尿醛固酮在原发性醛固酮增多症筛查中的价值及钠摄入对其筛查效率的影响

目的探讨尿醛固酮在原发性醛固酮增多症(PA)筛查中的作用并评估钠盐摄入对其筛查效率的影响。方法收集2006-09-2009-07中山大学附属第二医院内分泌科门诊和住院的高血压患者269例,其中包括原发性高血压患者237例(女119例,男118例,平均年龄47.4岁)和PA患者32例(女20例,男12例,平均年龄41.9岁)。所有入组的研究对象均测定立位1h血清醛固酮(SAC)和血浆肾素活性(PRA),并留取24h尿检测尿醛固酮、尿钠和尿钾。通过构建受试者工作特征曲线(ROC),评估尿醛固酮在PA筛查中的效率并确定最佳切点。以尿钠/尿钾作为钠盐摄入的评估方法,以尿钠/尿钾中位数作为切点将原发性高血压组分为高尿钠/尿钾组(n=119)和低尿钠/尿钾组(n=118),分别以这两组高血压人群作为阴性人群,以PA组作为阳性人群进行ROC分析,比较不同钠盐摄入情况对尿醛固酮水平及其PA筛查效率的影响。结果通过构建ROC曲线评估尿醛固酮在PA筛查中的作用,其曲线下面积为0.824(95%CI0.773～0.867,P<0.01),应用Youden’s指数确定尿醛固酮诊断PA的最佳切点值为11.6μg/24h,其敏感性和特异性分别为81.2%(95%CI63.3%～92.7%)和74.3%(95%CI68.2%～79.7%);在原发性高血压患者中,尿醛固酮与尿钠/尿钾呈负相关(r=-0.174,P<0.01)。高尿钠/尿钾组尿醛固酮、SAC和PRA水平明显低于低尿钠/尿钾组。结论尿醛固酮在PA筛查有一定意义,采用11.6μg/24h作为切点可取得较高的筛查效率。钠摄入可影响尿醛固酮水平,但不影响其筛查效率。     

Reconstitution of the human cytoplasmic dynein complex

Cytoplasmic dynein is the major motor protein responsible for microtubule minus-end–directed movements in most eukaryotic cells. It transports a variety of cargoes and has numerous functions during spindle assembly and chromosome segregation. It is a large complex of about 1.4 MDa composed of six different subunits, interacting with a multitude of different partners. Most biochemical studies have been performed either with the native mammalian cytoplasmic dynein complex purified from tissue or, more recently, with recombinant dynein fragments from budding yeast and Dictyostelium. Hardly any information exists about the properties of human dynein. Moreover, experiments with an entire human dynein complex prepared from recombinant subunits with a well-defined composition are lacking. Here, we reconstitute a complete cytoplasmic dynein complex using recombinant human subunits and characterize its biochemical and motile properties. Using analytical gel filtration, sedimentation-velocity ultracentrifugation, and negative-stain electron microscopy, we demonstrate that the smaller subunits of the complex have an important structural function for complex integrity. Fluorescence microscopy experiments reveal that while engaged in collective microtubule transport, the recombinant human cytoplasmic dynein complex is an active, microtubule minus-end–directed motor, as expected. However, in contrast to recombinant dynein of nonmetazoans, individual reconstituted human dynein complexes did not show robust processive motility, suggesting a more intricate mechanism of processivity regulation for the human dynein complex. In the future, the comparison of reconstituted dynein complexes from different species promises to provide molecular insight into the mechanisms regulating the various functions of these large molecular machines.     

醛固酮在乳鼠心肌细胞表达肿瘤坏死因子-α中的作用

目的:观察醛固酮对培养心肌细胞表达肿瘤坏死因子(TNF-α)的影响。方法:在原代培养的新生大鼠心肌细胞,采用免疫组化和RT-PCR,评价10-6M、10-5M醛固酮及10-5M醛固酮拮抗剂螺内酯对心肌细胞表达TNF-α的影响。用凝胶滞留实验(EMSA)验证NF-κB是否参与TNF-α的转录调控,筛选TNF-α可能启动子的位点。结果:TNF-α的免疫组化染色在对照组心肌细胞呈阴性,10-5和10-6M醛固酮作用心肌细胞48h,心肌细胞胞浆中出现棕色颗粒,10-5M醛固酮与螺内酯共同孵育的心肌细胞内TNF-α染色呈阴性。与单纯培养的对照组心肌细胞相比,10-5M,10-6醛固酮刺激心肌细胞24h致心肌细胞TNF-α表达的mRNA显著增高,与螺内酯共同培养的心肌细胞中TNF-αmRNA水平与对照组相比没有明显差异。醛固酮组心肌细胞的核蛋白与TNF-α上游含有潜在NF-κB结合位点的2段寡核苷酸(-619~-591 and-508~-481)的结合强于对照组,螺内酯能阻断这一效应。结论:正常情况下培养的新生鼠心肌细胞内无TNF-α合成,醛固酮通过醛固酮受体促使培养的心肌细胞表达TNF-α,NF-κB核转移是醛固酮激活心肌细胞表达TNF-α的一条通路。     

一氧化氮对小肠消化间期移行性复合运动作用的研究

目的研究一氧化氮（ＮＯ）在小肠消化间期移行性复合运动（ＭＭＣ）控制中的作用。方法在大鼠十二指肠、空肠分别埋置应变片,在动物清醒的状态下分别记录空腹和餐后静脉输注ＮＧ硝基Ｌ精氨酸甲酯（ＬＮＡＭＥ）、Ｌ精氨酸、Ｄ精氨酸、硝普钠和血管紧张素Ⅰ后十二指肠和空肠压力变化。结果在餐后注入一氧化氮合酶抑制剂ＬＮＡＭＥ,可诱发类似空腹状态下的ＭＭＣ运动形式;注入ＮＯ供体硝普钠,则中断空腹时的小肠ＭＭＣ周期,诱发进食后小肠运动形式;Ｌ精氨酸和ＬＮＡＭＥ同时输注,消除ＬＮＡＭＥ的作用,而Ｄ精氨酸无此作用。单独输注Ｌ精氨酸、Ｄ精氨酸或血管紧张素Ⅰ对小肠ＭＭＣ没有影响。结论小肠神经系统ＮＯ紧张性分泌的调节,可能与小肠消化间期和消化期之间小肠运动形式的转换有关,ＮＯ释放增加可导致Ⅱ相时间变长,中断或延长ＭＭＣ;抑制ＮＯ合成与小肠消化间期运动形式的产生有关。     

The complex that inserts lipopolysaccharide into the bacterial outer membrane forms a two-protein plug-and-barrel

The cell surfaces of Gram-negative bacteria are composed of lipopolysaccharide (LPS). This glycolipid is found exclusively in the outer leaflet of the asymmetric outer membrane (OM), where it forms a barrier to the entry of toxic hydrophobic molecules into the cell. LPS typically contains six fatty acyl chains and up to several hundred sugar residues. It is biosynthesized in the cytosol and must then be transported across two membranes and an aqueous intermembrane space to the cell surface. These processes are required for the viability of most Gram-negative organisms. The integral membrane β-barrel LptD and the lipoprotein LptE form an essential complex in the OM, which is necessary for LPS assembly. It is not known how this complex translocates large, amphipathic LPS molecules across the OM to the outer leaflet. Here, we show that LptE resides within the LptD β-barrel both in vitro and in vivo. LptD/E associate via an extensive interface; in one specific interaction, LptE contacts a predicted extracellular loop of LptD through the lumen of the β-barrel. Disrupting this interaction site compromises the biogenesis of LptD. This unprecedented two-protein plug-and-barrel architecture suggests how LptD/E can insert LPS from the periplasm directly into the outer leaflet of the OM to establish the asymmetry of the bilayer.     

CD147 is a regulatory subunit of the gamma-secretase complex in Alzheimer's disease amyloid beta-peptide production

gamma-Secretase is a membrane protein complex that cleaves the beta-amyloid precursor protein (APP) within the transmembrane region, after prior processing by beta-secretase, producing amyloid beta-peptides Abeta(40) and Abeta(42). Errant production of Abeta-peptides that substantially increases Abeta(42) production has been associated with the formation of amyloid plaques in Alzheimer's disease patients. Biophysical and genetic studies indicate that presenilin-1, which contains the proteolytic active site, and three other membrane proteins [nicastrin, anterior pharynx defective-1 (APH-1), and presenilin enhancer-2 (PEN-2)] are required to form the core of the active gamma-secretase complex. Here, we report the purification of the native gamma-secretase complexes from HeLa cell membranes and the identification of an additional gamma-secretase complex subunit, CD147, a transmembrane glycoprotein with two Ig-like domains. The presence of this subunit as an integral part of the complex itself was confirmed through coimmunoprecipitation studies of the purified protein from HeLa cells and of solubilized complexes from other cell lines such as neural cell HCN-1A and HEK293. Depletion of CD147 by RNA interference was found to increase the production of Abeta peptides without changing the expression level of the other gamma-secretase components or APP substrates whereas CD147 overexpression had no statistically significant effect on Abeta-peptide production, other gamma-secretase components or APP substrates, indicating that the presence of the CD147 subunit within the gamma-secretase complex down-modulates the production of Abeta-peptides.     

The role of sodium in hypertension is more complex than simply elevating arterial pressure

Excessive salt intake exacerbates hypertension and further increases left-ventricular mass in clinical essential and experimental hypertension. Additionally, a growing body of evidence strongly suggests that high dietary salt loading exerts detrimental cardiac effects independently of its hemodynamic load. The clinical evidence of cardiac structural and functional alterations associated with salt is, however, scarce. In order to explore the purported beliefs in humans, in this review we draw on our experimental studies in naturally occurring hypertension and discuss the clinical implications of the nonhemodynamic mechanisms underlying these salt-related changes.     

Herpes simplex virus-infected cell protein 0 blocks the silencing of viral DNA by dissociating histone deacetylases from the CoREST-REST complex

A preeminent phenotype of the infected cell protein 0 (ICP0) of herpes simplex virus 1 (HSV-1) is that it acts as a promiscuous transactivator. In most cell lines exposed to DeltaICP0 mutant virus at low ratios of virus per cell infection, alpha genes are expressed but the transition to beta and gamma gene expression does not ensue, but can be enhanced by inhibitors of histone deacetylases (HDACs). Earlier studies have shown that ICP0 interacts with CoREST and displaces HDAC1 from the CoREST-REST-HDAC1/2 complex. HDAC1 and CoREST are then independently translocated to the cytoplasm. Here, we test the hypothesis that ICP0 blocks the silencing of HSV DNA by displacing HDAC1 from the CoREST-REST complex. Specifically, first, mapping studies led us to construct a truncated CoREST (CoREST(146-482)) that in transfected cells displaced HDAC1 from the CoREST-REST complex. Second, we constructed two viruses. In BACs encoding the entire HSV-1, we replaced the gene encoding ICP0 with AmpR to yield a DeltaICP0 mutant R8501. We also replaced ICP0 with CoREST(146-482) to yield recombinant R8502. The yield of R8502 mutant virus in Vero, HEp-2, and human embryonic lung cells exposed to 0.1 pfu of virus per cell was 100-, 10-, and 10-fold higher, respectively, than those of R8501 mutant virus. In Vero cells, the yield of R8502 was identical with that of wild-type virus. We conclude that CoREST(146-482) functionally replaced ICP0 and that, by extension, ICP0 acts to block the silencing of viral DNA by displacing HDAC1/2 from the CoREST-REST complex.     

MAPK信号通路在肝癌发生发展及治疗中的作用

丝裂原活化蛋白激酶(MAPK)信号通路的异常活化与肝癌的发生、发展、转移密切相关。介绍了MAPK通路蛋白在肝癌中的表达及其在肝癌增殖、分化、转移中的作用,阐述了MAPK信号通路在肝癌治疗及预后评价中的价值。认为MAPK信号通路在肝癌的发生发展及治疗中发挥非常重要的作用,是肝癌治疗及预后评价的潜在分子靶点。     

Structure of the Dietzia Mrp complex reveals molecular mechanism of this giant bacterial sodium proton pump

Multiple resistance and pH adaptation (Mrp) complexes are sophisticated cation/proton exchangers found in a vast variety of alkaliphilic and/or halophilic microorganisms, and are critical for their survival in highly challenging environments. This family of antiporters is likely to represent the ancestor of cation pumps found in many redox-driven transporter complexes, including the complex I of the respiratory chain. Here, we present the three-dimensional structure of the Mrp complex from a Dietzia sp. strain solved at 3.0-Å resolution using the single-particle cryoelectron microscopy method. Our structure-based mutagenesis and functional analyses suggest that the substrate translocation pathways for the driving substance protons and the substrate sodium ions are separated in two modules and that symmetry-restrained conformational change underlies the functional cycle of the transporter. Our findings shed light on mechanisms of redox-driven primary active transporters, and explain how driving substances of different electric charges may drive similar transport processes.

In all life kingdoms, homeostasis of Na⁺ and H⁺ is essential for many aspects of cell physiology, including maintaining appropriate osmotic pressure, intracellular pH, and electrostatic membrane potential (ΔΨ) (1). A variety of Na⁺/H⁺ exchangers (also called antiporters) are directly involved in regulating the homeostasis of Na⁺ and H⁺, and form a major part of the superfamily of monovalent cation/proton antiporters (CPA) (2, 3). Members of the CPA superfamily have been identified and characterized in fungi, plants, and mammals (4). Usually, Na⁺/H⁺ antiporters utilize the electrochemical potential of protons, commonly called proton motive force (PMF), to catalyze efflux of Na⁺ as well as other monovalent cations. The stoichiometric ratio of protons to sodium ions may vary from transporter to transporter, primarily depending on the strength of PMF in which the transporter has been evolved to function optimally (see discussion in SI Appendix, Supplementary Material). One family of Na⁺/H⁺ antiporters, namely, the NhaA-like single-subunit antiporters, has been well studied (5, 6), and was shown to represent the canonical type of secondary active transporters, in which the proton influx and the Na⁺ efflux most likely share the same pathway. In contrast, multiple resistance and pH adaptation (Mrp) complexes are the most sophisticated known secondary active transporters. They alone form a unique family of atypical antiporters, termed CPA3 (7–10), in which protons and Na⁺ ions are likely to flow through distinct physical paths. Under physiological conditions, the driving force for Mrp transport can only come from protons but not Na⁺ ions (SI Appendix, Supplementary Material); therefore, protons are referred to as the driving substance. Moreover, Mrp complexes are essential for alkaliphilic and/or halophilic microorganisms to adapt to their extreme environments (11).A member of the Mrp family usually contains one copy of each of seven subunits called MrpA-F, all of which contribute to a total of ∼50 predicted transmembrane helices (TMs) (SI Appendix, Fig. S1). In the so-called group II Mrp complexes, subunit MrpB is fused to the C terminus of MrpA while keeping other subunits essentially unchanged, and the remaining nonfused complexes are termed group I (12). Amino acid sequence analysis showed that MrpA and MrpD are homologous (13), and their corresponding three-dimensional (3D) folding pattern is referred to as Mrp antiporter/pump folding. In addition, every subunit of the Mrp complex was shown to be important for the Na⁺/H⁺ antiporter activity (14). This notion raises an interesting question as to why a seemingly simple task requires such a complexed protein machinery.Furthermore, a number of important energy-converting protein complexes, including those from bacteria (15), plants (16), and mammalian animals (17), are phylogenetically related to the Mrp complex. For instance, components of the respiratory chain complex I (henceforth called complex I) are highly homologous to MrpA and MrpD subunits. In the membrane arm of complex I, three Mrp pump subunits are aligned in a head-to-tail fashion, and presumably carry out proton efflux, and their driving forces come from the redox reaction between NADH (nicotinamide adenine dinucleotide) and quinone (18). In some anaerobic archaea lacking complex I, the membrane-bound hydrogenase (MBH) complex replaces the functional role of complex I (19). In this type of ion-pumping complex, a subcomplex containing Mrp homologous subunits functions as a Na⁺ pump, and the driving force for this pump comes from the redox reaction between reduced ferredoxin and protons, subsequently generating hydrogen gas. In complex I, MBH complex, as well as other related redox-driven transporters, cellular redox energy (i.e., differential redox potential between electron donors and acceptors) is converted to transmembrane electrochemical potential of either protons or Na⁺; thus, these primary active transporters play essential roles in cellular energy homeostasis (20). It is generally accepted that, in these redox-driven ion pumps, electrostatic interactions between the transferred electrons and pumps drive the conformational changes that are required for the cation export (21, 22). While a number of 3D structures have been reported in recent years for such redox-driven transporters (15, 16, 23), the detailed mechanisms responsible for the energy conversion remain under debate. Taken together, the Mrp complex likely shares a common ancestor with the ion-pumping module(s) in a variety of redox-driven transporters, and thus understanding of the mechanism of Mrp antiporter complex should shed light on the energy-coupling mechanisms of all Mrp antiporter-containing transporters.Here, we report the cryoelectron microscopy (cryo-EM) structure of the group II Mrp complex from Dietzia sp. DQ12-45-1b, a strain of Gram⁺ bacteria isolated from the production water of a deep subterranean oil reservoir and which has also been found in a number of high-salinity environments (24). Our results from structural and functional analyses show that this Mrp complex contains two modules responsible for proton transport and Na⁺ pumping, respectively. On the basis of these findings, we propose a hitherto unknown mechanism for energy coupling between the two modules.     

Essential role of the linear ubiquitin chain assembly complex and TAK1 kinase in A20 mutant Hodgkin lymphoma

More than 70% of Epstein–Barr virus (EBV)-negative Hodgkin lymphoma (HL) cases display inactivation of TNFAIP3 (A20), a ubiquitin-editing protein that regulates nonproteolytic protein ubiquitination, indicating the significance of protein ubiquitination in HL pathogenesis. However, the precise mechanistic roles of A20 and the ubiquitination system remain largely unknown in this disease. Here, we performed high-throughput CRISPR screening using a ubiquitin regulator-focused single-guide RNA library in HL lines carrying either wild-type or mutant A20. Our CRISPR screening highlights the essential oncogenic role of the linear ubiquitin chain assembly complex (LUBAC) in HL lines, which overlaps with A20 inactivation status. Mechanistically, LUBAC promotes IKK/NF-κB activity and NEMO linear ubiquitination in A20 mutant HL cells, which is required for prosurvival genes and immunosuppressive molecule expression. As a tumor suppressor, A20 directly inhibits IKK activation and HL cell survival via its C-terminal linear-ubiquitin binding ZF7. Clinically, LUBAC activity is consistently elevated in most primary HL cases, and this is correlated with high NF-κB activity and low A20 expression. To further understand the complete mechanism of NF-κB activation in A20 mutant HL, we performed a specifically designed CD83-based NF-κB CRISPR screen which led us to identify TAK1 kinase as a major mediator for NF-κB activation in cells dependent on LUBAC, where the LUBAC-A20 axis regulates TAK1 and IKK complex formation. Finally, TAK1 inhibitor Takinib shows promising activity against HL in vitro and in a xenograft mouse model. Altogether, these findings provide strong support that targeting LUBAC or TAK1 could be attractive therapeutic strategies in A20 mutant HL.

Hodgkin lymphoma (HL) is the most common (6,000 to 7,000 new cases per year) lymphoma subtype in young adulthood (1). Although patients with this disease commonly respond well to current treatments, the survival rate for those at advanced stages or with relapsed/refractory disease remains low (2, 3), and novel treatments are clearly needed. Importantly, despite many recent advances and accumulating data (4), the pathogenic connection between the signaling input from the tumor microenvironment and genetic alterations within the malignant cells still needs to be further explored. The conceptual distillation of this connection into actionable interventions will be the key to new therapies for patients suffering with HL.Aberrant activation of the NF-κB pathway is one of the most striking oncogenic mechanisms in HL, which can be driven by signaling input from the tumor microenvironment. The malignant component of classical HL tumors, the Hodgkin and Reed/Sternberg (HRS) cells, represents a minority of the cells within these tumors, with the bulk of the tumor composed of various inflammatory cell types which provide abundant cytokines that can activate NF-κB. HRS cells express several TNFR family members on the surface that can stimulate the NF-κB–signaling pathways (5). NF-κB activity promotes HRS cell survival by up-regulating prosurvival genes, as well as cytokines and chemokines to shape the cellular microenvironment of HL (5).Genetic alterations are also found to contribute to the constitutive NF-κB activity of malignant HL cells and to amplify the input signal from the microenvironment. Up to 40% of HL cells are latently infected with Epstein–Barr virus (EBV) worldwide, although patients born in developed countries have a lower rate of positivity to EBV (6). EBV encodes viral oncoproteins that can promote NF-κB activation and latent membrane proteins 1 and 2A (LMP1 and LMP2A) (7). In EBV-negative HL, genetic or epigenetic mechanisms are emerging, i.e., REL amplification (8–10), frameshift/nonsense mutations in NFKBIA (encoding IκBα) (11–14), and NFKBIE (encoding IκBε) (15). In addition to the aforementioned genetic events, inactivation of the ubiquitin-editing protein TNFAIP3 (A20) by nonsense/deletions or missense mutations (mostly in C-terminal zinc fingers) is the most recurrent genetic alteration, occurring in 30 to 40% of all cases and 70 to 80% of EBV-negative HL cases (16, 17). A20 is a deubiquitinating or ubiquitin-modifying enzyme that counteracts the function of activated E3 ligases, thereby suppressing NF-κB activation. These observations strongly suggest that an E3 ligase(s) regulates ubiquitin-dependent signals to promote NF-κB activation and HRS cell proliferation, potentially regulating the microenvironment of HL. The identity of this ubiquitin enzyme(s) and its mechanism(s) of action is completely unknown. To address these gaps in knowledge, it will be important to gain a complete understanding of how the ubiquitin-modifying machinery regulates HL pathogenesis in order to identify and exploit critical therapeutic vulnerabilities.In this study, we decided to use the CRISPR library screening technologies to address these unresolved questions and to gain a complete understanding of how the NF-κB pathway and the ubiquitin-modifying machinery regulate HL pathogenesis.     

Genetics of inflammatory bowel disease： The role of the HLA complex

The human leucocyte antigen (HLA) complex on chromosome 6p21.3 is the most extensively studied genetic region in Inflammatory bowel disease (IBD). Consistent evidence of linkage to IBD3 (6p21.1-23), an area which encompasses the HLA complex, has been demonstrated for both Crohn's disease and ulcerative colitis, and a number of replicated associations with disease susceptibility and phenotype have recently emerged. However, despite these efforts the HLA susceptibility gene (s) for IBD remain elusive, a consequence of strong linkage disequilibrium, extensive polymorphism and high gene density across this region. This article reviews current knowledge of the role of HLA complex genes in IBD susceptibility and phenotype, and discusses the factors currently limiting the translation of this knowledge to clinical practice.     

Intermediates in the assembly of mitotic checkpoint complexes and their role in the regulation of the anaphase-promoting complex

The mitotic (or spindle assembly) checkpoint system prevents premature separation of sister chromatids in mitosis and thus ensures the fidelity of chromosome segregation. Kinetochores that are not attached properly to the mitotic spindle produce an inhibitory signal that prevents progression into anaphase. The checkpoint system acts on the Anaphase-Promoting Complex/Cyclosome (APC/C) ubiquitin ligase, which targets for degradation inhibitors of anaphase initiation. APC/C is inhibited by the Mitotic Checkpoint Complex (MCC), which assembles when the checkpoint is activated. MCC is composed of the checkpoint proteins BubR1, Bub3, and Mad2, associated with the APC/C coactivator Cdc20. The intermediary processes in the assembly of MCC are not sufficiently understood. It is also not clear whether or not some subcomplexes of MCC inhibit the APC/C and whether Mad2 is required only for MCC assembly and not for its action on the APC/C. We used purified subcomplexes of mitotic checkpoint proteins to examine these problems. Our results do not support a model in which Mad2 catalytically generates a Mad2-free APC/C inhibitor. We also found that the release of Mad2 from MCC caused a marked (although not complete) decrease in inhibitory action, suggesting a role of Mad2 in MCC for APC/C inhibition. A previously unknown species of MCC, which consists of Mad2, BubR1, and two molecules of Cdc20, contributes to the inhibition of APC/C by the mitotic checkpoint system.The mitotic (or spindle assembly) checkpoint system is a surveillance mechanism that monitors correct attachment of chromosomes to the mitotic spindle and thus ensures the fidelity of chromosome segregation (1–4). Kinetochores that are not attached, or are incorrectly attached to the spindle, generate an inhibitory signal that prevents progression into anaphase by inhibiting the Anaphase-Promoting Complex/Cyclosome (APC/C) ubiquitin ligase. APC/C ubiquitylates securin and cyclin, the degradation of which is necessary for chromosome segregation and for exit from mitosis. When the checkpoint is activated, a Mitotic Checkpoint Complex (MCC), which inhibits the APC/C, is assembled. MCC is composed of the checkpoint proteins BubR1, Bub3, and Mad2, associated with the APC/C coactivator Cdc20 (5). When the checkpoint is extinguished, MCC is disassembled (6–8). Disassembly of MCC is initiated by the release of Mad2, a process carried out by the joint action of the TRIP13 AAA-ATPase and the Mad2-binding protein p31^comet (9–12).The intermediary processes in the assembly of MCC and the possible role of different subcomplexes of MCC in the inhibition of APC/C are not sufficiently understood. When the checkpoint is activated, an early event that takes place on the kinetochore is the conversion of Mad2 from an open (O-Mad2) to a closed (C-Mad2) conformation that forms a tight complex with Cdc20 (1, 3). It has been proposed that the C-Mad2-Cdc20 (MC) subcomplex associates with BubR1-Bub3 to form the MCC (2, 4), but this has not yet been tested by direct experimentation. The roles of Cdc20-BubR1 intermediary subcomplexes are more obscure. [Bub3 is constitutively associated with BubR1, but has no appreciable influence on MCC function or structure (13, 14). Therefore, Bub3 was omitted from the subcomplexes of BubR1 and the mitotic checkpoint complexes described in this paper.] Cdc20 may bind to two different sites on BubR1. When the mitotic checkpoint is active, Cdc20 associates with a binding site at the N-terminal region of BubR1 in a process that requires Mad2 and that leads to MCC assembly (13, 15). Cdc20 also binds tightly to a second site at an internal region of BubR1 (15, 16, 17). The latter process does not require Mad2 and is not dependent on the mitotic checkpoint. The binding of Cdc20 to the internal site has been reported to inhibit the APC/C in vitro by the sequestration of Cdc20 (16, 17), but the role of this process in the regulation of APC/C with bound Cdc20 (APC/C^Cdc20) is not clear. It is also not clear whether or not Mad2-containing MCC is the major checkpoint inhibitor of APC/C. Nilsson et al. (18) reported that, in checkpoint complexes from HeLa cell extracts, only a small fraction of Cdc20 is associated with Mad2 and suggested that the role of Mad2 may be limited to the loading of Cdc20 onto BubR1. Cleveland and coworkers (19) furthermore proposed that Mad2-free Cdc20-BubR1 is the main checkpoint inhibitor of APC/C. These authors also suggested that C-Mad2 catalytically amplifies the production of the Cdc20-BubR1 inhibitor. According to this hypothesis, following the loading of Cdc20 onto BubR1, Mad2 dissociates and then binds another molecule of Cdc20 to produce the Cdc20-BubR1 inhibitor (19).In the present study, the roles of subcomplexes of mitotic checkpoint proteins in MCC assembly and in the regulation of APC/C activity were investigated by the use of purified subcomplexes. Our results do not support the catalytic model of C-Mad2 action. We furthermore find that the release of Mad2 from MCC decreases markedly, but not completely, its APC/C inhibitory action, suggesting that that the presence of Mad2 in MCC is important for the inhibition of APC/C^Cdc20. A previously unknown species of MCC, which consists of Mad2 and two molecules of Cdc20 bound to the two binding sites of BubR1, contributes to the inhibition of APC/C^Cdc20.     

原发性高血压病患者醛固酮逃逸现象的临床观察

目的 观察原发性高血压病患者使用血管紧张素转换酶抑制剂(ACEI)依那普利后并发的醛固酮逃逸现象.方法 65例原发性高血压病患者使用依那普利治疗,分别于治疗前及治疗后1、3、6个月采血,检测血管紧张素Ⅱ和醛固酮浓度,根据治疗3个月时的醛固酮浓度,判断有无并发醛固酮逃逸.结果 依那普利治疗后1个月,AngⅡ、Ald与治疗前相比均下降,但3个月时,AngⅡ有所升高,Ald明显增高.65例中有28例并发醛固酮逃逸,发生率约43%.结论 原发性高血压病患者长期(3个月以上)使用ACEI后,部分患者会出现醛固酮逃逸现象.