鸡疟原虫红细胞内期电镜观察

鸡疟原虫裂殖子侵入红细胞后，圆锥体、致密内膜和膜下微管均没立即消失。随着虫体发育，滋养体逐渐增大，形状不规则，由单层表膜包绕，虫体一侧有胞口，胞口直径与食物泡大小并非有关，食物泡形状各异，其内有结晶状疟色素，细胞质内具有一个大的有嵴线粒体和球形体。细胞核分裂从细胞核变长开始，核膜两极出现染色体团块，两极之间形成纺锤体，核分裂时虫体内细胞器也发生较大的变化，线粒体断裂成多个，裂殖体多处表膜形成致密内膜和膜下微管，内膜覆盖区向含虫空泡凸出形成裂殖子芽，棒状体、细胞核、线粒体和球形体相继进入裂殖子芽中，最终形成多个裂殖子。裂殖子由表膜复合物包绕，顶端呈截状圆锥形突起，有极环，虫体内有棒状体、细胞核、线粒体和球状体等结构。     

探究获得高成熟率伯氏疟原虫裂殖体的体外培养条件?

为获得大量有活力的伯氏疟原虫裂殖体,本研究对伯氏疟原虫ANKA虫株的体外培养条件主要从培养基的用量、培养基胎牛血清的含量、培养的细胞浓度、培养时间及培养的气体环境等方面进行了优化。当小鼠体内虫血率达到1％～3％,在红内期疟原虫处于环期或早期滋养体阶段时取血分组培养,观察在不同培养条件下裂殖体的状态并检测其活力。与既往的体外培养方法相比,优化后的培养方法,可将裂殖体的成熟率提高到80％,每个裂殖体含12～16个裂殖子,裂殖体尾静脉注射重新入侵红细胞4 h后的虫血率为1?57％,与对照组相比裂殖体的活力提高3?4倍。优化的方法可以提高裂殖体的得量和活力,为伯氏疟原虫的转染奠定基础。     

抗恶性疟原虫单克隆抗体的分析

在两次细胞融合中,经2—4次克隆化后已建成28株抗恶性疟原虫(FCC—1/HN)的杂交瘤。其中针对裂殖子和分裂体抗原的McAb有9株,仅对裂殖体特异的有4株,对裂殖体和滋养体期原虫产生荧光反应的有15株。 应用间接荧光抗体技术,检查这28株McAb对恶性疟原虫(安徽株),间日疟原虫、食蟹猴疟原虫,诺氏疟原虫、伯氏疟原虫、约氏疟原虫和鸡疟原虫的交叉反应。结果,仅有93D4和94B5属株特异性McAb,其余各株均对恶性疟原虫(安徽株)有明显荧光反应。有5     

抗恶性疟原虫全虫单克隆抗体的制备和鉴定

为建立分泌抗恶性疟原虫全虫单克隆抗体杂交瘤细胞株，研制简易、敏感、特异的快速诊断恶性疟试剂盒。本用恶性疟原虫全虫抗原免疫BALB/c小鼠，取其脾细胞与SP2／0细胞融合，用ELISA法筛选分泌抗体的阳性杂交瘤细胞克隆，测定单抗的效价和Ig亚类，并用SDS-PAGE、Western印迹、IFAT和IPA等方法对单抗进行鉴定。结果，获得4株(2H6，2A3，3B1和2C12)分泌抗恶性疟原虫单抗的杂交瘤细胞克隆，均属IgG类。其中2H6克隆产生的抗体滴度最高《腹水效价达1：32000)．属IgG1亚类。与伯氏疟愿虫、弓形虫无交卫反应；Western印迹分析显示2H6能被约：130、63、41、33和20kDa等抗原蛋白所识别,IFAT和IPA分析显示，2H6单抗的抗原主要定位于裂殖子、滋养体和裂殖体的表膜部分。从而提示所制备的抗恶性疟原虫全虫杂交癌细胞株能分越高滴度的抗体，其中2H6是针对恶性疟原虫裂殖于表面蛋白的特异性单抗，可用于怒性疟快速诊断试剂盘的研制。     

贝氏隐孢子虫在珍珠鸡体内发育的透射电镜观察

采用透射电镜观察了贝氏隐孢子虫在珍珠鸡气管和法氏囊的发育。贝氏隐孢子虫各期虫体均在上皮细胞微绒毛所形成的带虫空泡内发育，虫体基部有一营养器。滋养体呈圆形，有一个细胞核。胞质中有发达的粗面内质网。裂殖体经２或３次核分裂，以出芽方式形成４或８个裂殖子。成熟的裂殖子呈香蕉形，大小为２．８５×０．７０μｍ，被双层膜。小配子体由滋养体发育而来，内含多个缺少核仁的细胞核，细胞核移向胞质浅层，并进入胞质突起成为小配子的细胞核。大配子内可观察到二种成囊体和大量多糖颗粒，并在其胞质的空泡内发现小配子类似物。孢子生殖也在带虫空泡内进行，最终形成一个大残体和４个子孢子。子孢子有一个细胞核，富含微线和多糖颗粒。     

恶性疟人群免疫血清靶抗原的分析

本文报告利用乳过氧化物酶(LPO)催化的~(125)I放射性标记法,标记恶性疟原虫裂殖体(裂殖子)表面抗原。将标记的原虫制成TritonX—100提取物,然后分别与采自流行区的11份病人血清和非疟疾流行区正常人血清进行免疫沉淀,通过十二烷基硫酸钠—聚丙烯酰胺凝胶电泳(SDS PAGE)和放射自显影分析恶性疟患者血清的靶抗原。 结果指出,恶性疟裂殖体(裂殖子)的TritonX—100提取物中存在20多种表面膜抗原。分子量为270、240、183、160、137、104、98、74和63KD的多肽系免疫沉淀中的主要靶抗     

猪隐孢子虫内生发育虫体超微结构观察

用扫描电镜和透射电镜技术观察了猪隐孢子虫(Cryptosporidium suis)sucp分离株在5日龄仔猪体内内生发育虫体形态结构和寄生特点。结果发现,感染前期和高峰期,在盲肠、结肠和直肠有虫体寄生,而感染后期仅在直肠上发现虫体,并且多集中在肠腺区域。实验观察到滋养体、裂殖体、大配子体、合子和孢子化卵囊等不同阶段的虫体。虫体周围一定区域的微绒毛融合、萎缩或倒伏,肠粘膜上的微绒毛明显脱落。中期滋养体营养器和致密带之间有一层较宽的特殊结构,并且该结构到后期滋养体和裂殖体阶段消失,这层特殊结构尚未见文献报道。在带虫空泡内膜与虫体外膜形成的致密带上方存在典型的呈竖向或斜向排列的纤维塞状结构(microfilament plug)营养器。     

食蟹猴疟原虫B株配子体对大劣按纹感染性的活力周期

用食蟹猴疟原虫Ｂ株早期环状体接种１只恒河猴，接种后第２－１２天（血内仅存在１群原虫）测试了由第１－４代裂殖子形成的４代配子体（Ｇ）对大劣按蚊（海南株）感染性的活力周期。结果表明第１－４代裂殖子均形成了一定数量的Ｇ，由裂殖子发育到Ｇ功能成熟（可使蚊感染）各代有差异，第２代Ｇ至少需５５±１ｈ，第３代Ｇ则需６５±１ｈ；发育至７２±４ｈ时活力最旺，Ｇ寿命长者可达１００ｈ；生活期（对蚊有感染力的时间）第１、２代Ｇ约为１２－４２ｈ，第３代Ｇ约为１０－１９ｈ。Ｇ密度与蚊胃卵囊均数不成正比，第１、２代Ｇ对蚊的感染性比第３、４代强     

感染细胞内问号钩端螺旋体吞噬泡形成及其与溶酶体的融合

目的了解问号钩端螺旋体（简称钩体）感染宿主细胞后吞噬泡形成、吞噬泡与溶酶体融合及感染细胞超微结构改变。方法用问号钩体黄疸出血群赖型56601株感染小鼠单核巨噬样细胞j774A．1和猴肾成纤维细胞Cos-7。采用透射电镜观察J774A．1细胞和Cos-7细胞胞内钩体吞噬泡的形成及感染细胞超微结构的改变。采用免疫双荧光染色法和激光共聚焦显微镜，观察含钩体吞噬泡与溶酶体融合情况。结果问号钩体56601株感染J774A．1细胞30min、感染Cos-7细胞15min即可在胞浆中发现膜包绕的含钩体吞噬泡，吞噬泡内钩体均保持原有生理弯曲。56601株问号钩体感染Cos-7细胞2h后，钩体吞噬泡膜开始消失，但未发现j774A．1细胞内钩体吞噬泡膜消失的现象。J774A．1细胞内钩体吞噬泡与溶酶体发生共区域化，表明吞噬泡与溶酶体发生融合。J774A．1细胞感染钩体后出现染色质浓缩形成的凋亡小体样结构、细胞空泡变性、线粒体肿胀等超微结构病变，但Cos-7细胞感染钩体后其超微结构基本正常。结论问号钩体感染J774A．1细胞和Cos-7细胞后可迅速形成吞噬泡并可能与钩体Ⅲ型分泌系统产物有关。J774A．1细胞内钩体吞噬泡可与溶酶体发生融合。不同细胞的钩体吞噬泡膜消失及超微结构改变有明显差异。     

微波照射对兔精囊腺超微结构影响的研究

本研究用微波(2450MHz)照射兔阴囊区,使阴囊皮肤温度升至41～42℃,维持20分钟。用透射电镜观察照射后1小时至45天之间不同时间精囊腺粘膜层和肌层的超微结构改变。结果表明,主细胞和平滑肌细胞对微波照射敏感。照射后1小时,已见少数主细胞和平滑肌细胞呈现超微结构改变。3～7天后退变明显加重,至45天时呈减轻趋势。主细胞改变主要表现为粗面内质网池和高尔基复合体囊泡的扩大,胞质内出现许多髓样结构与脂滴、微绒毛肿胀及核的退变。平滑肌细胞的改变则表现为胞质内出现多个大空泡及髓样结构等。部分基细胞也呈现退变。固有膜增厚,向上皮不规则内凸。本文联系微波的抗生育作用,讨论其对精囊腺影响的意义。     

青蒿化学成份及其与青蒿素伍用对鼠疟的药效学研究

实验采用鼠疟模型及Peters4天抑制试验法,对从青蒿中分离得到的主要化学成份及其与青蒿素伍用进行了药效学评价。结果显示,青蒿素（QHS）的ED50和ED90分别为（10.2±1.3）mg/kg/d和（29.0±2.7）mg/kg/d。将青蒿素、青蒿酸、青蒿乙素、东莨菪内酯按1∶1∶1∶1混合得到QHH,其ED50和ED90分别为（12.6±1.1）mg/kg/d和（47.0±5.7）mg/kg/d。青蒿酸（QHA）、青蒿乙素（QHB）、东莨菪内酯（QHC）、青蒿黄酮1（QHD）、青蒿黄酮2（QHE）5种成份对鼠疟均有不同程度的抑制作用,但剂量高达500mg/kg/d时,抑制率最高也仅达到59%,远低于QHS的疗效。将分离的5种成份分别与QHS的半数有效剂量配伍进行药效测定,仅QHC的高剂量组显示一定的协同作用,其他各个组均未显示增效。QHH中QHS仅占1/4,但对鼠疟药效测定的结果ED50和ED90的值与QHS相近,提示QHA、QHB、QHC混合成份对QHS有一定的协同作用。因此,传统中药青蒿对疟疾的疗效是由以青蒿素为主的多组份共同作用的结果。     

Leishmania mexicana amazonensis: Attachment to the membrane of the phagocytic vacuole of macrophages in vivo

Intracellular forms ofLeishmania mexicana amazonensis divide inside the phagocytic vacuole of macrophages. Some parasites attach to the membrane of the phagocytic vacuole while others remain free in the vacuole. Examination of thin sections of the attachment region by electron microscopy revealed a space of 2 nm between the membrane of the phagocytic vacuole and the plasma membrane of the parasite. Freeze-fracture replicas showed an array of intramembranous particles in some areas of the parasite's plasma membrane resembling a gap junction which, in other cells, is involved in the process of intercellular communication.     

Experimental infection of mouse peritoneal mesothelium with scrub typhus rickettsiae: an ultrastructural study.

The infection cycle of Rickettsia tsutsugamushi in mouse peritoneal mesothelial cells, observed late in the course of an established infection, intimately involved the host cell plasma membrane. Organisms multiplied in the cytoplasm, moved to the cell periphery, and acquired a host-membrane coat as they budded from the cell surface. Rickettsiae enveloped by this membrane entered other mesothelial cells, apparently by a phagocytic mechanism. Organisms escaped from the phagocytic vacuole as the vacuole membrane and host membrane coat disintegrated. Free rickettsiae replicated by binary fission in the cell cytoplasm. Rickettsial infection of mesothelial cells induced conspicuous cellular hypertrophy with increased numbers of unaltered cytoplasmic organelles.     

Kinetics and pattern of organelle exocytosis duringToxoplasma gondii/host-cell interaction

The fate ofToxoplasma gondii dense-granule (GRA2, GRA3), rhoptry (ROP1), and surface (SAG1) proteins was followed by immunofluorescence assay (IFA) and immunoelectron microscopy at different stages after infection. Dense-granule exocytosis occurred in the apical area of the tachyzoite within minutes of invasion. Several exocytic events were found simultaneously in the same organism, both by serial sectioning and by freeze-fracture studies. Dense-granule contents were first found as a dense material trapped between parasite and vacuole membranes before either the vacuolar network or the vacuole membrane could be immunolabeled with specific antibodies. The vacuolar network was strongly labeled with dense-granule antibodies but not with the SAG1-specific probe, which suggests that the network is not enriched in membrane proteins. In addition to strongly labeling the vacuole membrane, GRA3 antibodies also labeled strands extending from the parasitophorous vacuoles into the host-cell cytoplasm.     

Biosynthesis, localization, and processing of falcipain cysteine proteases of Plasmodium falciparum

Falcipain-2 and -3 are cysteine proteases of erythrocytic Plasmodium falciparum parasites that appear to function principally as hemoglobinases. To better understand their biological roles, we analyzed the biosynthesis, localization, and processing of these enzymes in cultured parasites. Immunoprecipitation of metabolically labeled proteins indicated that falcipain-2 was synthesized through the trophozoite stage, falcipain-3 appeared in late trophozoites/early schizonts, and both proteases persisted for at least 6 h after synthesis. Falcipain-2 and -3 were localized to the food vacuole, the site of hemoglobin hydrolysis, by immunofluorescence and immunoelectron microscopy. Subcellular fractionation experiments indicated that both enzymes were synthesized as membrane bound proforms that were processed to soluble mature forms, but falcipain-2 was processed to the mature protease much more quickly than was falcipain-3. Cysteine protease inhibitors and brefeldin A, but not aspartic or serine protease inhibitors, blocked the processing of both enzymes, suggesting that falcipain-2 and -3 process by autohydrolysis after exiting the endoplasmic reticulum/Golgi network. However, although all tested cysteine protease inhibitors blocked hemoglobinase activity in the food vacuole, only lipophilic inhibitors (E-64d, Mu-Leu-Hph-VSPh, and ALLN), blocked intracellular processing of falcipain-2 and -3. More hydrophilic inhibitors (E-64 and leupeptin) did not block processing, suggesting that autocatalytic processing of falcipain-2 and -3 occurs in a specific cellular compartment before delivery to the food vacuole. Our results support overlapping but not fully redundant roles for falcipain-2 and -3, which are targeted to the food vacuole and activated sequentially to degrade hemoglobin in erythrocytic parasites.     

Plasmepsin 4, the food vacuole aspartic proteinase found in all Plasmodium spp. infecting man

Plasmepsins are aspartic proteinases of the malaria parasite, and seven groups of plasmepsins have been identified by comparing genomic sequence data available for the genes encoding these enzymes from Plasmodium falciparum, Plasmodium vivax, Plasmodium knowlesi, Plasmodium berghei, and Plasmodium yoelii. The food vacuole plasmepsins typified by plasmepsin 4 from P. falciparum (PfPM4) constitute one of these groups. Genes encoding the ortholog of PfPM4 have been cloned from Plasmodium ovale, Plasmodium malariae, and P. vivax. In addition, P. falciparum contains three paralagous food vacuole plasmepsins or plasmepsin-like enzymes that appear to have arisen by gene duplication, plasmepsins 1 (PfPM1), 2 (PfPM2) and HAP, and all four were localized to purified food vacuole preparations by two-dimensional gel electrophoresis and mass spectroscopic analysis. The three paralogs of PfPM4 do not have counterparts in the six other Plasmodium spp. examined by genomic DNA blot analysis and by review of available genomic sequence data. The presence of these paralogs among the food vacuole plasmepsins in P. falciparum as compared with the other three species causing malaria in man will impact efforts to rationally design antimalarials targeting the food vacuole plasmepsins.     

Labeled probes inserted in the macrophage membrane are transferred to the parasite surface and internalized during cell invasion by Toxoplasma gondii

Tachyzoites of Toxoplasma gondii attach to the macrophage surface and are internalized either by a phagocytic process, which can be inhibited by cytochalasin D, or by an active process, independent of host-cell actin. Previous studies have shown that parasite attachment induces the secretion of macromolecules found in the apical organelles (micronemes and rhoptries) and subsequent/concomitant parasite internalization with the formation of a membrane-bound vacuole known as the parasitophorous vacuole. In the present study we labeled the macrophage surface with fluorescent probes that bind to proteins (DiIC16) and lipids (DTAF) and then allowed control or cytochalasin-D-treated cells to interact with untreated or antibody-coated tachyzoites of T. gondii. The interaction was interrupted at different time points by fixation and the distribution of the probes was analyzed by confocal laser scanning microscopy. Following attachment of the parasites to the macrophage surface, intense labeling of the parasite surface was observed, suggesting transfer of components of the macrophage surface to the parasite surface. Nonadherent parasites were not labeled. Immediately after attachment, most of the parasites were internalized and labeling of the internalized parasites as well as of the parasitophorous vacuole, probably of its membrane, was evident, indicating that surface components of the macrophage are involved in the formation of the parasitophorous vacuole. Received: 30 April 1999 / Accepted: 21 July 1999     

Utrastructural observations on <Emphasis Type="Italic">Goussia metchnikovi</Emphasis> (Laveran, 1897) in the spleen of gudgeon, <Emphasis Type="Italic">Gobio gobio</Emphasis> L.

The ultrastructure of gamonts and sporulated oocysts of Goussia metchnikovi in the spleen of gudgeon, Gobio gobio from the river Lee, England is described. In developing microgamonts, small amylopectin granules were grouped centrally and nuclei were often arranged peripherally, close to the surface membrane. Nuclear chromatin condensed into peripheral dense portions that became the nuclei of flagellated microgametes, released to the parasitophorous vacuole. The cytoplasm of macrogametes had larger, scattered amylopectin granules, lipid globules and small electron-dense bodies, but no obvious wall forming bodies; peripheral vesicular structures with the appearance of mitochondria were also present and the parasitophorous vacuole contained flocculent material, but was otherwise free of structures. Sporulated oocysts contained four sporocysts and oocyst walls appeared to consist of a single membrane. Sporocyst walls showed a dehiscence suture, characteristic of the genus Goussia, which had filamentous extensions in places. The sporocyst wall comprised a dense inner layer and a thin outer layer with a fuzzy coat, separated by an electron lucent layer. Groups of oocysts were encapsulated by fibrous layers and inflammatory cells, and many sporocysts and their contained sporozoites showed evidence of elimination by the host.     

Plasmodium falciparum: inhibitors of lysosomal cysteine proteinases inhibit a trophozoite proteinase and block parasite development

Trophozoites of Plasmodium falciparum obtain free amino acids for protein synthesis by degrading host erythrocyte hemoglobin in an acidic food vacuole. We previously reported that leupeptin and L-trans-epoxysuccinyl-leucylamido(4-guanidino)butane (E-64), two inhibitors of the cysteine class of proteinases, blocked hemoglobin degradation in the trophozoite food vacuole, and we identified a 28-kDa trophozoite cysteine proteinase as a potential food vacuole hemoglobinase. We now report that the biochemical properties of the trophozoite cysteine proteinase closely resembled those of the lysosomal cysteine proteinases cathepsin B and cathepsin L. The trophozoite proteinase had a pH optimum of 5.5-6.0, near that of both lysosomal proteinases, and it was efficiently inhibited by highly specific diazomethylketone and fluoromethylketone inhibitors of cathepsin B and cathepsin L. The trophozoite proteinase preferred peptide substrates with arginine adjacent to hydrophobic amino acids, as does cathepsin L. Micromolar concentrations of the fluoromethylketone inhibitor Z-Phe-Ala-Ch2F blocked the degradation of hemoglobin in the trophozoite food vacuole and prevented parasite multiplication. In previous studies much higher concentrations of the inhibitor were not toxic for mice. Our results provide additional evidence that the 28-kDa trophozoite proteinase is a food vacuole hemoglobinase and suggest that specific inhibitors of the enzyme may have potential as antimalarial drugs.     

糖尿病大鼠味腺超微结构的研究

目的观察2型糖尿病大鼠味腺超微结构变化。方法雄性Wistar大鼠18只,随机分为对照组和实验组,高脂喂养加小剂量链脲佐菌素(STZ)腹腔注射制备2型糖尿病模型,饲养8周后取轮廓乳头,应用透射电镜观察味腺超微结构。结果与对照组大鼠比较,实验组大鼠味腺腺泡细胞的核膜凹陷、核固缩;线粒体嵴断裂、空泡变性;粗面内质网扩张,脱颗粒。结论糖尿病可导致味腺组织出现一系列超微结构病理改变,为进一步探讨糖尿病发生机制提供了形态学依据。