Damage-associated molecular pattern (DAMP) activation in melanoma: investigation of the immunogenic activity of 15-deoxy, Δ12,14 prostamide J2

Metastatic melanoma is the most deadly skin neoplasm in the United States. Outcomes for this lethal disease have improved dramatically due to the use of both targeted and immunostimulatory drugs. Immunogenic cell death (ICD) has emerged as another approach for initiating antitumor immunity. ICD is triggered by tumor cells that display damage-associated molecular patterns (DAMPs). These DAMP molecules recruit and activate dendritic cells (DCs) that present tumor-specific antigens to T cells which eliminate neoplastic cells. Interestingly, the expression of DAMP molecules occurs in an endoplasmic reticulum (ER) stress-dependent manner. We have previously shown that ER stress was required for the cytotoxic activity of the endocannabinoid metabolite, 15-deoxy, Δ^12,14 prostamide J₂ (15dPMJ₂). As such, the current study investigates whether 15dPMJ₂ induces DAMP signaling in melanoma. In B16F10 cells, 15dPMJ₂ caused a significant increase in the cell surface expression of calreticulin (CRT), the release of ATP and the secretion of high-mobility group box 1 (HMGB1), three molecules that serve as surrogate markers of ICD. 15dPMJ₂ also stimulated the cell surface expression of the DAMP molecules, heat shock protein 70 (Hsp70) and Hsp90. In addition, the display of CRT and ATP was increased by 15dPMJ₂ to a greater extent in tumorigenic compared to non-tumorigenic melanocytes. Consistent with this finding, the activation of bone marrow-derived DCs was upregulated in co-cultures with 15dPMJ₂-treated tumor compared to non-tumor melanocytes. Moreover, 15dPMJ₂-mediated DAMP exposure and DC activation required the electrophilic cyclopentenone double bond within the structure of 15dPMJ₂ and the ER stress pathway. These results demonstrate that 15dPMJ₂ is a tumor-selective inducer of DAMP signaling in melanoma.     

克百威及其代谢产物的小鼠骨髓红细胞微核试验研究

[目的]利用微核试验研究克百威及其4种代谢产物的遗传毒性。[方法]分别以0．1、0．2、0．4mg/kg3个不同剂量水平的克百威及其代谢产物3-羟基呋喃丹、3-酮基呋喃丹、呋喃酚和亚硝基呋喃丹腹腔注射染毒健康小鼠，24h后以同样剂量再次注射染毒，6h后脱颈椎处死动物，制片，观察并计数嗜多染红细胞的微核率，进行统计分析。[结果]克百威、呋喃酚及3-酮基呋喃丹小鼠胸骨骨髓嗜多染红细胞微核实验为阴性结果，高剂量处理组微核率分别为1．3‰、3．25‰和3．62‰，与阴性对照组(1．83‰)差异无显著性；而高剂量组3-羟基呋喃丹(7．00‰)和三个剂量组的亚硝基呋喃丹(微核率分别为3．62‰、5．00‰、7．85‰)均有明显微核效应。[结论]克百威、呋喃酚和3-酮基呋喃丹在受试剂量下微核试验阴性，3-羟基呋哺丹和亚硝基呋喃丹微核试验阳性，提示可能对染色体具有突变效应。     

阴道毛滴虫代谢产物体外对精子活力的影响

目的 :研究阴道毛滴虫代谢产物体外对人精子活力的影响。　方法 :阴道毛滴虫体外培养 ,除去虫体 ,留取培养液 ,稀释成三个浓度 ;将 10例正常生育男性的优化精子分为等体积的 4份 ,组成以下 4组 :A组加入未培养滴虫的空白培养液 ,B ,C ,D组依次加入前述浓度梯度的培养液 (1.2× 10 9/L、6× 10 8/L、1.2× 10 8/L) ,分别于 30s、1、2、4和 6h时采用计算机辅助精子分析系统 (CASA)分析精子运动参数。　结果 :阴道毛滴虫浓度在 6× 10 8/L以上时 ,精子的活率和活力与对照组相比都显著降低 (P <0 .0 5 ) ,并呈浓度依赖性和时间依赖性。　结论 :阴道毛滴虫代谢产物体外能抑制精子活力 ,可能是造成不孕不育的原因之一。     

Axial tissue diffusion can account for the disparity between current models of hepatic elimination for lipophilic drugs

An assumption of previous models of hepatic elimination is that there is negligible axial diffusion in the liver. We show, by construction of a stochastic model and analysis of published data, that compounds which are readily diffusible and partitioned into hepatocytes may undergo axial tissue diffusion. The compounds most likely to be affected by axial tissue diffusion are the lipophilic drugs for which the cell membranes provide little resistance and which are highly extracted, thereby creating steep concentration gradients along the sinusoid at steady state. This phenomenon greatly modifies the availability of the compound under conditions of altered hepatic blood flow and protein binding. For moderately diffusible compounds, these relationships are similar to those predicted by the simplistic venous-equilibrium model. Hence, the paradoxical ability of the venous-equilibrium model to describe the steady-state kinetics of lipophilic drugs such as lidocaine, meperidine, and propranolol may be finally resolved. The effects of axial tissue diffusion and vascular dispersion on hepatic availability of drugs are compared. Vascular dispersion is of major importance to the availability of poorly diffusible compounds, whereas axial tissue diffusion becomes increasingly dominant for highly diffusive and partitioned substances.This study was supported by the National Health and Medical Research Council of Australia.     

Involvement of the 12-lipoxygenase pathway of arachidonic acid metabolism in homosynaptic long-term depression of the rat hippocampus

Low-frequency stimulation is associated with long-term depression (LTD) of synaptic efficacy in various brain structures. Like long-term potentiation (LTP), homosynaptic LTD in area CA1 of the hippocampus appears to require NMDA receptor activation, changes in postsynaptic calcium concentration and phospholipase A₂ (PLA₂) activation. Arachidonic acid (AA) is released after the activation of calcium-dependent phospholipases and free AA is rapidly metabolized to a family of bioactive products (the eicosanoids) which are thought to be both intracellular and extracellular messengers. In the present study, we investigated the involvement of the cyclooxygenase and lipoxygenase pathways of AA metabolism in the formation of homosynaptic LTD in the rat hippocampus. Stimulation at 1 Hz for 15 min was used to produce homosynaptic depression in area CA1 of hippocampal slices. LTD induction was partially blocked by bromophenacyl bromide (50–100 μM), a selective PLA₂ inhibitor, and by the a nonselective lipoxygenase inhibitor nordihydroguaiaretic acid (NDGA; 100 μM). In contrast, the specific cyclooxygenase blocker indomethacin (100 μM) did not significantly reduce hippocampal LTD. Since NDGA interferes with LTD formation, we examined whether specific inhibitors of 5- and 12-lipoxygenases were capable of blocking LTD expression. The 12-lipoxygenase inhibitor baicalein at a concentration of 50 μM reduced LTP formation when given in the bath, an effect that was less pronounced with the 5-lipoxygenase inhibitor AA-861. These data suggest that the activation of endogenous PLA₂ and the formation of 12-lipoxygenase metabolites of AA may be important factors controlling the expression of hippocampal LTD.     

Concentration-dependent metabolism of diazepam in mouse liver

Previous mouse liver studies with diazepam (DZ),N-desmethyldiazepam (NZ), and temazepam (TZ) confirmed that under first-order conditions, DZ formed NZ and TZ in parallel. Oxazepam (OZ) was generatedvia NZ and not TZ despite that preformed NZ and TZ were both capable of forming OZ. In the present studies, the concentration-dependent sequential metabolism of DZ was studied in perfused mouse livers and microsomes, with the aim of distinguishing the relative importance of NZ and TZ as precusors of OZ. In microsomal studies, theK _ms andV _maxs, corrected for binding to microsomal proteins, were 34 μM and 3.6 nmole/min per mg and 239 μM and 18 nmole/min per mg, respectively, forN-demthylation andC ₃-hydroxylation of DZ. TheK _ms andV _maxs forN-demethylation andC ₃-hydroxylation of TZ and NZ, respectively, to form OZ, were 58 μM and 2.5 nmole/min per mg and 311 μM and 2 nmole/min per mg, respectively. The constants suggest that at low DZ concentrations, NZ formation predominates and is a major source of OZ, whereas at higher DZ concentrations, TZ is the important source of OZ. In livers perfused with DZ at input concentrations of 13 to 35 μM, the extraction ratio of DZ (E{DZ}) decreased from 0.83 to 0.60. NZ was the major metabolite formed although its appearance was less than proportionate with increasing DZ input concentration. By contrast, the formation of TZ increased disporportionately with increasing DZ concentration, whereas that for OZ decreased and paralleled the behavior of NZ. Computer simulations based on a tubular flow model and thein vitro enzymatic parameters provided a poorin vitro-organ correlation. TheE{DZ}, appearance rates of the metabolites, and the extraction ratio of formed NZ (E{NZ, DZ}) were poorly predicted; TZ was incorrectly identified as the major precursor of OZ. Simulations with optimized parameters imporved the correlations and identified NZ as the major contributor of OZ. Saturation of DZN-demethylation at higher DZ concentrations increased the role of TZ in the formation of OZ. The poor aqueous solubility (limiting the concentration range of substrates usedin vitro), avid tissue binding and the coupling of enzymatic reactions in liver, favoring sequential metabolism, are possible explanations for the poorin vitro-organ correlation. This work emphasizes the complexity of the hepatic intracellular milieu for drug metabolism and the need for additional modeling efforts to adequately describe metabolite kinetics. This work was supported by the Medical Research Council of Canada (MA-9104).     

磁共振波谱评价大脑半球代谢物的偏侧优势

为了利用无创性的磁共振波谱成像技术研究大脑半球代谢物的非对称性,本研究对56例正常成年人行磁共振波谱成像,主要对双侧大脑半球的4个解剖区域:额叶、颞叶、枕叶及顶叶的代谢物天门冬氨酸(NAA)、肌酸(Cr)、胆碱(Cho)进行测量,并以Cr为内参照物比较NAA和Cho相对含量(NAA/Cr、Cho/Cr)的侧别差异。结果表明:双侧额叶之间的NAA/Cr存在显著性差异,而Cho/Cr无显著性差异;在双侧颞叶之间,这2种比值均存在显著性差异;双侧枕叶之间Cho/Cr存在显著性差异,而NAA/Cr无显著性差异;双侧顶叶之间的NAA/Cr存在显著性差异,而Cho/Cr无显著性差异。本研究结果提示双侧大脑半球的代谢物存在偏侧优势。     

Pharmacokinetic and pharmacodynamic study of the combination of furosemide retard and triamterene

Summary The pharmacodynamics and pharmacokinetics of the combination of furosemide retard (30 mg)/triamterene (50 mg) were compared with furosemide (30 mg) in 18 healthy male volunteers aged 39.3±6.3 years. After the administration of furosemide the onset of its effect was very rapid, reaching a maximum between 1.5 to 3 h, and followed by rebound after 9 to 10.5 h. In contrast the combination furosemide retard/triamterene showed a protracted course with a duration of effect up to 12 h. The general effect over 12 h of the two preparations was equivalent with respect to the excretion of urine, sodium, chloride and calcium, but the combination caused significantly less excretion of potassium (p0.05) than furosemide. After a lag-phase of 33.9±5.4 min the maximum plasma concentration of furosemide was reached after 3.47±0.66 h, and the elimination half-life was approximately 2 h. After a lag-phase of 33.0±17.8 min the maximum plasma concentration of the main metabolite of triamterene, the OH-TA sulphuric acid ester, was reached after 1.7±0.59 h, and its elimination half-life amounted to 1.25±0.37 h. Because of the sustained release of furosemide from the retard-formulation, its principal pharmacokinetic parameters were better adapted to those of triamterene. The consequences were not only a protracted effect but also an improved electrolyte profile, especially with regard to reduced loss of potassium. In the case of renal insufficiency, however, the potassium level in serum might be increased to an undesirable extent.     

仙鹤草茎与叶的代谢组学比较分析

目的 通过代谢组学分析仙鹤草茎与叶中代谢物及其代谢通路差异,进一步阐明仙鹤草药效物质基础,促进仙鹤草合理开发利用。方法 取仙鹤草茎、叶新鲜样本,经液氮冷冻后提取代谢物,采用液相色谱-质谱法检测,所得数据运用统计学方法分析并与数据库比对鉴定。结果 筛选并鉴定出105个具有明显差异的代谢物,其中茎与叶相比有34个代谢物上调、71个代谢物下调。差异代谢物共注释到62条代谢通路中。茎中叶黄素、天冬酰胺、胍丁胺及多种氨基酸等物质含量更高,氨基酸代谢更强;叶中酚酸类、黄酮类物质积累更为丰富,次生代谢更强。结论 仙鹤草茎与叶均含有丰富的药效活性成分,但叶的次生代谢更强,次生代谢物积累更为丰富,此差异或可为仙鹤草针对性开发利用提供参考。     

骨碎补内生菌Lecanicillium sp. GZWMJZ-847中3个新3-氢化萘特特拉姆酸衍生物

目的 研究骨碎补内生蜡蚧菌Lecanicillium sp. GZWMJZ-847的次级代谢产物和抑菌活性。方法 采用大米培养基对菌株Lecanicillium sp. GZWMJZ-847发酵;采用凝胶柱色谱、正反相硅胶色谱等方法对化合物进行分离纯化;采用核磁共振谱、质谱、NMR计算等方法确定化合物的化学结构;采用营养肉汤稀释法测试化合物对病原细菌及酵母菌的抑菌活性、平板法测试化合物对病原霉菌的抑菌活性。结果 从菌株的发酵产物中分离鉴定了3个新的和3个已知的3-氢化萘特特拉姆酸衍生物,包括蜡蚧特特拉姆酸A（1）、蜡蚧特特拉姆酸B（2）、蜡蚧特特拉姆酸C（3）、Sch 210971（4）、Sch 210972（5）、Sch 213766（6）。化合物1和4～6对4株人体革兰阳性病原菌和1株植物革兰阴性病原菌具有抑制作用,最小抑菌浓度（minimun inhibitory concentration,MIC）为7.8～125 mmol/L。化合物6对油菜菌核病菌和小麦赤霉病菌表现出一定的抑制作用,半数抑制浓度分别为（22.05±0.62）、（55.54±1.72）μg/mL。结论 蜡蚧特特拉姆酸A～C（1～3）为新的3-氢化萘特特拉姆酸衍生物,骨碎补内生菌Lecanicillium sp. GZWMJZ-847产生的3-氢化萘特特拉姆酸对人体病原菌和植物病原菌可表现出抑制作用。