磷脂酶A_2激活在受体信号转导系统转变中的作用

与鸟核苷酸结合调节蛋白(G蛋白)相偶联的、具有7个跨膜区段的膜表面受体很多,但它们的信号转导系统只有两个:受体-G蛋白-腺苷酸环化酶(AC)系统和受体-G蛋白-磷脂酰肌醇(PI)系统。本研究以具有M_2亚型胆碱能受体(M_2受体)的人胚肺成纤维细胞证实,在激动剂氨甲酰胆碱(CCh)长时间作用下,原来与     

高渗诱导人气道上皮细胞系黏液高分泌

目的研究高渗条件对正常人气道上皮细胞(HBE)黏蛋白(MUC)5AC分泌的影响,以及蛋白激酶C(PKC)-热休克蛋白(HSP)70信号途径在其中的可能作用。方法 采用高渗盐水诱导培养HBE16细胞的方法复制黏液高分泌体外模型,分别用PKCμ抑制剂G 6976、PKCα抑制剂Safingol、PKCβ抑制剂LY333531和PKCδ抑制剂Rot-tlerin干预HBE16细胞。Western blot检测HSP70-2的蛋白含量;RT-PCR检测人HSP70-2转录水平;ELISA检测培养上清MUC5AC蛋白含量c各高渗组的培养上清MUC5AC蛋白含量、HSP70-2蛋白和转录水平较对照组显著升高,并随着培养时间的延长而逐渐增加(P<0.05)。G 6976处理组的上述指标显著降低(P<0.01),但仍高于对照组(P<0.05);而Safingol、LY333531和Rottlerin处理组均无改变。结论 高渗盐水可诱导人气道上皮细胞MUC5AC的高分泌,HSP70-2系通过PKC中的μ亚型在该过程中起重要作用的。     

重庆地区急性呼吸道感染患儿呼吸道合胞病毒流行特征及其G基因序列分析

目的 分析重庆地区2008-2009年度急性呼吸道感染住院患儿呼吸道合胞病毒(respiratory syncytial virus,RSV)的亚型流行情况,并了解优势流行株BA株的G蛋白基因特征.方法 采集2008年4月-2009年3月全年于重庆医科大学附属儿童医院因急性呼吸道感染住院的508例患儿鼻咽深部分泌物,用RT-PCR方法检测RSV并进行亚型鉴定,选取29例B亚型和10例A亚型RSV阳性标本,用RT-PCR的方法扩增全长G蛋白并测序.结果 在508例标本中,RSV阳性126例(24.8%),其中检测出A亚型43例(34.1%),B亚型80例(63.5%),A、B亚型混合感染3例(2.4%).所测的10株A亚型的G基因与标准株A2的核苷酸同源性为91.4%～92.0%,均属GA2基因型;29株B亚型的G基因与标准株CH18537的核苷酸同源性为92.0%～93.0%,其中19株均为具有60个高度重复核苷酸插入的BA株.B亚型流行株与CH18537标准株相比,G基因有多种核苷酸变异如缺失、插入等,尤其在G蛋白近C端1/3处的高变区.结论 2008-2009年RSV仍是重庆地区儿童急性呼吸道感染的主要病原,与既往两年A亚型优势流行不同,2008-2009年度B亚型毒株流行占优;近年新发现的BA株可能已成为本地区优势流行株,BA株G基因变异是否导致G蛋白功能增强,进而促进其优势流行尚有待研究.     

乙型肝炎病毒C启动子区C1673T/C1799G联合突变的意义

目的 探讨HBV(乙型肝炎病毒)C启动子区(CP) C1673T/C1799G联合变异的生物学和临床意义.方法 136名慢性HBV感染者,包括无症状携带者(ASC) 25例,慢性乙型肝炎(CHB)38例,慢性重型乙型肝炎(CSHB) 24例,乙肝肝硬化(LC) 36例,肝细胞肝癌(HCC)13例,用半巢氏PCR的方法结合直接测序法检测HBV基因亚型及CP区变异,分析C1673T/C1799G联合变异在不同基因亚型中的发生率及与HBV复制、e抗原表达和不同慢性HBV感染疾病谱的关系.结果 本组病例中,Ba亚型110例,Bj亚型1例,C1亚型7例,C2亚型8例,C1673T/C1799G联合变异发生率为80.9％,其中在Ba亚型中为96.4％,C1亚型为14.3％,C2亚型为12.5％,Ba亚型与C1和C2亚型比较,差异有统计学意义(P ＜0.0001).变异组HBV DNA载量与非变异组比较差异无统计学意义(P＞0.05);e抗原阳性组变异率为71.4％,e抗原阴性组为87.5％,两组比较差异有统计学意义(P＜0.05).从ASC、CHB、CSHB、LC到HCC组,变异的发生率差异无统计学意义(P＞0.05).结论 C1673T/C17991G联合变异常见于Ba基因亚型,该变异不影响HBV DNA的复制水平,可能与不同慢性HBV感染疾病谱无关.     

H4亚型禽流感血凝素单克隆抗体的研制及夹心ELISA的建立

目的制备H4亚型禽流感病毒(AIV)血凝素单克隆抗体(mAb),基于mAb建立H4亚型AIV夹心ELISA检测方法。方法用灭活H4亚型AIV免疫BALB/c小鼠,通过细胞融合、血凝抑制(HI)试验筛选和亚克隆,得到了一株抗H4亚型AIV mAb(6G4)。免疫荧光细胞化学染色和Western blot法验证6G4与H4亚型AIV的反应性, HI试验鉴定6G4特异性、广谱性和稳定性,试剂盒鉴定6G4亚类。以鸡抗H4亚型AIV多克隆抗体作为包被抗体, 6G4作为捕获抗体, HRP标记的山羊抗小鼠IgG作为酶标抗体,通过条件优化和一系列验证,建立H4亚型AIV夹心ELISA检测方法。结果 6G4亚类为IgG1,轻链为κ链,能稳定分泌抗体,具有良好的反应性、特异性、广谱性和稳定性;基于6G4建立的ELISA特异性强、敏感性高、重复性好,适合大批量检测。结论成功制备了抗H4亚型AIV的mAb,建立了检测H4亚型AIV的夹心ELISA。     

多巴胺D1受体信号通路及其在睡眠剥夺中的作用

脑内多巴胺(dopamine,DA)能神经元主要集中于嗅球、下丘脑、中脑腹侧被盖区(VTA)和黑质致密部(SNc)等区域,参与了机体的学习记忆、认知和思维能力等高级神经功能调控,同时还与运动控制、情绪、警觉性及药物成瘾等神经活动密切相关[2]. 多巴胺受体(dopamine receptor,DAR)属于G蛋白偶联受体,与其偶联的G蛋白为异三聚体蛋白,由α、β、γ3个亚单位组成,其中α亚基本身具有GTP酶活性,其结合的GDP被GTP取代后变为活化型而催化腺苷酸环化酶(AC)和磷脂酶C(PLC)等效应酶.γ亚基根据其序列的同源程度至少可以分为12种亚型,在敲除内源性G蛋白γ7亚基表达的HEK293细胞株中,多巴胺D1受体激动剂SKF38393对腺苷酸环化酶的活化作用被明显抑制;同时抑制γ7亚基的表达则导致了细胞内G蛋白β1亚基表达量的减少[2].这两个结果表明,Gβ1(n)二聚体在多巴胺D1受体活化腺苷酸环化酶过程中也具有非常重要的作用.     

湖北省HIV-1流行株gag基因序列测定及亚型分析

目的 了解湖北省HIV-1感染者流行毒株亚型分布和流行趋势.方法 随机采集湖北省HIV-1感染者的抗凝全血标本,分离血浆,提取病毒RNA,用套式聚合酶链反应扩增HIV-1病毒gag基因,并进行序列测定和亚型分析.结果 对80份HIV-1感染者的样品进行扩增,得到了62份样品的HIV-1基因片段.共发现7种HIV-1亚型和流行重组株.泰国B'亚型占11.3％ (7/62),欧美B亚型占4.8％ (3/62),G亚型占4.8％ (3/62),CRF07-BC占22.6％ (14/62),CRF08-BC占6.5％(4/62),CRF01-AE占48.4％(30/62),CRF15-01B占1.6％ (1/62).在湖北省发现了CRF15-01B和G亚型.结论 湖北省存在多种HIV-1亚型和流行重组型,CRF01-AE、CRF07-BC两种重组亚型毒株为目前湖北省HIV-1流行优势毒株,应加强对HIV-1毒株亚型变异的监测,及时调整防治策略.     

生长抑素受体亚型在消化系肿瘤诊断及治疗中的研究与应用进展

生长抑素受体 (SSTR)是一种G蛋白偶联受体 ,包括 5个亚型SSTR1 5 ,各亚型通过不同的传导途径发挥抗肿瘤细胞增殖作用。人类消化系统肿瘤组织可表达多种SSTR ,且大多情况下同时表达两种以上的SSTR ,其中以SSTR2和SSTR5最为常见 ,同一肿瘤不同分期状态下SSTR的表达状态也有所不同。SSTR5’C末端缺失长度与抗细胞增殖作用成正比。临床应用与不同亚型SSTR具有高亲和力的生长抑素类似物有助于某些消化系统肿瘤的诊断与治疗     

26例有偿献血员HIV-1基因分型与变异特征

目的 分析某地区有偿献血人员中流行的人免疫缺陷病毒1型( HIV-1) gag、pol、env基因亚型及基因变异特征.方法 提取HIV-1感染者外周血单核细胞(PBMC) DNA,经巢式PCR( Nested PCR)扩增gag(p17-p24)、pol( PR-RT)、env(C2-V5)基因片段,纯化测序后用MEGA5.0等生物学软件对核苷酸序列进行分析.结果 23份样本为B亚型,2份为B亚型与C亚型重组,1份为CRF01_AE与B亚型重组.PR区未发现蛋白酶抑制剂主要耐药性突变,RT区检测到核苷类逆转录酶抑制剂耐药性突变M184V和非核苷类逆转录酶抑制剂耐药性突变K101E,G190A.结论 流行于该地区的HIV-1毒株以B亚型为主.大多数毒株对常规抗病毒药物仍然敏感,使用HARRT治疗方案依然有效.CXCR4型辅助受体的毒株顶端四肽多为GPGR(91.7％),提示GPGR可能与疾病的进展有关.     

人类白细胞抗原G(HLA-G)与非小细胞肺癌的研究进展

人类白细胞抗原G(HLA-G)属于非经典主要组织相容性复合体Ⅰb(MHCⅠb)类分子,包括膜结合型和可溶性HLA-G两种形式,通过相应受体调节多种免疫细胞的功能,是形成母胎耐受、肿瘤免疫逃逸的重要免疫学机制之一。非小细胞肺癌(NSCLC)在肺癌中占比最高且预后差,研究发现HLA-G的基因多态性及表达水平与NSCLC发生发展密切相关,提示HLA-G可作为NSCLC早期诊断、亚型区分、治疗及预后等潜在的生物标志物,具有辅助诊断依据的临床价值,对其机制的深入研究更可能提供NSCLC诊疗的新策略。     

Higher TNFα responses in young males compared to females are associated with attenuation of monocyte adenylyl cyclase expression

Tumor necrosis factor α (TNFα) expression is strongly attenuated by the intracellular signaling mediator cyclic adenosine monophosphate (cAMP), which is synthesized by adenylyl cyclase (AC) enzymes. We have compared AC regulation and TNFα production in male and female monocytes, and characterized the role of monocyte AC isoforms in TNFα regulation.Males and females, age groups 20–30 years and 50–70 years donated blood for this study. In lipopolysaccharide-stimulated blood from young male donors, we observed significantly higher TNFα responses (6 h, p = 0.03) compared to females of the same age, a difference not observed in the older donors. Rapid down-regulation of the monocyte AC isoforms AC4, AC7 and AC9 were observed in young males. AC-directed siRNA experiments in the human monocyte cell line THP-1 demonstrated that AC7 and AC9 knock-down significantly induced TNFα release (p = 0.01 for both isoforms). These data indicate that the stronger TNFα-responses in young males may be partly associated with male-specific down-regulation of adenylyl cyclases.     

Temporal expression of calcium/calmodulin-dependent adenylyl cyclase isoforms in rat articular chondrocytes: RT-PCR and immunohistochemical localization

A multitude of signalling cascades are implicated in the homeostasis of articular chondrocytes. However, the identity of these signalling pathways is not fully established. The 3, 5′‐cyclic AMP‐mediated signalling system is considered to be a prototype. Adenylyl cyclase (AC) is an effector enzyme responsible for the synthesis of cAMP. There are 10 mammalian AC isoforms and some of these are differentially regulated by calcium/calmodulin (Ca²⁺/CaM). Ca²⁺ is known to play an important role in the development and maintenance of skeletal tissues. Ca²⁺/CaM‐dependent AC isoforms and their temporal expression in articular chondrocytes in rats were identified using RT‐PCR and immunohistochemistry techniques. All Ca²⁺/CaM‐dependent AC isoforms were expressed in chondrocytes from all age groups examined. Each isoform was differentially expressed in developing and adult articular chondrocytes. Generally, expression of AC isoforms was observed to increase with age, but the increase was not uniform for all Ca²⁺/CaM‐dependent AC isoforms. Expression of Ca²⁺/CaM‐dependent AC isoforms along with other signalling molecules known to be present in articular chondrocytes indicate complicated and multifactorial signalling cascades involved in the development and homeostasis of articular cartilage. The significance of these findings in terms of articular chondrocyte physiology is discussed.     

Identification of an adenylyl cyclase inhibitor for treating neuropathic and inflammatory pain

Neuropathic pain, often caused by nerve injury, is commonly observed among patients with different diseases. Because its basic mechanisms are poorly understood, effective medications are limited. Previous investigations of basic pain mechanisms and drug discovery efforts have focused mainly on early sensory neurons such as dorsal root ganglion and spinal dorsal horn neurons, and few synaptic-level studies or new drugs are designed to target the injury-related cortical plasticity that accompanies neuropathic pain. Our previous work has demonstrated that calcium-stimulated adenylyl cyclase 1 (AC1) is critical for nerve injury-induced synaptic changes in the anterior cingulate cortex. Through rational drug design and chemical screening, we have identified a lead candidate AC1 inhibitor, NB001, which is relatively selective for AC1 over other adenylate cyclase isoforms. Using a variety of behavioral tests and toxicity studies, we have found that NB001, when administered intraperitoneally or orally, has an analgesic effect in animal models of neuropathic pain, without any apparent side effects. Our study thus shows that AC1 could be a productive therapeutic target for neuropathic pain and describes a new agent for the possible treatment of neuropathic pain.     

Uterine Quiescence: The Role of Cyclic AMP

It is accepted that whilst hormones such as oxytocin, vasopressin and prostaglandin F2alpha induce myometrial contractions, essentially via an elevation of intracellular calcium, other ligands, such as beta-adrenoceptor agonists, calcitonin gene-related peptide, and prostaglandin E2, promote uterine quiescence via their ability to increase intracellular cyclic AMP levels. At present, the exact factors initiating human parturition remain unknown, and labour may occur due to a loss of uterine quiescence, an increase in uterine contractility, or a combination of both. Whilst many studies have aimed to understand the mechanisms underlying uterine contractility there is a relative paucity of data regarding myometrial relaxation. We have verified the presence of mRNA encoding adenylyl cyclase (AC) isoforms I, II, III, V, VI, VII, VIII and IX in both non-pregnant and pregnant human myometrium, and in isolated myometrial cells maintained in cell culture. Furthermore, by means of immunoblotting and immunocytochemistry, we have demonstrated the expression of these isoforms as membrane-associated AC proteins, and identified changes in individual AC isoform expression during gestation. These findings illustrate the diversity of potential cAMP generating pathways in human myometrium, and the complexity of the signal transduction systems underlying uterine quiescence. Experimental Physiology (2001) 86.2, 265-272.     

Reduced adenylyl cyclase immunolabeling and activity in postmortem temporal cortex of depressed suicide victims

BACKGROUND: Previous studies have found altered receptor/G protein-modulated adenylyl cyclase (AC) activity in subjects with mood disorders. METHODS: To investigate whether these effects are associated with altered levels of specific isoforms of AC, we measured AC isoform I, IV and V/VI immunoreactivities in postmortem temporal cortex from nine depressed suicide victims, nine subjects with bipolar disorder (BD) and 18 age-matched non-psychiatric controls. Basal, GTPgammaS- and forskolin-stimulated AC activities were measured in the temporal cortex from the nine depressed suicide victims and their controls. RESULTS: Western blotting revealed significant reductions in immunolabeling in AC type IV (-49%; p < 0.05) in depressed suicide subjects compared to age-matched controls, but no differences were found in AC type I or type V/VI. There were no statistically significant differences in AC type I, IV or V/VI immunoreactivities between BD and matched control subjects. Functionally, there was a significant reduction in forskolin-stimulated AC activity in depressed suicide subjects compared to controls, which may be, in part, related to higher basal AC activity in the former group. LIMITATIONS: Our sample size was small with diverse subject characteristics. CONCLUSIONS: These preliminary findings suggest altered levels and/or function in AC type IV may contribute to disturbances in the postreceptor cAMP signaling cascade in depression.     

First messenger regulation of capacitation via G protein-coupled mechanisms: a tale of serendipity and discovery

When placed in a suitable environment, mammalian spermatozoa begin to capacitate and continue until fully capacitated; in vitro, some will 'over-capacitate' and undergo spontaneous acrosome loss, undesirable since acrosome-reacted cells are non-fertilizing. Seminal plasma contains several molecules able to bind to specific receptors on spermatozoa, thereby activating/regulating important intracellular signalling pathways. Three such 'first messengers' are fertilization promoting peptide (FPP), adenosine and calcitonin, all of which stimulate capacitation and then inhibit spontaneous acrosome reactions by regulating adenylyl cyclase (AC)/cAMP. A recent study has reported the presence in spermatozoa of several membrane-associated AC isoforms, mainly smaller in size than the corresponding ACs in somatic cells, and evidence suggests that more than one of these isoforms may be involved in responses to these first messengers. To regulate AC, FPP receptors appear to interact initially with stimulatory A(2A) adenosine receptors, which function only in uncapacitated cells, and then with inhibitory A(1) receptors, which function only in capacitated cells. In contrast, there appears to be a single population of calcitonin receptors. Responses to cholera and pertussis toxins suggest involvement of G proteins and G(s) plus several G(i) subunits have been identified in both mouse and human spermatozoa. In particular, Galpha(s) and Galpha(i2) are found in the same regions as FPP, adenosine and calcitonin receptors, supporting biochemical evidence for G protein involvement in these responses. In vivo, these first messengers could have a significant effect, helping to maximize the number of capacitated, acrosome-intact (i.e. potentially fertilizing) spermatozoa by regulating what is clearly an important signalling pathway.     

Implication of adenylyl cyclase 8 in pathological smooth muscle cell migration occurring in rat and human vascular remodelling

Recently, we discovered on primary cell cultures that adenylyl cyclase type 8 (AC8) was involved in the transition of rat vascular smooth muscle cells (VSMCs) to an inflammatory phenotype. Here we demonstrate, in human vessels displaying early or advanced atherosclerotic lesions, that: (a) only intimal VSMCs strongly express AC8; and (b) very few AC8‐positive VSMCs were detected in the medial layer, either in atherosclerotic or healthy arteries. Furthermore, over‐expressing AC8 in primary rat VSMC cultures triggered the recolonization of a wounded zone and similar results were obtained in the presence of mitomycin, a potent inhibitor of proliferation. This phenomenon was prevented by silencing AC8. Indeed, in IL‐1β‐treated cells, AC8 silencing halted migration and decreased the matrix‐metalloproteinases 2/9 secretion, known to be involved in VSMC migration. In vivo, we showed: (a) a pronounced up‐regulation of AC8 expression in highly migrating VSMCs of the injured rat carotid artery; (b) an undetectable AC8 labelling in re‐endothelized vessels where neo‐intimal thickening had stopped. From our data, we conclude that AC8 expression appears closely linked to the properties developed by VSMCs in atherosclerosis and post‐angioplasty neo‐intima formation leading to restenosis. In addition, it reinforces the idea that VSMC responses to their cell environment greatly depend on the AC isoforms expressed and attributes a new role for AC8 in these pathological vascular processes. Copyright © 2010 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Selective reduction in type I adenylyl cyclase after microsphere embolism in rat brain.

Alterations of adenylyl cyclase (AC) subtypes after cerebral ischemia remain unclear. The purpose of the present study was to characterize alterations in AC after sustained cerebral ischemia. Sustained cerebral ischemia was induced by injection of 900 microspheres into the right (ipsilateral) internal carotid artery of rats. Microsphere embolism (ME) decreased the Ca(2+)/calmodulin-sensitive AC activity in the ipsilateral hippocampus examined up to 7 days after the embolism, whereas basal and 5'-guanylyl imidodiphosphate-sensitive AC activities were not altered. An immunoreactivity of type I adenylyl cyclase (AC-I) was decreased in the ipsilateral hippocampus during these periods, whereas type V/VI AC and VIII AC immunoreactivities were not altered. These results suggest that a selective reduction in the AC-I level and activity is induced by ME, which may lead to dysfunction of AC signal transduction.     

Regulation of cAMP levels in area CA1 of hippocampus by Gi/o-coupled receptors is stimulus dependent in mice

The cAMP signaling cascade plays a critical role in regulating synaptic efficacy and cellular excitability in hippocampus. Adenylyl cyclase (AC) activation and subsequent generation of cAMP occurs through a number of mechanisms in area CA1 of hippocampus, including Galpha(s)-mediated stimulation upon G-protein coupled receptor (GPCR) activation and Ca2+ -mediated stimulation upon NMDA receptor activation. In addition, activation of Gi/o-coupled receptor subtypes can regulate cAMP levels through modulation of AC activity. Multiple Gi/o-coupled receptor subtypes are expressed in area CA1, where they are commonly thought to dampen synaptic transmission and excitability, in part through inhibition of AC activity and cAMP generation. However, a large family of ACs exists, and in recombinant systems, the cAMP signals generated by these AC isoforms are both inhibited and enhanced by Gi/o-coupled receptors in a manner dependent on the AC isoform and stimulus. Thus, we have assessed the effects of activating several different Gi/o-coupled receptors on the generation of cAMP induced either by NMDA receptor activation or by beta-adrenergic receptor activation within area CA1 of mouse hippocampus. We find that in situ the effect of Gi/o-coupled receptor activation does indeed depend upon the means by which ACs are activated. In addition, the effects are also Gi/o-coupled receptor-specific, suggesting additional complexity. These data indicate that Gi/o-coupled receptors may play roles in "routing" second messenger signaling in area CA1.     

Transgenic rescue of defective Cd36 enhances myocardial adenylyl cyclase signaling in spontaneously hypertensive rats

Dysfunction or abnormalities in the regulation of fatty acid translocase Cd36, a multifunctional membrane protein participating in uptake of long-chain fatty acids, has been linked to the development of heart diseases both in animals and humans. We have previously shown that the Cd36 transgenic spontaneously hypertensive rat (SHR-Cd36), with a wild type Cd36, has higher susceptibility to ischemic ventricular arrhythmias when compared to spontaneously hypertensive rat (SHR) carrying a mutant Cd36 gene, which may have been related to increased β-adrenergic responsiveness of these animals (Neckar et al., 2012 Physiol. Genomics 44:173–182). The present study aimed to determine whether the insertion of the wild type Cd36 into SHR would affect the function of myocardial G protein-regulated adenylyl cyclase (AC) signaling. β-Adrenergic receptors (β-ARs) were characterized by radioligand-binding experiments and the expression of selected G protein subunits, AC, and protein kinase A (PKA) was determined by RT-PCR and Western blot analyses. There was no significant difference in the amount of trimeric G proteins, but the number of β-ARs was higher (by about 35 %) in myocardial preparations from SHR-Cd36 as compared to SHR. Besides that, transgenic rats expressed increased amount (by about 20 %) of the dominant myocardial isoforms AC5/6 and contained higher levels of both nonphosphorylated (by 11 %) and phosphorylated (by 45 %) PKA. Differently stimulated AC activity in SHR-Cd36 significantly exceeded (by about 18–30 %) the enzyme activity in SHR. Changes at the molecular level were reflected by higher contractile responses to stimulation by the adrenergic agonist dobutamine. In summary, it can be concluded that the increased susceptibility to ischemic arrhythmias of SHR-Cd36 is attributable to upregulation of some components of the β-AR signaling pathway, which leads to enhanced sensitization of AC and increased cardiac adrenergic responsiveness.