高浓度葡萄糖诱导红细胞磷脂酰丝氨酸暴露的机制研究

本研究探讨高浓度葡萄糖诱导红细胞磷脂酰丝氨酸（PS）暴露的机制。将红细胞于葡萄糖缓冲液中孵育后用流式细胞术分析PS标记率和前向角值；检测caspase-3和easpase-8的活化情况；用流式细胞术和荧光显微镜观察亮抑酶肽对葡萄糖引起的红细胞PS暴露的抑制效果。结果表明：葡萄糖可以诱导红细胞PS暴露，而且其效率和葡萄糖浓度密切相关。随着葡萄糖浓度的增加，红细胞的PS标记率呈逐步增加的趋势，当葡萄糖浓度为0．8mol／L时，红细胞的PS标记率在80％以上。然而，在葡萄糖引起的红细胞PS暴露过程中，caspase-3和caspase-8没有活化，而亮抑酶肽却可以很好地抑制红细胞PS暴露和使其体积缩小。随着亮抑酶肽浓度的增加，红细胞PS标记率呈逐步下降的趋势，而细胞体积却呈逐步增加的趋势。结论：高浓度葡萄糖可以导致严重的红细胞PS暴露，而且这种PS暴露和caspase无关，推测葡萄糖诱导的红细胞PS暴露是受一条对亮抑酶肽高度敏感的、未知的通路调控的．     

Some examples of the clinical importance of the basic and heparin-induced phospholipase A in human plasma

Phospholipase A activity was determined in human plasma with biological ³²P-labelled phosphatidylethanolamine from rat liver following cardiac operations with the aid of the heart-lung-machine. The basic activity before heparin and the initiation of bypass was 72 nmol β ml^?1 β h^?1 plasma; thereby large differences in the basic activity were detected between cyanotic and acyanotic heart diseases. A sharp increase of phospholipase activity was observed after heparin (a smaller one in intensive care patients with low-dose heparin), and a further increase up to the end of perfusion. Normal values are reached after giving protamine at the end of the operation. An important role for plasma phospholipase in the regulation of physiological and pathophysiological processes may be supposed from these results.     

On the molecular mechanisms for the highly procoagulant pattern of C6 glioma cells

BACKGROUND: That there is a correlation between cancer and procoagulant states is well-known. C6 glioma cell line was originally induced in random-bred Wistar-Furth rats and is morphologically similar to glioblastoma multiforme, the most common aggressive glioma resistant to therapeutic interventions. OBJECTIVES: In this study we analyzed the molecular mechanisms responsible for the highly procoagulant properties of C6 glioma cells. METHODS: The presence of tissue factor (TF) and phosphatidylserine (PS) in C6 cells was investigated by flow-cytometric and functional analyses. The assembly of extrinsic tenase, intrinsic tenase and prothrombinase complexes on these cells was studied using enzymatic assays employing plasma or purified proteins. RESULTS: TF was identified by flow-cytometric and functional [factor (F) Xa formation in the presence of cells and FVIIa] assays. Alternatively, conversion of FX into FXa was also observed in the presence of C6 cells, FIXa and FVIIIa. This effect was both cell- and FVIIIa-dependent, being consistent with formation of the intrinsic tenase complex. C6 cells were also able to activate prothrombin in the presence of FXa and FVa, thus supporting formation of the prothrombinase complex. This ability was similar to positive controls performed with PS-containing vesicles. Accordingly, exposure of PS on C6 cells was demonstrated by flow cytometry employing specific anti-PS antibodies. In addition, annexin V, which blocks PS binding sites, inhibited FX and prothrombin conversion by their respective C6-assembled activating complexes. CONCLUSION: C6 glioma cells support all procoagulant reactions leading to robust thrombin formation. This ability results from concomitant TF exposure and from the presence of the anionic lipid PS at the outer leaflet of cell membrane. Therefore, this animal cell line may be used to explore new aspects concerning the role of blood coagulation proteins in tumor biology, especially those affecting the central nervous system.     

流式细胞术联合测定保存血小板表达的磷脂酰丝氨酸和CD62p

人类血小板通过不同的技术进行离体保存后，其特性改变差异较大。建立适用于各种保存血小板特性分析的流式细胞术（FCM）对保存血小板的质量监测和新保存方法研究有重要意义。本研究通过对FCM分析方法主要条件的优化评估，建立了联合测定血小板膜表面包括磷脂酰丝氨酸在内的多参数FCM定量分析方法。在标本制作中，血小板不需要洗涤即可直接进行标记，减少了血小板离心洗涤过程中的激活。除了FCM常用的同型对照外，中还将新鲜富含血小板血浆（FPRP）、凝血酶激活FPRP和液氮处理FPRP作为血小板标准阴性、阳性对照，直接应用于FCM分析，且效果良好。该方法中Gly-Pro-Arg-Pro乙酸盐（GPRP）的选择应用，既防上了血小板聚集和纤维蛋白的形成，同时可稳定血小板，减少制备过程中人为激活，克服了在血小板、Ca^2 和血浆共存条件下FCM定量分析血小板的困难，尤其是为深低温处理血小板包括PS在内的多参数分析提供了良好的方法基础。研究表明该方法标本处理简单，结果分析简便、准确，重复性好。作还通过低温、低渗或激活剂的方法制备了几种膜表面分子标记显不同的血小板应用于FCM分析，不仅证实了所建立的FCM分析方法在各种不同膜表面分子标记的血小板分析中的实用性，又表明了中制备的4种不同膜表面分子标记的血小板在FCM分析保存血小板方法学中的实用价值。     

Antiproliferative activity and standardization of Tecomella undulata bark extract on K562 cells

Ethnopharmacological relevance

The bark of Tecomella undulata is primarily used in the treatment of syphilis, painful swellings and cancer by traditional healers. Also, it is claimed to be useful in treating urinary discharges, enlargement of spleen, leucorrhoea, leukoderma, tumors, liver disorders, gonorrhea, gout and promotes wound healing in Indian traditional system of medicine.

Aim

To establish a scientific validation for the antitumor effects of Tecomella undulata bark and explore the mechanistic pathway in chronic myeloid leukemia cell line, K562. The study was further extended to standardize the extract using quercetin as biomarker.

Methods

Induction of apoptosis by chloroform extract of Tecomella undulata bark (CTUB) was determined by MTT, Annexin V and caspase activation assays. The cell cycle analysis was done by flow cytometer and nuclear staining by DAPI. The standardization of the extract was performed through reverse phase-HPLC method under PDA detection.

Result

Results clearly showed the induction of apoptosis by CTUB in K562 cells. The effect was found to be dose dependent, having IC₅₀ of 30 μg/ml with activation of FAS, FADD, caspase 8, caspase 3/7 and fragmentation of DNA. The bioactive CTUB was determined to possess 0.03% (w/w) of quercetin.

Conclusion

The investigation clearly demonstrated the potential antitumor effect of CTUB, thereby validating the traditional claim. Quercetin, known to have anticancer activity is being reported and quantified for the first time from the bark of Tecomella undulata.     

Effects of carbonyl cyanide p-(trifluoromethoxy) phenylhydrazone on the growth inhibition in human pulmonary adenocarcinoma Calu-6 cells

Carbonyl cyanide p-(trifluoromethoxy) phenylhydrazone (FCCP) is an uncoupler of mitochondrial oxidative phosphorylation in eukaryotic cells. Here, we evaluated the in vitro effects of FCCP on the growth of Calu-6 lung cancer cells. FCCP inhibited the growth of Calu-6 cells with an IC₅₀ of approximately 6.64 ± 1.84 μM at 72 h, as shown by MTT. DNA flow cytometric analysis indicated that FCCP induced G1 phase arrest below 20 μM of FCCP. Treatment with FCCP decreased the level of CDKs and cyclines in relation to G1 phase. In addition, FCCP not only increased the p27 level but also enhanced its binding with CDK4, which was associated with hypophosphorylation of Rb protein. While transfection of p27 siRNA inhibited G1 phase arrest in FCCP-treated cells, it did not enhance Rb phosphorylation. FCCP also efficiently induced apoptosis. The apoptotic process was accompanied with an increase in sub-G1 cells, annexin V staining cells, mitochondria membrane potential (MMP) loss and cleavage of PARP protein. All of the caspase inhibitors (caspase-3, -8, -9 and pan-caspase inhibitor) markedly rescued the Calu-6 cells from FCCP-induced cell death. However, knock down of p27 protein intensified FCCP-induced cell death. Moreover, FCCP induced the depletion of GSH content in Calu-6 cells, which was prevented by all of the caspase inhibitors. In summary, our results demonstrated that FCCP inhibits the growth of Calu-6 cells in vitro. The growth inhibitory effect of FCCP might be mediated by cell cycle arrest and apoptosis via decrease of CDKs and caspase activation, respectively. These findings now provide a better elucidation of the mechanisms involved in FCCP-induced growth inhibition in lung cancer.     

Lactadherin blocks thrombosis and hemostasis in vivo: correlation with platelet phosphatidylserine exposure

Summary.  Background:  Platelet membrane phosphatidylserine (PS) is considered to be essential for hemostasis and thrombosis, but the in vivo topography of platelet PS has not been characterized. We hypothesized that platelet PS exposure would be identified on adherent platelets at the site of vascular injury and that blockade of PS would impede hemostasis and thrombosis. Objective:  To localize and estimate the extent of platelet PS exposure and evaluate the impact of PS blockade in vivo . Methods:  Lactadherin, a PS-binding milk protein, was utilized together with annexin  V to detect both partial and complete membrane PS exposure on platelets in a mouse model of thrombosis and to evaluate the functional need for PS. Preliminary experiments were performed with synthetic membranes and with purified platelets. Results:  The number of lactadherin-binding sites on synthetic membranes was proportional to PS content, whereas annexin  V required a threshold of 2.5–8% PS. Approximately 95% of thrombin-stimulated platelets exposed PS, but the quantity was below the threshold for annexin  V binding at physiologic Ca²⁺ concentrations. In mice, most adherent and aggregated platelets on the walls of ferric chloride-treated mesenteric veins exposed low levels of PS, rather than having complete exposure. In mice, blockade of PS with lactadherin inhibited platelet prothrombinase and factor Xase activity, and prolonged tail bleeding time and the time to carotid artery thrombosis. Conclusions:   In vivo PS exposure contributes to both hemostasis and thrombosis. In this model of vascular injury, most platelets exhibit partial rather than complete PS exposure.     

Alcohol induced burr cell (echinocytic) haemolytic anaemia and haemochromatosis

Summary A 51-year-old man with chronic alcoholic liver disease developed a severe haemolytic anaemia characterized by the presence of circulating burrshaped cells (echinocytes). Several transfusions of packed red cells were ineffective in raising the haemoglobin concentration, showing that the abnormality was acquired by the transfused cells. Liver biopsies revealed haemochromatosis. Haematological parameters normalized four months after the patient stopped drinking alcohol, but burr cells were still present and erythrocyte life-span was still markedly shortened at one year follow-up. Since serum cholesterol, HDL-cholesterol, and Apo-Al and Apo-B lipoproteins were considerably decreased, the lipid composition of the red cell membrane was studied. Findings showed that echinocytosis occurred with no change in membrane cholesterol content, nor in cholesterol: phospholipid ratio, but with an alteration in the phosphatidylserine and phosphatidylinositol concentrations. While haemochromatosis was most likely the cause of the erythrocyte anomaly, alcohol intake was probably responsible for the acute onset of haemolytic anaemia with effects directly on the erythrocyte membrane as well as mediated by the progressive hepatic injury, with alterations in the plasma and successively in the intramembrane lipid composition.     

L-carnitine inhibits a subset of platelet activation responses in chronic uraemia.

BACKGROUND: Activated uraemic platelets expose the aminophospholipid phosphatidylserine (PS) at their outer surface, which generates a cell procoagulant phenotype and seems at least partly due to an increase in cell caspase-3 activity. L-carnitine (LC) may decrease surface-exposed PS in stored apheresis platelets and inhibit the activity of recombinant caspases, but its effects on platelet activation response with PS externalization have not been ascertained in chronic renal failure. In the present study, we investigated in vitro and in vivo the effects of LC on PS exposure in platelets from chronic uraemic patients. METHODS: Platelet PS-exposure was assayed by flow cytometry using annexin V. Caspase activity in platelets was determined by the cleaving activity of the specific substrate DEVD-pNA and by a flow cytometric assay using rhodamine-fluorescence. The effects of LC in vivo were examined in a prospective cross-over trial including 10 patients on maintenance haemodialysis (HD) who were randomly allocated to two different treatment groups: LC (2 g i.v.) for 4 months followed by placebo (2 g i.v.) for another 4 months (group A), or placebo followed by LC (group B). RESULTS: PS-exposing platelets in blood samples obtained from HD patients were significantly higher than in healthy subjects (P<0.001) under both unstimulated and agonist-stimulated conditions. When uraemic platelets were pre-incubated with LC before agonist stimulation, platelet PS exposure proved to be significantly reduced (-13.7% for 0.5 mM LC and -25% for 5 mM LC). Pre-incubation of uraemic platelets with LC again significantly decreased the cells' caspase activity (P<0.05). In HD patients (Group A), LC supplementation was associated with a significant decrease (P<0.05) in platelet PS exposure followed by a progressive increase during treatment with placebo. In the other group of patients, while no change in platelet PS exposure was observed during the first 4 months of treatment with placebo, a significant reduction (P<0.05) in PS-positive platelets occurred after 2 and 4 months of LC therapy. CONCLUSION: Our data show that LC may reduce, possibly via inhibition of caspase activity, the exposure of PS in activated uraemic platelets. These findings may have implications for the thrombophilic tendency of uraemia.