Histone Deacetylase Inhibition Induces Apoptosis in Neuroblastoma

Histone deacetylase inhibitors constitute a promising new treatment for cancer due to their novel site of action and low toxicity. Almost all histone deacetylase inhibitors currently in clinical development have anti-proliferate activities against cells in cultures, and specially cause cell cycle arrest, differentiation and apoptosis. Interestingly, despite their rapid advance into clinical use, the cellular responses leading to these effects remain unclear. We recently reported that histone deacetylase inhibitor treatment induces apoptosis of neuroblastoma cells by increasing the acetylation of Ku70 in the cytoplasm, resulting in the release of Bax from Ku70. Subsequently, Bax releases cytochrome c from mitochondria causing apoptosis. Here we will discuss these findings and the implications of our model for the further clinical development of histone deacetylase inhibitors in the treatment of cancer.     

牛α—sl酪蛋白基因序列指导htPA突变体小基因在小鼠乳腺中的表达

将包括α－sl酪蛋白基因的启动子、LAtPA小基因、α－sl酪蛋白基因下游调控序列的融合基因经显微注射至小鼠的受精卵中,获得了5只转基因阳性鼠,其中1只转基因阳性雌鼠乳清中LAtPA的含量为0．18αg/ml,说明构建的LAtPA小基因能够正确表达出有生物活性的LAtPA, 在酪蛋白基因调控序列的指导下在小鼠乳汁中表达。     

Improved Medium for Extraction of Plasminogen Activator from Tissue

Quantitation of plasminogen activators present in tissue may depend to a large extent on the extraction procedure used to solubilize the enzymes. Potassium thiocyanate solution is known to be an efficient solubilizer, but it can inhibit assay systems other than fibrin plates. An equally effective acetate-detergent extractant is reported here which can be used with the highly sensitive azocase inolytic assay procedure. The results indicate that a threefold increase in activator activity can be extracted from selected tissues relative to that previously reported for a phosphate-detergent extractant. The extraction medium contains 75 mM K acetate, 0.3 M NaCl, 0.1 M L-arginine, 10 mM EDTA, 0.25% Triton X-100, final pH 4.2.     

Activation and Inhibition of Histone Deacetylase 8 by Monovalent Cations

The metal-dependent histone deacetylases (HDACs) catalyze hydrolysis of acetyl groups from acetyllysine side chains and are targets of cancer therapeutics. Two bound monovalent cations (MVCs) of unknown function have been previously observed in crystal structures of HDAC8; site 1 is near the active site, whereas site 2 is located >20 Å from the catalytic metal ion. Here we demonstrate that one bound MVC activates catalytic activity (K_1/2 = 3.4 mm for K⁺), whereas the second, weaker-binding MVC (K_1/2 = 26 mm for K⁺) decreases catalytic activity by 11-fold. The weaker binding MVC also enhances the affinity of the HDAC inhibitor suberoylanilide hydroxamic acid by 5-fold. The site 1 MVC is coordinated by the side chain of Asp-176 that also forms a hydrogen bond with His-142, one of two histidines important for catalytic activity. The D176A and H142A mutants each increase the K_1/2 for potassium inhibition by ≥40-fold, demonstrating that the inhibitory cation binds to site 1. Furthermore, the MVC inhibition is mediated by His-142, suggesting that this residue is protonated for maximal HDAC8 activity. Therefore, His-142 functions either as an electrostatic catalyst or a general acid. The activating MVC binds in the distal site and causes a time-dependent increase in activity, suggesting that the site 2 MVC stabilizes an active conformation of the enzyme. Sodium binds more weakly to both sites and activates HDAC8 to a lesser extent than potassium. Therefore, it is likely that potassium is the predominant MVC bound to HDAC8 in vivo.     

Hemorrhagic Transformation After Tissue Plasminogen Activator Treatment in Acute Ischemic Stroke

Cellular and Molecular Neurobiology - Hemorrhagic transformation (HT) is a common complication after thrombolysis with recombinant tissue-type plasminogen activator (rt-PA) in ischemic stroke. In...     

Twin-Arginine Translocation of Active Human Tissue Plasminogen Activator in Escherichia coli

When eukaryotic proteins with multiple disulfide bonds are expressed at high levels in Escherichia coli, the efficiency of thiol oxidation and isomerization is typically not sufficient to yield soluble products with native structures. Even when such proteins are secreted into the oxidizing periplasm or expressed in the cytoplasm of cells carrying mutations in the major intracellular disulfide bond reduction systems (e.g., trxB gor mutants), correct folding can be problematic unless a folding modulator is simultaneously coexpressed. In the present study we explored whether the bacterial twin-arginine translocation (Tat) pathway could serve as an alternative expression system for obtaining appreciable levels of recombinant proteins which exhibit complex patterns of disulfide bond formation, such as full-length human tissue plasminogen activator (tPA) (17 disulfides) and a truncated but enzymatically active version of tPA containing nine disulfides (vtPA). Remarkably, targeting of both tPA and vtPA to the Tat pathway resulted in active protein in the periplasmic space. We show here that export by the Tat translocator is dependent upon oxidative protein folding in the cytoplasm of trxB gor cells prior to transport. Whereas previous efforts to produce high levels of active tPA or vtPA in E. coli required coexpression of the disulfide bond isomerase DsbC, we observed that Tat-targeted vtPA and tPA reach a native conformation without thiol-disulfide oxidoreductase coexpression. These results demonstrate that the Tat system may have inherent and unexpected benefits compared with existing expression strategies, making it a viable alternative for biotechnology applications that hinge on protein expression and secretion.     

Fibrin Targeting of Echogenic Liposomes with Inactivated Tissue Plasminogen Activator

Fibrin-specific molecular targeting strategies are desirable for site-specific imaging and treatment of late stage atheroma, but fibrin-specific antibodies are difficult to produce and present immunogenicity problems. Tissue plasminogen activator (tPA) is an endogenous protein that has been shown to bind fibrin with high affinity and may circumvent antibody difficulties. Use of tPA-derived proteins or peptides, however, requires that the plasminogen-activating proteolytic activity be neutralized or removed. As an initial step in determining the feasibility of this targeting strategy, human recombinant tPA (Activase®) was irreversibly inhibited with D-phe-L-pro-L-arg-chloromethyl ketone (PPACK) and conjugated to intrinsically echogenic liposomes (ELIP) by a thioether coupling protocol. Fibrin-binding affinities were assessed with a novel two‐stage fibrin pad ELISA. We achieved 95–99% inactivation, while retaining both tPA fibrin-binding activities of K_D ～ 2 nM and 33 nM. Thermodynamic analysis of the PPACK-inactivated tPA (tPA(P)) revealed highly exothermic interactions, indicative of ionic associations, especially for the higher affinity. The conjugation efficiency of tPA(P) to ELIP was within the range of that previously achieved for IgG and exhibited satisfactory fibrin targeting, characterized by striking increases of enthalpy and entropy increments. Evidence for coupling of noncovalent association energetics with the phosphatidylethanolamine major phase transition, observed in previous IgG antibody conjugations, was also evident in this case, but the nature of the transduction mechanism was different. These results demonstrate that tPA-derived components lacking proteolytic activity can be employed as fibrin-targeting agents for delivery of therapeutic and diagnostic formulations.     

The Release of Factor Viii and Tissue Plasminogen Activator can not be Blocked by Specific Antagonists to Vasopressin

Abstract

Vasopressin and in particular 1-deamino-8-D-arginine vasopressin (DDAVP) can release factor VIII (FVIII) and tissue plasminogen activator (tPA) to the blood. In the present study DDAVP was injected in conscious dogs which had been preloaded with specific antagonists either against vasopressin's vasopressor response (V₁-receptors) or its antidiuretic response (V₂-receptors). The presence in the blood of either of the antagonists had no effect on the increase of FVIII or tPA following stimulation with DDAVP. It is therefore concluded that the effect of DDAVP on coagulation and fibrinolysis is elicited via a new class of receptors different from the known V₁- and V₂-receptors.     

Plasminogen Activator and Plasminogen Activator Inhibitor in Mouse Ovaries during Periovulatory Periods

Changes of ovarian tPA,uPA and PA inhibitor activities were examined in PMSG-and hCG-treatedimmature mice during periovulatory periods.The results show that 15% of the gonadotropin-treatedanimals ovulated 8 hrs after hCG administration,about 6-8 hrs earlier than in rat.It is also shownthat not only tPA activity,but also uPA activity,was regulated by gonadotropins in ovarianhomogenates and granulosa cells,and they reached maximum prior to ovulation.No measurableamount of PAI-1 activity could be detected in mouse granulosa cell conditioned medium andfollicular fluid,but considerable amount of α_2-antiplasmin,a specific inhibitor for plasmin,wasfound in follicular fluid.Cumulus-oocyte complexes contain mainly tPA.Since the ovulated eggsstill have high tPA activity,it is thought that the enzyme in the oocyte may play an important rolein implantation.     

组织纤溶酶原激活剂突变体在转基因小鼠乳腺中的表达

组织纤溶酶原激活剂(tPA)是一种较理想的溶血栓药物,本研究采用其突变体——长效组织纤溶酶原激活剂(LAtPA)的cDNA作为目的基因,将它插入羊β-乳球蛋白(BLG)基因起始密码之前,使LAtPA的转录、翻译受控于BLG基因的5′、3′序列,再将所构建的BLG-LAtPA用显微注射方法建立转基因鼠,经点杂交筛选和Southern印迹鉴定,获得2只LAtPA基因整合阳性鼠,并在阳性母鼠乳汁中检测到有溶纤活性的LAtPA表达,表达水平1.5μg/ml。在这两只转基因鼠的9只F1代子鼠中,有5只是阳性的,tPA表达水平维持在1～2μg/ml,BLGtPA融合基因整合到小鼠基因组,能稳定地遗传给子代。     

Plants Release Precursors of Histone Deacetylase Inhibitors to Suppress Growth of Competitors

To secure their access to water, light, and nutrients, many plant species have developed allelopathic strategies to suppress competitors. To this end, they release into the rhizosphere phytotoxic substances that inhibit the germination and growth of neighbors. Despite the importance of allelopathy in shaping natural plant communities and for agricultural production, the underlying molecular mechanisms are largely unknown. Here, we report that allelochemicals derived from the common class of cyclic hydroxamic acid root exudates directly affect the chromatin-modifying machinery in Arabidopsis thaliana. These allelochemicals inhibit histone deacetylases both in vitro and in vivo and exert their activity through locus-specific alterations of histone acetylation and associated gene expression. Our multilevel analysis collectively shows how plant-plant interactions interfere with a fundamental cellular process, histone acetylation, by targeting an evolutionarily highly conserved class of enzymes.     

组织型纤溶酶原活化物的纯化及性质研究

新鲜猪心组织制成丙酮粉后,用0.45mol/L,pH4.2醋酸钾抽提组织型纤溶酶原活化物(t-PA)。抽提液经硫酸铵盐析,Benzamidine和血纤维蛋白亲和层析,Sephadex G-150凝胶过滤,纯化得到t-PA。比活11000IU/mg,经SDS-聚丙烯酰胺凝胶电泳鉴定,分子量为67000。 本文比较了t-PA、高分子量尿激酶(H-UK)和低分子量尿激酶(L-UK)的热稳定性及抑制剂对它们的抑制作用。结果表明,抑制剂对H-UK的抑制作用最强,L-UK次之,t-PA最弱;三者的热稳定性相似。     

人黑色素瘤细胞分泌的组织纤溶酶原激活剂(t-PA)的纯化

 本文报道了培养的人黑色素瘤细胞分泌的组织纤溶酶原激活剂(t-PA)的纯化方法。Bowes株人黑色素瘤细胞的分泌产物,经CM-Sephadex C--50层析,赖氨酸-Sepharose 4B,苯甲眯-sepharose 4B亲和层析后,即可得到纯化470倍的蛋白纯品。样品经聚丙烯酰胺凝胶电泳鉴定为均一单带,测得其分子量约为72kD。纯化的t-PA与尿激酶相比较,发现前者有更高亲和纤维蛋白的能力。     

导向性纤溶酶原激活剂的研究

溶栓疗法是血栓治疗中的一种重要措施.研制具有高选择性的导向性纤溶酶原激活剂有着重大的理论意义和实用价值.采用血栓特异的单克隆抗体及其片段来介导溶栓剂已展示出较好的应用前景.双功能抗体以及同时具有抗栓,抗凝活性的小肽正逐渐拓宽人们有关导向分子研制的视野.所有这一切都将随着分子生物学技术的不断完善而付诸实现.     

Tissue Plasminogen Activator in Trabecular Meshwork Attenuates Steroid Induced Outflow Resistance in Mice

Tissue plasminogen activator, a serine protease encoded by the PLAT gene is present in the trabecular meshwork (TM) and other ocular tissues and has been reported to be downregulated by treatment with steroids in vitro. Steroids are known to cause changes in outflow facility of aqueous humor in many species. In the present study, we tested whether overexpression of PLAT can prevent and/or reverse the outflow facility of mouse eyes treated with steroids. Animals received bilateral injection with 20 µl of triamcinolone acetonide (TA) (40mg/ml) suspension subconjunctivally to induce outflow facility changes. Some animals received unilateral intracameral injection with 2 µl of adenoviral suspension [3-4x10¹² virus genomes per milliliter (vg/ml)] carrying sheep PLAT cDNA (AdPLAT) either concurrently with TA injection or one week after TA injection, whereas others received bilateral intracameral injection with 2µl of adenoviral suspension (9x10¹² vg/ml) carrying no transgene (AdNull) concurrently with TA injection. Animals were sacrificed one week after AdPLAT or AdNull treatment. Endogenous mRNA expression levels of mouse PAI-1 and MMP-2, -9 and -13 were also measured using qRT-PCR. Outflow facility one week after AdPLAT administration was increased by 60% and 63% respectively for animals that had not or had been pretreated with steroids. Overexpression of PLAT significantly upregulated expression of PAI-1, MMP-2, -9 and -13 compared to the levels found in TA only treated eyes. These findings suggest that overexpression of PLAT in TM of mouse eyes can both prevent and reverse the decrease in outflow facility caused by steroid treatment and is associated with upregulation of MMPs.     

Tissue Plasminogen Activator Enhancing Activity of Vaso-Pressin Analogues in Monkeys: Structure-Activity Study

Abstract

Marmocets were used in a structure activity study of the ability of vaso-pressin analogues to activate plasminogen activator (tPA). In evaluation of dDAVP analogues with L – alanine migrating from position 2 to 9 we found [L – Ala⁴]dDAVP and [L – Ala⁵]dDAVP to be potent activators of tPA. Double substitutions in dDAVP showed that combinations of a modification in position 4 valine with a change at position 2 (2 – O – methyltyrosine) generated tPA releasing activity. On the other hand enlargement of the substituent at position 2 (2 – O – ethyltyrosine) completely eliminated the activity of [L – Val⁴]dDAVP. The tPA activity is dependent on the position of a positively charged group at the amino acid in position 8 of the peptide chain. A shift of the guanido group further away from the backbone (D – arginine to D – homoarginine) resulted in a loss of tPA activating properties.     

抗t-PA抗体水平和血栓的相关性研究

目的:研究自身免疫性疾病病人抗t-PA抗体水平和病人血栓形成之间的关系。方法:用酶联免疫吸附法(ELISA)检测原发性抗磷脂综合症和红斑狼疮患者(32例狼疮样抗凝物阳性,32例狼疮样抗凝物阴性)与40例健康对照的IgG类抗t-pA抗体的水平,用Pearson Chi-Square test的方法分析了病人体内IgG类抗t-PA抗体水平和血栓之间的相关性。结果:本试验研究的病人群体中IgG类抗t-PA抗体阳性的有13(20.3%)个,并且我们的研究表明IgG类抗t-PA抗体阳性和血栓病史显著相关(P=0.04)。结论:原发性抗磷脂综合症和红斑狼疮病人群体中有较高的IgG类抗t-PA抗体水平,它们可能和病人体内血栓的发生有关。