence of platelets was significantly diminished by membrane impermeant thiol blockers or by the thiol isomerase-inhibitors bacitracin and anti-PDI antibody RL90, respectively. Platelet thrombin formation depends on binding of coagulation factors to the platelet surface. Therefore, involvement of thiol isomerases in this binding was investigated. As shown by confocal microscopy and flow cytometry, thrombin-stimulated platelets exhibited increased surface-associated PDI as well as extracellular disulfide reductase activity compared with unstimulated platelets. Flow cytometric analysis revealed that membrane impermeant thiol blockers or PDI inhibitors, which had been added after platelet stimulation and after phosphatidylserine exposure to exclude their influence on primary platelet activation, significantly inhibited binding of all coagulation factors to thrombin-stimulated platelets. CONCLUSIONS: Thus, surface-associated PDI is an important regulator of coagulation factor ligation to thrombin-stimulated platelets and of subsequent feedback activation of platelet thrombin generation. Cell surface thiol isomerases might be therefore powerful targets to control hemostasis and thrombosis.
Assuntos
Fatores de Coagulação Sanguínea/metabolismo , Plaquetas/metabolismo , Retroalimentação Fisiológica , Isomerases de Dissulfetos de Proteínas/fisiologia , Trombina/biossíntese , Espaço Extracelular/metabolismo , Humanos , Ativação Plaquetária , Ligação Proteica , Isomerases de Dissulfetos de Proteínas/metabolismoRESUMO
OBJECTIVE: To explore the effect of maternal systemic lupus erythematosus (SLE) on first-trimester screening markers for Down syndrome. METHODS: A retrospective study was conducted on 1150 normal singleton fetuses that underwent first-trimester combined screening for Down syndrome. Fetal delta nuchal translucency (NT), maternal serum PAPP-A and free beta-hCG were compared between pregnancies with SLE (n = 10) and without preexisting maternal disease (n = 1140). RESULTS: The medians +/- SD for delta NT, log(10) MoM of PAPP-A and free beta-hCG +/- SD in pregnancies with SLE and without maternal disease were - 0.18 +/- 0.29 versus - 0.18 +/- 0.33, 0.005 +/- 0.32 versus 0.02 +/- 0.26, and 0.22 +/- 0.19 versus - 0.014 +/- 0.28, with a p value of 0.7, 0.98 and 0.03, respectively. CONCLUSIONS: Patients with preexisting SLE have increased maternal serum-free beta-hCG levels in the first-trimester. But, because of the multimodal procedure of risk calculation there is no significant difference in the screen-positive rate after the combined first-trimester screening for trisomy 21.
Assuntos
Gonadotropina Coriônica Humana Subunidade beta/sangue , Síndrome de Down/diagnóstico , Lúpus Eritematoso Sistêmico/sangue , Complicações na Gravidez/sangue , Primeiro Trimestre da Gravidez/sangue , Proteína Plasmática A Associada à Gravidez/metabolismo , Adolescente , Adulto , Biomarcadores/sangue , Feminino , Humanos , Pessoa de Meia-Idade , Gravidez , Estudos RetrospectivosRESUMO
OBJECTIVE: To explore if maternal serum free beta-hCG and pregnancy-associated plasma protein A (PAPP-A) levels in the first-trimester of pregnancy are altered in patients with habitual abortions and if there is an effect on first-trimester screening for Down syndrome. METHODS: A retrospective study was conducted on 913 normal singleton fetuses that underwent first-trimester combined screening for Down syndrome. Maternal serum PAPP-A and free beta-hCG were compared between patients with (n = 64) and without habitual abortions (n = 849). RESULTS: The medians +/- SD log(10) MoM of PAPP-A and free beta-hCG +/- SD in patients with and without habitual abortions were 0.063 +/- 0.28 versus - 0.014 +/- 0.27 and - 0.001 +/- 0.27 versus - 0.018 +/- 0.31, with a p value of 0.042 and 0.87, respectively. The screen positive rate setting the cut off at 1:350 looking at the background risk for trisomy 21 was 71.4% in women with and 81.2% in women without habitual abortion, after combined first-trimester screening it was 7.8% in women with and 10.1% in women without recurrent abortion. CONCLUSIONS: Patients with habitual abortions have slightly increased maternal serum PAPP-A levels in the first-trimester. This marginal difference seems not to effect risk calculation in combined first-trimester screening for trisomy 21.
Assuntos
Aborto Habitual/sangue , Gonadotropina Coriônica Humana Subunidade beta/sangue , Aberrações Cromossômicas , Cromossomos Humanos Par 21 , Testes Genéticos , Primeiro Trimestre da Gravidez/sangue , Proteína Plasmática A Associada à Gravidez/análise , Adolescente , Adulto , Síndrome de Down/diagnóstico , Síndrome de Down/etiologia , Feminino , Humanos , Pessoa de Meia-Idade , Gravidez , Diagnóstico Pré-Natal/métodos , Fatores de RiscoRESUMO
OBJECTIVES: To examine the effect of early vaginal bleeding on first-trimester screening markers for Down syndrome. METHODS: A retrospective study was conducted on 1755 normal singleton fetuses that underwent first-trimester combined screening for Down syndrome on the basis of ultrasound and maternal serum markers. Fetal delta-nuchal translucency (NT), maternal serum pregnancy-associated plasma protein A (PAPP-A) and free beta-hCG were compared between pregnancies with (n = 252) and without (n = 1503) an episode of vaginal bleeding. Subgroup analysis for the intensity of bleeding (spotting n = 191; light n = 32; heavy n = 29) was performed. RESULTS: The median +/- SD (log(10)) for delta-NT, multiple of medians (MoM) PAPP-A and MoM free beta-hCG (corrected for maternal weight, smoking and ethnicity) was - 0.17 +/- 0.62, 1.10 +/- 0.28, 1.1 +/- 0.28 and - 0.15 +/- 0.51, 0.98 +/- 0.26, 0.94 +/- 0.3 in pregnancies with and without a history of early vaginal bleeding, which were not significantly different. Exclusion of patients with spotting from the vaginal bleeding group revealed significantly higher maternal serum free beta-hCG MoM values (median +/- SD (log(10))) compared to patients without bleeding, 1.29 +/- 0.27 vs 0.96 +/- 0.3(p = 0.011). Screen-positive (cut off of 1:350) rate after combined first-trimester screening was 28.1% in patients with light vaginal bleeding and 8.4% in patients without bleeding (p = 0.001). CONCLUSIONS: Light vaginal bleeding before first-trimester combined screening for Down syndrome leads to a higher screen-positive rate after combined first trimester screening, without a significant difference in serum levels of the screening markers.
Assuntos
Gonadotropina Coriônica Humana Subunidade beta/sangue , Síndrome de Down/diagnóstico , Medição da Translucência Nucal , Proteína Plasmática A Associada à Gravidez/análise , Diagnóstico Pré-Natal , Hemorragia Uterina , Adolescente , Adulto , Biomarcadores/sangue , Feminino , Humanos , Pessoa de Meia-Idade , Valor Preditivo dos Testes , Gravidez , Primeiro Trimestre da Gravidez , Risco Ajustado , Ultrassonografia Pré-Natal , Hemorragia Uterina/sangueRESUMO
The purinergic receptor system plays an important role in the regulation of both vascular and tubular functions within the kidney; however, the release of purinergic agonists other than ATP by renal tissue is not known. In this investigation, we determine if kidney tissue is a source of diadenosine polyphosphates, which have high affinity for the P(2X) and P(2Y) receptors. Both diadenosine pentaphosphate and hexaphosphate were identified by matrix-assisted laser desorption ionization-mass spectrometry in extracts purified from both whole porcine kidney and from cloned cells of the LLC-PK1 cell line. Both polyphosphates in nanomolar concentrations were found to significantly stimulate the proliferation of vascular smooth muscle cells derived from rat thoracic aortas. The purinergic-receptor antagonist, suramin, did not significantly affect the growth-stimulatory properties of the polyphosphates. The growth stimulation of vascular smooth muscle cells by platelet-derived growth factor was potentiated by both diadenosine polyphosphates. We conclude that diadenosine polyphosphates are endogenous purinergic agonists of the kidney that have physiologic and pathophysiologic relevance. These epithelial cell metabolic products have vasoregulatory properties while linking the energy supply and tubular function.