Antisense RNA inactivation of myosin heavy chain gene expression in Dictyostelium discoideum

The role of myosin in the contraction of striated muscle cells is well known, but its importance in nonmuscle cells is not yet clear. The function of myosin in Dictyostelium discoideum has been investigated by isolating cells which specifically lack myosin heavy chain (MHC A) protein. Cells were transformed with a vector encoding RNA complementary to mhcA messenger RNA (antisense RNA). Stable transformants have a dramatic reduction in the amount of MHC A protein, grow slowly, and generate giant multinucleated progeny, indicating an impairment in cytokinesis. Surprisingly, the cells adhere to surfaces, extend pseudopods and are capable of ameboid locomotion. The developmental sequence that is initiated by starving cells is severely impaired by the lack of myosin. The cells are unable to form multicellular aggregates normally and do not undergo subsequent morphogenesis. By changing the food source from liquid medium to bacteria, expression of the endogenous mhcA messenger RNA can be increased relative to expression of antisense RNA. When grown in this way, the transformed cells accumulate MHC A protein, remain mononucleate, and proceed through development normally.     

Disruption of the Dictyostelium myosin heavy chain gene by homologous recombination

The phenomenon of homologous recombination, which allows specific gene conversion and gene insertion, can be a powerful system for the study of eukaryotic cell biology. Data are presented demonstrating that integration of a transfected plasmid by homologous recombination occurs in the motile eukaryotic cell Dictyostelium discoideum. A plasmid carrying a G418 resistance gene and the amino terminal half of the myosin heavy chain gene was used to transfect Dictyostelium. A large fraction of the resultant G418-resistant cells had the plasmid integrated into the single genomic copy of the heavy chain gene. These cells, which fail to express the native myosin but express the myosin fragment, are defective in cytokinesis and become large and multinucleate. In spite of the absence of native myosin, these cells, termed hmm cells, exhibit many forms of cell movement, including membrane ruffling, phagocytosis, and chemotaxis. The hmm cells can aggregate but are blocked at a later stage in the Dictyostelium developmental cycle. The hmm cells revert to the wild-type phenotype. Reversion of the hmm phenotype is due to excision and loss of the transforming plasmid. The revertant cells express native myosin, are G418 sensitive, and have a normal developmental cycle. These results constitute genetic proof that the intact myosin molecule is required for cytokinesis and not for karyokinesis.     

The signal to move: D. discoideum go orienteering

Cells migrating directionally toward a chemoattractant source display a highly polarized cytoskeletal organization, with F-actin localized predominantly at the anterior and myosin II at the lateral and posterior regions. Dictyostelium discoideum has proven a useful system for elucidating signaling pathways that regulate this chemotactic response. During development, extracellular adenosine 3', 5' monophosphate (cAMP) functions as a primary signal to activate cell surface cAMP receptors (cARs). These receptors transduce different signals depending on whether or not they are coupled to heterotrimeric guanine nucleotide-binding proteins (G proteins) (see the STKE Connections Maps). Multiple G protein-stimulated pathways interact to establish polarity in chemotaxing D. discoideum cells by localizing F-actin at their leading edge and by regulating the phosphorylation state and assembly of myosin II. Many of the molecular interactions described are fundamental to the regulation of chemotaxis in other eukaryotic cells.     

应用细胞培养方法分离猪源弓形虫虫株

为获得猪源弓形虫野生虫株,对细胞培养过程中的培养基种类、血清用量及培养佐剂等条件进行优化,选择最佳分离条件。对临床血清学检测呈阳性猪场的19份血液样品进行弓形虫虫株的培养检测,共获得野生虫株10株。运用PCR方法进行初步鉴定,证实分离虫株为弓形虫野生虫株。结果表明,细胞培养方法是一种分离弓形虫虫株较为理想的方法。     

Interferon-gamma: the major mediator of resistance against Toxoplasma gondii

Mice were injected with a monoclonal antibody to interferon-gamma to examine the importance of endogenous production of this lymphokine in resistance against infection with the sporozoan parasite Toxoplasma gondii. Mice with intraperitoneal infections of T. gondii that received no antibody survived and developed chronic T. gondii infection, whereas the infected mice that received the monoclonal antibody died of toxoplasmosis. The activation of macrophages, which kill T. gondii in vivo, was inhibited by administration of the monoclonal antibody, but the production of antibodies to T. gondii was not suppressed. The fact that an antibody to interferon-gamma can eliminate resistance to acute Toxoplasma infection in mice suggests that this lymphokine is an important mediator of host resistance to this parasite.     

小菜蛾肌球蛋白重链部分cDNA序列克隆、表达概况及分析

肌肉肌球蛋白重链在生物运动过程中具有至关重要的作用,但在昆虫中此类基因极少被报道。首次克隆和分析小菜蛾肌球蛋白重链部分cDNA序列,GenBank登录号为FJ462712,其长408个碱基,推测编码135个氨基酸。同源分析表明:此多肽序列与凤蝶、家蚕、果蝇的同源性分别为95%,93%,82%。半定量RT-PCR研究表明:小菜蛾MHC基因在不同的生长发育阶段的表达皆不相同,在老熟幼虫和成虫中的表达量显著高于低龄幼虫和蛹。为进一步鉴定小菜蛾肌球蛋白重链的生理特征提供基因序列信息。     

Expression and characterization of a functional myosin head fragment in Dictyostelium discoideum

The isolated head fragment of myosin is a motor protein that is able to use energy liberated from the hydrolysis of adenosine triphosphate to cause sliding movement of actin filaments. Expression of a myosin fragment nearly equivalent to the amino-terminal globular head domain, generally referred to as subfragment 1, has been achieved by transforming the eukaryotic organism Dictyostelium discoideum with a plasmid that carries a 2.6-kilobase fragment of the cloned Dictyostelium myosin heavy chain gene under the control of the Dictyostelium actin-15 promoter. The recombinant fragment of the myosin heavy chain was purified 2400-fold from one of the resulting cell lines and was found to be functional by the following criteria: the myosin head fragment copurified with the essential and regulatory myosin light chains, decorated actin filaments, and displayed actin-activated adenosine triphosphatase activity. In addition, motility assays in vitro showed that the recombinant myosin fragment is capable of supporting sliding movement of actin filaments.     

弓形虫AMA1蛋白真核表达载体的构建及免疫活性检测

[目的]构建弓形虫AMA1基因的真核表达质粒,研究其在体外细胞中的表达情况。[方法]运用PCR方法和克隆技术,构建pcD-NA3.1-AMA1重组质粒;利用脂质体介导转染法,将该重组质粒导入HEK-293细胞,用Western blotting法检测其在体外的表达情况。[结果]从弓形虫RH株cDNA中扩增出AMA1基因片段,成功构建重组表达质粒pcDNA3.1-AMA1,转染AMA1基因的HEK-293细胞表达产物经Western blotting鉴定,其分子量约为70kD,能被特异性免疫血清所识别。[结论]构建的真核表达质粒pcDNA3.1-AMA1能在HEK-293细胞中表达,为下一步弓形虫DNA疫苗的研究奠定了基础。     

转染技术在顶复门原虫研究中的应用

 自1993年转染技术首次在弓形虫中成功报道以来,顶复门原虫的转染研究得到了快速发展,不仅在弓形虫、疟原虫和住肉孢子虫等实现了稳定转染,而且在体外细胞上生长效率较低的柔嫩艾美耳球虫也相继实现了瞬时转染和稳定转染。顶复门原虫转染技术的日渐成熟及其在顶复门原虫研究中的应用,为顶复门原虫基因功能和免疫学等领域研究提供了新思路和新的有力工具。本文结合本课题组近几年来的研究成果,综述了转染技术及其在顶复门原虫基因表达调控、基因功能、耐药性分子机理、药物敏感性试验和免疫学等研究中应用的最新进展。     

TLR11 activation of dendritic cells by a protozoan profilin-like protein

Mammalian Toll-like receptors (TLRs) play an important role in the innate recognition of pathogens by dendritic cells (DCs). Although TLRs are clearly involved in the detection of bacteria and viruses, relatively little is known about their function in the innate response to eukaryotic microorganisms. Here we identify a profilin-like molecule from the protozoan parasite Toxoplasma gondii that generates a potent interleukin-12 (IL-12) response in murine DCs that is dependent on myeloid differentiation factor 88. T. gondii profilin activates DCs through TLR11 and is the first chemically defined ligand for this TLR. Moreover, TLR11 is required in vivo for parasite-induced IL-12 production and optimal resistance to infection, thereby establishing a role for the receptor in host recognition of protozoan pathogens.     

Myosin I can act as a molecular force sensor

The ability to sense molecular tension is crucial for a wide array of cellular processes, including the detection of auditory stimuli, control of cell shape, and internalization and transport of membranes. We show that myosin I, a motor protein that has been implicated in powering key steps in these processes, dramatically alters its motile properties in response to tension. We measured the displacement generated by single myosin I molecules, and we determined the actin-attachment kinetics with varying tensions using an optical trap. The rate of myosin I detachment from actin decreases >75-fold under tension of 2 piconewtons or less, resulting in myosin I transitioning from a low (<0.2) to a high (>0.9) duty-ratio motor. This impressive tension sensitivity supports a role for myosin I as a molecular force sensor.     

Polarized myosin produces unequal-size daughters during asymmetric cell division

Asymmetric positioning of the mitotic spindle before cytokinesis can produce different-sized daughter cells that have distinct fates. Here, we found an asymmetric division in the Caenorhabditis elegans Q neuroblast lineage that began with a centered spindle but generated different-sized daughters, the smaller (anterior) of which underwent apoptosis. During this division, more myosin II accumulated anteriorly, suggesting that asymmetric contractile forces might produce different-sized daughters. Indeed, partial inactivation of anterior myosin by chromophore-assisted laser inactivation created a more symmetric division and allowed the survival and differentiation of the anterior daughter. Thus, the balance of myosin activity on the two sides of a dividing cell can govern the size and fate of the daughters.     

PCR检测急·慢性弓形虫感染小鼠的研究

[目的]建立灵敏、特异、稳定的检测弓形虫感染的PCR法。[方法]检测弓形虫急、慢性感染鼠血清及组织中弓形虫速殖子DNA,并对检测感染鼠血清中弓形虫速殖子的灵敏度进行评估。[结果]急性感染鼠接种后18 h,40%的感染鼠被检测为阳性;接种后21 h,80%的感染鼠被检测为阳性;24 h以后100%的感染鼠被检测为阳性。慢性感染鼠接种后18 d可从肝、肺、脾、肾、脑组织中检测到弓形虫DNA。试验中检测到1个弓形虫,相当于0.2 pg的弓形虫DNA含量。[结论]结果为运用PCR法检测弓形虫感染提供了参考。     

Polarity and velocity of sliding filaments: control of direction by actin and of speed by myosin

Myosin filaments, which are responsible for a large repertoire of motile activities in muscle and nonmuscle cells, can translocate actin filaments both toward and away from their central bare zone. This bidirectional movement suggests that there is enough flexibility in the head portion of the tightly packed myosin molecules in the native myosin filaments to move actin filaments not only in the expected direction, but also in the direction opposite to that predicted by the regular structure of muscle--away from the center of the myosin filament.     

弓形虫GRA3基因真核表达质粒的构建与鉴定

以pGEX-GRA3为模板,经PCR扩增获得弓形虫GRA3基因,克隆于pMD-18T simple载体上。重组质粒经PCR鉴定、PstⅠ和XhoⅠ双酶切鉴定后测序分析,再将其定向插入真核表达载体pVAXⅠ,构建真核表达重组质粒pVAX-GRA3。经PCR和酶切鉴定后,将重组质粒pVAX-GRA3转染Hela细胞,然后进行RT-PCR检测。RT-PCR结果表明,检测到了目的基因的存在,说明pVAX-GRA3成功转入Hela细胞。     

刚地弓形虫RH株SAG3蛋白抗体检测的ELISA方法建立

[目的]利用纯化的刚地弓形虫RH株表面蛋白SAG3的原核重组蛋白为基础,建立抗体间接ELISA检测方法。[方法]采用棋盘滴定法,确定间接ELISA的最佳条件。采用鼠抗新孢子虫阳性血清、鼠抗安氏隐孢子虫阳性血清、鼠抗柔嫩艾美尔球虫阳性血清、鼠抗蓝氏贾第虫阳性血清以及鼠抗微小隐孢子虫阳性血清和健康小鼠阴性血清作对照,检测血清之间是否有交叉反应。[结果]最佳包被条件为:抗原包被浓度0.25μg/孔,被检血清稀释度1∶400,酶标二抗稀释度1∶3 000,封闭剂5%脱脂奶粉的PBST溶液;血清之间没有交叉反应。该方法的灵敏度为1∶1 600。[结论]该方法灵敏度高,特异性较强,可重复性较好,可用于对弓形虫感染的检测及流行病学调查。     

不同宿主源弓形虫ROP16基因的遗传变异分析

为研究不同分离株弓形虫棒状体蛋白ROP16基因的遗传变异,扩增9种不同来源的弓形虫样品的ROP16基因,并对其序列进行比对及构建进化树的分析。结果表明,弓形虫不同分离株ROP16序列的同源性均在990%以上,但存在限制性位点多态性;用弓形虫ROP16作为PCR-RFLP的标记分子,未能鉴别出传统的弓形虫Ⅰ型、Ⅱ型、Ⅲ型,却鉴定出一株特殊的猪源弓形虫分离株。弓形虫ROP16基因的株间保守性提示其是一个用于预防弓形虫病的潜在疫苗候选分子,值得对其进行深入研究。     

猪弓形虫病PCR诊断方法的建立

以具有35个拷贝的重复序列B1基因作为弓形虫种特异的遗传标记,建立诊断弓形虫病特异性的PCR方法.敏感性和特异性试验结果表明:该PCR方法最低能检测到10个弓形虫速殖子DNA,且与寄生于猪体内的6种常见微生物无交叉反应,说明该PCR方法具有高度的敏感性和特异性.动物感染试验检测结果表明,在试验动物出现明显临床症状时,对取材的7个组织样品用该PCR方法检测,有6个组织样品为阳性,其中扩增结果最清晰的的组织为肺和肝组织.     

弓形虫分子免疫学研究进展

弓形虫病是由刚第弓形虫引起的一种危害严重的全球性人兽共患寄生虫病,全世界各国学者对此原虫的研究十分重视,尤其是近年来对其免疫学的研究成果对深入了解该原虫的致病机理和进行有效的免疫预防非常重要。从宿主感染弓形虫后的免疫效应、免疫诊断及其疫苗等方面阐述了弓形虫分子免疫学研究进展。