Cell cycle induction in post-mitotic neurons proceeds in concert with the initial phase of programmed cell death in rat

Neuronal programmed cell death (PCD) is increasingly becoming recognized as a dynamic process that may be amenable to resolution. Critical to this resolution is the identification of the cellular pathways that modulate the initial stages of apoptotic death. In this regard, we examined whether the activation of a latent cell cycle was associated with the initial phase of PCD. We demonstrate that free radical nitric oxide induced PCD results in the rapid generation of membrane phosphatidylserine residue exposure. This early phase of PCD functions in parallel with an untoward attempt to enter the cell cycle in the same population of post-mitotic neurons. We therefore offer an attractive molecular target to prevent or reverse neuronal PCD by elucidating a novel mechanism through which the majority of neurons meet their demise by attempting to enter a latent cell cycle.     

Calcium phosphate precipitation in aqueous suspensions of phosphatidylserine-containing anionic liposomes

Summary Liposomes prepared from 6.3∶1.8∶0.9∶ 1.0 molar mixtures of phosphatidylcholine, dicetyl phosphate, cholesterol, and phosphatidylserine, respectively, (PS(+) liposomes) were compared with similarly prepared liposomes without the phosphatidylserine (PS(−) liposomes) for their effect on calcium phosphate precipitate formation in aqueous solutions at pH 7.4 and 22°C. The liposomes, encapsulated with 50 mM phosphate (P_I), were suspended in buffered 2.2 mM CaCl₂, 0 or 1.5 mM KH₂PO₄ solutions and made permeable to Ca²⁺ fluxes with the ionophore, X-537A. External solution Ca²⁺ losses were found to be small in both PS(+) and PS(−) liposome suspensions when no ionophore was added. Even with 1.5 mM P_I in the external solution, these losses did not exceed 0.2 mM. However, inoculating both liposome preparations with X-537A resulted in rapid, appreciable losses in solution Ca²⁺. Previous studies showed that in PS(−) liposomes, these latter losses were due to calcium phosphate precipitation, with the precipitate confined to the interior of the liposomes when no external P_I was present, but extending to outside the liposomes when the suspending medium was rendered metastable. In the present study, Ca²⁺ losses resulting from intraliposomally confined precipitation were found to be marginally greater in PS(+) liposomes due primarily to a larger volume of entrapped P_I available for reaction in these liposomes. However, with the addition of P_I to the external solution, the reverse was observed, i.e., considerably less Ca²⁺ was lost in PS(+) than in PS(−) suspensions, a result of markedly less X-537A-induced precipitate forming outside PS(+) liposomes. The most probable explanation for this latter decrease was a PS-induced adherence of the outer liposome membranes to the surfaces of developing crystals, restricting the avialability of these surfaces as sites for further growth.     

Phosphatidylserine is a marker of tumor vasculature and a potential target for cancer imaging and therapy

: (1) To determine whether exposure of phosphatidylserine (PS) occurs on vascular endothelium in solid tumors in mice. (2) To determine whether PS exposure can be induced on viable endothelial cells in tissue culture by conditions present in the tumor microenvironment.

: Externalized PS in vivo was detected by injecting mice with a monoclonal anti-PS antibody and examining frozen sections of tumors and normal tissues for anti-PS antibody bound to vascular endothelium. Apoptotic cells were identified by anti-active caspase-3 antibody or by TUNEL assay. PS exposure on cultured endothelial cells was determined by ¹²⁵I-annexin V binding.

: Anti-PS antibody bound specifically to vascular endothelium in six tumor models. The percentage of PS-positive vessels ranged from 4% to 40% in different tumor types. Vascular endothelium in normal organs was unstained. Very few tumor vessels expressed apoptotic markers. Hypoxia/reoxygenation, acidity, inflammatory cytokines, thrombin, or hydrogen peroxide induced PS exposure on cultured endothelial cells without causing loss of viability.

: Vascular endothelial cells in tumors, but not in normal tissues, externalize PS. PS exposure might be induced by tumor-associated oxidative stress and activating cytokines. PS is an abundant and accessible marker of tumor vasculature and could be used for tumor imaging and therapy.     

胎盘广泛性微血栓在子痫前期发病中的作用——一种新的子痫前期动物模型评价

目的:应用磷脂酰丝氨酸/磷脂酰胆碱(PS/PC)微团反复静脉注射诱导妊娠小鼠胎盘形成广泛性微血栓,观察是否引起孕鼠高血压、蛋白尿、胎仔宫内生长受限等子痫前期(PE)样表现。方法:随机将27只妊娠ICR鼠分为2组,造模组于妊娠5.5~16.5天,连续尾静脉注射PS/PC 1mg/d,对照组则同期注射生理盐水0.1ml/d,观察两组的收缩压、尿蛋白、胎盘重、胎仔重、死胎数、血小板计数、血浆AT-Ⅲ含量和D-二聚体含量变化,并分析胎盘、肝、肾等脏器纤维蛋白沉积和血栓形成情况。结果:与对照组相比,造模组表现出高血压(124.06mmHg vs 96.75mmHg,P<0.005)、蛋白尿(622.02mg/L vs273.29mg/L,P<0.005)、胎仔宫内生长受限(0.998g vs 1.182g,P<0.005)、死胎发生率增高;胎盘广泛纤维蛋白沉积和微血栓形成,但肝、肾未发现类似改变。造模组还表现为血浆AT-Ⅲ含量降低(87.71mg/L vs 155.81mg/L,P<0.005)、D-二聚体含量升高(2.87mg/L vs 0.27mg/L,P<0.05)及血小板数量下降(83.94×1010/L vs 111.28×1010/L,P<0.05)。结论:PS/PC微团静脉注射能诱导ICR孕小鼠胎盘广泛纤维蛋白沉积和微血栓形成,引起ICR孕小鼠PE样表现,故胎盘广泛纤维蛋白沉积和微血栓形成可能是PE发病的重要机制之一。这是一个新的从胎盘病理和止血功能方面研究PE病因的模型。     

Ethanol induces Fas/Apo [apoptosis]-1 mRNA and cell suicide in the developing cerebral cortex

INTRODUCTION: Animal studies modeling fetal alcohol syndrome have demonstrated that developmental exposure to alcohol is associated with decreased brain weight and significant neuronal loss in multiple regions of the developing brain. Our previous data suggest that the Fas/Apo [apoptosis]-1 receptor is transiently expressed in the developing cerebral cortex during the peak period of naturally occurring apoptotic cell death and maximum sensitivity to alcohol. Therefore, we hypothesized that ethanol increases the expression of suicide receptors such as Fas/Apo-1 in the developing fetal cerebral cortex and leads to an upregulation or extension of the normal period of apoptosis and consequent disorganization of the neural circuitry. METHODS: Ethanol was administered in one of four doses (120, 320, 630, and 950 mg/dl) to organotypic explant cultures of the developing cerebral cortex established from postnatal day 2 rats and maintained for 6 days in vitro. The number of cells expressing Fas/Apo-1 receptor mRNA was counted. Apoptosis was measured by the use of two independent assays; a cell death enzyme-linked immunosorbent assay for DNA fragmentation and flow cytometric analysis of Annexin-V binding to phosphatidylserine externalized to the outer leaflet of the plasma membrane. Necrosis was also estimated by two independent measures, the amount of lactate dehydrogenase released into culture medium and flow cytometric analysis of cells that were positive for both Annexin-V and propidium iodide. RESULTS: A significantly larger number of developing cortical cells expressed Fas/Apo-1 mRNA at the lower doses (120 and 320 mg/dl) than at the higher doses (630 and 950 mg/dl). Furthermore, ethanol induced apoptosis in a dose-related manner, with peak apoptosis observed at a dose of 630 mg/dl in the case of DNA fragmentation and at 630 and 950 mg/dl in the case of phosphatidylserine translocation to the outer leaflet of the plasma membrane. Ethanol did not induce necrosis at any of the administered doses of ethanol. CONCLUSIONS: Our data suggest that ethanol induces a susceptibility to apoptotic signals at low doses by upregulating the expression of mRNAs for cytotoxic receptors such as Fas/Apo-1 in the developing cerebral cortex. However, ethanol itself specifically induces apoptosis in the developing cerebral cortex only at higher doses.     

Effect of miltefosine on erythrocytes

BackgroundMiltefosine, an alkylphosphocholine drug with antiparasite, antibacterial, antifungal and antineoplastic potency, is the only oral drug that can be used to treat visceral and cutaneous leishmaniasis. The effect of miltefosine is at least partially due to triggering of apoptosis. Similar to apoptosis of nucleated cells, erythrocytes may enter suicidal death or eryptosis, which is characterized by cell shrinkage and by cell membrane scrambling with phosphatidylserine-exposure at the erythrocyte surface. Eryptosis may be triggered following increase of cytosolic Ca²⁺-level ([Ca²⁺]_i). The present study explored, whether miltefosine elicits eryptosis.MethodsCell volume has been estimated from forward scatter, phosphatidylserine-exposure from annexin-V-binding, hemolysis from hemoglobin release, [Ca²⁺]_i from Fluo3-fluorescence.ResultsA 48 h exposure to miltefosine (?4.9 μM) was followed by significant decrease of forward scatter and significant increase of annexin-V-binding. The effect was paralleled by significant increase of [Ca²⁺]_i. The annexin-V-binding following miltefosine treatment was significantly blunted in the nominal absence of extracellular Ca²⁺.ConclusionMiltefosine stimulates eryptosis, an effect at least partially due to stimulation of Ca²⁺ entry.     

Effect of honokiol on erythrocytes

Honokiol ((3,5-di-(2-propenyl)-1,1-biphenyl-2,2-diol), a component of Magnolia officinalis, stimulates apoptosis and is thus considered for the treatment of malignancy. In analogy to apoptosis of nucleated cells, erythrocytes may enter eryptosis, a suicidal death characterized by cell shrinkage and by breakdown of cell membrane phosphatidylserine asymmetry with phosphatidylserine-exposure at the erythrocyte surface. Eryptosis may be triggered following increase of cytosolic Ca²⁺-activity ([Ca²⁺]_i). The present study explored, whether honokiol elicits eryptosis. Cell volume has been estimated from forward scatter, phosphatidylserine-exposure from annexin V binding, hemolysis from hemoglobin release, [Ca²⁺]_i from Fluo3-fluorescence, and ceramide from fluorescent antibodies. As a result, a 48 h exposure to honokiol was followed by a slight but significant increase of [Ca²⁺]_i (15 μM), significant decrease of forward scatter (5 μM), significant increase of annexin-V-binding (5 μM) and significant increase of ceramide formation (15 μM). Honokiol further induced slight, but significant hemolysis. Honokiol (15 μM) induced annexin-V-binding was significantly blunted but not abrogated in the nominal absence of extracellular Ca²⁺. In conclusion, honokiol triggers suicidal erythrocyte death or eryptosis, an effect at least in part due to stimulation of Ca²⁺ entry and ceramide formation.     

Safety of Soy-derived Phosphatidylserine in Elderly People

Abstract

Phosphatidylserine (PS) is a phospholipid which has been claimed to enhance neuronal membrane function, and can be derived from several sources. Earlier studies used brain cortex derived PS, of which the human tolerability of 300 mg daily in 130 patients has been shown. The human tolerability of PS derived from soy-bean has not been reported, although it is widely sold as a nutritional supplement which may improve cognitive function in the elderly. We report the results of a study of the safety of two dosages of soy-phosphatidylserine (S-PS) in elderly.

Subjects were 120 elderly of both sexes who fulfilled the more stringent criteria for age-associated memory impairment; some also fulfilled the criteria for age-associated cognitive decline. Subjects were allocated at random to one of the three treatment groups: placebo, 300 or 600 mg S-PS daily. Standard biochemical and hematological safety parameters, blood pressure, heart rate and adverse events were assessed at baseline, after 6 and 12 weeks of treatment.

No significant differences were found in any of the outcome variables between the treatment groups after Bonferonni-Holme correction.

In conclusion, soy derived PS is a safe nutritional supplement for older persons if taken up to a dosage of 200 mg three times daily.     

A lipophilic free radical initiator, 2,2′-azobis (2,4-dimethylvaleronitrile) (AMVN) enhances caspase-dependent apoptosis induced by hyperthermia

Purpose: A free radical initiator, 2,2'-azobis (2-amidinopropane) dehydrochloride (AAPH), was previously found to enhance apoptosis by hyperthermia. Here, but more lipophilic free radical initiator, 2,2'-azobis (2,4-dimethylvaleronitrile) (AMVN) was investigated for its effects as a possible heat sensitizer. Materials and methods: Human myelogenous monocytic leukaemia U937 cells were treated with hyperthermia combined with a various concentration of AMVN for investigating its ability to induce apoptosis and various parameters to identify the pathway. Results: Combined treatment of hyperthermia and AMVN induced DNA fragmentation markedly, while hyperthermia or AMVN alone induced marginal DNA fragmentation. Fractions of cells showed low mitochondrial membrane potential and increased superoxide production after the combined treatment. Experiments using various caspase inhibitors and a fluorogenic monitor of caspase 3 activities indicated that caspase acts both up- and down-stream of mitochondria. Conclusions: AMVN is suggested to be a potential heat sensitizer effective at a lower concentration than AAPH. The possible mechanism is discussed.     

Enhanced efficacy of curcumin with phosphatidylserine-decorated nanoparticles in the treatment of hepatic fibrosis

Hepatic macrophages have been considered as a therapeutic target for liver fibrosis treatment, and phosphatidylserine (PS)-containing nanoparticles are commonly used to mimic apoptotic cells that can specifically regulate macrophage functions, resulting in anti-inflammatory effects. This study was designed to test the efficacy of PS-modified nanostructured lipid carriers (mNLCs) containing curcumin (Cur) (Cur-mNLCs) in the treatment of liver fibrosis in a rat model. Carbon tetrachloride-induced liver fibrosis in rats was used as an experimental model, and the severity of the disease was examined by both biochemical and histological methods. Here, we showed that mNLCs were spherical nanoparticles with decreased negative zeta potentials due to PS decoration, and significantly increased both mean residence time and area under the curve of Cur. In the rats with liver fibrosis, PS-modification of NLCs enhanced the nanoparticles targeting to the diseased liver, which was evidenced by their highest accumulation in the liver. As compared to all the controls, Cur-mNLCs were significantly more effective at reducing the liver damage and fibrosis, which were indicated by in Cur-mNLCs-treated rats the least increase in liver enzymes and pro-inflammatory cytokines in the circulation, along with the least increase in collagen fibers and alpha smooth muscle actin and the most increased hepatocyte growth factors (HGF) and matrix metalloprotease (MMP) two in the livers. In conclusion, PS-modified NLCs nanoparticles prolonged the retention time of Cur, and enhanced its bioavailability and delivery efficiency to the livers, resulting in reduced liver fibrosis and up-regulating hepatic expression of HGF and MMP-2.