斯氏按蚊血淋巴游离氨基酸和蛋白的研究

用氨基酸自动分析仪测定斯氏按蚊血淋巴游离氨基酸的变化,用紫外吸收差法测定其蛋白浓度。感染约氏疟原虫蚊与正常蚊相比,吸血后第4d,6种氨基酸含量降低,5种氨基酸含量增加;吸血后第7d,4种氨基酸含量降低,7种氨基酸含量增加;吸血后第11d,9种氨基酸含量降低,4种氨基酸含量增加。感染蚊血淋巴蛋白浓度均高于同期正常蚊。     

两种按蚊对食蟹猴疟原虫B株敏感性的比较

用食蟹猴疟原虫B株接种恒河猴后,在最佳感染时机血餐,第7d胃感染率,大劣按蚊平均为94.9％(74/78),斯氏按蚊平均为94.6％(70/74)。第12d涎腺感染率,大劣按蚊平均为93.9％(62/66),斯氏按蚊平均为92.2％(59/64),两者的胃和涎腺感染率无显著性差异。卵囊均数,大劣按蚊为124.2个,斯氏按蚊为47.6个,前者为后者的2.6倍。在欠佳感染时机血餐,大劣按蚊的胃和涎腺感染率均高于斯氏按蚊,大劣按蚊的卵囊均数为斯氏按蚊的3倍余。     

大劣按蚊含硫脂蛋白基因在约氏疟原虫感染中的作用

目的分析大劣按蚊含硫脂蛋白(TEP1)基因在约氏疟原虫感染过程中的作用。方法根据GenBank中大劣按蚊TEP1基因序列设计带有T7启动子的引物,以大劣按蚊cDNA为模板,进行PCR扩增,纯化产物,体外转录试剂盒合成AdTEP1双链RNA。羽化1～2日的雌性大劣按蚊分3组(200只/组):TEP1干扰组、绿色荧光蛋白(EGFP)干扰组和对照组。TEP1、EGFP干扰组按蚊分别进行胸部微量注射AdTEP1双链RNA、EGFP双链RNA各147 ng,对照组未处理。干扰后3d,每组取15只按蚊,去头,以大劣按蚊核糖蛋白S7(AdS7)为内参进行半定量PCR,检测干扰效果。干扰后4d,用约氏疟原虫BY256荧光株感染3组大劣按蚊。感染后9d,每组各解剖25只蚊胃,记录感染率和感染度。结果对照组和EGFP干扰组AdTEP1的表达条带亮度基本一致,而TEP1干扰组AdTEP1的表达条带亮度非常微弱。感染后9 d,蚊胃卵囊数统计学分析表明,对照组、EGFP干扰组和TEP1干扰组感染率分别为(24±2.83)%、(24±0.71)%和(80±3.54)%;感染度分别为0.32±0.7、0.44±0.85和5.52±4.84...     

三种蚊虫血淋巴游离氨基酸的研究

用离心法收集雌蚊血淋巴,用氨基酸自动分析仪检测血淋巴游离氨基酸的组成,并对三种蚊虫作了比较。羽化时,中华按蚊分别与斯氏按蚊和白纹伊蚊相比,各有14种氨基酸有显著性差异;斯氏按蚊与白纹伊蚊相比,有12种氨基酸有显著性差异。吸鼠血后第4d,中华按蚊与斯氏按蚊和白纹伊蚊相比,分别有15和16种氨基酸有显著性差异;斯氏按蚊与白纹伊蚊相比,有14种氨基酸有显著性差异。白纹伊蚊吸鼠血后第4d与吸鸡血后第4d相比,有17种氨基酸有显著性差异。这些差异是由蚊种和脊推动物宿主的不同引起的。     

斯氏按蚊和白纹伊蚊中肠蛋白及蛋白酶分析

取出吸血前后不同时间的雌蚊中肠,分别测定其蛋白含量、组分和蛋白酶活性。所有雌蚊吸血后中肠蛋白含量随时间增加,感染疟原虫的雌蚊中肠蛋白含量均高于同期正常雌蚊。中肠蛋白 SDS—PAGE 表明:斯氏按蚊羽化时和吸血后 d7正常蚊及感染蚊均为15条蛋白区带,吸血后 d10正常蚊为16条区带,感染蚊为14条区带。白纹伊蚊吸血后 d6和 d9正常蚊和感染蚊均为19条蛋白区带。感染雌蚊中肠蛋白酶活性均高于同期正常雌蚊。     

按蚊中肠前酚氧化酶原与约氏疟原虫卵囊黑化的关系

目的　分析约氏疟原虫卵囊黑化期间按蚊中肠内前酚氧化酶原 (Prophenoloxidase ,PPO)的变化 ,探讨中肠PPO与疟原虫卵囊黑化的关系。　方法　解剖分离约氏疟原虫感染前和感染后 3、5、7、11和 15d斯氏与大劣按蚊中肠 ,匀浆后经SDS PAGE和转膜 ,以抗烟草天蛾PPO的抗体进行免疫印迹分析 ,并于感染后第 5d开始在镜下观察约氏疟原虫卵囊黑化情况 ,直至第 15d止。　结果　斯氏与大劣按蚊中肠在约氏疟原虫感染前和感染后的不同时期经免疫印迹均可检测到 1条分子质量约为 67ku的PPO阳性蛋白带 ,但感染后的PPO蛋白带比感染前明显增强 ,而且同时间点大劣按蚊中肠PPO蛋白带比斯氏按蚊明显 ,尤其在感染的后期大劣按蚊中肠PPO蛋白带明显比斯氏按蚊增强 ;在感染后 7d可见大劣按蚊中肠内发育的约氏疟原虫卵囊部分黑化 ,在 15d时可见疟原虫卵囊完全黑化。　结论　按蚊中肠PPO含量增加 ,尤其是长时间内维持在较高水平与约氏疟原虫卵囊黑化密切相关。     

感染约氏疟原虫的斯氏按蚊组织化学观察

目的 观察蚊媒感染疟原虫后的组织化学变化。方法 应用组织化学技术观察对照组和感染约氏疟原虫的斯氏按蚊体内乳酸脱氢酶（LDH）、珀酸脱氢酶（SDH）、三磷酸腺苷酸（Mg^2 -ATPase)活性反应与含量以及糖原含量的变化。结果 与对照组相比,感染约氏疟原虫后,蚊体内LDH活性与含量显著增加,SDH和Mg^2 -ATPase活性与含量及糖原含量显著降低。结论 感染约氏疟原虫可明显影响蚊媒的营养代谢和吸收。     

按蚊中肠前酚氧化酶原与约氏疟原虫卵囊黑化的关系

目的 分析约氏疟原虫卵囊黑化期间按蚊中肠内前酚氧化酶原(Prophenoloxidase，PPC))的变化，探讨中肠PPO与疟原虫卵囊黑化的关系。方法 解剖分离约氏疟原虫感染前和感染后3、5、7、11和15d斯氏与大劣按蚊中肠，匀浆后经SDS-PAGE和转膜，以抗烟草天蛾PPO的抗体进行免疫印迹分析，并于感染后第5d开始在镜下观察约氏疟原虫卵囊黑化情况，直至第15d止。结果 斯氏与大劣按蚊中肠在约氏疟原虫感染前和感染后的不同时期经免疫印迹均可检测到1条分子质量约为67ku的PPO阳性蛋白带，但感染后的PPO蛋白带比感染前明显增强，而且同时间点大劣按蚊中肠PPO蛋白带比斯氏按蚊明显，尤其在感染的后期大劣按蚊中肠PPO蛋白带明显比斯氏按蚊增强；在感染后7d可见大劣按蚊中肠内发育的约氏疟原虫卵囊部分黑化，在15d时可见疟原虫卵囊完全黑化。结论 按蚊中肠PPO含量增加，尤其是长时间内维持在较高水平与约氏疟原虫卵囊黑化密切相关。     

硝喹对斯氏按蚊体内不同时期约氏疟原虫发育的影响

目的　观察硝喹对斯氏按蚊体内不同时期约氏疟原虫发育的影响。方法　在感染约氏疟原虫1 d前给予常规蔗糖水或硝喹蔗糖水（含100 μmol/L硝喹）供斯氏按蚊吸食,停糖水24 h后用感染约氏疟原虫的昆明鼠血餐,观察硝喹处理后约氏疟原虫在蚊胃内的卵囊数量变化。感染6、14 d后停常规蔗糖水24 h,随后给予常规蔗糖水或硝喹蔗糖水供斯氏按蚊吸食,观察斯氏按蚊血淋巴及唾液腺中的约氏疟原虫子孢子数量变化。结果　感染前1 d将斯氏按蚊暴露于硝喹蔗糖水后,感染第7天蚊胃中约氏疟原虫卵囊数量[（119.2 ± 16.1）只]较常规蔗糖水组[（207.3 ± 21.8）只]显著降低（t = 3.207,P < 0.05）。感染第6天停常规蔗糖水24 h,将斯氏按蚊暴露于硝喹蔗糖水后,按蚊血淋巴中约氏疟原虫子孢子数量峰值[（952.3 ± 22.7）只]在感染第14天出现,常规蔗糖水组按蚊血淋巴中子孢子数量峰值[（1 287.0 ± 39.0）只]在感染第12天出现;感染第17天,硝喹蔗糖水和常规蔗糖水组按蚊唾液腺中子孢子数量分别为（9 467.0 ± 1 304.0）只和（10 533.0 ± 758.7）只,差异无统计学意义（t = 0.707,P = 0.506）。感染第14天停常规蔗糖水24 h,将斯氏按蚊暴露于硝喹蔗糖水后,按蚊唾液腺中约氏疟原虫子孢子数量[（21 900.0 ± 2 613.0）只]较常规蔗糖水处理组[（10 533.0 ± 732.3）只]显著增加（t = 4.188,P < 0.05）。结论　在斯氏按蚊感染疟原虫不同时期给予硝喹处理对其体内约氏疟原虫发育影响不一,未感染斯氏按蚊经硝喹处理后可减少疟原虫传播。     

大劣按蚊丝氨酸蛋白酶与疟原虫感染相关性研究

目的探讨大劣按蚊血淋巴中丝氨酸蛋白酶与约氏疟原虫感染的相关性。方法首先利用二维电泳分离感染约氏疟原虫的和吸食正常血的大劣按蚊血淋巴蛋白;然后进行Western blot,用丝氨酸蛋白酶抗体进行识别,并比较感染组与正常对照组间的区别;选择感染后不同时相点对大劣按蚊蚊胃和唾液腺进腺镜检,以观察蚊体内疟原虫情况。结果Western blot显示,正常对照组无阳性蛋白点,感染组有2个阳性蛋白点,分子质量单位分别为44ku和27ku,等电点分别为8.0和5.0;镜检显示,感染7d时可见卵囊颗粒样病变,11d时卵囊发育不同步且黑化明显,之后检出的卵囊数逐渐减少。蚊唾液腺内未见子孢子。结论大劣按蚊血淋巴中的丝氨酸蛋白酶与约氏疟原虫感染相关,可能参与蚊抗疟原虫感染的黑化包被反应。     

Analysis of amino acids in hemolymph and acid hydrolysates of midguts of Anopheles dirus infected with Plasmodium cynomolgi

Quantitative determinations of free amino acids in hemolymph and acid hydrolysates of midguts of female Anopheles dirus infected with Plasmodium cynomolgi bastianellii were carried out and the results were compared with those of noninfected mosquitoes. On day 10 after infected blood meal, the contents of methionine, isoleucine, leucine, ornithine, lysine in the hemolymph of infected mosquitoes markedly decreased as compared with those in the controls. However, the quantitative analysis of the amino acids of the acid hydrolysates of the midguts from infected mosquitoes on day 9 after an infected blood meal showed that the content of their total amino acids was 70% more than that in the controls, with special reference to aspartic acid, glutamic acid, valine, methionine, isoleucine, leucine, phenylalanine, tryptophan.     

Modification of the effects of blood on amino acid metabolism by intravenous isoleucine

The absence of isoleucine in the hemoglobin molecule has been suggested to contribute to increased urea production after a blood meal. To unravel the underlying mechanism, the effects of isoleucine infusion after blood ingestion in the healthy pig were studied. The isoleucine dose was chosen to induce an arterial isoleucine increase comparable to those observed for leucine or valine after blood ingestion. For the experiments, 10 female overnight-fasted pigs (20-25 kg) received 250 mL bovine erythrocytes intragastrically 1 week after catheter implantation for measuring hepatic, splanchnic, portal-drained viscera, and hindquarter fluxes of amino acids, urea, and ammonia. After the administration of erythrocytes, isoleucine or saline was administered i.v. for 6 hours. The data obtained show that the increase in arterial levels of urea and almost all amino acids was significantly greater in the control group (P less than 0.001) than in the isoleucine group. The net efflux of nearly all amino acids by the portal-drained viscera increased significantly less (P less than 0.001) in the isoleucine group. The liver uptake of amino acids increased after the blood meal, but the difference was not significant except for glutamine (P less than 0.001). Hindquarter amino acid net influx increased marginally. Splanchnic urea production increased more in the control group than in the isoleucine group (P less than 0.05). The data strongly suggest that i.v. administration of isoleucine enhanced the biological value of a blood meal, possibly by promoting amino acid retention in the portal drained viscera.     

嗜人按蚊血淋巴微量元素与常量元素的研究

应用ＩＣＡＰ－９０００型等离子原子发射光谱仪检测嗜人按蚊血淋巴的元素组成，检出铁、锌、铜、锰、铬、钼、钴、镍、钒、锶、硼、铝、钡、锆、铅、镉、镓、锂等１８种微量元素及钙、镁、钾、钠、磷、硫等６种常量元素。初羽化雌蚊血淋巴中６种常量元素含量均比吸血后５ｄ蚊为高，且有非常显著性差异；微量元素铁的含量也较高，有非常显著性差异，锌和铝含量则较低，有非常显著性差异和显著性差异。比较嗜人按蚊和中华按蚊血淋巴中１６种元素含量，显示初羽化雌蚊和吸血后５ｄ的蚊血淋巴中分别为１４种和１３种元素，有非常显著性差异。嗜人按蚊与白纹伊蚊比较结果，初羽化雌蚊和吸血后５ｄ蚊血淋巴均为１３种元素，有非常显著性差异。结果提示，蚊虫血淋巴元素组成与其营养代谢、生长发育有密切关系，不同蚊种以及对疟原虫敏感性不同者也表现差异。     

荷肝癌小鼠血浆和肿瘤组织游离氨基酸代谢变化及其与肿瘤体积相关性研究

目的探讨荷肝癌小鼠游离氨基酸代谢变化,为肝癌患者氨基酸失衡疗法提供理论依据。方法采用HITACHIL8800型氨基酸分析仪,检测10例荷肝癌小鼠血浆和肿瘤组织游离氨基酸,并以10例正常小鼠做为对照组,研究氨基酸代谢变化与肿瘤体积的关系。结果荷瘤组血浆游离精氨酸、缬氨酸、丝氨酸、谷氨酸明显降低;肿瘤组织游离蛋氨酸、精氨酸、缬氨酸、亮氨酸与对应血浆游离氨基酸呈负相关;肿瘤组织游离异亮氨酸、缬氨酸、蛋氨酸、酪氨酸、亮氨酸、谷氨酸、丝氨酸与肿瘤体积呈正相关。结论肝癌在其生长过程中与某些特定氨基酸关系密切,这将为针对肝癌的氨基酸失衡处理提供实验和理论依据。     

Serum amino acids in hepatic encephalopathy--effects of branched chain amino acid infusion on serum aminogram

Encephalopathic patients with cirrhosis of the liver consistently showed elevated levels of the aromatic amino acids, phenylalanine, tyrosine and free tryptophan as well as methionine in serum, whereas levels of the branched chain amino acids, valine, leucine and isoleucine, were depressed. Comatose patients with fulminant hepatitis had markedly elevated levels of all amino acids, the results being greatly different from those of cirrhotic patients. Molar ratios of (valine + leucine + isoleucine)/(phenylalanine + tyrosine) decreased both in cirrhotics with and without encephalopathy and in cases with fulminant hepatitis. Infusion of a commercially available L-amino acid solution in a cirrhotic patient induced a strikingly abnormal aminogram documented in hepatic encephalopathy. Therefore, effects of branched chain amino acid infusion on the deranged amino acid pattern were primarily studied for the purpose of improvement in hepatic encephalopathy by normalization of serum amino acid patterns. Elevated levels of the aromatic amino acids and methionine could be apparently depressed in a cirrhotic patient by this type of infusion but not in a case of fulminant hepatitis probably because of the poor utilization of these amino acids in severely impaired liver.     

Reduction of large neutral amino acid concentrations in plasma and CSF of patients with maple syrup urine disease during crises

Neurological dysfunction is common in patients with maple syrup urine disease (MSUD). However, the mechanisms underlying the neuropathology of this disorder are poorly understood. We determined the concentrations of all amino acids in plasma of patients with MSUD during crises (with severe CNS symptoms) and after recovery in the hope of detecting possible alterations of these levels during metabolic decompensation. Blood samples obtained from 11 children with MSUD aged 1 month to 7 years and from 10 age-matched controls (5 months to 6 years) with no evidence of metabolic disease were examined for their amino acid content by high-performance liquid chromatography. We observed that leucine, isoleucine and valine concentrations were respectively 30, 9 and 3 times higher than normal values, whereas the concentrations of the large neutral amino acids (LNAA) phenylalanine, tyrosine, tryptophan and methionine were significantly lower during metabolic decompensation as compared to the controls. In addition, concentrations of leucine, but not of valine or isoleucine, were inversely related to the LNAA concentrations in plasma. The concentrations of these amino acids in plasma returned to normal values when patients were clinically well. CSF amino acid concentrations also showed decreased amounts of LNAA and increased concentrations of branched-chain amino acids. It is possible that the decrease in plasma concentrations of LNAA may lead to a deficit of these essential amino acids in the brain as well as of their products such as proteins and neurotransmitters, a fact that might be related to the neurological dysfunction of MSUD.     

斯氏按蚊血淋巴酚氧化酶与约氏疟原虫卵囊黑化的关系

[目的 ]探讨酚氧化酶 (phenoloxidase,PO)与疟原虫卵囊黑化的关系。 [方法 ]以斯氏按蚊 /约氏疟原虫为模型 ,对 4组斯氏按蚊 (不吸血组、吸正常血组、吸感染血组和硝喹组 )血淋巴进行聚丙烯酰胺凝胶电泳 (PAGE)和凝胶图像分析 ,检测单酚氧化酶 (monophenoloxidase ,MPO)和二酚氧化酶 (diphenoloxidase,DPO)活性。 [结果 ]吸正常血组和不吸血组蚊血淋巴中MPO及o DPO活性无明显差异 ;与吸正常血组或不吸血组相比 ,感染组MPO及o DPO活性无明显变化 ,但用药组d1 0 则显著增加 ,d1 5 显著降低。 [结论 ]斯氏按蚊血淋巴中PO活性变化与约氏疟原虫卵囊黑化在时间上一致。     

Amino acid profiles in early diabetic and non-diabetic pregnancy

Plasma amino acid concentrations were measured in six insulin-dependent diabetic women and seven non-diabetic women in early pregnancy while fasting and one hour after a standard meal. Fasting plasma levels of total amino acids and individual amino acids were similar in the two groups, excepting isoleucine, which was raised in the diabetics. One hour post-prandially total amino acid concentrations were similar in the two groups; however, mean concentrations of total branched chain amino acids and mean concentration of the individual amino acids, serine, valine, isoleucine, leucine and tyrosine were elevated in the diabetics. Amino acids are important in early islet development and in insulin secretion from fetal pancreas in vitro. The elevated post-prandial amino acid levels found in pregnant diabetics in early pregnancy may contribute to fetal islet hypertrophy and hyperinsulinaemia.