利用细胞培养基或动物血浆中的荧光标记产物测定鞘磷脂合酶活性

鞘磷脂合酶（SMS）催化神经酰胺（ceramide,Cer．）转变为鞘磷脂（sphingomyelin,SM）。近来的研究表明神经酰胺和鞘磷脂参与了代谢综合征的过程,因而鞘磷脂合酶被认为是开发抗代谢综合征药物的潜在靶点。鞘磷脂合酶有两个同工酶,分别称为鞘磷脂合酶1（SMS1）和鞘磷脂合酶2（SMS2）。这两种同工酶的亚细胞定位不同,在不同组织中的表达水平也有差异。到目前为止,已发表有多种方法测定组织和细胞匀浆中的总SMS活性,这些方法通过分析总反应体系或细胞内的酶促反应产物来衡量SMS活性。本文介绍一种测定SMS活性或筛选SMS抑制剂的新方法。我们将荧光标记的神经酰胺（NBD-Cer．）作为底物与细胞孵育,或者将该底物注射到小鼠体内,然后监测在细胞培养基或小鼠血浆中出现的荧光标记的鞘磷脂（NBD-SM）的含量。采用这种办法可有效检测出D609（一种鞘磷脂合酶抑制剂）对细胞和小鼠整体SMs活性的抑制作用。我们进一步采用该方法检测了SMS1基因敲除小鼠和sMs2基因敲除小鼠的SMS活性,结果发现注射底物后,SMS2基因敲除小鼠（而不是SMS1基因敲除小鼠）血浆中NBD-SM的堆积被明显阻断。因而该方法可用于检测生理或药理条件下在体或离体组织的SMS活性、筛选SMS抑制剂、甚至筛选SMS2特异性抑制剂。     

mPGES-1——一个新的药物开发靶点

综述膜结合型前列腺素E2合酶-1（mPGES-1）的生物学性质、生理和病理作用,以及将其作为一个新的药物作用靶点用于药物开发的可能性。mPGES-1是3种前列腺素E2合酶之一,属于诱导型表达的酶,能被致炎因子诱导而大量表达,在多种疾病,如关节炎、炎症相关性发热和疼痛、动脉粥样硬化及癌症的病理生理过程中均发挥着重要作用。     

酸性鞘磷脂酶在酒精性肝病中的作用CSCD

鞘磷脂酶(sphingomyelinase,SMase)是鞘磷脂代谢的关键酶,通过水解磷酸二酯键产生第二信使神经酰胺(ceramide,Cer),参与调节机体多种生化反应。依据SMase生物活性的最适宜pH环境,将其分为酸性鞘磷脂酶(acid sphingomyelinase,ASMase)、中性SMase和碱性SMase。其中生物体内ASMase的含量及生物学活性均占比最高,是SMase最重要也是分布最广的亚型^([1])。     

“鞘磷脂变阻器”在细胞中的作用机制研究

鞘磷脂代谢产物在细胞中具有重要的生理学意义。其代谢产物中神经酰胺和鞘氨醇具有抑制细胞生长、促进细胞凋亡的作用。而1-磷酸鞘氨醇（S1P）则具有促进细胞增殖、分化的作用。鞘磷脂代谢产物的动态平衡决定着细胞的生存和死亡。据此有人提出了“鞘磷脂变阻器”的概念。深入探讨鞘磷脂代谢产物．不但能从分子层面去更多了解细胞的生存机制,而且能为临床提供疾病治疗的新靶点。     

酸性鞘磷脂酶活性调控及相关药物研究进展

酸性鞘磷脂酶(ASM)是鞘磷脂代谢的关键酶,调节细胞增殖、分化、衰老和凋亡等。ASM过少或缺陷易引起尼曼-匹克病,过多会导致神经性病变、抑郁症、脓毒症和糖尿病等。研究发现,ASM的激活受环境因素、药物因素和其他因素的影响,细菌、病毒、电离辐射、重金属、心理压力、乙醇和细胞因子等可激活ASM,诱发上述疾病发生。应用ASM直接抑制或功能性抑制药物可减轻相关疾病带来的损伤。ASM活性增强,可促使肿瘤细胞凋亡,具有抗肿瘤作用,有望成为新的抗肿瘤药物靶标及肿瘤预测因子。本文就ASM活性调控及相关药物研究进展进行简要综述。     

猪血红细胞膜中鞘磷脂的提取及含量测定

目的从红细胞中提取鞘磷脂。方法采用分离血液、取血球、溶血、脱水等方法提取鞘磷脂 ,并用高效液相色谱分析其含量。结果可提取到红细胞膜中的鞘磷脂 ,其含量为 5 .2 %。结论此法工艺可行 ,为工业化生产提供了依据     

脂蛋白相关磷脂酶A2及其抑制剂的研究进展

脂蛋白相关磷脂A2(Lp-PLA2)也称血浆血小板激活因子乙酰水解酶,是介导氧化低密度脂蛋白引起炎症和动脉粥样硬化的一个关键性酶。Lp-PLA2作为一个动脉粥样硬化药物发现的新靶点,已发现了许多化合物对其具有选择性抑制作用。本文综述了Lp-PLA2的生物学性质、与动脉粥样硬化的关系及其抑制剂用于治疗动脉粥样硬化的研究进展。     

二萜合酶的研究进展

植物二萜类化合物广泛存在于自然界中,是一类重要的天然化合物。随着一些具有重要经济价值的二萜不断被发现,其生物合成中的二萜合酶也倍受关注。二萜合酶催化活性的多样性决定了终产物(二萜化合物)的丰富性。本文重点探讨了近年来二萜生物合成途径和二萜合酶的类型、克隆、催化机制及其合成生物学应用等研究进展。     

内源性一氧化氮合酶抑制物与动脉粥样硬化及药物的作用机制

一氧化氮（nitric oxide，NO）是机体重要的气体信息分子，具有多种生物学效应。NO由L-精氨酸在一氧化氮合酶（nitric oxide synthase，NOS）催化作用下生成。非对称二甲基精氨酸（asymmetric dimethylarginine，ADMA）是内源性的L-精氨酸类似物，能竞争性抑制NOS活性而减少NO生成，与动脉粥样硬化等多种心血管疾病密切相关，是一种新的心血管疾病危险因子。[第一段]     

Nrf2-ARE通路及其在缺血性心肌损伤中的作用

Nrf2-ARE通路是近年来发现的尤为重要的抗氧化应激通路,其信号转导机制复杂而广泛。近年来,研究发现其在缺血性心肌损伤的发病机理中发挥着重要作用,有望成为治疗缺血性心肌损伤的一个新的治疗靶点。本文就其生物学功能及其在缺血性心肌损伤中的作用作一阐述。     

Regulation of death and growth signals at the plasma membrane by sphingomyelin synthesis: implications for hematological malignancies

Resistance to death receptor ligands (such as FasL and TRAIL) and anticancer treatments is a hallmark of cancer cells. Ceramide, a biologically active sphingolipid, antagonizes cell growth and promotes apoptosis and non-apoptotic forms of cell death. The intracellular levels of ceramide are highly regulated via complex metabolic pathways. Sphingomyelin synthases (SMS) 1 and 2 convert ceramide to sphingomyelin (SM), a ubiquitous phospholipid in mammals. A growing body of evidence in the literature indicates that SMSs likely modulate hematological cell growth and sensitivity to stress-induced apoptosis. On one hand, complete and sustained inhibition of SMS activity is likely to alter membrane composition and properties through membrane SM depletion, perturbing intracellular signaling pathways and leukemia cell growth and conferring partial resistance to death receptor ligands. On the other hand, different patents & reports point to anti-apoptotic functions for SMSs. In patients with chemoresistant leukemia, a decreased intracellular ceramide level was associated with a higher SMS activity. Thus, SMSs and cofactors may constitute original pharmacological targets to treat leukemia.     

The effect of sphingomyelin synthase 2 (SMS2) deficiency on the expression of drug transporters in mouse brain

Sphingomyelin synthase (SMS), the last enzyme involved in the biosynthesis of sphingomyelin (SM), plays a critical role in the constitution of cell membrane and has impact on the expression of membrane proteins. SMS2, one of two SMS enzymes, is predominantly located in the plasma membrane, and is mainly expressed in the brain. Therefore, it is conceivable that SMS2 deficiency may have impact on expression of some membrane proteins, such as membrane-bound drug transporters. Using SMS2 gene deficient mouse brain tissues, we studied the gene and protein expression profiles of drug transporters, ERM proteins (ezrin/radixin/moesin) and the cytoskeleton protein, β-actin, in mouse brain by RT-PCR, western blot and immunohistochemistry analysis. We found that the mRNA expression of Mdr1 rather than the other drug transporters was significantly decreased in the SMS2 deficient brain. Accordingly, the expression and the function of Pgp (Mdr1/P-glycoprotein) were significantly downregulated in brain. In addition, the substantially downregulated expression of ezrin and β-actin was also observed in the SMS2 deficient brain. The immunohistochemistry analysis further revealed the suppressed expression of Pgp, ezrin and β-actin in both cortex and paraventricular areas of SMS2 knockout mice. Furthermore, both Pgp and β-actin were found to be co-immunoprecipitated with ezrin from the total brain lysate, suggesting the association between Pgp, ezrin and β-actin in the brain. These results indicate that SMS2 participates in the expression regulation of drug transporters, particularly Pgp, and suggest that SMS2 may be a potential target for enhancing drug access to the brain.     

1-磷酸鞘氨醇受体的研究进展

1-磷酸鞘氨醇S1P(Sphingosine 1-phosphate,S1P)是调节细胞内外多种生物学功能的重要信号分子之一,作用于S1P受体后,在很多生理和病理过程中发挥重要的调节作用。Sphk-S1P-S1PR信号通路及其在炎症、肿瘤、动脉粥样硬化、自体免疫系统疾病、神经系统疾病等多种疾病中的作用已成为目前研究的热点之一。越来越多的S1P受体调节剂被研发。     

肝细胞癌相关因子与泛素-蛋白酶体通路关系的研究

肝细胞癌因为其治疗方法的局限性,成为世界上致死率最高的恶性肿瘤疾病之一。泛素-蛋白酶体通路（ubiquitin-proteasome pathway,UPP）是调节多种细胞生物学过程的重要机制,也是恶性肿瘤相关疾病调节异常的潜在靶点。本文就泛素-蛋白酶体通路与肝细胞癌发生相关因子[乙型肝炎病毒（HBV）、P27、NF-κB等]的关系加以综述,以寻找新的方法对肝细胞癌的发生发展加以干预。     

Pharmacological activities of a new glycosaminoglycan,acharan sulfate isolated from the giant African snail Achatina fulica

Acharan sulfate (AS) is a glycosaminoglycan (GAG) prepared from the giant African snail, Achatina fulica. In this study, some biological activities of AS were evaluated on the basis of structural similarities to heparin/heparan sulfate and the biological functions of GAGs. We demonstrated that it exhibited strong immunostimulating activities as measured by carbon clearance test in mice and in vivo phagocytosis. It also exhibited a significant hypoglycemic activity in epinephrine (EP)-induced hyperglycemia as well as antifatigue effects by weight-loaded forced swimming test. And it showed hypolipidemic activities in cholesterol-rich mixture induced hyperlipidemia in rats. The above results indicate that AS has diverse biological activities and suggest therapeutically important target molecules.     

Calmodulin mediates sulfur mustard toxicity in human keratinocytes

Sulfur mustard (SM) causes blisters in the skin through a series of cellular changes that we are beginning to identify. We earlier demonstrated that SM toxicity is the result of induction of both death receptor and mitochondrial pathways of apoptosis in human keratinocytes (KC). Because of its importance in apoptosis in the skin, we tested whether calmodulin (CaM) mediates the mitochondrial apoptotic pathway induced by SM. Of the three human CaM genes, the predominant form expressed in KC was CaM1. RT-PCR and immunoblot analysis revealed upregulation of CaM expression following SM treatment. To delineate the potential role of CaM1 in the regulation of SM-induced apoptosis, retroviral vectors expressing CaM1 RNA in the antisense (AS) orientation were used to transduce and derive stable CaM1 AS cells, which were then exposed to SM and subjected to immunoblot analysis for expression of apoptotic markers. Proteolytic activation of executioner caspases-3, -6, -7, and the upstream caspase-9, as well as caspase-mediated PARP cleavage were markedly inhibited by CaM1 AS expression. CaM1 AS depletion attenuated SM-induced, but not Fas-induced, proteolytic processing and activation of caspase-3. Whereas control KC exhibited a marked increase in apoptotic nuclear fragmentation after SM, CaM1 AS cells exhibited normal nuclear morphology up to 48h after SM, indicating that suppression of apoptosis in CaM1 AS cells increases survival and does not shift to a necrotic death. CaM has been shown to activate the phosphatase calcineurin, which can induce apoptosis by Bad dephosphorylation. Interestingly, whereas SM-treated CaM1-depleted KC expressed the phosphorylated non-apoptotic sequestered form of Bad, Bad was present in the hypophosphorylated apoptotic form in SM-exposed control KC. To determine if pharmacological CaM inhibitors could attenuate SM-induced apoptosis via Bad dephosphorylation, KC were pretreated with the CaM-specific antagonist W-13 or its less active structural analogue W-12. Following SM exposure, KC exhibited Bad dephosphorylation, which was inhibited in the presence of W-13, but not with W-12. Consequently, W-13 but not W-12 markedly suppressed SM-induced proteolytic processing and activation of caspase-3, as well as apoptotic nuclear fragmentation. Finally, while the CaM antagonist W-13 and the calcineurin inhibitor cyclosporin A attenuated SM-induced caspase-3 activation, inhibitors for CaM-dependent protein kinase II (KN62 and KN93) did not. These results indicate that CaM, calcineurin, and Bad also play a role in SM-induced apoptosis, and may therefore be targets for therapeutic intervention to reduce SM injury.     

Sphingomyelinase D/Ceramide 1-Phosphate in Cell Survival and Inflammation

Sphingolipids are major constituents of biological membranes of eukaryotic cells. Many studies have shown that sphingomyelin (SM) is a major phospholipid in cell bilayers and is mainly localized to the plasma membrane of cells, where it serves both as a building block for cell architecture and as a precursor of bioactive sphingolipids. In particular, upregulation of (C-type) sphingomyelinases will produce ceramide, which regulates many physiological functions including apoptosis, senescence, or cell differentiation. Interestingly, the venom of some arthropodes including spiders of the genus Loxosceles, or the toxins of some bacteria such as Corynebacterium tuberculosis, or Vibrio damsela possess high levels of D-type sphingomyelinase (SMase D). This enzyme catalyzes the hydrolysis of SM to yield ceramide 1-phosphate (C1P), which promotes cell growth and survival and is a potent pro-inflammatory agent in different cell types. In particular, C1P stimulates cytosolic phospholipase A2 leading to arachidonic acid release and the subsequent formation of eicosanoids, actions that are all associated to the promotion of inflammation. In addition, C1P potently stimulates macrophage migration, which has also been associated to inflammatory responses. Interestingly, this action required the interaction of C1P with a specific plasma membrane receptor, whereas accumulation of intracellular C1P failed to stimulate chemotaxis. The C1P receptor is coupled to Gi proteins and activates of the PI3K/Akt and MEK/ERK1-2 pathways upon ligation with C1P. The proposed review will address novel aspects on the control of inflammatory responses by C1P and will highlight the molecular mechanisms whereby C1P exerts these actions.     

三七皂苷对持续炎症加速兔动脉粥样硬化形成中氧化应激影响的研究

目的:初步探讨三七皂苷(PNS)对持续炎症加速兔动脉粥样硬化(AS)形成中氧化应激的影响。方法:将24只日本大耳兔随机均分为对照组、高脂组、炎症组和PNS组,各组给予相应治疗。动态监测各组动物血清中C反应蛋白(CRP)、一氧化氮(NO)、丙二醛(MDA)含量和超氧化物歧化酶(SOD)活性;测定胸主动脉AS斑块面积。结果:炎症组血清CRP、MDA含量显著增高,SOD活性和NO含量显著下降,胸主动脉AS斑块面积显著增加,且CRP、MDA含量与斑块面积均呈显著性正相关,与高脂组有显著性差异(P<0.01)。与炎症组比较,PNS组可减轻炎症AS症状,防止AS形成。结论:持续炎症增强氧化应激可能是加速高脂饮食兔AS的形成机制之一。PNS可降低氧化应激,减轻炎症反应,防治AS。     

Established and emerging vascular risk factors and the development of aortic stenosis: an opportunity for prevention?

Background: Aortic stenosis (AS) is the commonest valvular heart disease in the developed world. It is becoming increasingly accepted that the pathogenesis of AS and of its preceding abnormalities, aortic valve sclerosis (AVS) and aortic valve calcification (AVC), shares many characteristics with the atherosclerotic process. Objective: To assess the contribution of established and emerging vascular risk factors in the development of AS and to evaluate the potential of pharmacological intervention to modify the natural history of AS. Methods: We reviewed the epidemiological data that link AS and atherosclerosis and studies of vasculoprotective agents in patients with AS. Results/conclusions: AS, AVS and AVC share many common risk factors with atherosclerosis and are possible markers of preclinical vascular disease. Statins appear to delay the progression of AS. However, more studies are needed before introducing such pharmacologic treatment for AS. The future may point towards targeted prevention of AS.     

细菌ClpP蛋白酶功能及其抗菌药物靶点的研究进展

摘要：ClpP(Casein lytic proteinase P)是一种广泛存在于真核细胞和原核生物中的丝氨酸蛋白酶，可与多种类型AAA+ (ATPase associated with various cellular activity)超家族ATP酶组成多种ClpP蛋白酶复合物，其主要功能是清除或降解细菌胞内合成不当、受损伤、变性聚集或无用的蛋白，并维持正常代谢和压力刺激下胞内蛋白质的动态平衡。近年研究表明，细菌ClpP可协助病原菌在宿主体内生存、繁殖和播散，ClpP在细菌致病过程中发挥重要作用，其相关致病机制也受到广泛关注。近年，细菌对抗生素耐药性不断增强，已严重威胁人类健康，ClpP蛋白酶因其独特的蛋白水解作用而成为抗菌药物研究的新靶点。本文综述了ClpP在不同病原体中的结构、分子功能和不同作用以及相应药物开发方面的研究进展。