Regulation of carbamoyl phosphate synthetase by MAP kinase

The de novo synthesis of pyrimidine nucleotides is required for mammalian cells to proliferate. The rate-limiting step in this pathway is catalysed by carbamoyl phosphate synthetase (CPS II), part of the multifunctional enzyme CAD. Here we describe the regulation of CAD by the mitogen-activated protein (MAP) kinase cascade. When phosphorylated by MAP kinase in vitro or activated by epidermal growth factor in vivo, CAD lost its feedback inhibition (which is dependent on uridine triphosphate) and became more sensitive to activation (which depends upon phosphoribosyl pyrophosphate). Both these allosteric regulatory changes favour biosynthesis of pyrimidines for growth. They were accompanied by increased epidermal growth factor-dependent phosphorylation of CAD in vivo and were prevented by inhibition of MAP kinase. Mutation of a consensus MAP kinase phosphorylation site abolished the changes in CAD allosteric regulation that were stimulated by growth factors. Finally, consistent with an effect of MAP kinase signalling on CPS II activity, epidermal growth factor increased cellular uridine triphosphate and this increase was reversed by inhibition of MAP kinase. Hence these studies may indicate a direct link between activation of the MAP kinase cascade and de novo biosynthesis of pyrimidine nucleotides.     

Abscisic acid controls calcium-dependent egress and development in Toxoplasma gondii

Calcium controls a number of critical events, including motility, secretion, cell invasion and egress by apicomplexan parasites. Compared to animal and plant cells, the molecular mechanisms that govern calcium signalling in parasites are poorly understood. Here we show that the production of the phytohormone abscisic acid (ABA) controls calcium signalling within the apicomplexan parasite Toxoplasma gondii, an opportunistic human pathogen. In plants, ABA controls a number of important events, including environmental stress responses, embryo development and seed dormancy. ABA induces production of the second-messenger cyclic ADP ribose (cADPR), which controls release of intracellular calcium stores in plants. cADPR also controls intracellular calcium release in the protozoan parasite T. gondii; however, previous studies have not revealed the molecular basis of this pathway. We found that addition of exogenous ABA induced formation of cADPR in T. gondii, stimulated calcium-dependent protein secretion, and induced parasite egress from the infected host cell in a density-dependent manner. Production of endogenous ABA within the parasite was confirmed by purification (using high-performance liquid chromatography) and analysis (by gas chromatography-mass spectrometry). Selective disruption of ABA synthesis by the inhibitor fluridone delayed egress and induced development of the slow-growing, dormant cyst stage of the parasite. Thus, ABA-mediated calcium signalling controls the decision between lytic and chronic stage growth, a developmental switch that is central in pathogenesis and transmission. The pathway for ABA production was probably acquired with an algal endosymbiont that was retained as a non-photosynthetic plastid known as the apicoplast. The plant-like nature of this pathway may be exploited therapeutically, as shown by the ability of a specific inhibitor of ABA synthesis to prevent toxoplasmosis in the mouse model.     

Virulent strains of Toxoplasma gondii comprise a single clonal lineage.

The protozoan Toxoplasma gondii is a prevalent parasite in wild and domestic animals worldwide, being transmitted through the food chain by carnivorous feeding and scavenging. Toxoplasma normally divides asexually to yield a haploid form that can infect virtually any vertebrate but it also has a well defined sexual cycle that occurs exclusively in cats. Toxoplasma has become important as an often fatal opportunistic pathogen in patients with AIDS, although the 15-85% of adult human populations that are chronically infected with T. gondii are typically asymptomatic. Infections in immunocompromised hosts have variable outcomes. For example, only 30 to 50% of AIDS patients that are chronically infected with the parasite develop toxoplasmic encephalitis and only about half of acute maternal infections result in congenital disease of the newborn. T. gondii strains differ in their virulence in animals, but the extent to which different strains are related has not been determined. Here we analyse 28 strains from a variety of hosts on five continents and find that the ten virulent strains have an essentially identical genotype, whereas the nonvirulent strains are moderately polymorphic. These data strongly suggest that virulent strains of T. gondii originated from a single lineage which has remained genetically homogeneous despite being globally widespread, and despite the ability of this organism to reproduce sexually.     

Uptake of apoptotic cells drives the growth of a pathogenic trypanosome in macrophages

After apoptosis, phagocytes prevent inflammation and tissue damage by the uptake and removal of dead cells. In addition, apoptotic cells evoke an anti-inflammatory response through macrophages. We have previously shown that there is intense lymphocyte apoptosis in an experimental model of Chagas' disease, a debilitating cardiac illness caused by the protozoan Trypanosoma cruzi. Here we show that the interaction of apoptotic, but not necrotic T lymphocytes with macrophages infected with T. cruzi fuels parasite growth in a manner dependent on prostaglandins, transforming growth factor-beta (TGF-beta) and polyamine biosynthesis. We show that the vitronectin receptor is critical, in both apoptotic-cell cytoadherence and the induction of prostaglandin E2/TGF-beta release and ornithine decarboxylase activity in macrophages. A single injection of apoptotic cells in infected mice increases parasitaemia, whereas treatment with cyclooxygenase inhibitors almost completely ablates it in vivo. These results suggest that continual lymphocyte apoptosis and phagocytosis of apoptotic cells by macrophages have a role in parasite persistence in the host, and that cyclooxygenase inhibitors have potential therapeutic application in the control of parasite replication and spread in Chagas' disease.     

Golgi biogenesis in Toxoplasma gondii

Two models have been put forward to explain the growth of new Golgi during the cell cycle. The first suggests that a new Golgi grows out of the endoplasmic reticulum by de novo synthesis. The second suggests that a pre-existing Golgi is needed for the growth of a new one, that is, the Golgi is an autonomously replicating organelle. To resolve this issue, we have exploited the simplicity of the apicomplexan parasite Toxoplasma gondii, which has only a single Golgi stack. Here we show, by using video fluorescence microscopy and three-dimensional reconstructions of serial thin sections, that the Golgi grows by a process of lateral extension followed by medial fission. Further fission leads to the inheritance by each daughter of a pair of Golgi structures, which then coalesce to re-form a single Golgi. Our results indicate that new Golgi grow by autonomous duplication and raise the possibility that the Golgi is a paired structure that is analogous to centrioles.     

FGFR2IIIc及其突变型腺病毒对小鼠乳腺癌作用的研究

目的：研究FGFR2IIIc及其突变型腺病毒对小鼠乳腺癌的作用。方法：确定腺病毒颗粒Ad、Ad-FGFR2IIIc及Ad-FGFR2IIIcS252W感染4T1细胞的最佳MOI,并通过RT-PCR检测外源目的基因的表达。建立BALB/c小鼠的4T1乳腺癌模型,在此模型上原位注射重组腺病毒颗粒治疗17天,观察其对小鼠肿瘤的作用。结果：当MOI=60时腺病毒对4T1细胞的感染率可达到95%以上,RT-PCR结果显示,FGFR2IIIc和FGFR2IIIcS252W可在4T1细胞中成功表达。与对照组和Ad组比较,Ad-FGFR2IIIc对小鼠肿瘤无明显作用,Ad-FGFR2IIIcS252W能显著减缓小鼠肿瘤体积和质量的增长。结论：FGFR2IIIc对BALB/c小鼠4T1乳腺癌无显著作用,FGFR2IIIcS252W能够显著抑制BALB/c小鼠4T1乳腺癌的生长。     

Minor but not major histocompatibility antigens of thymus epithelium tolerize precursors of cytolytic T cells

Treatment of fetal thymuses with 2-deoxyguanosine depletes these organs of many haematopoietic cells, and if such thymuses are transplanted into allogeneic athymic nude mice, intrathymic development of cytolytic T-lymphocyte precursors (CTL-P) occurs, including those which are specific for class I major histocompatibility complex (MHC) antigens expressed by the thymus epithelium. Thus, T cells from BALB/c (H-2d) nude mice transplanted with allogeneic C57BL/6 (H-2b) thymic epithelium can be stimulated in vitro to produce CTL specific for H-2b class I MHC antigens. We report here that thymocytes and lymph node T cells from such mice are responsive in mixed leukocyte reaction in the absence of exogenous growth factors, indicating that lack of tolerance is manifest at the level of CTL-P and proliferating T cells. We also show that T cells from such mice are tolerant to minor histocompatibility antigens of the thymus donor in the context of MHC antigens of the recipient. The results indicate that haematopoietic rather than epithelial cells tolerize CTL-P and that donor-type minor but not major histocompatability antigens can be presented in tolerogenic form by haematopoietic cells expressing recipient-type MHC antigens.     

绿色荧光裸鼠部分免疫学指标的测定及分析

摘要: 目的检测绿色荧光蛋白( Green Fluorescence Protein,简称GFP) 转基因裸鼠的部分免疫学指标,分析GFP 转基因裸鼠与其他小鼠在免疫学方面的优缺点,为将来的研究提供基础参考值。方法① 实验选用6 ～ 8 周GFP 转基因裸鼠、BALB/c 裸鼠和BALB/c 小鼠,活体称体质量,剖取脾脏称其质量,之后进行脾淋巴细胞增殖能力的测定。② 取外周血,测定部分血常规指标及免疫球蛋白指标。结果① GFP 转基因裸鼠脾脏质量、脾脏系数以及T、B 淋巴细胞增殖反应与BALB/c 裸鼠相近,但与BALB/c 小鼠差异显著。② GFP 转基因裸鼠、BALB/c 裸鼠两种裸鼠品系外周血白细胞水平、淋巴细胞以比例及免疫球蛋白IgG、IgM 都远低于BALB/c 小鼠,差异显著。结论从脏器系数、白细胞水平,免疫球蛋白水平等角度发现,GFP 转基因裸鼠和BALB/c 裸鼠的免疫系统水平较BALB/c 小鼠相弱,其中GFP 转基因裸鼠的免疫系统水平最为低下。     

Protective effect of DNA-mediated immunization with liposome-encapsulated GRA4 against infection of Toxoplasma gondii

The dense granule protein 4 (GRA4) is a granular protein from Toxoplasma gondii, and is a candidate for vaccination against this parasite. In this study, the plasmid pcDNA3.1-GRA4 (pGRA4), encoding for the GRA4 antigen, was incorporated by the dehydration-rehydration method into liposomes composed of 16 mmol/L egg phosphatidylcholine (PC), 8 mmol/L dioleoyl phosphatidylethanolamine (DOPE), and 4 mmol/L 1,2-diodeoyl-3-(trimethylammonium) propane (DOTAP). C57BL/6 mice and BALB/c mice were immunized intramuscularly three times with liposome-encapsulated pGRA4 to determine whether DNA immunization could elicit a protective immune response to T. gondii. Enzyme-linked immunosorbent assay (ELISA) of sera from immunized mice showed that liposome-encapsulated pGRA4 generated high levels of IgG antibodies to GRA4. Production of primary interferon (IFN)-γ and interleukin (IL)-2 in GRA4-stimulated splenocytes from vaccinated mice suggested a modulated Th1-type response. 72.7% of C57BL/6 mice immunized with liposome-encapsulated pGRA4 survived the challenge with 80 tissue cysts of ME49 strain, whereas C57BL/6 mice immunized with pGRA4 had only a survival rate of 54.5%. When immunized BALB/c mice were intraperitoneally challenged with 10³ tachyzoites of the highly virulent RH strain, the survival time of mice immunized with liposome-encapsulated pGRA4 was markedly longer than that of other groups. Our observations show that liposome-encapsulated pGRA4 enhanced the protective effect against infection of T. gondii.     

不同光照时间对BALB/c裸小鼠生长发育及繁殖影响的比较研究

目的比较不同光照时间对BALB/c裸小鼠的影响。方法观察裸小鼠出生后在不同光照时间里的生长情况;记录雌鼠的交配分娩间隔、生产胎数、产仔数及育成率;测定血液生化指标。结果各组裸小鼠的生长差异不显著(P>0.05);中光照组的平均育成率和离乳裸鼠育成率与长光照组比较差异显著(P<0.05);中光照组的ALT、TB、SUA与长光照组比较差异显著(P<0.05)。结论中光照和短光照适宜BALB/c裸小鼠的生长发育和繁殖。     

饲料硬度对BALB/c裸小鼠生长性能的影响

目的裸小鼠是免疫缺陷动物,饲料硬度对其采食性能及生长影响较大。研究和生产适合裸小鼠采食、硬度适宜并可提高利用率的颗粒饲料,以满足其正常生长。方法 SPF级10周龄BALB/c裸鼠240只,雌雄各半,随机分为实验Ⅰ组、实验Ⅱ组、实验Ⅲ组、实验Ⅳ、实验Ⅴ和实验Ⅵ组。实验Ⅰ-Ⅵ分别给予硬度为83.3N、96.0N、112.7N、132.3N、149.0N和181.3N的颗粒饲料,观察不同饲料硬度对裸鼠体质量增加的效果。结果各实验组动物生长情况显示,83.3N、96.0N、112.7N等3个实验组裸小鼠的体质量增加明显;随着饲料硬度增加,裸小鼠体质量增加量呈下降趋势。结论饲料硬度控制在108-137N范围,BALB/c裸小鼠生长性能良好,还可提高裸小鼠对饲料的利用率。     

裸小鼠的繁殖及在肿瘤学中的应用

目的　总结了八年实验动物裸小鼠繁殖和应用。方法　用随机抽取 2 0窝BALB/c裸小鼠繁殖生育情况分析和 5只BALB/c母鼠 (nu/ +)四胎生育情况比较。结果　BALB/c裸小鼠平均每胎产仔 9 35只 ,用nu/ +雌与nu/nu雄交配方法繁殖 ,平均每胎所得纯合子仔鼠能占仔数的 5 5 0 % ,离乳率 97% ,1～ 4胎生育恒定 ,一年存活率达10 0 % ,最长生存期可达 75 2d。微生物检测结果未发现致病菌 ,寄生虫等。结论　空气过滤十万级环境加上生物层流柜达一万级的条件 ,只要严格执行无菌操作 ,裸小鼠繁殖可基本达到SPF要求。     

Susceptibility of beta 2-microglobulin-deficient mice to Trypanosoma cruzi infection.

The beta 2-microglobulin (beta 2m) protein associates with the products of the class I major histocompatibility (MHC) loci; this combination functions in the thymic development of and antigen presentation to CD8+ T cells. Mice in which the beta 2m gene has been disrupted by homologous recombination fail to express class I MHC gene products, and therefore lack CD8+ T cells and measurable cytotoxic T-cell responses. However, beta 2m- mice appear to have normal development of both CD4+ alpha/beta T-cell receptor (TCR+) and gamma/delta TCR+ T cells and are not overtly more susceptible than beta 2m+ mice to potential environmental agents of infection or to experimental viral infection. Here we show that beta 2m- mice suffer high parasitaemias and early death when infected with the obligate cytoplasmic protozoan parasite Trypanosoma cruzi. Despite this increased susceptibility, the beta 2m- mice are more responsive than their beta 2m+ littermates in terms of lymphokine production, making higher levels of both interleukin-2 and interferon-gamma in response to mitogen stimulation. In addition, the beta 2m- mice show essentially no inflammatory response in parasite-infected tissues. These results confirm previous experiments on mice depleted of CD8+ cells using antibody treatment in demonstrating the importance of CD8+ T cells in immune protection in T. cruzi infection. They also implicate CD8+ T cells and/or class I MHC molecules in regulation of lymphokine production and recruitment of inflammatory cells.     

Failure of wild-type or a mutant form of protein kinase C-alpha to transform fibroblasts

A mutant form of the alpha-isoform of protein kinase C (PKC) was recently isolated from an ultraviolet radiation-induced murine fibrosarcoma cell line and reported to transform mouse BALB/c 3T3 fibroblasts on transfection. Four point mutations in the regulatory domain were assumed to be responsible for its oncogenicity and unusual preference for membrane localization. Here, we report that overexpression of the reported mutant PKC alpha complementary DNA in three fibroblast cell lines, including BALB/c 3T3, does not enable these cells to grow in soft agar or nude mice. In addition, this mutant PKC alpha form seems to be indistinguishable from the wild-type PKC alpha with respect to its dependence on cofactors, phorbol ester binding, subcellular distribution and its effects on growth and morphology. These results fail to confirm the previous study and indicate that overexpression of either the wild-type or the reported mutant form of PKC alpha does not transform rodent fibroblasts.     

KM小鼠与BALB／c小鼠五项免疫学指标的初步观察

采用五种免疫功能测定法对８３２只健康ＫＭ小鼠和８３只ＢＡＬＢ／ｃ小鼠进行免疫学功能指标的测定,结果表明ＣｏｎＡ诱导小鼠脾淋巴细胞转化能力ＯＤ值分别为０．０１４８－０．０１９２和０．０１２０１～０．０２０６,二硝基氟苯诱导小鼠迟发型变态反应（耳肿胀法）小鼠耳重差分别为１０．３９～１２．１７ｍｇ和１１．４６８～１６．２３４ｍｇ;血清溶血素抗体积数分别为７８．９８～９１．３０和１０６．７３～１１６．０７     

小鼠结核分枝杆菌感染模型用于评价重组HBHA疫苗免疫效果的实验研究

目的通过复制小鼠结核病模型评价重组HBHA疫苗的免疫效果。方法将重组肝素结合血凝素(HBHA)疫苗接种BALB/c小鼠,观察其诱导的细胞免疫应答水平,并以MTB H37Rv毒株攻击免疫小鼠,研究重组疫苗诱导的保护力。结果重组HBHA免疫BALB/c小鼠后可诱发细胞毒作用,将体内MTB裂解。重组疫苗不仅能刺激CD4+T,CD8+T增殖、活化,还可诱导脾细胞分泌IFN-γ、IL-2等细胞因子。H37Rv毒株攻击后,被免疫小鼠的组织病理学症状减轻。结论本研究通过检测小鼠体内细胞免疫应答和肺脏组织病理学改变等指标,客观的反映了重组疫苗免疫小鼠结核模型的保护效果。     

IVC系统和屏障系统对SPF级小鼠繁殖能力影响的比较研究

目的比较相对洁净环境下使用独立通风笼盒（IVC）系统和传统屏障饲养环境条件对SPF级BALB/c和C57BL/6两种近交系小鼠繁殖性能和生长发育的影响。方法将8周龄的SPF级BALB/c和C57BL/6小鼠以雌雄2∶1比例随机饲养在IVC饲养环境和传统屏障饲养环境下7个月,观察比较两种品系小鼠不同胎次产仔数、不同周龄的体质量变化、离乳率和交配间隔时间等繁殖和生长能力指标。结果在IVC饲养环境下两种品系的小鼠不同胎次产仔数、哺乳期及离乳后仔鼠体质量,离乳存活率和交配分娩间隔时间等指标与传统的屏障饲养环境下的相同指标相比无显著差异（P〉0.05）。结论在相对洁净环境下使用IVC系统与屏障环境相比,小鼠繁殖能力无显著差异,小规模的实验动物可以在IVC系统中进行生产繁殖。     

黑龙江省实验小鼠、大鼠遗传学质量调查

目的根据最新颁布的国家标准,对黑龙江省的实验小鼠、大鼠的遗传学质量进行调查。方法参照国家标准《实验动物近交系小鼠、大鼠生化标记检测方法》GB/T 14927.1-2008,应用乙酸纤维素板,对来自4个单位的BALB/c和C57BL/6实验小鼠及3个单位的SD和Wistar实验大鼠进行生化标记位点的检测。结果被检的BALB/c小鼠和大鼠中均存在杂合。结论大鼠符合封闭群遗传概貌,但近交系小鼠出现了遗传漂变。     

BALB/c和昆明（KM）小鼠脓毒症盲肠结扎穿孔模型的比较

目的比较应用BALB/c和昆明（KM）小鼠建立脓毒症盲肠结扎穿孔（Cecal Ligation and Puncture,CLP）模型的区别。方法 BALB/c和KM小鼠各30只,分别建立假手术组和CLP模型组,术后6 h进行血常规、生化检测、病理切片检查,术后72 h比较两种品系小鼠盲肠结扎模型的生存率。结果术后72 h两品系小鼠假手术组存活率均为100%,CLP组BALB/c小鼠存活率为40%,KM小鼠存活率为60%,BALB/c小鼠72 h生存能力低于KM小鼠（P〈0.05）。与各自品系的假手术组相比,BALB/c小鼠和KM小鼠在术后6 h白细胞上升,血小板下降,谷丙转氨酶、谷草转氨酶升高（p〈0.05）,肺损伤明显,而红细胞、尿素氮和肌酐无明显差异。结论 BALB/c小鼠较KM小鼠对脓毒症盲肠结扎模型的敏感度更高。     

BABL/c突变卷毛小鼠免疫学特性指标的分析

目的 探讨BALB/c突变卷毛小鼠与BALB/c小鼠之间的免疫学指标是否存在差异。方法 选择6周龄的BALB/c突变卷毛小鼠和BALB/c小鼠各20只(雌雄各半),分别使用全自动血细胞分析仪检测6项血液免疫细胞指标;使用全自动生化仪检测4项抗体和补体指标;ELISA法检测血清细胞因子TNF-α、IL-2、IL-4水平;并对两组结果进行对比分析。结果 两组小鼠NE、NE#和MO的雌性及组间结果相比较具有显著性差异(P<0.05,P<0.01),两组小鼠LY的雌性结果相比较具有显著性差异(P<0.05);两组小鼠IgM的性别及组间结果相比具有显著性差异(P<0.05,P<0.01),两组小鼠的IgG和C3的雄性及组间结果相比较具有显著性差异(P<0.01),两组小鼠IL-4的雌性及组间结果相比较具有显著性差异(P<0.05)。结论 BALB/c突变卷毛小鼠和BALB/c小鼠的血液免疫学指标存在差异,提示突变基因可能对小鼠的免疫系统产生了一定的影响。