BACKGROUND & AIMS: :As an initial step towards the characterization of replicative DNA polymerases of trypanosomes, we have cloned, sequenced and examined the expression of the Trypanosoma (Trypanozoon) brucei brucei gene that encodes the DNA polymerase alpha catalytic core (pol alpha). The protein sequence contains the six conserved regions that have been recognized previously in eukaryotic and viral replicative DNA polymerases. In addition, we have identified a seventh region which appears to be conserved primarily in alpha-type DNA polymerases. The T.brucei DNA pol alpha core N-terminus is 123 and 129 amino acids smaller than that of the human and yeast homologue, respectively. The gene is separated by 386 bp from an upstream open reading frame (ORF) of 442 codons. Stable transcripts of the upstream sequence are detected in both dividing and non-dividing forms, while pol alpha transcripts are detected principally in dividing forms. Allelic copies of the T.brucei pol alpha region exhibit restriction site polymorphisms; one such sequence polymorphism affects the amino acid sequence of the T.brucei DNA pol alpha core. The T.brucei pol alpha region cross-hybridizes weakly with that of T.(Nannomonas) congolense and T.(Duttonella) vivax.

背景与目标： : 作为表征锥虫复制型DNA聚合酶的第一步，我们已经克隆，测序并检查了编码DNA聚合酶 α 催化核心 (pol α) 的锥虫 (Trypanozoon) brucei brucei基因的表达。蛋白质序列包含六个保守的区域，这些区域以前在真核和病毒复制DNA聚合酶中已被识别。此外，我们已经确定了第七个区域，该区域似乎主要在 α 型DNA聚合酶中保守。T.brucei DNA pol α 核心N-末端分别比人和酵母同源物小123和129个氨基酸。该基因通过386 bp与442密码子的上游开放阅读框 (ORF) 分离。以分裂和非分裂形式检测上游序列的稳定转录本，而pol α 转录本主要以分裂形式检测。T.br ucei pol alpha区域的等位基因拷贝表现出限制性位点多态性; 一种这样的序列多态性会影响T.br ucei DNA pol alpha核心的氨基酸序列。T.Br ucei pol alpha区与T.(Nannomonas) congolense和T.(Duttonella) vivax弱杂交。

BACKGROUND & AIMS: :Histone post-translational modification, mediated by histone acetyltransferases and deacetylases, is one of the most studied factors affecting gene expression. Recent data showing differential histone acetylation states during the Trypanosoma cruzi cell cycle suggest a role for epigenetics in the control of this process. As a starting point to study the role of histone deacetylases in the control of gene expression and the consequences of their inhibition and activation in the biology of T. cruzi, two inhibitors for different histone deacetylases: trichostatin A for class I/II and sirtinol for class III and the activator resveratrol for class III, were tested on proliferative and infective forms of this parasite. The two inhibitors tested caused histone hyperacetylation whereas resveratrol showed the opposite effect on both parasite forms, indicating that a biologically active in vivo level of these compounds was achieved. Histone deacetylase inhibitors caused life stage-specific effects, increasing trypomastigotes infectivity and blocking metacyclogenesis. Moreover, these inhibitors affected specific transcript levels, with sirtinol causing the most pronounced change. On the other hand, resveratrol showed strong anti-parasitic effects. This compound diminished epimastigotes growth, promoted metacyclogenesis, reduced in vitro infection and blocked differentiation and/or replication of intracellular amastigotes. In conclusion, the data presented here supports the notion that these compounds can modulate T. cruzi gene expression, differentiation, infection and histones deacetylase activity. Furthermore, among the compounds tested in this study, the results point to Resveratrol as promising trypanocidal drug candidate.

背景与目标： : 由组蛋白乙酰转移酶和脱乙酰酶介导的组蛋白翻译后修饰是影响基因表达的研究最多的因素之一。最近的数据显示克氏锥虫细胞周期中组蛋白乙酰化状态的差异表明，表观遗传学在控制这一过程中起作用。作为研究组蛋白去乙酰化酶在控制基因表达中的作用及其在克鲁氏T (T. cruzi) 生物学中的抑制和激活后果的起点，两种不同组蛋白去乙酰化酶的抑制剂: 曲古抑制素a用于I/II类和sirtinol用于III类和激活剂白藜芦醇用于III类，对该寄生虫的增殖和感染形式进行了测试。测试的两种抑制剂引起组蛋白高乙酰化，而白藜芦醇对两种寄生虫形式均表现出相反的作用，表明这些化合物在体内达到了生物活性水平。组蛋白去乙酰化酶抑制剂引起生命阶段特异性作用，增加了锥虫的感染性并阻止了代谢生成。此外，这些抑制剂会影响特定的转录水平，其中sirtinol引起最明显的变化。另一方面，白藜芦醇表现出很强的抗寄生虫作用。该化合物可减少外生菌的生长，促进元环化，减少体外感染并阻止细胞内单胞菌的分化和/或复制。总之，此处提供的数据支持以下观点: 这些化合物可以调节T. cruzi基因的表达，分化，感染和组蛋白脱乙酰基酶活性。此外，在本研究中测试的化合物中，结果表明白藜芦醇是有希望的杀锥虫药物候选者。

BACKGROUND & AIMS: :Cell invasion by the protozoan parasite Trypanosoma cruzi involves activation of host signaling pathways and the recruitment and fusion of lysosomes at the parasite entry site. A major signaling pathway regulating invasion of fibroblasts, epithelial cells, and myoblasts involves mobilization of Ca(2+) from intracellular stores and requires the activity of a T. cruzi serine peptidase, oligopeptidase B (OPB). Deletion of the OPB gene results in a marked defect in trypomastigote virulence, consistent with a greatly reduced cell invasion capacity. Here we show that uptake by macrophages, on the other hand, is largely independent of OPB expression and sensitive to inhibition of by cytochalasin D. The residual invasion capacity of OPBnull trypomastigotes in fibroblasts still involves lysosome recruitment, although in a significantly delayed fashion. Transient elevations in intracellular Ca(2+) concentrations were observed in host cells exposed to both wild-type and OPBnull trypomastigotes, but the signals triggered by the mutant parasites were less vigorous and delayed. The capacity of triggering elevation in host cell cyclic AMP (cAMP), however, was unaltered in OPBnull trypomastigotes. Modulation in cAMP levels preferentially affected the residual cell invasion capacity of OPBnull parasites, suggesting that this signaling pathway can play a dominant role in promoting cell invasion in the absence of the major OPB-dependent pathway.

背景与目标： : 原生动物寄生虫克氏锥虫的细胞入侵涉及宿主信号通路的激活以及溶酶体在寄生虫进入部位的募集和融合。调节成纤维细胞，上皮细胞和成肌细胞侵袭的主要信号通路涉及从细胞内存储中动员Ca(2)，并且需要T. cruzi丝氨酸肽酶，寡肽酶B (OPB) 的活性。OPB基因的缺失导致锥虫毒力明显缺陷，与细胞侵袭能力大大降低一致。另一方面，在这里，我们表明巨噬细胞的摄取在很大程度上独立于OPB表达，并且对细胞松弛素D的抑制敏感。尽管成纤维细胞中OPBnull类锥虫的残余侵袭能力仍涉及溶酶体募集，尽管其延迟方式明显。在暴露于野生型和OPBnull锥虫的宿主细胞中观察到细胞内Ca(2) 浓度的瞬时升高，但是由突变寄生虫触发的信号不那么活跃和延迟。然而，在OPBnull锥虫中触发宿主细胞循环AMP (cAMP) 升高的能力并未改变。cAMP水平的调节优先影响了OPBnull寄生虫的残余细胞入侵能力，这表明在缺乏主要的OPB依赖性途径的情况下，该信号通路可以在促进细胞入侵中发挥主导作用。

BACKGROUND & AIMS: :Heme is an essential cofactor for many biological processes in aerobic organisms, which can synthesize it de novo through a conserved pathway. Trypanosoma cruzi, the etiological agent of Chagas disease, as well as other trypanosomatids relevant to human health, are heme auxotrophs, meaning they must import it from their mammalian hosts or insect vectors. However, how these species import and regulate heme levels is not fully defined yet. It is known that the membrane protein TcHTE is involved in T. cruzi heme transport, although its specific role remains unclear. In the present work, we studied endogenous TcHTE in the different life cycle stages of the parasite to gain insight into its function in heme transport and homeostasis. We have confirmed that TcHTE is predominantly detected in replicative stages (epimastigote and amastigote), in which heme transport activity was previously validated. We also showed that in epimastigotes, TcHTE protein and mRNA levels decrease in response to increments in heme concentration, confirming it as a member of the heme response gene family. Finally, we demonstrated that T. cruzi epimastigotes can sense intracellular heme by an unknown mechanism and regulate heme transport to adapt to changing conditions. Based on these results, we propose a model in which T. cruzi senses intracellular heme and regulates heme transport activity by adjusting the expression of TcHTE. The elucidation and characterization of heme transport and homeostasis will contribute to a better understanding of a critical pathway for T. cruzi biology allowing the identification of novel and essential proteins.

背景与目标： : 血红素是需氧生物中许多生物过程必不可少的辅因子，可以通过保守的途径从头合成。克氏锥虫是恰加氏病的病原体，以及与人类健康相关的其他锥虫，是血红素营养缺陷型动物，这意味着它们必须从哺乳动物宿主或昆虫媒介中进口。但是，这些物种如何导入和调节血红素水平尚未完全确定。众所周知，膜蛋白TcHTE参与了克氏T。克氏血红素的转运，尽管其具体作用尚不清楚。在目前的工作中，我们研究了寄生虫不同生命周期阶段的内源性TcHTE，以了解其在血红素转运和体内平衡中的功能。我们已经确认，TcHTE主要在复制阶段 (epimastigote和amastigote) 检测到，其中血红素运输活性先前已得到验证。我们还表明，在epimastigotes中，TcHTE蛋白和mRNA水平随着血红素浓度的增加而降低，这证实了它是血红素反应基因家族的成员。最后，我们证明了克氏T. cruzi epimastigotes可以通过未知机制感知细胞内血红素，并调节血红素转运以适应不断变化的条件。基于这些结果，我们提出了一个模型，其中T。cruzi通过调节TcHTE的表达来感知细胞内血红素并调节血红素转运活性。血红素转运和稳态的阐明和表征将有助于更好地理解克鲁氏杆菌生物学的关键途径，从而可以鉴定新的和必需的蛋白质。

BACKGROUND & AIMS: :While growing in the tsetse fly, Trypanosoma brucei expresses a major surface glycoprotein, the procyclic acidic repetitive protein (PARP). The parp genes are transcribed by an alpha-amanitin-resistant RNA polymerase. We have determined the sequence requirements for parp promoter activity. Studies of RNA produced from input DNA in transiently transfected trypanosomes indicate that the RNA is correctly processed by trans-splicing and polyadenylation. Deletion analyses show that 330 bp are sufficient for full promoter and splicing activity and that the promoter structure is complex, involving at least three elements whose mutual spacing is important. Mutagenesis pin-pointed two sequences vital for promoter activity; neither bears any resemblance to known prokaryotic or eukaryotic promoter elements.

背景与目标： : 在采采蝇中生长时，布氏锥虫表达一种主要的表面糖蛋白，即原环酸性重复蛋白 (PARP)。parp基因由抗 α-amanitin的RNA聚合酶转录。我们已经确定了parp启动子活性的序列要求。对瞬时转染的锥虫中输入DNA产生的RNA的研究表明，通过反式剪接和多聚腺苷酸化可以正确处理RNA。缺失分析表明，330 bp足以实现完整的启动子和剪接活性，并且启动子结构复杂，涉及至少三个相互间隔重要的元素。诱变针尖的两个对启动子活性至关重要的序列; 都没有与已知的原核或真核启动子元件有任何相似之处。

BACKGROUND & AIMS: :RNA editing is an essential post-transcriptional process that creates functional mitochondrial mRNAs in Kinetoplastids. Multiprotein editosomes catalyze pre-mRNA cleavage, uridine (U) insertion or deletion, and ligation as specified by guide RNAs. Three functionally and compositionally distinct editosomes differ by the mutually exclusive presence of the KREN1, KREN2 or KREN3 endonuclease and their associated partner proteins. Because endonuclease cleavage is a likely point of regulation for RNA editing, we elucidated endonuclease specificity in vivo. We used a mutant gamma ATP synthase allele (MGA) to circumvent the normal essentiality of the editing endonucleases, and created cell lines in which both alleles of one, two or all three of the endonucleases were deleted. Cells lacking multiple endonucleases had altered editosome sedimentation on glycerol gradients and substantial defects in overall editing. Deep sequencing analysis of RNAs from such cells revealed clear discrimination by editosomes between sites of deletion versus insertion editing and preferential but overlapping specificity for sites of insertion editing. Thus, endonuclease specificities in vivo are distinct but with some functional overlap. The overlapping specificities likely accommodate the more numerous sites of insertion versus deletion editing as editosomes collaborate to accurately edit thousands of distinct editing sites in vivo.

背景与目标： : RNA编辑是一个重要的转录后过程，在运动质体中产生功能性线粒体mrna。多蛋白editosomes催化前mRNA切割，尿苷 (U) 插入或缺失以及指导rna指定的连接。通过KREN1，KREN2或KREN3核酸内切酶及其相关伴侣蛋白的互斥存在，三个功能和组成上不同的编辑体不同。由于核酸内切酶切割可能是RNA编辑的调控点，因此我们阐明了体内核酸内切酶的特异性。我们使用突变的 γ ATP合酶等位基因 (MGA) 来规避编辑核酸内切酶的正常必要性，并创建了细胞系，其中删除了一个，两个或全部三个核酸内切酶的两个等位基因。缺乏多种核酸内切酶的细胞在甘油梯度上的editosome沉降发生了改变，并且在整体编辑中存在实质性缺陷。来自此类细胞的rna的深度测序分析显示，通过编辑体在缺失位点与插入编辑位点之间具有明显的区别，并且对插入编辑位点具有优先但重叠的特异性。因此，体内的核酸内切酶特异性是不同的，但具有一些功能重叠。重叠的特异性可能会容纳更多的插入和删除编辑位点，因为editosomes协作以在体内准确编辑数千个不同的编辑位点。

BACKGROUND & AIMS: :Trypanosoma cruzi (Y strain)-infected interleukin-4(-/-) (IL-4(-/-)) mice of strains 129/J, BALB/c, and C57BL/6 showed no significant difference in parasitemia levels or end point mortality rates compared to wild-type (WT) mice. Higher production of gamma interferon (IFN-gamma) by parasite antigen (Ag)-stimulated splenocytes was observed only for C57BL/6 IL-4(-/-) mice. Treatment of 129/J WT mice with recombinant IL-4 (rIL-4), rIL-10, anti-IL-4, and/or anti-IL-10 monoclonal antibodies (MAbs) did not modify parasitism. However, WT mice treated with rIL-4 and rIL-10 had markedly increased parasitism and suppressed IFN-gamma synthesis by spleen cells stimulated with parasite Ag, concanavalin A, or anti-CD3. Addition of anti-IL-4 MAbs to splenocyte cultures from infected WT 129/J, BALB/c, or C57BL/6 mice failed to modify IFN-gamma synthesis levels; in contrast, IL-10 neutralization increased IFN-gamma production and addition of rIL-4 and/or rIL-10 diminished IFN-gamma synthesis. We conclude that endogenous IL-4 is not a major determinant of susceptibility to Y strain T. cruzi infection but that IL-4 can, in association with IL-10, modulate IFN-gamma production and resistance.

背景与目标： : 克氏锥虫 (Y株) 感染的interleukin-4(-/-) (IL-4(-/-)) 129/J，BALB/c和C57BL/6株小鼠与野生型 (WT) 小鼠相比，寄生虫血症水平或终点死亡率没有显着差异。仅在C57BL/6 IL-4(-/-) 小鼠中观察到由寄生虫抗原 (Ag) 刺激的脾细胞产生的较高 γ 干扰素 (IFN-γ)。用重组IL-4 (rIL-4) 、rIL-10、anti-IL-4和/或anti-IL-10单克隆抗体 (mab) 处理129/jwt小鼠不会改变寄生虫。然而，用rIL-4和rIL-10处理的WT小鼠的寄生虫显着增加，并抑制了被寄生虫Ag，伴刀豆球蛋白A或anti-CD3刺激的脾细胞的IFN-γ 合成。向感染的WT 129/J、BALB/c或C57BL/6小鼠的脾细胞培养物中添加anti-IL-4 mab不能改变IFN-γ 的合成水平; 相反，IL-10中和增加IFN-γ 的产生和添加rIL-4和/或rIL-10减少IFN-γ 的合成。我们得出的结论是，内源性IL-4不是Y株T. cruzi感染易感性的主要决定因素，但IL-4可以与IL-10相关地调节IFN-γ 的产生和抗性。

BACKGROUND & AIMS: :To determine which fat body genes were differentially expressed following infection of Glossina morsitans morsitans with Trypanosoma brucei brucei we generated four suppression subtractive hybridisation (SSH) libraries. We obtained 52 unique gene fragments (SSH clones) of which 30 had a known orthologue at E-05 or less. Overall the characteristics of the orthologues suggest: (i) that trypanosome infection has a considerable effect on metabolism in the tsetse fly; (ii) that self-cured flies are mounting an oxidative stress response; and (iii) that self-cured flies are displaying increased energy usage. The three most consistently differentially expressed genes were further analysed by gene knockdown (RNAi). Knockdown of Glossina transferrin transcripts, which are upregulated in self-cured flies compared with flies infected with trypanosomes, results in a significant increase in the number of trypanosome infections establishing in the fly midgut, suggesting transferrin plays a role in the protection of tsetse flies from trypanosome infection.

背景与目标： : 为了确定在布鲁氏锥虫感染Glossina morsitans morsitans后，哪些脂肪体基因差异表达，我们生成了四个抑制消减杂交 (SSH) 文库。我们获得了52个独特的基因片段 (SSH克隆)，其中30个具有E-05或更少的已知直系同源基因。总的来说，直系同源物的特征表明 :( i) 锥虫感染对采采蝇的代谢有相当大的影响; (ii) 自固化的果蝇正在产生氧化应激反应; (iii) 自固化的果蝇显示出增加的能量消耗。通过基因敲除 (RNAi) 进一步分析了三个最一致的差异表达基因。与感染锥虫的果蝇相比，在自固化的果蝇中被上调的Glossina转铁蛋白转录本的敲除导致在果蝇中肠中建立的锥虫感染数量显着增加，表明转铁蛋白在保护采采蝇中发挥作用免受锥虫感染。

BACKGROUND & AIMS: :Synthetic peptides modelled according to the amino acid sequences derived from the repeated domains of five Trypanosoma cruzi antigens were used in an immunoradiometric assay to detect antibodies appearing after natural human infections. An enzyme-linked immunosorbent assay and an indirect immunofluorescence assay performed with a complex antigenic mixture from parasites were used as controls. The results indicate that the synthetic peptides were recognized by a large proportion of serum samples collected from 34 patients with Chagas' disease in Chile and point to their possible use in diagnosis.

背景与目标： : 根据源自五种克氏锥虫抗原的重复结构域的氨基酸序列建模的合成肽用于免疫放射测定，以检测自然人类感染后出现的抗体。使用来自寄生虫的复杂抗原混合物进行的酶联免疫吸附测定和间接免疫荧光测定作为对照。结果表明，从智利的34例南美锥虫病患者中收集的大量血清样品识别了合成肽，并指出了它们在诊断中的可能用途。

BACKGROUND & AIMS: :1. The dependence of V and V/K(m) for threonine transport into Trypanosoma brucei upon the external concentration of H(+) was studied. 2. Two ionizing groups, the alpha-amino group of the substrate and a group at the substrate-binding site of the carrier, were found to influence the observed kinetic behaviour of transport. 3. The pK of the group at the substrate-binding site on the free carrier was found to be 6.95 at 30 degrees C and to be temperature-dependent; its heat of ionization was -63.8kJ, which is outside the range for most proton dissociations and suggests a significant contribution from some other source, possibly the remainder of the carrier or the membrane environment. 4. Binding of substrate caused the pK of its alpha-amino group to shift to a higher value, whereas that of the carrier group shifted to a lower value (6.65 at 30 degrees C). 5. The ionic interaction between substrate and carrier appeared to be involved in the stabilizing of the protonated substrate and the species of the carrier-substrate complex required for the membrane-translocation step. 6. The same ionic species of carrier-substrate complex is required for both substrate dissociation and translocation of the substrate through the membrane. 7. H(+) symport or antiport did not occur during threonine uptake.

背景与目标： : 1。研究了苏氨酸向布氏锥虫转运的V和V/K(m) 对外部H () 浓度的依赖性。2.发现两个电离基团，即底物的 α-氨基基团和载体的底物结合位点的基团，会影响观察到的运输动力学行为。3.发现自由载体上的底物结合位点的基团的pK在30 ℃ 下是6.95的，并且是温度依赖性的; 其电离热为-63.8kj，这超出了大多数质子解离的范围，并暗示了其他来源的重大贡献，可能是载体的其余部分或膜环境。4.底物的结合使其 α-氨基的pK转变为较高的值，而载体基团的pK转变为较低的值 (在30 ℃ 下6.65)。5.底物与载体之间的离子相互作用似乎参与质子化底物的稳定以及膜转运步骤所需的载体-底物复合物的种类。6.底物解离和底物通过膜的转运都需要相同离子种类的载体-底物复合物。7.苏氨酸摄取过程中未发生H(+) 共生或反端口。

BACKGROUND & AIMS: :Trypanosoma cruzi is the agent of Chagas disease, and the finding that this parasite possesses a mitochondrial calcium uniporter (TcMCU) with characteristics similar to that of mammalian mitochondria was fundamental for the discovery of the molecular nature of MCU in eukaryotes. We report here that ablation of TcMCU, or its paralog TcMCUb, by clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 led to a marked decrease in mitochondrial Ca2+ uptake without affecting the membrane potential of these cells, whereas overexpression of each gene caused a significant increase in the ability of mitochondria to accumulate Ca2+ While TcMCU-knockout (KO) epimastigotes were viable and able to differentiate into trypomastigotes, infect host cells, and replicate normally, ablation of TcMCUb resulted in epimastigotes having an important growth defect, lower rates of respiration and metacyclogenesis, more pronounced autophagy changes under starvation, and significantly reduced infectivity. Overexpression of TcMCUb, in contrast to what was proposed for its mammalian ortholog, did not result in a dominant negative effect on TcMCU.IMPORTANCE The finding of a mitochondrial calcium uniporter (MCU) in Trypanosoma cruzi was essential for the discovery of the molecular nature of this transporter in mammals. In this work, we used the CRISPR/Cas9 technique that we recently developed for T. cruzi to knock out two components of the uniporter: MCU, the pore subunit, and MCUb, which was proposed as a negative regulator of MCU in human cells. In contrast to what occurs in human cells, MCU is not essential, while MCUb is essential for growth, differentiation, and infectivity; has a bioenergetic role; and does not act as a dominant negative subunit of MCU.

背景与目标： : 克氏锥虫是恰加斯病的病原体，发现该寄生虫具有与哺乳动物线粒体相似的特征的线粒体钙单转运蛋白 (TcMCU)，对于发现真核生物中MCU的分子性质至关重要。我们在这里报告说，通过聚集的规则间隔的短回文重复序列 (CRISPR)/Cas9消融TcMCU或其paralog TcMCUb导致线粒体Ca2吸收显着减少，而不影响这些细胞的膜电位，尽管每个基因的过表达导致线粒体积累Ca2的能力显着增加，而TcMCU基因敲除 (KO) epimastigotes存活并能够分化为锥虫，感染宿主细胞并正常复制，但TcMCUb的消融导致epimastigotes具有重要的生长缺陷，呼吸和代谢生成率较低，饥饿下自噬变化更明显，感染性明显降低。TcMCUb的过表达，与其哺乳动物直系同源物相比，并未对TcMCU产生明显的负面影响。重要性克氏锥虫中线粒体钙单转运蛋白 (MCU) 的发现对于发现哺乳动物中这种转运蛋白的分子性质至关重要。在这项工作中，我们使用了最近为T. cruzi开发的CRISPR/Cas9技术来敲除uniporter的两个组件: MCU，pore亚基和MCUb，后者被提议作为人类细胞中MCU的负调节器。与人类细胞中发生的情况相反，MCU不是必不可少的，而MCUb对于生长，分化和感染性至关重要; 具有生物能作用; 并且不充当MCU的主要负亚基。

BACKGROUND & AIMS: :We evaluated Trypanosoma cruzi infection in 397 wild Mepraia gajardoi specimens from five coastal localities in northern Chile by detection of minicircle DNA by polymerase chain reaction. The wild capture sites were classified into two ecotopes: a fully wild ecotope (ecotope 1) and a wild ecotope near human dwellings (ecotope 2). Infection rates varied between 11% and 27%. Minicircle hybridization assays showed that T. cruzi lineages Tc II and Tc VI were commonly detected in ecotope 1 and ecotope 2, respectively. These results are discussed in the context of insect proximity to human dwellings, the alimentary profile of Mepraia sp., T. cruzi lineages detected in the past in the same disease-endemic area circulating in humans, and Triatoma infestans.

背景与目标： : 我们通过聚合酶链反应检测小圆DNA，评估了智利北部五个沿海地区的397个野生Mepraia gajardoi标本中的克氏锥虫感染。野生捕获地点分为两个生态群落: 一个完全野生的生态群落 (ecotope 1) 和一个靠近人类住所的野生生态群落 (ecotope 2)。感染率在11% 和27% 之间变化。小圆杂交试验表明，克氏T。克氏谱系Tc II和Tc VI分别在生态环境1和生态环境2中被普遍检测到。这些结果是在昆虫靠近人类住所，过去在人类中传播的同一疾病流行地区检测到的Mepraia sp。，T. cruzi谱系的饮食特征以及Triatoma infestans的背景下进行讨论的。

BACKGROUND & AIMS: :Trypanosoma brucei expresses two hexokinases that are 98% identical, namely, TbHK1 and TbHK2. Homozygous null TbHK2-/- procyclic-form parasites exhibit an increased doubling time, a change in cell morphology, and, surprisingly, a twofold increase in cellular hexokinase activity. Recombinant TbHK1 enzymatic activity is similar to that of other hexokinases, with apparent Km values for glucose and ATP of 0.09 +/- 0.02 mM and 0.28 +/- 0.1 mM, respectively. The k(cat) value for TbHK1 is 2.9 x 10(4) min(-1). TbHK1 can use mannose, fructose, 2-deoxyglucose, and glucosamine as substrates. In addition, TbHK1 is inhibited by fatty acids, with lauric, myristic, and palmitic acids being the most potent (with 50% inhibitory concentrations of 75.8, 78.4, and 62.4 microM, respectively). In contrast to TbHK1, recombinant TbHK2 lacks detectable enzymatic activity. Seven of the 10 amino acid differences between TbHK1 and TbHK2 lie within the C-terminal 18 amino acids of the polypeptides. Modeling of the proteins maps the C-terminal tails near the interdomain cleft of the enzyme that participates in the conformational change of the enzyme upon substrate binding. Replacing the last 18 amino acids of TbHK2 with the corresponding residues of TbHK1 yields an active recombinant protein with kinetic properties similar to those of TbHK1. Conversely, replacing the C-terminal tail of TbHK1 with the TbHK2 tail inactivates the enzyme. These findings suggest that the C-terminal tail of TbHK1 is important for hexokinase activity. The altered C-terminal tail of TbHK2, along with the phenotype of the knockout parasites, suggests a distinct function for the protein.

背景与目标： : 布氏锥虫表达两种98% 相同的己糖激酶，即TbHK1和tbhk2。纯合的空TbHK2-/-原环形式的寄生虫表现出倍增时间增加，细胞形态发生变化，令人惊讶的是，细胞己糖激酶活性增加了两倍。重组TbHK1酶活性与其他己糖激酶相似，葡萄糖和ATP的表观Km值分别为0.09 +/- 0.02 mM和0.28 +/- 0.1 mM。TbHK1的k(cat) 值为2.9 × 10(4) min(-1)。TbHK1可以使用甘露糖，果糖，2-脱氧葡萄糖和氨基葡萄糖作为底物。此外，TbHK1被脂肪酸抑制，月桂酸、肉豆蔻酸和棕榈酸是最有效的 (分别具有50% 的75.8、78.4和62.4 microM的抑制浓度)。与TbHK1相反，重组TbHK2缺乏可检测的酶活性。TbHK1和TbHK2之间的10个氨基酸差异中有7个位于多肽的C端18个氨基酸内。蛋白质的建模映射酶的结构域间裂隙附近的C末端尾巴，该酶在底物结合时参与酶的构象变化。用TbHK1的相应残基代替TbHK2的最后18个氨基酸，产生活性重组蛋白，其动力学特性与TbHK1相似。相反，用TbHK2尾巴代替TbHK1的C末端尾巴会使酶失活。这些发现表明，TbHK1的C末端尾部对己糖激酶活性很重要。TbHK2的C末端尾部改变，以及敲除寄生虫的表型，表明该蛋白具有明显的功能。

BACKGROUND & AIMS: :Kinetoplastid mRNAs possess a unique hypermethylated cap 4 structure derived from the standard m7GpppN cap structure, with 2'-O methylations on the first four ribose sugars and additional base methylations on the first adenine and the fourth uracil. While the enzymes responsible for m7GpppN cap 0 formations has been characterized in Trypanosoma brucei, the mechanism of cap 4 methylation and the role of the hypermethylated structure remain unclear. Here, we describe the characterization of a 48 kDa T.brucei 2'-O nucleoside methyltransferase (TbCom1). Recombinant TbCom1 transfers the methyl group from S-adenosylmethionine (AdoMet) to the 2'-OH of the second nucleoside of m7GpppNpNp-RNA to form m7GpppNpNmp-RNA. TbCom1 is also capable of converting cap 1 RNA to cap 2 RNA. The methyl transfer reaction is dependent on the m7GpppN cap, as the enzyme does not form a stable interaction with GpppN-terminated RNA. Mutational analysis establishes that the TbCom1 and vaccinia virus VP39 methyltransferases share mechanistic similarities in AdoMet- and cap-recognition. Two aromatic residues, Tyr18 and Tyr187, may participate in base-stacking interactions with the guanine ring of the cap, as the removal of each of these aromatic side-chains abolishes cap-specific RNA-binding.

背景与目标： : kinetopplastid mrna具有源自标准m7GpppN cap结构的独特的超甲基化cap 4结构，在前四个核糖上具有2 '-O甲基化，在第一个腺嘌呤和第四个尿嘧啶上具有其他碱基甲基化。尽管在布氏锥虫中已经表征了负责m7GpppN cap 0形成的酶，但cap 4甲基化的机制和超甲基化结构的作用仍不清楚。在这里，我们描述了48 kDa T.br ucei 2 '-O核苷甲基转移酶 (TbCom1) 的表征。重组TbCom1将甲基从S-腺苷甲硫氨酸 (AdoMet) 转移至m7GpppNpNp-RNA第二核苷的2 '-OH形成m7GpppNpNmp-RNA。TbCom1还能够将cap 1 RNA转换为cap 2 RNA。甲基转移反应取决于m7GpppN帽，因为该酶不会与GpppN终止的RNA形成稳定的相互作用。突变分析表明，TbCom1和痘苗病毒VP39甲基转移酶在AdoMet和cap识别中具有机械相似性。两个芳香族残基Tyr18和Tyr187可能参与与cap的鸟嘌呤环的碱基堆积相互作用，因为这些芳香族侧链中的每一个的去除消除了cap特异性RNA的结合。

BACKGROUND & AIMS: :We have isolated a cDNA clone corresponding to a single-copy nuclear gene that is upregulated at the mRNA level during in vitro differentiation of bloodstream trypomastigotes of strains of both Trypanosoma brucei brucei and Trypanosoma brucei rhodesiense to procyclic forms. Transcript levels begin to increase within minutes of introduction of bloodstream forms into culture and peak well before cultures exhibit a procyclic morphology. This increase in transcript levels was found to occur both in the absence of protein synthesis and in a nontransforming strain blocked very early in the developmental program, both conditions under which accumulation of procyclic acidic repetitive protein (PARP) transcripts did not occur in control experiments. DNA sequence analysis reveals an open reading frame sufficient to encode a protein of approximately 50 kDa within the cDNA, but data base searches for homology at either the amino acid or nucleotide level revealed no related sequences. A high density of kinase consensus target sites in the deduced amino acid sequence suggests that the gene product may be a phosphoprotein.

背景与目标： : 我们已经分离出一个对应于单拷贝核基因的cDNA克隆，该基因在布鲁氏锥虫和布鲁氏锥虫的菌株的血流锥虫的体外分化过程中在mRNA水平上调至原环形式。转录本水平在将血流形式引入培养物后的几分钟内开始增加，并在培养物表现出原环形态之前达到峰值。发现在没有蛋白质合成的情况下以及在发育程序早期阻断的非转化菌株中都会发生这种转录水平的增加，在这两种条件下，在对照实验中均未发生原环酸性重复蛋白 (PARP) 转录本的积累。DNA序列分析揭示了一个开放阅读框，足以在cDNA中编码约50 kDa的蛋白质，但是数据库在氨基酸或核苷酸水平上搜索同源性，没有发现相关序列。推导的氨基酸序列中激酶共有靶位点的高密度表明该基因产物可能是磷蛋白。