胆碱能M受体拮抗剂：2α－（2＇，2＇－二取代基－2＇－羟基乙氧基）托品烷光学异构体合成

胆碱能Ｍ受体拮抗剂：２α－（２＇，２＇－二取代基－２＇－羟基乙氧基）托品烷光学异构体合成王林，恽榴红，张其楷（军事医学科学院毒物药物研究所，北京１００８５０）目前临床所用托品类抗胆碱药一般为３位取代托品酯类衍生物，这类药物作用较广泛，但选择性不高。Ａ...     

肾康宁片中黄芪甲苷的薄层扫描法测定

目的:建立肾康宁片中黄芪甲甙含量测定方法。方法:双波长薄层扫描法,经水饱和正丁醇液冷浸超声提取,再上D101大树脂纯化,以氯仿-甲醇-水(65:30:10)下层液为展开剂,检测波长为520nm,参比波长为700nm。结果:平均加样回收率为97.8％(RSD=1.4％,n=6),标准曲线r=0.9996。结论:方法结果可靠、操作简单,可作为肾康宁片的质量控制标准。     

压电频移中和萃取法测定微克量枸橼酸维静宁

用涂圈压电石英晶体频移一中和萃取法测定枸橼酸维静宁(咳必清),检出限0.02μg,在0.05～1.6μg范围内呈线性,相关系数0.999,变异系数为2.3%。与经典的中和萃取法相比,本法具有明显的优越性。     

ProNGF,a cytokine induced after myocardial infarction in humans,targets pericytes to promote microvascular damage and activation

Treatment of acute cardiac ischemia focuses on reestablishment of blood flow in coronary arteries. However, impaired microvascular perfusion damages peri-infarct tissue, despite arterial patency. Identification of cytokines that induce microvascular dysfunction would provide new targets to limit microvascular damage. Pro–nerve growth factor (NGF), the precursor of NGF, is a well characterized cytokine in the brain induced by injury. ProNGF activates p75 neurotrophin receptor (p75^NTR) and sortilin receptors to mediate proapoptotic responses. We describe induction of proNGF by cardiomyocytes, and p75^NTR in human arterioles after fatal myocardial infarction, but not with unrelated pathologies. After mouse cardiac ischemia-reperfusion (I-R) injury, rapid up-regulation of proNGF by cardiomyocytes and p75^NTR by microvascular pericytes is observed. To identify proNGF actions, we generated a mouse expressing a mutant Ngf allele with impaired processing of proNGF to mature NGF. The proNGF-expressing mouse exhibits cardiac microvascular endothelial activation, a decrease in pericyte process length, and increased vascular permeability, leading to lethal cardiomyopathy in adulthood. Deletion of p75^NTR in proNGF-expressing mice rescues the phenotype, confirming the importance of p75^NTR-expressing pericytes in the development of microvascular injury. Furthermore, deficiency in p75^NTR limits infarct size after I-R. These studies identify novel, nonneuronal actions for proNGF and suggest that proNGF represents a new target to limit microvascular dysfunction.The primary therapeutic goal after acute myocardial infarction (MI) is to limit the duration of ischemia and to establish reperfusion using angioplasty or thrombolysis. However, even with improved arterial flow, a significant proportion of patients experience microvascular damage that leads to decreased microvascular perfusion and chronically impaired heart function (Eltzschig and Collard, 2004; Bekkers et al., 2010). At this time, the proinflammatory cytokines induced by ischemia that mediate microvascular dysfunction or apoptosis after cardiac ischemia remain largely unknown.We considered whether nerve growth factor (NGF), and specifically its uncleaved precursor proNGF, could act as a potential proapoptotic and proinflammatory cytokine in the ischemic heart. NGF is initially synthesized as proNGF, which is normally cleaved intracellularly to release mature NGF (Heymach and Shooter, 1995). Mature NGF binds to the TrkA receptor tyrosine kinase to mediate survival and differentiative effects in neurons (Reichardt, 2006). Under pathological conditions, proNGF is secreted and acts as a distinct ligand to promote neuronal apoptosis by binding to the p75 neurotrophin receptor (p75^NTR), a member of the tumor necrosis factor receptor family, and the transmembrane receptor sortilin (Lee et al., 2001; Nykjaer et al., 2004). This receptor complex activates stress and apoptotic signaling molecules, such as JNK (c-Jun N-terminal kinase) and caspase-3 (Nykjaer et al., 2005; Jansen et al., 2007; Volosin et al., 2008; Hempstead, 2009).ProNGF and p75^NTR are present at low to undetectable levels in normal, uninjured adult tissues (Fanburg-Smith and Miettinen, 2001; Harrington et al., 2004; Lommatzsch et al., 2005; Hempstead, 2009). However, they are rapidly induced after acute neuronal injury and mediate cell death or degeneration after seizures or axotomy (Harrington et al., 2004; Volosin et al., 2008). In addition, proNGF is up-regulated in neurodegenerative diseases and aging (Pedraza et al., 2005; Jansen et al., 2007). However, ngf mRNA is expressed in many organs, and secreted mature NGF promotes sympathetic innervation during development and regulates sympathetic tone in the adult (Donovan et al., 1995; Glebova and Ginty, 2005; Habecker et al., 2008). In the adult heart, mature NGF is secreted tonically by cardiac myocytes to modulate synaptic transmission by sympathetic neurons (Luther and Birren, 2006). Additionally, within hours of cardiac ischemia-reperfusion (I-R) injury in rodents, ngf mRNA is induced (Hiltunen et al., 2001), and immunoreactivity to the mature NGF domain increases in human hearts after acute MI (Meloni et al., 2010). These studies, however, do not distinguish whether mature NGF or proNGF is induced after cardiac ischemia. P75^NTR expression is also induced in the vasculature after acute injury to the aorta (Donovan et al., 1995), and p75^NTR activation promotes vascular smooth muscle and endothelial cell death in vitro (Wang et al., 2000; Kim et al., 2004). Genetic deletion of p75^NTR in mice (p75^−/−) results in reduced apoptosis of vascular smooth muscle cells after vascular injury, suggesting that locally produced neurotrophins regulate this response (Kraemer, 2002).The induction of ngf mRNA by cardiomyocytes and of p75^NTR by vascular cells after injury suggests a potential paracrine role for NGF or proNGF in modulating vascular integrity. Microvascular endothelial survival depends on reciprocal interactions with neighboring pericytes during development, and pericytes maintain microvascular structure and function in the adult animal (Gaengel et al., 2009). Disruption of endothelial cell–pericyte communication during development leads to vascular hemorrhage and embryonic death, as is readily observed in platelet-derived growth factor B (Pdgfb)– or platelet-derived growth factor receptor β (Pdgfrb)–deficient mice, where pericyte recruitment to specific vascular beds is impaired (Lindahl et al., 1997; Hellström et al., 1999; Bjarnegård et al., 2004). In adult mice, TGF-β and bone morphogenetic proteins play critical roles in maintaining pericyte survival and promoting microvascular integrity (El-Bizri et al., 2008; Walshe et al., 2009). These observations suggest that disruption of endothelial cell–pericyte interactions during cardiac microvascular maturation may lead to cardiac dysfunction later in life.In this study, we examined the expression of proNGF and its receptors, p75^NTR and sortilin-related VPS10 domain containing receptor 2 (SorCS2), a sortilin family member (Willnow et al., 2008), in the infarcted myocardium after cardiac ischemia in human autopsy material and in an established mouse model of I-R injury. We observed the induction of proNGF by cardiac myocytes and arterioles, and of p75^NTR and SorCS2 by mural cells of arterioles in the peri-infarct region of individuals who died after a recent MI. Comparable induction patterns were observed in mouse tissue after I-R injury. To investigate the consequences of proNGF expression on the cardiac vasculature, we generated and analyzed a knockin mouse that expresses one allele of cleavage-resistant proNGF (proNgf-hemagglutinin [HA]/⁺). We found that proNGF overexpression targets p75^NTR- and SorCS2-immunoreactive pericytes in the perinatal period, leading to a reduction of pericyte processes, microvascular endothelial activation, and increased vascular permeability in young adulthood, culminating in a dilated cardiomyopathy and premature lethality. This phenotype is rescued in mice that are deficient in p75^NTR. Furthermore, deficiency in p75^NTR limits the infarct size expansion after myocardial I-R injury, when compared with infarct size in wild-type mice. These observations identify proNGF and p75^NTR as potential therapeutic targets to limit microvascular dysfunction in the ischemic heart.