帕金森病患者脑脊液蛋白质组学分析

帕金森病（Parkinson′s disease,PD）是一种中枢神经系统慢性进展性疾病.本研究采用双向凝胶电泳（two-dimensional gel electrophoresis,2-DE）分离脑脊液（cerebrospinal fluid,CSF）蛋白,获得2-DE图谱,通过ImageMaster 2D Elite软件分析寻找两组的差异蛋白点.结果显示,PD患者CSF中有4个蛋白点丰度下降,22个蛋白点丰度上升.还利用电喷雾质谱（electrospray ionization-tandem mass spectrometric,ESI-MS）对差异蛋白点进行鉴定,发现丰度上升的蛋白点有电压依赖性钙通道α2/δ1亚基,结合珠蛋白,β2-微球蛋白和阿朴脂蛋白A-IV前体,丰度下降的蛋白点为转铁蛋白和转甲状腺蛋白.研究发现,PD患者与对照组CSF蛋白质表达有明显差异,对差异蛋白进行质谱鉴定并了解它们的功能,为以后进一步研究他们在PD发病机制和病程进展中的作用奠定基础.     

雪莲愈伤组织蛋白质提取及双向电泳分析

以产自西藏绵头雪莲为材料,分别采用TCA/丙酮法、尿素法和酚法,提取雪莲愈伤组织蛋白质并进行双向电泳,对蛋白产量和纯度以及电泳图谱进行比较。结果表明:(1)酚法较TCA/丙酮法和尿素法获得的蛋白质更纯,杂质少,蛋白点多且分辨率较高,在二维电泳图谱中背景清晰,横纹和纵纹较少。(2)对酚法提取的蛋白质样品进行上样量的比较结果显示,17cm胶条500μg上样量二维图谱的背景和蛋白点分布较好。(3)用0℃处理雪莲愈伤组织12h,利用建立的双向电泳体系分析结果发现,有33个蛋白点比常温(23℃)上调1.5倍表达,有5个蛋白点的表达下调2倍。(4)质谱鉴定结果表明,低温诱导的蛋白包括NADP-依赖型异柠檬酸脱氢酶、腺苷高半胱氨酸酶、抗性RPP8类蛋白、蛋白点gi|13129470、α-微管蛋白,分别与新陈代谢、植物防御、能量代谢、细胞的结构蛋白相关。     

Fluorometrical Analysis of Guanidino Compounds in Human Cerebrospinal Fluid

Abstract: Guanidino compounds in CSF of 57 human subjects were determined fluorometrically after reaction with phenanthrenequinone in alkali solution, using HPLC. Creatinine (65.2 ± 13.4 nmol/ml), arginine (24.7 ± 6.4 nmol/ml), and homoarginine (0.7 ± 0.3 nmol/ml) were found in all subjects. Trace amounts of guanidinosuccinic acid and guanidinoacetic acid were detected in some of the subjects. Brain guanidino compounds, taurocyamine, N -acetylarginine, and methylguanidine were not detected in CSF.     

Chronic exposure of primates to 60-Hz electric and magnetic fields: II. Neurochemical effects

We exposed Macaca nemestrina (pig-tailed macaques) to electric (E) and magnetic (B) fields ranging in intensity from 3 kV/m and 0.1 G to 30 kV/m and 0.9 G for three 21-day (d) periods. Experimental animals were exposed to sham E and B fields for two 21-d periods, one prior to and one following actual exposure to E and B fields, resulting in a total of five 21-d periods. Control animals were exposed to sham E and B fields for the entire 105-d interval. At the end of each 21-d period cerebrospinal fluid (CSF) was obtained by lumbar puncture and analyzed for concentrations of homovanillic acid (HVA) and 5-hydroxyindoleacetic acid (5-HIAA), metabolites of dopamine and serotonin neurotransmitters, respectively, by high-performance liquid chromatography with electrochemical detection (HPLC-ECD). Results are based on an examination of six experimental and four control animals. Exposure to E and B fields at all strengths was associated with a significant decline in CSF concentrations of both HVA and 5-HIAA when statistical comparisons were made against values obtained at the end of the preexposure interval. However, HVA returned to preexposure levels during the postexposure period, while 5-HIAA did not. No significant change in the concentrations of HVA or 5-HIAA was noted in the control animals. These results strongly suggest that exposure of the nonhuman primate to E and B fields can significantly affect specific biochemical estimates of nervous system function. These effects may involve alterations either in neuronal activity or in the activity of enzymes that catabolize the neurotransmitters.     

Clinical relevance of measuring GABA concentrations in cerebrospinal fluid

Determination of GABA concentrations in human cerebrospinal fluid can be used to assess GABA-ergic activity in the central nervous system. As CSF free GABA concentrations may vary with age, sex, CSF fraction, and collection and storage conditions, careful attention to these factors are necessary to allow interpretation of results. Longitudinal studies to investigate the influence of pharmacological agents on CSF GABA have proven especially useful to define clinical biochemical activity and have been utilized to attribute the anti-epileptic action of vigabatrin, a selective inhibitor of GABA-transaminase, to its effects on brain GABA metabolism.Special issue dedicated to Dr. Sidney Udenfriend.     

Fluids of the brain and the pathogenesis of MS

The plaques of multiple sclerosis are generally thought to spread outwards from central veins. We propose that periventricular plaques in the cerebral hemispheres, and superficial plaques in the brain stem and spinal cord, point to the importance of demylinating factors in the cerebrospinal fluid.Special issue dedicated to Dr. Alan N. Davison.     

Transport of quinolinic acid into rabbit and rat brain

The transport metabolism of [³H]quinolinic acid in the central nervous system of rabbits and rats were studied. In vitro [³H]quinolinic acid was not readily accumulated by isolated choroid plexus. After the intraventricular injection of tracer quantities of [³H]quinolinic acid, the [³H]quinolinic acid did not enter the brain as readily as concurrently injected [¹⁴C]mannitol and was not metabolized, The permeability-surface area constant for [³H]quinolinic acid at the rat blood-brain barrier was 1.5±1.3×10^–5 sec^–1 compared to 2.8±0.4×10^–5 sec^–1 for [³H]mannitol. Our results suggest that: 1) [³H]quinolinic acid is transported in the CNS by passive diffusion and 2) is not metabolized.     

D-amino acid levels in human physiological fluids

Plasma, urine, cerebrospinal fluid (CSF), and amniotic fluid were examined to determine whether free D-amino acids were present and if so at what levels. It was found that D-amino acids exist in all physiological fluids tested, but that their level varied, considerably. The lowest levels of D-amino acids were usually found in amniotic fluid or CSF (almost always <1% of the corresponding L-amino acid). The highest levels were found in urine (usually tenth percent to low percent levels). Pipecolic acid seemed to be different from the other amino acids tested in that it was excreted primarily as the D-enantiomer (often >90%). Correspondingly high levels of D-pipecolic acid were not found in plasma. Some of the trends found in this work seemed to be analogous to those found in a recent rodent study. © 1993 Wiley-Liss, Inc.     

经皮穿刺抽取大鼠脑脊液方法的改良

目的:改进传统经皮穿刺抽取脑脊液的方法,提高采集大鼠脑脊液动物存活率。方法:90只SD大鼠随机分为对照组、传统穿刺组和改良穿刺组(每组30只)。对照组不进行穿刺。传统穿刺组按反握抓持法进行穿刺取样。改良穿刺组采用左手固定大鼠头部,将其抬高至约135°,右手正握改良1 ml注射器,以枕骨隆突和第一颈椎为标志定位枕骨大孔,在枕骨大孔中心偏下平行进针,抽取脑脊液。分别于第1、4、7日抽取大鼠脑脊液,每次抽取记录取样时间、采集量、脑脊液性状,抽取后3 d观察大鼠存活情况,选取存活大鼠进行重复采样。结果:传统经皮穿刺取样法采样成功率为63.33%,三次重复采样动物存活率为10%;改良经皮穿刺取样法采样成功率为86.67%,三次重复采样动物存活率为50%,相比传统采样有显著升高(P<0.01)。结论:改良经皮穿刺取样法操作简单,耗时少,成功率高。操作无需定位设备,单人可以完成。动物反复取样存活率高,方便进行脑脊液重复采样。     

SEREX identification of the autoantibodies that are prevalent in the cerebrospinal fluid of patients with moyamoya disease

We performed SEREX (serological analysis of recombinant cDNA expression library) to identify autoantibodies that are prevalent in the cerebrospinal fluid of patients with moyamoya disease. These autoantibodies include PC326 (of unknown function), SRY (sex determining region Y), and peroxisomal D3,D2-enoyl-CoA isomerase.     

Some Possible Evidences for an Alteration in the Actin-Myosin Interaction in Stored Muscle

A two-dimensional mapping analysis was performed by HPLC for 4 kinds of standard galactosyllactoses (GLs, trisaccharide) which were assumed to be produced from lactose (galactopyranosylβ1→4 glucopyranose) in yogurt during the fermentation of lactic acid bacteria. After the pyridylamination of GLs, they were analyzed by HPLC in the reverse-phase (RP) and anion-exchange (AE) modes. The retention times of each peak obtained were converted to glucose units (GU) in RP mode for the pyridylaminated isomaltooligosaccharides (G1-3) and to relative retention time (RRT) in AE mode against pyridylaminated-isomaltotriose, and then the address data [GU, RRT] were plotted on a graph. This two-dimensional mapping method was found useful for a rapid qualitative evaluation of the chemical structure of trisaccharides formed in yogurt.     

Multiplex real-time PCR for the simultaneous detection of herpes simplex virus, human herpesvirus 6, and human herpesvirus 7

A simultaneous detection system to quantify HSV, HHV-6, and HHV-7 DNA via multiplex real-time PCR using different fluorochromes was developed. The minimum quantitative level established via this multiplex assay was four copies per reaction for HSV type 1, four copies for HHV-6, and three copies for HHV-7, respectively. The dynamic range encompassed at least six orders of magnitude. The system was specific and reproducible even in the presence of large amounts of other viral DNA. We then applied this multiplex real-time PCR assay to 105 CSF specimens obtained from subjects less than 15 years old in whom a diagnosis of viral encephalitis/encephalopathy was suspected on clinical grounds. The detection rate for each viral DNA was 6.7% for HSV, 9.5% for HHV-6, and 1.9% for HHV-7. These results indicate that our system is reliable and may be useful for the rapid diagnosis of viral encephalitis/encephalopathy.     

Albumin heterogeneity in low-abundance fluids. The case of urine and cerebro-spinal fluid

Background

Serum albumin is a micro-heterogeneous protein composed of at least 40 isoforms. Its heterogeneity is even more pronounced in biological fluids other than serum, the major being urine and cerebrospinal fluid. Modification ‘in situ’ and/or selectivity of biological barriers, such as in the kidney, determines the final composition of albumin and may help in definition of inflammatory states.

Scope of review

This review focuses on various aspects of albumin heterogeneity in low ‘abundance fluids’ and highlights the potential source of information in diseases.

Major conclusions

The electrical charge of the protein in urine and CSF is modified but with an opposite change and depending on clinical conditions.In normal urine, the bulk of albumin is more anionic than in serum for the presence of ten times more fatty acids that introduce equivalent anionic charges and modify hydrophobicity of the protein. At the same time, urinary albumin is more glycosylated compared to the serum homolog. Finally, albumin fragments can be detected in urine in patients with proteinuria.For albumin in CSF, we lack information relative to normal conditions since ethical problems do not allow normal CSF to be studied. In multiple sclerosis, the albumin charge in CSF is more cationic than in serum, this change possibly involving structural anomalies or small molecules bindings.

General significance

Massively fatty albumin could be toxic for tubular cells and be eliminated on this basis. Renal handling of glycosylated albumin can alter the normal equilibrium of filtration/reabsorption and trigger mechanisms leading to glomerulosclerosis and tubulo-interstitial fibrosis. This article is part of a Special Issue entitled Serum Albumin.     

Increased activin levels in cerebrospinal fluid of rabbits with bacterial meningitis are associated with activation of microglia

Activin, a member of the transforming growth factor superfamily, is upregulated in a number of inflammatory episodes such as septicemia and rheumatoid arthritis. In the CNS, activin has been predominantly assessed in terms of a neuroprotective role. In this report we characterized the activin response in the CNS in a rabbit model of meningitis. In normal animals, cerebrospinal fluid (CSF) activin levels were higher than those in serum, indicating an intracranial secretion of this cytokine. Following intracisternal inoculation with Streptococcus pneumoniae, activin in CSF was unchanged for the first 12 h and then rose progressively; levels were increased approximately 15-fold within 24 h. Activin levels were correlated positively with CSF protein content and with the number of apoptotic neurons in the dentate gyrus. No apparent correlation was observed between CSF activin concentrations and bacterial titer, lactate concentrations or leukocyte density. Using immunohistochemistry, activin staining was localized to epithelial cells of the choroid plexus, cortical neurons and the CA3 region of the hippocampus, with similar staining intensities in both normal and meningitic brains. However, in meningitic brains there was also strong staining in activated microglia and infiltrating macrophages. Taken together, these results demonstrate that activin forms part of the CNS response to immune challenge and may be an important mediator to modulate inflammatory processes in the brain.