蜱类唾液腺结构与功能的研究进展

唾液腺是蜱类的重要器官 ,在其生命活动中起重要作用。它不仅能维持机体离子、水分平衡 ,使蜱成功地吸血 ,而且是各类疾病传播的窗口。蜱类唾液腺的研究 ,对于揭示唾液腺结构与功能、功能与适应以及开展有效的防治均有重要意义。本文针对国内外的研究成果 ,对蜱类唾液腺结构、分泌物成分和在蜱类生命活动中的作用做了较详细分析 ,供有关研究者参考。1　唾液腺结构关于蜱类唾液腺结构 ,在早期光学显微镜阶段 ,已对具肢扇头蜱Ｒｈｉｐｉｃｅｐｈａｌｕｓａｐｐｅｎ ｄｉｃｕｌａｔｕｓ (Ｔｉｌｌ,1 96 1 )和微小牛蜱Ｂｏｏｐｈｉｌｕｓｍｉｃ…     

长角血蜱唾液腺Apyrase的生物学特性和功能的研究

长角血蜱在吸血时 ,其唾液腺中的 apyrase可明显地抑制由 A DP所诱导的血小板聚集反应 ,并呈剂量反应关系 ,1/ 2对唾液腺可以完全抑制血小板的聚集反应。通过 apyrase的动力学、 HPLC分子筛、等电聚焦电泳和温度敏感性实验证明 ,水解 ATP和 ADP并不是 ATP酶、 ADP酶或几种酶共同作用的结果 ,而是一种酶即 apyrase的作用结果。Apyrase对 Ca2 具有依赖性。而 Mg2 、Zn2 、Mn2 对之影响较小 ,Hg2 和EDTA是 apyrase的抑制剂。吸血对蜱唾液腺 apyrase活性影响较大 ,吸血 6天时其活性最大 ,吸血后活力明显下降。A pyrase与哺乳动物的 5′-核苷酸酶相似 ,它是一种糖基 -磷脂酰肌醇 ( GPI)膜锚结构蛋白。     

镰形扇头蜱IgG结合蛋白基因的克隆与表达分析

本实验以镰形扇头蜱吸血前后雄蜱唾液腺的抑制消减杂交cDNA文库中的EST数据为基础,应用RACE方法从镰形扇头蜱体内克隆出一个IgG(IgG binding protein,IGBP)基因的全长序列,该基因全长683bp,编码178个氨基酸,预测分子量为19.5kDa,经同源性比较该基因与具尾扇头蜱的IGBP-MB基因序列的同源性高达92%。用RT-PCR方法分析了该基因表达的性别特异性、各个器官、不同发育阶段的表达情况,结果表明,该基因表达没有性别特异性;IGBP基因在唾液腺、壳这2个器官有所表达,但在肠中却没有表达;在蜱的各个发育阶段均有表达。     

p38 MAPK信号途径参与大鼠系膜细胞MMP2表达

目的探讨系膜细胞中调节MMP2表达的信号通路。方法鼠系膜细胞培养,分别加入酪氨酸激酶抑制剂（Herbimycin A）,p38MAPK抑制剂（SB203580）,MEK抑制剂（U0126）,ERK抑制剂（PD098059）,P13K抑制剂（Y294002）,用酶谱法分析MMP2酶活性、用RT-PCR及Westem方法从基因及蛋白水平,观察阻断剂对MMP2表达的影响。结果酪氨酸激酶抑制剂、MEK抑制剂、ERK抑制剂及P13K抑制剂对MMP2酶活性、mRNA及蛋白水平无影响,在72hp38MAPK抑制剂SB203580抑制MMP2活性25％。在48h当SB203580浓度分别为10、20、30及40μmol／L时,系膜细胞中MMP2的抑制率分别为7．2％、13．5％、21．3％及28％。结论大鼠系膜细胞中p38MAPK信号传导途径参与了MMP2的表达,并且p38MAPK抑制剂SB203580对MMP2的抑制有剂量依赖性。     

以结核分枝杆菌H_(37)Rv莽草酸脱氢酶为靶点的高通量筛选模型的建立及应用

目的建立以结核分枝杆菌莽草酸脱氢酶为靶点的新型抗结核药物高通量筛选模型;用此模型筛选莽草酸脱氢酶抑制剂;进一步评价化合物对莽草酸脱氢酶活性的影响。方法表达并纯化结核分枝杆菌H37Rv莽草酸脱氢酶;利用还原型辅酶II(NADPH)在溶液中的光吸收,测定酶的活性,构建了该酶抑制剂的高通量筛选模型;用Z′因子法评价该模型的可靠性,并对5万余个化合物进行筛选;测定了各抑制剂的IC50并对抑制剂6186050的酶抑制动力学进行了研究;用菌液稀释法评价了抑制剂对某些临床分离菌株包括耐药菌株的影响。结果得到了重组莽草酸脱氢酶;测得比活力为20987U/mg,所建的莽草酸脱氢酶高通量筛选模型Z′因子为0.76,符合高通量筛选的要求;对5万余个化合物进行筛选得到9个抑制率较高的化合物;抑制剂6186050为竞争性可逆抑制剂;抑制剂6230384和6186050对海分枝杆菌的最低抑菌浓度(MIC)都是32μg/ml。结论建立了稳定性好、灵敏度较高的结核分枝杆菌莽草酸脱氢酶抑制剂高通量药物筛选模型,应用该模型筛选得到的抑制剂可能具有抑菌活性。     

抗蜱免疫研究Ⅰ.中华硬蜱叮咬免疫接种宿主后其吸血…

本文选用中华硬蜱雌性成虫唾液腺抗原对家兔进行人工免疫接种，在按常规方法免疫接种三次后，用中华硬蜱成虫进行感染，分别观察唾液腺抗原接种组、佐剂对照组和空白对照组中华硬蜱的吸血、生殖情况。结果显示，中华硬蜱叮咬唾液抗原免疫接种兔后其吸血量，产卵量均较对照组显著降低，其中大部分雌蜱不能达到饱食状态，蜱的吸血量较佐剂和空白对照组分别下降６１．４８％和６６．０４％，中华硬蜱吸血时间略有延长，产卵量指数降低，     

抗蜱免疫研究Ⅰ．中华硬蜱叮咬免疫接种宿主后其吸血、生殖能力的变化

本文选用中华硬蜱（Ｉｘｏｄｅｓｓｉｎｅｎｓｉｓ，Ｔｅｎｇ）雌性成虫唾液腺抗原（ＳＧＡ）对家兔进行人工免疫接种，在按常规方法免疫接种三次后，用中华硬蜱成虫进行感染（叮咬），分别观察唾液腺抗原接种组、佐剂对照组和空白对照组中华硬蜱的吸血、生殖情况。结果显示，中华硬蜱叮咬唾液抗原免疫接种兔后其吸血量、产卵量均较对照组显著降低，其中大部分雌蜱不能达到饱食状态，蜱的吸血量较佐剂和空白对照组分别下降６１．４８％和６６．０４％；中华硬蜱吸血时间略有延长，产卵量指数降低。并对蜱抗原免疫接种诱导宿主产生特异性抵抗力进行了分析讨论。     

用16S rRNA基因序列分析从西藏的微小牛蜱中检出的埃立克体

用埃立克体属特异性套式PCR和DNA序列测定从西藏微小牛蜱检出边缘无形体和埃立克体;用半套式PCR扩得该埃立克体的16S rRNA基因,并测定了它的序列;将该埃立克体的16S rRNA基因序列与其它埃立克体的16S rRNA基因序列进行对比分析,结果证明它的16S rRNA基因与埃立克体属的犬埃立克体群16S rRNA基因相似水平最高(97%～98%);用16S rRNA基因序列做系统发育分析,结果显示该采自西藏的微小牛蜱的埃立克体可能是与查菲埃立克体密切相关的一种新埃立克体.     

卡斯帕酶-3抑制剂的高通量筛选及活性化合物F03ZA-575的初步研究

目的筛选并初步研究微生物来源的卡斯帕酶-3(Caspases-3)抑制剂活性化合物。方法使用E.coli异源表达的卡斯帕酶-3作为靶酶,建立基于Caspase-3酶抑制剂的体外高通量筛选模型并对微生物来源的共计10026个次级代谢产物提取物进行了筛选。使用有机溶剂萃取、硅胶柱和LH-20柱层析、高压液相分离等方法从代谢产物中分离活性化合物并经各种理化性质及NMR分析等对活性化合物的结构进行确定。结果从10026个微生物来源的代谢产物中筛选获得了10个阳性样品。其中,从1株真菌的代谢产物中分离得到的化合物F03ZA-575对Caspase-3酶显示出较强的抑制活性,结构解析确认该化合物与Duclauxin同质。结论该化合物对Caspase-3酶的抑制活性将有助于揭示其抗肿瘤作用的机制。     

ALK和ROS1：肺癌治疗的联合靶点

间变性淋巴瘤激酶（anaplasticlymphomakinase，ALK）基因重排可见于包括非小细胞肺癌non-smallcelllungcancer，NSCLC）在内的多种恶性肿瘤中。ALK融合基因使激酶具有异常活性，而野生型ALK激酶域突变也可使它被激活。ALK基因重排使得NSCLC中出现了新的分子亚型，该亚型对ALK抑制剂高度耐药。克唑替尼（crizotinib）是一个口服小分子ATP模拟化合物，它最初作为MET抑制剂被开发，随后被发现具有抗ALK活性的脱靶效应（off-target），并被美国FDA批准用于治疗ALK阳性的NSCLC患者。近来在NSCLC患者中还发现了ROSl受体酪氨酸激酶染色体重排，而克唑替尼正处于治疗该分子亚型NSCLC患者的临床试验中。任何计算机辅助药物设计都是依赖其分子结构和配体的药物设计方法，每种方法的详细信息中均应重点强调利用这二者，以开发多靶点小分子激酶抑制剂。此类多靶点小分子激酶抑制剂均可对ROS1和ALK重排的NSCLC有抑制增殖作用。因此，本综述重点强调了关于靶向这些激酶的重要性，以及在优化出效能更佳、选择性更强的ROS1和ALK激酶抑制剂中所取得的进步。     

Baculovirus expression of BmAChE3, a cDNA encoding an acetylcholinesterase of Boophilus microplus (Acari: Ixodidae)

The complete cDNA sequence encoding a Boophilus microplus (Canestrini) (Acari: Ixodidae) acetylcholinesterase (AChE3) was expressed in the baculovirus system. The recombinant AChE3 protein (rBmAChE3) was secreted as a soluble form into the cell culture medium and was identified as a functional AChE by substrate specificity and by inhibition with the AChE-specific inhibitors eserine sulfate and BW284c51. Inhibition kinetics of rBmAChE3, in the presence of the organophosphate paraoxon, revealed sensitivity comparable with that of adult, organophosphate-susceptible neural AChE. To our knowledge, this is the first report of the cloning and successful expression of a functional ixodid AChE.     

Dermacentor variabilis and boophilus microplus (Acari: Ixodidae): experimental vectors of Babesia equi to equids

The experimental vector competence of five laboratory-reared ixodid tick species representing three genera [Amblyomma americanum (L.), Boophilus microplus (Canestrini), D. andersoni Stiles, D. occidentalis Marx, and D. variabilis (Say)] for Babesia equi (Laveran 1901) was evaluated by delayed transfer of male ticks from infected to susceptible equids or by infesting the latter animals with adult ticks previously fed as nymphs on infected equids. After feeding for 5, 6, or 13 d on acquisition hosts, ticks were forcibly removed and held off the host at 26 degrees C, approximately 93% RH, and a photoperiod of 14:10 (L:D) h for 6, 12, or 27 d. Intrastadial transmission to susceptible ponies by D. variabilis males, and transstadial transmission to susceptible burros by B. microplus adults, was demonstrated by blood smear and indirect immunofluorescence serology. The data indicated that male D. variabilis and adult B. microplus, tick species that occur on equids in North America and, in the case of the latter tick, also extensively in tropical and subtropical regions of the world, may be competent natural vectors of B. equi     

Mutation of antitrypsin to antithrombin. alpha 1-antitrypsin Pittsburgh (358 Met leads to Arg), a fatal bleeding disorder

Our previous studies predicted a functional relationship between the plasma proteins alpha 1-antitrypsin and antithrombin III. To elucidate this relationship we investigated the plasma of a 14-year-old boy who had died from an episodic bleeding disorder. A variant alpha 1-antitrypsin was identified in which the methionine at position 358 had been replaced by an arginine. This had converted the alpha 1-antitrypsin from its normal function as an inhibitor of elastase to that of an inhibitor of thrombin. This finding indicates that the reactive center of alpha 1-antitrypsin is methionine 358, which acts as a bait for elastase, just as the normal reactive center of antithrombin III is arginine 393, which acts as a bait for thrombin. The independence of the new thrombin inhibitor from heparin control explains the bleeding disorder; it also indicates that heparin normally acts directly on antithrombin III, revealing its inherent inhibitory activity. The episodic nature of the bleeding was a consequence of the mutant protein's being an acute-phase reactant, the level of which increased several-fold after trauma.     

Simultaneous detection of Anaplasma marginale and a new Ehrlichia species closely related to Ehrlichia chaffeensis by sequence analyses of 16S ribosomal DNA in Boophilus microplus ticks from Tibet

To identify ehrlichial agents in Boophilus microplus ticks, DNA samples of B. microplus collected from the Tibet Autonomous Region and Sichuan Province of China were screened by a nested PCR. Sixteen of 43 (37%) DNA samples of B. microplus from Tibet were positive in nested PCR analysis. All 27 samples from Sichuan were negative. The screen identified two ehrlichial agents based on different 16S rRNA genes that were found after amplifying and sequencing the 5'-end fragments of the 16S rRNA genes. One sequence was identical to that of the gene of Anaplasma marginale, an etiological agent of animal anaplasmosis. The other sequence was most similar to that of the gene of Ehrlichia chaffeensis, an etiological agent of human monocytic ehrlichiosis. The sequence of 1,501 bases from the novel ehrlichial agent was obtained and showed the greatest levels of sequence similarity (97 to 98%) to 16S rRNA gene sequences of the members of the E. canis group of the genus EHRLICHIA: Sequence comparison of the 16S rRNA gene with the members of the genus Ehrlichia reveals that the novel ehrlichial agent detected in B. microplus ticks is a new species of the genus Ehrlichia and is most closely related to E. chaffeensis.     

Expression of equi merozoite antigen 2 during development of Babesia equi in the midgut and salivary gland of the vector tick Boophilus microplus

Equi merozoite antigens 1 and 2 (EMA-1 and EMA-2) are Babesia equi proteins expressed on the parasite surface during infection in horses and are orthologues of proteins in Theileria spp., which are also tick-transmitted protozoal pathogens. We determined in this study whether EMA-1 and EMA-2 were expressed within the vector tick Boophilus microplus. B. equi transitions through multiple, morphologically distinct stages, including sexual stages, and these transitions culminate in the formation of infectious sporozoites in the tick salivary gland. EMA-2-positive B. equi stages in the midgut lumen and midgut epithelial cells of Boophilus microplus nymphs were identified by reactivity with monoclonal antibody 36/253.21. This monoclonal antibody also recognized B. equi in salivary glands of adult Boophilus microplus. In addition, quantification of B. equi in the mammalian host and vector tick indicated that the duration of tick feeding and parasitemia levels affected the percentage of nymphs that contained morphologically distinct B. equi organisms in the midgut. In contrast, there was no conclusive evidence that B. equi EMA-1 was expressed in either the Boophilus microplus midgut or salivary gland when monoclonal antibody 36/18.57 was used. The expression of B. equi EMA-2 in Boophilus microplus provides a marker for detecting the various development stages and facilitates the identification of novel stage-specific Babesia proteins for testing transmission-blocking immunity.     

Biochemical detection of esterases in the adult female integument of organophosphate-resistant Boophilus microplus (Acari: Ixodidae)

Esterase activity was present in the integument of adult female Boophilus microplus (Canestrini) ticks that are resistant to organophosphates (OP). Three esterases were purified from adult integument, which hydrolyze the substrates p-nitrophenylacetate and beta-naphthyl acetate after comparison of OP-resistant strain and an OP-susceptible strains. The esterases purified by ion-exchange chromatography were characterized using different esterase inhibitors; eserine sulfate, diethyl p-nitrophenyl phosphate (paraoxon), para-hydroxyl-mercuribenzoate (pHMB), and diisopropylphosphofluoridate (DFP). All of the esterases had a molecular mass of 64 Kd (PAGE), but were characterized based on the esterase inhibitor effects as a B-esterase with beta-naphthyl acetate affinity, a carboxylesterase with beta-naphthyl acetate and p-nitrophenyl acetate affinity, and one A-Esterase (nonspecific esterase) with p-nitrophenyl acetate affinity. The described esterases are an important detoxification mechanism in B. microplus ticks at the integument. We describe also a microplate biochemical assay for the detection of esterase activity in the tick integument, potentially a useful tool to detect esterase-mediated OP resistance in B. microplus ticks.     

A mathematical model of lipid-mediated thrombin generation.

Thrombin is an enzyme that is generated in both vascular and non-vascular systems. In blood coagulation, a fundamental process in all species, thrombin induces the formation of a fibrin clot. A dynamical model of thrombin generation in the presence of lipid surfaces is presented. This model also includes the self-regulating thrombin feedback reactions, the thrombomodulin-protein C-protein S inhibitory system, tissue factor pathway inhibitor (TFPI), and the inhibitor, antithrombin (AT). The dynamics of this complex system were found to be highly lipid dependent, as would be expected from experimental studies. Simulations of this model indicate that a threshold lipid level is required to generate physiologically relevant amounts of thrombin. The dependence of the onset, the peak levels, and the duration of thrombin generation on lipid was saturable. The lipid concentration affects the way in which the inhibitors modulate thrombin production. A novel feature of this model is the inclusion of the dynamical protein C pathway, initiated by thrombin feedback. This inhibitory system exerts its effects on the lipid surface, where its substrates are formed. The maximum impact of TFPI occurs at intermediate vesicle concentrations. Inhibition by AT is only indirectly affected by the lipid since AT irreversibly binds only to solution phase proteins. In a system with normal plasma concentrations of the proteins involved in thrombin formation, the combination of these three inhibitors is sufficient both to effectively stop thrombin generation prior to the exhaustion of its precursor, prothrombin, and to inhibit all thrombin formed. This model can be used to predict thrombin generation under extreme lipid conditions that are difficult to implement experimentally and to examine thrombin generation in non-vascular systems.     

R86Q, a mutation in BmAChE3 yielding a Rhipicephalus microplus organophosphate-insensitive acetylcholinesterase

Mutations were identified in the cDNA sequence encoding the acetylcholinesterase BmAChE3 in strains of Rhipicephalus (Boophilus) microplus (Canestrini) resistant or susceptible to organophosphate (OP) acaricide. The mutation that occurred most frequently in the OP-resistant San Román strain resulted in a substitution of glutamine (Q) for arginine (R) at position 86 in BmAChE3 (position 66 in mature BmAChE). Clones containing the mutant and wild-type cDNA sequences were expressed in the baculovirus system. Enzyme kinetics of recombinant BmAChE3 containing or lacking the R86Q mutation demonstrated that the R86Q mutation increased substrate affinity and conferred insensitivity to paraoxon inhibition. This is the first demonstration of a mutation in a gene encoding an ixodid acetylcholinesterase resulting in OP insensitivity. A restriction fragment length polymorphism assay was developed and used to diagnose the frequency of the R86Q mutation in BmAChE3 genomic DNA from seven laboratory-colonized strains. Use of the R86Q diagnostic assay detected an increased frequency of the R86Q mutation in OP-resistant tick strains compared with that of OP-susceptible strains; however, the R86Q mutation was also present in OP-susceptible strains at unexpectedly high frequency. Because the R86Q mutation generates an OP-resistant enzyme in vitro and it is present at an elevated frequency in laboratory strains selected for OP resistance, we conclude that the data are consistent with a potential role for BmAChE3 in development of OP resistance; however, because the R86Q mutation has a high frequency in susceptible strains, the R86Q mutation alone is insufficient to generate the OP-resistant phenotype at the organismal level. There are likely to be additional mutations in BmAChE3, mutations in additional acetylcholinesterase genes, or additional resistance mechanisms (e.g., oxidative metabolism) that contribute to expression of the OP-resistant phenotype.     

Bioengineered surfaces to improve the blood compatibility of biomaterials through direct thrombin inactivation

Thrombus formation, due to thrombin generation, is a major problem affecting blood-contacting medical devices. This work aimed to develop a new strategy to improve the hemocompatibility of such devices by the immobilization of a naturally occurring thrombin inhibitor into a nanostructured surface. Boophilin, a direct thrombin inhibitor from the cattle tick Rhipicephalus microplus, was produced as a recombinant protein in Pichia pastoris. Boophilin was biotinylated and immobilized on biotin-terminated self-assembled monolayers (SAM) via neutravidin. In order to maintain its proteinase inhibitory capacity after surface immobilization, boophilin was biotinylated after the formation of a boophilin–thrombin complex to minimize the biotinylation of the residues involved in thrombin–boophilin interaction. The extent of boophilin biotinylation was determined using matrix-assisted laser desorption/ionization-time of flight/time of flight mass spectrometry. Boophilin immobilization and thrombin adsorption were quantified using quartz crystal microbalance with dissipation. Thrombin competitive adsorption from human serum was assessed using ¹²⁵I-thrombin. Thrombin inhibition and plasma clotting time were determined using spectrophotometric techniques. Boophilin-coated SAM were able to promote thrombin adsorption in a selective way, inhibiting most of its activity and delaying plasma coagulation in comparison with boophilin-free surfaces, demonstrating boophilin’s potential to improve the hemocompatibility of biomaterials used in the production of blood-contacting devices.