顶复门原虫糖酵解代谢及其关键酶:己糖激酶的研究进展

顶复门原虫是包括刚地弓形虫、疟原虫及球虫等在内的一大类寄生性原虫的总称,引起重要的人畜寄生虫病。由于药物的长期使用,对于原有的药物产生了明显的抗药性,急需新型抗寄生虫药物的出现。顶复门原虫不能利用脂肪和蛋白质作为能量来源,只能依赖己糖激酶(Hex-okinase,HK)参与的糖酵解途径提供ATP。顶复门原虫HK的基因序列、结构特征与植物等一些低等生物更为接近,而明显区别于脊椎动物和大多数的真核生物,成为研究开发药物的理想靶位。本文对顶复门原虫糖酵解途径及其关键酶进行了综述。     

顶复门原虫类异戊二烯生物合成途径及其关键酶的研究进展

顶复门原虫包括疟原虫(Plasmodium spp.)、刚地弓形虫(Toxoplasma gondii)、艾美耳球虫(Eimeria spp.)、锥虫(Trypanosoma spp.)、泰勒虫(Theileria spp.)及巴贝斯虫(Babesia spp.)等一大类引起严重人畜疾病的寄生性原虫。顶复门原虫利用2C-甲基-D-赤藓糖醇-4-磷酸(MEP)途径合成类异戊二烯前体物质,这些化合物对于维持顶复门原虫的生存具有十分重要的作用。1-脱氧-D-木酮糖-5-磷酸(DOXP)还原异构酶是MEP途径的关键酶,对其作用机理及抑制剂的筛选研究已取得重要进展。论文对顶复门原虫类异戊二烯的MEP途径,DOXP还原异构酶的作用机理及靶标研究进展进行综述。     

顶复门原虫γ-氨基丁酸代谢旁路研究进展

顶复门原虫包括一大类单细胞寄生性原虫,是人和动物的重要病原,会对人类健康以及畜牧业生产造成严重危害。γ-氨基丁酸是一种四碳非蛋白质氨基酸,广泛存在于生物体内。顶复门原虫的γ-氨基丁酸代谢旁路主要参与能量代谢,为虫体生长提供短期的能量储备,在虫体适应不同寄生环境过程中起着关键性作用。寄生原虫的γ-氨基丁酸代谢明显区别于哺乳动物宿主,仅限于中枢神经系统中的代谢,可在虫体入侵宿主细胞及有性生殖阶段发挥重要的作用。本文对顶复门原虫γ-氨基丁酸代谢途径及其生物学作用进行综述。     

顶复门原虫多胺合成研究进展

多胺是一类聚阳离子脂肪族化合物,广泛存在于生物体内,其在顶复门原虫的生长、发育和繁殖等多个阶段发挥着重要作用。顶复门原虫的多胺合成途径存在多样性,其与宿主细胞的经典路径不同,且顶复门原虫多胺生物合成限速酶(鸟氨酸脱羧酶与S-腺苷甲硫氨酸脱羧酶)的半衰期与宿主细胞限速酶的半衰期存在显著差异。因此,顶复门原虫多胺合成限速酶及转运载体成为抗原虫药物研发的候选靶标,以顶复门原虫多胺合成限速酶及多胺转运载体为靶标的抗寄生原虫抑制剂研究取得一定进展。论文综述了顶复门原虫多胺的生物学功能、生物合成途径及其抑制剂研究进展,以期为抗原虫药物靶标研究及新型抑制剂研发提供参考。     

顶复门原虫醛缩酶的生物学功能与应用进展

顶复门原虫是一类专一性细胞内寄生原虫,不仅危害畜禽的健康,有些种类还是人兽共患病原,威胁人类的健康。醛缩酶是一类可逆醇醛裂合酶,根据作用机理可分为Ⅰ型与Ⅱ型醛缩酶。研究发现,顶复门原虫存在Ⅰ型醛缩酶,它们主要参与顶复门原虫的能量代谢、虫体黏附及入侵宿主细胞等过程,或可成为抗原虫药物研发的候选靶标。论文主要对顶复门原虫醛缩酶的生物学功能与应用研究现状进行综述,以期为顶复门原虫感染的防控提供参考。     

顶复门原虫电子转移链代谢及Ⅱ型NADH脱氢酶研究进展

顶复门原虫是包括刚地弓形虫、疟原虫及球虫等在内的一大类寄生性原虫的总称,可引起重要的人畜寄生虫病.抗顶复门原虫药物的长期使用,甚至是滥用,使得这类寄生虫对现有药物产生了明显的抗药性,急需开发新型药物.Ⅱ型NAD(P)H脱氢酶是电子转移链途径中的关键酶,由于其仅存在于某些植物、细菌、真菌和寄生原虫等一些低等生物体内,而在高等动物体内缺失,是研发新型抗感染性药物的重要靶标.笔者主要针对顶复门原虫线粒体电子转移链代谢途径以及Ⅱ型NAD(P)H脱氢酶的研究概况进行综述.     

miRNA在顶复门原虫感染中的作用机制

<正>顶复门原虫是一类专一性的细胞内寄生原虫,如疟原虫、弓形虫和隐孢子虫,严重危害人类和动物的健康,而且给全球公共卫生和畜牧业带来巨大的经济损失。宿主通过启动免疫反应、凋亡及自噬等一系列细胞反应清除体内寄生原虫,而顶复门原虫也形成了多种机制逃避宿主的识别和清除,如干扰宿主一些重要信号通路、调节宿主细胞凋亡、细胞因子的产生、细胞周期及脂代谢等。宿主清除胞内寄生原虫和顶复门原虫逃避机制的能力主要是     

自噬通路及其在顶复门原虫入侵发育中的作用

自噬(autophagy)是细胞通过降解自身大分子物质或破损细胞器等来维持胞内稳态的一种平衡机制.近年来在顶复门原虫与宿主细胞相互作用研究中发现,顶复门原虫不但可诱导宿主细胞产生自噬并启动天然免疫途径清除寄生虫,亦可进化出独特的机制来抵抗宿主细胞自噬甚至利用其为自身生长提供条件.不同种类顶复门原虫与宿主细胞互作方式不同...     

顶复门原虫钙依赖蛋白激酶的研究进展

钙依赖蛋白激酶(Calcium-dependent protein kinases,CDPKs)是一类大的蛋白激酶家族,属于丝氨酸/苏氨酸类蛋白激酶,广泛存在于各种植物和原生动物中,参与多种生命活动的调控.随着生物信息学、分子生物学及基因工程的迅速发展,对顶复门原虫CDPKs的研究日益增多.研究结果表明,这类蛋白家族成员参与调控寄生虫入侵、外出宿主细胞、配子形成、宿主体内移行等顶复门原虫生活史的多个重要时期,其特殊的分子结构或可成为研究抗寄生虫疫苗或药物的候选靶标.本文以疟原虫、弓形虫和艾美耳球虫CDPKs为重点,综述了顶复门原虫CDPKs的结构、功能及生物学意义,展望了顶复门原虫CDPKs的研究和应用前景,以期为研究顶复门原虫的致病机理及研发抗原虫生物制剂提供参考.     

顶复门原虫与细菌Ⅱ型丙二酰单酰辅酶A:ACP转酰基酶研究进展

大部分顶复门原虫与人畜重要寄生虫病密切相关.近年来,由于各类药物在寄生虫与细菌病防治中的广泛应用,甚至是滥用,造成了这类寄生虫与细菌的严重抗药性,急需一些新型抗感染性药物的出现.由于在一些顶复门原虫与细菌中广泛存在的Ⅱ型脂肪酸代谢途径在其脊椎动物宿主中并不存在,而丙二酰单酰辅酶A∶ACP转酰基酶(malonyl-CoA...     

牧草害虫优势天敌伞裙追寄蝇飞行中代谢相关酶活性的变化

昆虫在飞行过程伴随着较高的代谢速率和能源物质消耗.为明确伞裙追寄蝇飞行肌对能源物质的利用,测定了与能源物质代谢相关的5种酶,即3-磷酸甘油醛脱氢酶(GAPDH)、3-磷酸甘油脱氢酶(GDH)、3-羟酰辅酶A脱氢酶(HOAD)、乳酸脱氢酶(LDH)和柠檬酸合酶(CS)的活性变化.结果表明:GAPDH活性在吊飞过程中呈先升...     

箭箸豌豆甘油醛-3-磷酸脱氢酶基因片段的克隆及序列分析

本研究预期通过同源克隆获得了箭菩豌豆(Vicia sativa)甘油醛-3-磷酸脱氢酶(GAPDH)基因的部分片段,为研究箭菩豌豆适应高海拔环境的分子机制奠定基础.根据近缘物种GAPDH基因序列设计1对引物,通过RT-PCR技术克隆获得了一段长度1141 bp的序列.生物信息学分析表明,该序列编码的381个氨基酸,与其...     

Cloning and purification of the Streptococcus suis serotype 2 glyceraldehyde-3-phosphate dehydrogenase and its involvement as an adhesin

Streptococcus suis serotype 2 is a swine pathogen responsible for diverse diseases and may be present in the tonsils of pigs which show no sign of illness. Because adhesion to host cells may be important in the carrier state, this study was undertaken to characterize a 39 kDa surface protein identified as a glyceraldehyde-3-phosphate dehydrogenase (GAPDH), possibly implicated in the adhesion of the bacteria. The gene encoding for the GAPDH of S. suis was cloned and sequenced. The DNA sequence contained an open reading frame encoding for a 336 amino acid polypeptide exhibiting 95% sequence identity with the GAPDH from Streptococcus pyogenes and from other streptococci. Using the Qiaexpress expression plasmids, the gapdh gene was inducibly overexpressed in E. coli to produce GAPDH with a hexahistidyl N-terminus to permit its purification. The (His)6GAPDH protein was found to possess functional GAPDH enzymatic activity after the purification. An adherence assay with S. suis and porcine tracheal rings pre-incubated with (His)6GAPDH and non-incubated rings was showed a significant reduction in the adhesion of S. suis in the (His)6GAPDH pre-incubated rings compared to the non-incubated rings. The GAPDH protein of S. suis seems to be involved in the first steps of the bacterial adhesion to host cells.     

Characterization of two proteins of Staphylococcus aureus isolated from bovine clinical mastitis with homology to glyceraldehyde-3-phosphate dehydrogenase

Staphylococcus aureus is the most common causative agent of bovine mastitis and vaccines developed to control this disease showed limited protection due in part to the lack of common antigens among the mastitis isolates. We isolated and identified two genes encoding proteins with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) activity from a S. aureus strain isolated from bovine clinical mastitis. The GapB and GapC proteins share considerable homology to the GapB and GapC products of human strains of S. aureus. These two proteins could be distinguished by their different GAPDH activities and binding to bovine transferrin properties. Both gapB and gapC genes were conserved in 11 strains tested, and the GapC protein was present on the surface of all S. aureus strains.     

Role of GapC in the pathogenesis of Staphylococcus aureus

Staphylococcus aureus is recognized worldwide as a major pathogen causing clinical or subclinical intramammary infections in lactating cows, sheep and goats. S. aureus produces a wide arsenal of cell surface and extracellular proteins involved in virulence. Among these are two conserved proteins with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) activity named glyceraldehyde-3-phosphate dehydrogenase-B (GapB) and -C (GapC). In this study, we used the S. aureus wild type strain RN6390 and its isogenic gapC mutant H330 in in vitro and in vivo studies and determined that the S. aureus GapC protein plays a role on adherence to and internalization into bovine mammary epithelial (MAC-T) cells. In addition, we found that S. aureus H330 did not caused mastitis after an experimental infection of ovine mammary glands. Together, these results show that GapC is important in the pathogenesis of S. aureus mastitis.     

Identification and validation of housekeeping genes as internal control for gene expression in an intravenous LPS inflammation model in chickens

Real-time PCR has become a powerful tool for the detection of inflammatory parameters, including cytokines. Reference or housekeeping genes are used for the normalization of real-time RT-PCR results. In order to obtain reliable results, the stability of these housekeeping genes needs to be determined. In this study the stability of five genes, including beta-actin, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), hypoxanthine phophoribosyl-transferase (HPRT), ubiquitin (UB) and glucose-6-phosphate dehydrogenase (G6PDH), was determined in a lipopolysaccharide inflammation model in chickens. beta-Actin appeared to be the most stable single gene in our model. Because the use of a single gene for normalization can lead to relatively large errors, the use of the geometric mean of multiple reference genes or normalization factor is preferred. The most stable combination for gene expression analysis in this lipopolysaccharide inflammation model in chickens is G6PDH and UB, since their correlation coefficients were 0.953 and 0.969, respectively (BestKeeper) and an M value of 0.34 and a low V(2/3) value of 0.155 (geNorm) were obtained. The use of HPRT and GAPDH should be avoided. The stable housekeeping genes, G6PDH and UB together, can be used to normalize the expression of pro-inflammatory cytokines in a lipopolysaccharide inflammation model in chickens.     

Adhesion activity of glyceraldehyde-3-phosphate dehydrogenase in a Chinese Streptococcus suis type 2 strain

A total of 36 streptococcal strains, including seven S. equi ssp.zooepidemicus, two S. suis type 1 (SS1), 24 SS2, two SS9, and one SS7, were tested for glyceraldehyde-3-phosphate dehydrogenase gene (gapdh). Except from non-virulent SS2 strain T1 5, all strains harboured gapdh.The gapdh of Chinese Sichuan SS2 isolate ZY05719 and Jiangsu SS2 isolate HA9801 were sequenced and then compared with published sequences in the GenBank.The comparison revealed a 99.9 % and 99.8 % similarity of ZY05719 and HA9801, respectively, with the published sequence. Adherence assay data demonstrated a significant ((p<0.05)) reduction in adhesion of SS2 in HEp-2 cells pre-incubated with purified GAPDH compared to non pre-incubated controls, suggesting the GAPDH mediates SS2 bacterial adhesion to host cells.     

鹿茸组织中内参基因的筛选和验证

为筛选在不同生长时期鹿茸组织中稳定表达的内参基因,试验以不同生长时期(分别为脱盘后10、20、40和60 d)的鹿茸组织为材料,采用实时荧光定量PCR(qRT-PCR)方法分析甘油醛-3-磷酸脱氢酶(GAPDH)、β2-微球蛋白(B2M)、还原型辅酶Ⅰ(NADH)、60S 核糖体蛋白L40(RPL40)、谷胱甘肽还原酶7(GPx)和β肌动蛋白(ACTB)6个看家基因的表达情况,并运用 geNorm和NormFinder 两个程序综合分析6个看家基因的表达稳定性.结果显示,GAPDH、ACTB、RPL40表达稳定性较好,可用作鹿茸基因表达研究的内参基因,而NADH和GPx的稳定性最差,不适合作内参基因.通过对鹿茸生长相关基因(ANXA5、HSP27、PRD2、CRABP1、LGALS1)表达分析,进一步验证了上述结果,并且发现这5种基因均在脱盘后10 d的鹿茸组织中高表达.该研究结果为鹿茸快速生长及骨化相关基因的研究奠定了一定基础.