Purine Metabolism in Trypanosomatids*

SYNOPSIS. Purine nucleotide biosynthesis was studied in culture forms of Trypanosoma cruzi strain Y, Crithidia deanei (a reduviid trypanosomatid with an endosymbiote) and an aposymbiotic strain of C. deanei (obtained by curing C. deanei with chloramphenicol). Trypanosoma cruzi was found to synthesize purine nucleotides only from the preformed bases adenine and guanine (“salvage” pathway), adenine being incorporated into both adenine and guanine nucleotides. Similar results were obtained with guanine, indicating that this flagellate has a system for the interconversion of purine nucleotides. Crithidia deanei was able to synthesize purine and pyrimidine nucleotides from glycine (“de novo” pathway) and purine nucleotides from adenine and guanine (“salvage” pathway). Adenine was incorporated into both adenine and guanine nucleotides, while guanine was incorporated into guanine nucleotides only, indicating the presence of a metabolic block at the level of GMP reducaase. The aposymbiotic C. deanei strain was unable to utilize glycine for the synthesis of purine nucleotides, although glycine was utilized for synthesizing pyrimidine nucleotides. These results suggest that the endosymbiote is implicated in the de novo purine nucleotide pathway of the C. deanei-endosymbiote complex. The incorporation of adenine and guanine by aposymbiotic C. deanei strain followed a pattern similar to that observed for C. deanei.     

On the Fine Structure of the Nucleus in Trypanosoma cruzi in Tissue Culture Forms. Spindle Fibers in the Dividing Nucleus*

The fine structure of the dividing nucleus in the intracellular amastigote forms of Trypanosoma cruzi from tissue cultures has been described. In the first phase of division the nucleus shows a homogenous structure owing to the dispersion of its chromatin and nucleolar material. Microtubules similar to those of a mitotic spindle in metozoan cells then appear, running from one pole to the other. They disappear when the division of the nucleus is complete and the chromatin and the nucleolar material reorganize into their former positions.     

Studies on Nuclei of Paramecium aurelia. II. Amino Acid Composition and Electrophoretic Properties of the Chromosomal Basic Proteins*

SYNOPSIS. The basic proteins of Paramecium aurelia nucleus were extracted from isolated nuclei and deoxyribonucleoprotein (DNP) of such nuclei. About 35–40% of the nuclear protein, predominantly a lysine-rich histone, is acid soluble. Five major components of the histone can be distinguished by polyacrylamide gel electrophoresis. Some components of Paramecium histone are similar to those of mammalian histones in their electrophoretic mobility, but they differ from the latter in the electrophoretic velocity and relative levels. The basic to acidic amino acid ratio of the histone from the ciliate is ～1.1–1.5, and its amino acid composition resembles closely that of yeast histone. Through the use of Sephadex G-200 gel filtration for purification of the histones extracted directly from isolated nuclei 2 basic proteins were resolved—component I, with an elution volume of 1.4, constitutes ～20% and component II, with an elution volume of 1.9, ～80%.     

Comparison and Evolutionary Analysis of the Glycosomal Glyceraldehyde-3-Phosphate Dehydrogenase from Different Kinetoplastida

In this work, we present the sequences and a comparison of the glycosomal GAPDHs from a number of Kinetoplastida. The complete gene sequences have been determined for some species (Crithidia fasciculata, Herpetomonas samuelpessoai, Leptomonas seymouri, and Phytomonas sp), whereas for other species (Trypanosoma brucei gambiense, Trypanosoma congolense, Trypanosoma vivax, and Leishmania major), only partial sequences have been obtained by PCR amplification. The structure of all available glycosomal GAPDH genes was analyzed in detail. Considerable variations were observed in both their nucleotide composition and their codon usage. The GC content varies between 64.4% in L. seymouri and 49.5% in the previously sequenced GAPDH gene from Trypanoplasma borreli. A highly biased codon usage was found in C. fasciculata, with only 34 triplets used, whereas in T. borreli 57 codons were employed. No obvious correlation could be observed between the codon usage and either the nucleotide composition or the level of gene expression. The glycosomal GAPDH is a very well-conserved enzyme. The maximal overall difference observed in the amino acid sequences is only 25%. Specific insertions and extensions are retained in all sequences. The residues involved in catalysis, substrate, and inorganic phosphate binding are fully conserved, whereas some variability is observed in the cofactor-binding pocket. The implications of these data for the design of new trypanocidal drugs targeted against GAPDH are discussed. All available gene and amino acid sequences of glycosomal GAPDHs were used for a phylogenetic analysis. The division of the Kinetoplastida into two suborders, Bodonina and Trypanosomatina, was well supported. Within the letter group, the Trypanosoma species appeared to be monophyletic, whereas the other trypanosomatids form a second clade. Received: 23 February 1998/Accepted: 26 March 1998     

Biosynthesis of Castor Oil: Effect of Polyamines on the Acylation of Lysophosphatidic Acid at the sn-2 Position with Ricinoleic Acid

Polyamines with diamine structures of chain length longer than 3C were essential for the synthesis of phosphatidic acid (PA) from ricinoleoyl-CoA and lysophosphatidic acid (LPA) by the castor LPA acyltransferase reaction, suggesting that polyamines modulate enzyme affinity for the acyl-CoA substrate in vivo.     

Analysis of Genetic Diversity at the arom Locus in Isolates of Pneumocystis carinii

ABSTRACT. The DNA sequences of a portion of the 5-enolpyruvyl shikimate phosphate synthase domain of the arom gene, encoding the pentafunctional AROM protein, were determined from isolates of Pneumocystis carinii from five mammalian host species (rat, human, ferret, rabbit and mouse). High levels of genetic divergence were found among P. carinii derived from different hosts species, 7–22% at the DNA sequence level, and 7–26% at the derived amino acid sequence level. Two separate and distinct sequences were isolated from infected ferret lungs. Low levels of divergence were seen in human-derived organisms.     

冠心病患者血清尿酸、高敏C 反应蛋白、纤维蛋白原水平变化 的临床研究

目的:观察冠心病患者血清中尿酸、高敏C反应蛋白、纤维蛋白原水平的变化.方法:选取2010年11月至2011年11月于我院就诊的68例冠心病患者(稳定型心绞痛21例,不稳定型心绞痛24例,急性心肌梗死13例)作为研究对象,并选取同期于我院体检中心体检的62例健康人为对照组,检测受试者血清中尿酸、高敏C反应蛋白、纤维蛋白原的水平.结果:研究组患者血清中UA、CRP和FBG水平显著高于对照组(P＜0.05).与稳定型心绞痛组比,不稳定型心绞痛的CRP水平增高(5.34±1.98 mg/L vs.11.36±2.73 mg/L,P＜0.05),急性心肌梗死组的UA (345.63±86.4 μmol/L vs.493.76±101.2 μmol/L,P＜0.05)、CRP (5.34±1.98mg/L vs.21.3±2.24 mg/L,P＜0.05)和FBG(3.86±1.34 g/L vs.6.85±2.36 g/L,P＜0.05)水平显著增高,与不稳定型心绞痛组比,急性心肌梗死组的UA(378.91±89.7 μmol/L vs.493.76±101.2 μmol/L,P＜0.05)、CRP(11.36±2.73 mg/L vs.21.3±2.24 mg/L,P＜0.05)和FBG(4.27±2.08 g/L vs.6.85±2.36 g/L,P＜0.05)水平显著增高(P＜0.05).结论:冠心病患者血清中尿酸、高敏C反应蛋白和纤维蛋白原的水平升高,3个指标可用于评估治疗效果和预后.     

急性脑梗死患者血浆同型半胱氨酸水平及其相关因素分析*

目的:探讨急性脑梗死患者血浆同型半胱氨酸水平及相关因素。方法:采用高效液相色谱法检测232例急性脑梗死患者血浆Hcy水平,并对其相关因素进行统计学分析。结果:急性脑梗死组患者血浆Hcy水平明显升高(P<0.05);并与叶酸及维生素B12水平显著相关。不同年龄段急性脑梗死患者血浆Hcy水平具有差异。并与颈动脉粥样硬化斑块相关联。结论:血浆Hcy水平增高是脑梗死的危险因素之一,并与患者年龄、血液中叶酸、维生素B12水平及脑梗死性质相关联。     

Arachidonic Acid Inhibition of Muscarinic Receptor-Mediated Nitric Oxide Production Occurs at the Level of Calcium Mobilization in Chinese Hamster Ovary Cells

Strong evidence supports that nitric oxide (NO) alters cell signaling pathways involving arachidonic acid (AA). Little is known, however, about the reciprocal modulation of nitrergic pathways by AA. The effects of exogenous AA on signal transduction of M₁ muscarinic acetylcholine receptors were investigated in a model system of stably transfected Chinese hamster ovary cells. AA concentration-dependently inhibited the effects of carbachol in producing NO (IC₅₀ = 191 M) but did not alter inositol phosphate production or M₁ receptor binding. AA inhibited both carbachol-induced transient and sustained increase in intracellular calcium concentration ([Ca²⁺]_i; IC₅₀ = 11 and 12 M, respectively). Furthermore, AA-induced increase in [Ca²⁺]_i cross-desensitizes with thapsigargin, but AA does not inhibit Ca²⁺-ATPase activity. These data support the concept that AA concentration-dependently inhibits receptor-mediated NO production at the level of calcium mobilization.     

Octanoic Acid Produces Accumulation of Monoamine Acidic Metabolites in the Brain: Interaction with Organic Anion Transport at the Choroid Plexus

Effects of octanoic acid on monoamines and their acidic metabolites in the rat brain were analyzed by HPLC. Octanoic acid (1,000 mg/kg i.p.) elevated homovanillic acid levels by 54% in the caudate and 338% in the hypothalamus but increased 5-hydroxyindoleacetic acid (5-HIAA) levels in both the caudate and the hypothalamus by approximately 50% compared with the control. A lower dose of octanoic acid (500 mg/kg) increased 5-HIAA levels by 29% in the caudate and 20% in the hypothalamus. However, it did not produce any changes in the concentration of homovanillic acid in either the caudate or the hypothalamus. Treatment with octanoic acid also failed to change the level of dopamine, serotonin, and 3,4-dihydroxyphenylacetic acid in the caudate and the hypothalamus. The role of carrier-mediated transport in the clearance of 5-HIAA from the rabbit CSF was also evaluated in vivo by ventriculocisternal perfusion. Steady-state clearance of 5-HIAA from CSF exceeded that of inulin and was reduced in the presence of octanoic acid. Because this transport system in the choroid plexus is normally responsible for the excretion of the serotonin metabolite from the brain to the plasma, accumulation of endogenously produced organic acids in the brain, secondary to reduced clearance by the choroid plexus, could be a contributing factor in the development of encephalopathy in children with medium-chain acyl-CoA dehydrogenase deficiency who have elevated levels of octanoic acid systematically.     

AECOPD患者血清降钙素原与其它炎症指标水平的相关性分析

目的：分析慢性阻塞性肺疾病急性加重（AECOPD）患者血清降钙素原与其它炎症指标水平的相关性。方法：选择2012年1月至2012年12月我院重症医学科收治的195例AECOPD患者作为研究对象,所有患者均采血后行降钙素原（procalcitonin,PCT）、超敏c反应蛋白（C-reactiveprotein,hs-CRP）、血常规（blood General,WBC）、血沉（erythrocyte sedimentationrate,ESR）、白介素六（interleukin-6,IL-6）检测,同时采集痰标本后行痰培养和药敏试验检测,分析其血清PCT水平与其它炎症指标的相关性。结果：195例AECOPD患者中,PCT增高者43例（22．1％）,正常者152例（77．9％）,其血清PCT水平与hs-CRP、WBC、中性粒细胞百分比（neutrophilpercentage,NEUT％）均显著相关（P〈0．05）,相关系数分别为0．262、0．382、0．333,与ESR、IL-6、痰培养结果无明显相关性（P〉0．05）。结论：血清PCT水平判定AECOPD患者是否合并感染有一定的局限性,联合检测hs-CRP、WBC、NEUT％,可作为诊断AECOPD感染的有力补充。     

高血压患者颈动脉粥样硬化与血脂、血压和血尿酸水平的相关性分析

目的:探讨高血压患者颈动脉粥样硬化(CAS)与血脂、血压以及血尿酸水平的相关性。方法:选择2017年2月至2018年8月在我院就诊的高血压患者117例作为研究组,另选择同期在我院进行体检的健康志愿者50例作为对照组。采用彩色多普勒超声诊断仪测定所有受试者的颈动脉内中膜厚度(IMT),并根据研究组患者的颈动脉IMT将其分为斑块组(IMT≥1.3 mm,33例)、IMT增厚组(1.0 mm≤IMT1.3 mm,49例)和IMT正常组(IMT1.0 mm,35例)。比较研究组与对照组受试者IMT,同时分别比较研究组与对照组受试者以及不同IMT高血压患者平均收缩压(SBP)、平均舒张压(DBP)、血清总胆固醇(TC)、甘油三酯(TG)、低密度脂蛋白胆固醇(LDL-C)、高密度脂蛋白胆固醇(HDL-C)以及尿酸水平,并采用Pearson相关性分析法分析高血压患者IMT与各指标的相关性。结果:与对照组比较,研究组IMT、SBP、DBP、TC、TG、LDL-C、血尿酸水平升高,HDL-C水平降低,差异均有统计学意义(P0.05)。斑块组患者SBP、DBP、TC、TG、LDL-C、血尿酸水平高于IMT增厚组和IMT正常组,HDL-C水平低于IMT增厚组和IMT正常组,差异均有统计学意义(P0.05);IMT增厚组患者SBP、DBP、TC、TG、LDL-C、血尿酸水平高于IMT正常组,HDL-C水平低于IMT正常组,差异均有统计学意义(P0.05)。Pearson相关性分析显示,高血压患者的IMT与SBP、DBP、TC、TG、LDL-C、血尿酸均呈正相关,与HDL-C呈负相关(P0.05)。结论:高血压患者IMT与血脂、血压和血尿酸水平均有明显相关性,血压、血脂、血尿酸参与了高血压患者CAS的发生与发展。     

Identification of Novel Site‐Specific Alterations in the Modification Level of Myelin Basic Protein Isolated from Mouse Brain at Different Ages Using Capillary Electrophoresis–Mass Spectrometry

Myelin basic protein (MBP) is a multifunctional protein involved in maintaining the stability and integrity of the myelin sheath by a variety of interactions with membranes and other proteins. MBP is subjected to extensive posttranslational modifications (PTMs) that are known to be crucial for the regulation of these interactions. Here, we report capillary electrophoresis–mass spectrometric (CE–MS) analysis for the separation and identification of MBP peptides that incorporate the same PTM at different sites, creating multiple localization variants, and the ability to analyze challenging modifications such as asparagine and glutamine deamidation, isomerization, and arginine citrullination. Moreover, we observed site‐specific alterations in the modification level of MBP purified from brain of mice of different age. In total, we identified 40 modifications at 33 different sites, which include both previously reported and seven novel modifications. The identified modifications include Nα‐terminal acetylation, mono‐ and dimethylation, phosphorylation, oxidation, deamidation, and citrullination. Notably, some new sites of arginine methylation overlap with the sites of citrullination. Our results highlight the need for sensitive and efficient techniques for a comprehensive analysis of PTMs.     

高尿酸血症患者血清尿酸水平与糖耐量异常、炎症因子及红细胞参数的相关性分析

摘要 目的：探讨高尿酸血症（HUA）患者血清尿酸（UA）水平与糖耐量异常、炎症因子及红细胞参数的相关性。方法：选取2020年1月~2021年1月期间我中心在健康体检中诊断的HUA患者240例（观察组）,另选取同期在我中心体检的健康志愿者200例（对照组）,分别检测血清UA、炎症因子：超敏C反应蛋白（hs-CRP）、单核细胞趋化蛋白1（MCP-1）、白介素-6（IL-6）、肿瘤坏死因子-α（TNF-α）、红细胞参数：红细胞分布宽度（RDW）、红细胞平均体积（MCV）及红细胞计数、糖耐量指标：空腹血糖（FPG）、餐后2h血糖（2hPG）及空腹胰岛素（FINS）,计算稳态模型胰岛素抵抗指数（HOMA-IR）和稳态模型胰岛素敏感指数（HOMA-ISI）。结果：观察组的UA、RDW及红细胞计数均高于对照组,MCV低于对照组（P<0.05）。观察组的hs-CRP、MCP-1、IL-6及TNF-α均高于对照组（P<0.05）。观察组的FPG、2hPG、FINS及HOMA-IR均高于对照组,HOMA-ISI低于对照组（P<0.05）。Pearson相关系数指出,HUA患者血清UA水平与红细胞计数、RDW、hs-CRP、MCP-1、IL-6、TNF-α、FPG、2hPG、FINS及HOMA-IR呈正相关,而与HOMA-ISI、MCV呈负相关（P<0.05）。结论：高尿酸水平可引起HUA患者炎症反应增强、糖耐量异常及红细胞参数改变,控制UA水平可减少多种并发症的发生风险。     

纳络酮对脑梗塞患者血清降钙素原及叶酸水平及临床疗效的影响

目的:探讨纳络酮对脑梗塞患者血清降钙素原、叶酸水平及临床疗效的影响。方法:收集我院收治的110例急性脑梗死患者,随机分为实验组和对照组,每组55例。两组患者入院后根据实际情况给予抗血小板聚集,保护脑细胞,调控血压,脱水降低颅内压降颅压等对症治疗。对照组患者给予疏血通注射液6 m L+0.9%氯化钠注射液250 m L静脉滴注,1次/d;实验组患者在对照组基础上给予盐酸纳洛酮注射液3.2 g/d,+0.9%氯化钠注射液250 m L静脉滴注,治疗疗程为14 d。观察并比较两组患者治疗前后血清降钙素原(PCT)、同型半胱氨酸(Hcy)、叶酸水平以及临床治疗有效率。结果:与治疗前相比,两组患者治疗后的血清PCT、Hcy水平均显著下降,叶酸水平均明显升高,差异均具有统计学意义(P0.05);与对照组相比,实验组患者的PCT、Hcy水平较低,差异具有统计学意义(P0.05);与对照组相比,实验组患者治疗后的血清叶酸水平、临床治疗有效率均较高,差异具有统计学意义(P0.05)。结论:纳络酮能够显著提高脑梗塞患者的临床疗效,可能与其减轻炎症反应,降血清Hcy水平,升高血清叶酸水平有关。     

左房内径、血清尿酸水平与老年心房颤动的相关性分析

目的:探讨左房内径(LAD)、血清尿酸(UA)水平与老年心房颤动的相关性。方法:选择2013年1月至2016年7月在我院住院的60岁以上的非瓣膜性房颤患者,共166例,其中持续性房颤组85例,阵发性房颤组81例,选择同期无房颤的高血压、冠心病老年患者83例作对照组。通过心脏彩超检查检测LAD、左心室舒张末期内径(LVDD)、左心室收缩末期内径(LVDS)、左心室射血分数(LVEF),以≥40 mm为左房内径增大。并采用生化分析检测患者血清UA水平。结果:(1)持续性房颤组LAD、LVEF、左心房增大发生率均显著高于阵发性房颤组和对照组,而阵发性房颤组以上指标均明显高于对照组,差异具有统计学意义(P0.05)。(2)持续性房颤组和阵发性房颤组患者血清UA水平均显著高于对照组,但持续性房颤组和阵发性房颤组之间血清UA水平比较差异无统计学意义(P0.05)。结论:左心房内径大小、血尿酸水平与老年患者心房颤动的发生密切相关。     

Crystal structures of recombinant human purple Acid phosphatase with and without an inhibitory conformation of the repression loop

The crystal structure of human purple acid phosphatase recombinantly expressed in Escherichia coli (rHPAP(Ec)) and Pichia pastoris (rHPAP(Pp)) has been determined in two different crystal forms, both at 2.2A resolution. In both cases, the enzyme crystallized in its oxidized (inactive) state, in which both Fe atoms in the dinuclear active site are Fe(III). The main difference between the two structures is the conformation of the enzyme "repression loop". Proteolytic cleavage of this loop in vivo or in vitro results in significant activation of the mammalian PAPs. In the crystals obtained from rHPAP(Ec), the carboxylate side-chain of Asp145 of this loop acts as a bidentate ligand that bridges the two metal atoms, in a manner analogous to a possible binding mode for a phosphate ester substrate in the enzyme-substrate complex. The carboxylate side-chain of Asp145 and the neighboring Phe146 side-chain thus block the active site, thereby inactivating the enzyme. In the crystal structure of rHPAP(Pp), the enzyme "repression loop" has an open conformation similar to that observed in other mammalian PAP structures. The present structures demonstrate that the repression loop exhibits significant conformational flexibility, and the observed alternate binding mode suggests a possible inhibitory role for this loop.     

神经节苷脂联合咪达唑仑治疗重症颅脑损伤的临床疗效及对患者血清MMP-9、GFAP、MBP、NSE水平的影响

目的:探讨神经节苷脂联合咪达唑仑治疗重症颅脑损伤的临床疗效及对患者血清基质金属蛋白酶-9(MMP-9)、胶质纤维酸性蛋白(GFAP)、髓鞘碱性蛋白(MBP)、神经元特异性烯醇化酶(NSE)水平的影响。方法:选择2016年3月到2017年9月于我院进行治疗的85例重症颅脑损伤患者,将其按随机数表法分为观察组(n=45)和对照组(n=40),对照组使用神经节苷脂治疗,观察组采用神经节苷脂联合咪达唑仑治疗。比较两组治疗后疗效及血清GFAP、中枢神经特异蛋白(S100β)、NSE、MMP-9、MBP、C反应蛋白(CRP)、白细胞介素-6(IL-6)、肿瘤坏死因子α(TNF-α)水平、格拉斯哥昏迷评分(Glasgow Coma Scale,GCS)评分的变化及不良反应的发生情况。结果:治疗后,观察组临床疗效总有效率(93.33%)显著高于对照组(75.00%,P0.05);两组血清GFAP、S100β、NSE、MMP-9、MBP、CRP、IL-6及TNF-α水平均较治疗前明显下降,且观察组以上指标均显著低于对照组(P0.05);观察组GCS评分明显高于对照组(P0.05),且不良反应发生率明显低于对照组(6.67%vs. 30.00%,P0.05)。结论:神经节苷脂联合咪达唑仑治疗重症颅脑损伤患者的临床效果显著,明显优于单用神经节苷脂治疗,可能与其有效改善患者血清MMP-9、GFAP、MBP、NSE水平及抑制炎症因子的生成有关。     

Primary structure of a photoactive yellow protein from the phototrophic bacterium Ectothiorhodospira halophila,with evidence for the mass and the binding site of the chromophore.

The complete amino acid sequence of the 125-residue photoactive yellow protein (PYP) from Ectothiorhodospira halophila has been determined to be MEHVAFGSEDIENTLAKMDDGQLDGLAFGAIQLDGDGNILQYNAAEGDITGRDPKEVIGKNFFKDVAP+ ++ CTDSPEFYGKFKEGVASGNLNTMFEYTFDYQMTPTKVKVHMKKALSGDSYWVFVKRV. This is the first sequence to be reported for this class of proteins. There is no obvious sequence homology to any other protein, although the crystal structure, known at 2.4 A resolution (McRee, D.E., et al., 1989, Proc. Natl. Acad. Sci. USA 86, 6533-6537), indicates a relationship to the similarly sized fatty acid binding protein (FABP), a representative of a family of eukaryotic proteins that bind hydrophobic molecules. The amino acid sequence exhibits no greater similarity between PYP and FABP than for proteins chosen at random (8%). The photoactive yellow protein contains an unidentified chromophore that is bleached by light but recovers within a second. Here we demonstrate that the chromophore is bound covalently to Cys 69 instead of Lys 111 as deduced from the crystal structure analysis. The partially exposed side chains of Tyr 76, 94, and 118, plus Trp 119 appear to be arranged in a cluster and probably become more exposed due to a conformational change of the protein resulting from light-induced chromophore bleaching. The charged residues are not uniformly distributed on the protein surface but are arranged in positive and negative clusters on opposite sides of the protein. The exact chemical nature of the chromophore remains undetermined, but we here propose a possible structure based on precise mass analysis of a chromophore-binding peptide by electrospray ionization mass spectrometry and on the fact that the chromophore can be cleaved off the apoprotein upon reduction with a thiol reagent. The molecular mass of the chromophore, including an SH group, is 147.6 Da (+/- 0.5 Da); the cysteine residue to which it is bound is at sequence position 69.