Biochemical And Biophysical Parasitology

生物化学和生物物理寄生虫学

• 批准号: 8745271
• 负责人: Fuyuki Tokumasu
• 金额: $ 33.73万
• 依托单位: NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8745271
• 关键词: Area; Biochemical; Biological Process; Cell membrane; Cell physiology; Cells; Cellular Membrane; Characteristics; Cholesterol; Cleaved cell; Collaborations; Collection; Complex; Computer Assisted; Data; Development; Dimensions; Drug resistance; Dyes; Electron Microscopy; Electronics; Erythrocyte Membrane; Erythrocytes; Fatty Acids; Fluorescence; Genotype; Gluconeogenesis; Glycerol; Goals; Hemoglobin; Human; Image; Imagery; Invaded; Light; Lipids; Malaria; Manuscripts; Measures; Membrane; Membrane Potentials; Membrane Protein Traffic; Metabolic; Metabolism; Methods; Microscopy; Mitochondria; Modification; Molecular; Monitor; Optics; Parasites; Parasitology; Phospholipids; Physics; Physiological; Plasmodium falciparum; Play; Process; Property; Proteins; Protocols documentation; Publishing; Reactive Oxygen Species; Relative (related person); Research Project Grants; Resolution; Role; Scanning; Shapes; Sickle Hemoglobin; Signal Transduction; Solutions; Source; Staging; Structure; Techniques; Technology; Time; Vacuole; Work; absorption; asexual; cholesterol analog; design; imaging modality; improved; insight; light intensity; lipid metabolism; mitochondrial membrane; quantum; trafficking; trend; Area; Biochemical; Biological Process; Cell membrane; Cell physiology; Cells; Cellular Membrane; Characteristics; Cholesterol; Cleaved cell; Collaborations; Collection; Complex; Computer Assisted; Data; Development; Dimensions; Drug resistance; Dyes; Electron Microscopy; Electronics; Erythrocyte Membrane; Erythrocytes; Fatty Acids; Fluorescence; Genotype; Gluconeogenesis; Glycerol; Goals; Hemoglobin; Human; Image; Imagery; Invaded; Light; Lipids; Malaria; Manuscripts; Measures; Membrane; Membrane Potentials; Membrane Protein Traffic; Metabolic; Metabolism; Methods; Microscopy; Mitochondria; Modification; Molecular; Monitor; Optics; Parasites; Parasitology; Phospholipids; Physics; Physiological; Plasmodium falciparum; Play; Process; Property; Proteins; Protocols documentation; Publishing; Reactive Oxygen Species; Relative (related person); Research Project Grants; Resolution; Role; Scanning; Shapes; Sickle Hemoglobin; Signal Transduction; Solutions; Source; Staging; Structure; Techniques; Technology; Time; Vacuole; Work; absorption; asexual; cholesterol analog; design; imaging modality; improved; insight; light intensity; lipid metabolism; mitochondrial membrane; quantum; trafficking; trend

In this FY, we accomplished and advanced several projects. They are (1) Identifications of unique properties of parasite-induced membranes, including vacuole membrane (PVM) and Maurers Cleft, and differences in properties of parasitized erythrocyte membranes between HbA and HbS erythrocytes, (2) Direct visualizations of whole MC by electron microscopy, (3) Microscopy study of gametocyte metabolism and drug resistance relationship, and (4) Identifications of hemoglobin distributions and their modifications using hyperspectral imaging combined with optical phantom. (1) We continued studying parasite-derived membranes with unique biophysical properties using fluorescence lifetime microscopy (FLIM). During invasion process, PVM surrounds invading parasite. PVM continuously increase in its size as parasites mature. In addition, new membranes emerge from PVM and form Maurer's cleft (MC). This structure seems to play crucial roles in trafficking and delivery processes for parasite-derived proteins. However, the source of lipids enough to support the size increase in the new membrane structure is not known. A key to solve this question is cholesterol content as parasite does not have a cholesterol synthesis mechanism. In addition, cholesterol is one of the most important molecules for membrane biophysical properties. The potentially unique properties of PVM and MC could provide important insights for their functions responsible for efficient trafficking of parasite proteins to erythrocyte membrane. In previous FYs, we identified the lipid exchange/transfer between parasite and host erythrocyte membranes during intraerythrocytic stages monitored by the translocations of membrane dye, Di4-ANEPPDHQ, and cholesterol analogue, bodipy-cholesterol. These results suggest that lipids are transferred between erythrocyte and parasites, and transfer mechanism may not be the same between cholesterol and non-sterol lipids such as phospholipids. We also found that hemoglobin S erythrocytes, both heterozygous (AS) and homozygous (SS) non-parasitized erythrocytes, had lower Di-4 lifetime compared to non-parasitized HbA erythrocytes. Differences in Di-4 lifetime were also observed in parasitized erythrocytes while the same trend of lifetime differences between erythrocyte, PV and parasite membranes within a same genotype. These data indicate that HbS erythrocytes have significantly different membrane property than that of normal HbA erythrocytes, which may control protein assembly and distributions on the host erythrocyte membrane. In this FY, we found that that cholesterol gradient inside parasitized erythrocytes are created by constant flow of non-sterol parasite lipids from parasite to PVM and small flux of cholesterol to PVM from surrounding host erythrocyte membrane to compensate exceeding reduction of relative cholesterol level at PVM. (2) We continued studying gametocyte metabolism using microscopy approach. In previous FY, we identified (a) a high lipid content, but similar cholesterol level, (b) reduced reactive oxygen species, and (d) similar mitochondrial membrane potential. We also studied requirement of mitochondrial activity for gametocyte developments and found complex physiological variability exist in gametocyte developments. In this FY, we focused on studying level of metabolic products on lipid metabolism and potential gluconeogenesis. Using fluorescence indicators, we are currently measuring (a) glycerol, (b) fatty acid, and (c) triacylglycerides and comparing these levels with asexual stages of parasites to study relative activities of each metabolic cascade. Using neutral lipids indictor, we found that neutral lipids in gametocytes are stored in droplet-like compartments along with the parasite plasma membrane. This distribution is very different from asexual stages of parasite in which neutral lipids distribute diffusedly in the parasite with some concentrated areas. These data should provide important information on the mechanism of gametocytogenesis and following sexual stage. (3) In previous FY, we initiated a project studying hemoglobin distributions within a erythrocyte using hyperspectral imaging. Hemoglobin molecules have specific absorption spectra with several characteristic peaks, which change to different spectra as hemoglobin degrades. Hyperspectral imaging uses a specially designed optics and changes wavelength of incident light, and scans from UV to near IR regions. Intensities of captured images at each wavelength were compared to original light intensities and absorption spectra were produced. This technique has multiple advantages as absorption spectra of any area of cell can be analyzed that potentially help estimating hemoglobin amount and the level of degradations. We established technical analysis protocol to estimate hemoglobin concentration in terms of hyperspectral imaging signals. We published this work to J. of Selected Topics in Quantum Electronics. This FY, we used optical phantom with confined hemoglobin solution with triangular prism shape. Known dimension of hemoglobin solution provide accurate information of optical absorbance data with respect to estimated volume of hemoglobin solution. We are now preparing a manuscript for this work.

在此FY中，我们完成并提前了几个项目。它们是（1）鉴定寄生虫诱导的膜的独特性能，包括液泡膜（PVM）和毛呼室裂缝，以及HBBA和HBS hBS erythropytes之间的寄生寄生的红细胞膜的性能差异，（2）通过电子显微镜的整个MC研究（2）启示性的MIC（2）gamecopy（3）gamecopy（3）gamecopy（3）gamecopy（3） （4）使用高光谱成像与光学幻像结合使用血红蛋白分布及其修饰。 （1）我们继续研究具有荧光寿命显微镜（FLIM）的独特生物物理特性的寄生虫衍生的膜。在入侵过程中，PVM围绕入侵寄生虫。随着寄生虫的成熟，PVM的大小不断增加。此外，新的膜从PVM出现并形成Maurer's Cleft（MC）。这种结构似乎在寄生虫衍生的蛋白质的运输和交付过程中起着至关重要的作用。然而，尚不清楚脂质的源源足以支持新膜结构的尺寸增加。解决这个问题的关键是胆固醇含量，因为寄生虫没有胆固醇的合成机制。此外，胆固醇是膜生物物理特性最重要的分子之一。 PVM和MC的潜在独特特性可以为其功能提供重要的见解，以使寄生虫蛋白有效地运输到红细胞膜上。 在以前的FYS中，我们确定了通过膜染料，DI4-ANEPPDHQ和胆固醇类似物，Bodipy-Cholesterol的易位，寄生虫和宿主红细胞膜之间的脂质交换/转移。这些结果表明，脂质是在红细胞和寄生虫之间转移的，并且胆固醇和非脂肪脂质（如磷脂）之间的转移机制可能不相同。 我们还发现，与非寄存的HBA红细胞相比，杂合（AS）和纯合（SS）非寄生的红细胞的血红蛋白红细胞的生命周期都较低。 在寄生的红细胞中也观察到DI-4寿命的差异，而同一基因型内的红细胞，PV和寄生虫膜之间终生差异的趋势相同。 这些数据表明，HBS红细胞具有与正常HBA红细胞的膜特性明显不同，后者可能控制宿主红细胞膜上的蛋白质组装和分布。 在此FY中，我们发现，寄生的红细胞内胆固醇梯度是通过从寄生虫到PVM到PVM的持续流动而产生的，而胆固醇的小通量从周围的宿主红细胞膜上从宿主的红细胞膜上流向PVM，以补偿超过PVM下的胆固醇的相对降低。 （2）我们继续使用显微镜方法研究配子细胞代谢。 在以前的FY中，我们确定了（a）高脂质含量，但相似的胆固醇水平，（b）减少活性氧和（d）类似的线粒体膜电位。 我们还研究了对配子细胞发展的线粒体活性的要求，并发现配子细胞发展中存在复杂的生理变异性。 在此FY中，我们专注于研究代谢产物水平在脂质代谢和潜在的糖异生中。 使用荧光指标，我们目前正在测量（a）甘油，（b）脂肪酸和（c）三酰基甘油酸酯，并将这些水平与寄生虫的无性阶段进行比较，以研究每个代谢级联的相对活性。 使用中性脂质指控器，我们发现配子细胞中的中性脂质与寄生虫质膜一起存储在类似液滴的隔室中。 这种分布与寄生虫的无性阶段大不相同，中性脂质分布在寄生虫中，并具有一些浓缩区域。 这些数据应提供有关配子细胞发生机制和性阶段之后的重要信息。 （3）在以前的FY中，我们启动了一个使用高光谱成像在红细胞内研究血红蛋白分布的项目。 血红蛋白分子具有特定的吸收光谱，具有几个特征峰，它们会变成不同的光谱，因为血红蛋白降解。 高光谱成像使用特殊设计的光学和变化的入射光波长，并从紫外线到附近的IR区域进行扫描。 将每个波长处捕获的图像的强度与原始光强度进行比较，并产生吸收光谱。 该技术具有多个优势，因为可以分析任何细胞区域的吸收光谱，从而有助于估计血红蛋白量和降解水平。 我们建立了技术分析方案，以高光谱成像信号来估计血红蛋白浓度。 我们将此工作发布给J.的J. Quantum Electronics中的选定主题。 我们使用具有三角形棱镜形状的密闭血红蛋白溶液的光学幻像。 血红蛋白溶液的已知维度提供了相对于估计体积的血红蛋白溶液的准确信息。 我们现在正在为这项工作准备手稿。