空间记忆相关蛋白的蛋白质组学研究

C57BL/6J小鼠在评价空间记忆的多T迷宫(MTM)和Barnes迷宫(BM)中表现不同,本研究拟寻找导致这种行为差异的海马蛋白.应用双向凝胶电泳结合质谱鉴定和数据库检索,分析比较经两种迷宫训练测试后小鼠海马蛋白水平的不同,发现经过BM和MTM训练的小鼠有29种蛋白表达存在明显差异.其中,在BM训练组中5种蛋白表达上调,而在MTM组中24种蛋白表达上调.与空间记忆相关的蛋白按功能可分为如下6类:(1)能量代谢相关蛋白;(2)细胞骨架相关蛋白;(3)分子伴侣;(4)神经发育相关蛋白;(5)转录因子和蛋白合成相关蛋白;(6)信号转导蛋白.本研究结果丰富了空间记忆的机制,对于神经科学的进一步发展具有启发意义.     

嗅觉记忆相关蛋白的蛋白质组学研究

应用双向凝胶电泳结合质谱鉴定和数据库检索, 分析比较了C57BL/6J小鼠在嗅觉训练和记忆测试后嗅球蛋白表达的差异, 探讨了与嗅觉记忆相关的蛋白质. C57BL/6J小鼠经嗅觉训练后, 可对相应的气味保持记忆能力, 其嗅球蛋白表达存在明显差异, 5种蛋白与嗅觉记忆形成显著相关. 上述蛋白功能涉及神经发育, 信号转导及细胞骨架和核苷酸代谢, 其中神经发育和信号转导相关蛋白表达上调, 而细胞骨架和核苷酸代谢相关蛋白表达水平降低. 这些与嗅觉记忆形成相关的蛋白深化了对嗅觉记忆机制的认识, 为研究和治疗认知相关疾病提供了新靶标.     

快速老化模型小鼠海马蛋白质组学初步研究

应用双向凝胶电泳结合质谱鉴定, 分析比较6月龄和12月龄快速老化模型小鼠(Senescence-accele-rated mouse, SAM)的快速老化亚系SAM-prone/8(SAMP8)及抗快速老化亚系SAM-resistance/1(SAMR1)海马蛋白表达的差异, 从蛋白质水平初步探讨与老化相关的学习记忆功能障碍的发生机制. 结果表明, 与同龄SAMR1比较, 6月龄SAMP8海马中有15个蛋白点表达显著上调, 5个蛋白点表达显著下调; 12月龄SAMP8海马中有12个蛋白点表达显著上调, 2个蛋白点表达显著下调, 2个蛋白点只在SAMP8中有表达. 应用质谱分析结合数据库检索, 共鉴定了22种蛋白质. 6月龄和12月龄SAMP8与SAMR1海马中表达有明显变化的蛋白按功能可分为如下4类: (1) 能量代谢相关蛋白; (2) 线粒体功能相关蛋白; (3) 信号转导相关蛋白; (4) 其它蛋白. 研究结果表明, SAMP8和SAMR1海马蛋白表达存在明显差异, 其中一些蛋白与SAMP8随龄出现的学习记忆功能减退相关, 并可能为研究或发现促智药物作用的新蛋白靶标提供线索.     

快速老化模型小鼠血清蛋白质组学分析比较

以6月及12月龄SAMP 8及同龄SAMR 1为研究对象, 应用双向凝胶电泳法, 分析比较了快速老化模型小鼠(Senescence accelerated mice, SAM)的快速老化亚系SAMP 8及抗快速老化亚系SAMR 1血清蛋白表达的差异. 与同龄SAMR 1比较, 6月龄SAMP 8血清中有15个蛋白点表达显著上调, 3个蛋白点表达显著下调, 7个蛋白点只在SAMP 8中有表达; 12月龄SAMP 8血清中有9个蛋白点表达显著上调, 7个蛋白点表达显著下调, 12个蛋白点只在SAMP 8中有表达. 应用质谱进行肽质量指纹图谱分析和数据库检索共鉴定了19种蛋白质. 其中6个蛋白只在6月龄SAMP 8中表达, 4个蛋白只在12月龄SAMP 8中表达. 此外, 在6月龄及12月龄SAMP 8血清差异蛋白中, 存在9个共同的差异蛋白, 按照功能可分为4类: (1) 免疫相关蛋白; (2) 老化相关蛋白; (3) 糖代谢及神经元凋亡相关蛋白; (4) 其它蛋白. 上述研究结果显示, SAMP 8和SAMR 1血清蛋白表达存在明显差异, 其中一些差异蛋白可能是SAMP 8老化进程中相关病理生理变化的重要原因.     

鼠肝癌淋巴道转移细胞模型的蛋白质组学研究

对2株来源于同一亲本细胞但淋巴道转移力显著不同的小鼠肝癌腹水型细胞株Hca-F(淋巴结转移率75%)和Hca-P(淋巴结转移率25%), 采用荧光差异双向凝胶电泳(2D DIGE)和DeCyder定量分析软件及HPLC-nESI-MS/MS技术, 定量分析和鉴定了小鼠肝癌细胞Hca-F和Hca-P的差异表达蛋白. 结果显示, 有116个蛋白质点表达水平存在明显差异(p<0.05), 在Hca-F中表达上调蛋白质点62个, 下调蛋白质点54个. 对所有116个蛋白质点进行了电喷雾串联质谱鉴定, 共鉴定出109种单一(Unique)蛋白. 其中部分蛋白已被报道与不同类型肿瘤的发生、浸润和转移相关, 多数蛋白质被首次报道与肝癌的淋巴道转移过程直接相关.     

细胞介导的抑制效应在防止抗胎免疫反应中的作用

本文采用 GVHR和 MLR两项实验方法,对 C57BL×BG1(H-2~b×H-2~k)和C57BL×SW1(H-2~b×H-2~?)两种交配组合的60只经产妊娠小鼠进行研究.证实同种妊娠小鼠的子宫引流淋巴结和脾脏中都存在着抑制细胞,由这些细胞介导的抑制性免疫调节效应可能在成功地保护胎儿同种移植物免受排斥中起重要作用.本文还对淋巴细胞亚群发生的这种改变的机理进行了初步探讨。     

气相色谱-质谱结合随机森林鉴定糖尿病小鼠经诺和龙治疗代谢轨迹

采用气相色谱-质谱法(GC-MS)同时测定经诺和龙治疗的2型糖尿病KK-ay小鼠和C57BL/6J健康对照组小鼠尿液中的多种代谢物。利用随机森林算法对经诺和龙治疗后不同周期的2型糖尿病(T2DM)KK-ay小鼠的GC-MS数据进行统计分析,获得小鼠经治疗后的代谢轨迹图。样品预处理中,以核糖醇为内标,用甲醇除蛋白,尿酶除去尿素,经N2吹干后用肟化-硅烷化法进行衍生;GC-MS测定中,采用DB-5MS毛细管柱程序升温分离尿样中的多种成分,并结合NIST标准质谱库和标准品对尿液中代谢物进行定性定量分析,共鉴定出氨基酸、脂肪酸、有机酸、酯类和糖类等40种内源性代谢物质。将得到的数据输入随机森林进行建模分析,得到其治愈轨迹图。研究结果表明,诺和龙能有效地改善糖尿病小鼠的血糖代谢。     

基于1H NMR代谢组学方法分析马兜铃酸I诱导的雌雄小鼠急性肾毒性

基于¹H NMR的代谢组学方法, 分析了C57BL/6J小鼠尿样的代谢特征, 揭示出马兜铃酸I(AAI)导致急性肾毒性的分子机理及其在雌性和雄性小鼠上差异的根源. 研究结果表明, AAI的急性肾毒性主要来自AAI给药后抑制了体内的三羧酸(TCA)循环和能量代谢, 破坏了体内肠道菌群的生态平衡, 改变了肾脏细胞内外的渗透压, 从而引起了肾小管的损伤. 代谢模式分析显示雄性小鼠对AAI的肾毒性比雌性小鼠更敏感, 可能源于雄性小鼠本身更低的能量代谢. 结果表明, 基于尿样¹H NMR代谢组学方法有可能为药物的毒性机制和毒性性别差异研究提供新思路.     

重组鼠疫汉坦钩端多抗原位点同源合成基因的表达及活性鉴定

将人工合成的含鼠疫耶尔森氏菌的2个抗原位点、汉坦病毒的5个抗原位点和钩端螺旋体的5个抗原位点的串联基因片段克隆到原核表达载体pET-20b中,构建重组表达质粒pET-rYHL.转化E.coli BL21(DE3)后获得表达菌株.表达菌株经IPTG诱导后,可高效表达带组氨酸标签的以包涵体形式存在的融合蛋白.包涵体蛋白经尿...     

维拉帕米作用下急性心梗大鼠比较蛋白质组学研究

以急性心梗大鼠为研究对象, 应用双向凝胶电泳法(2-DE)分析比较了维拉帕米作用下急性心梗大鼠心肌蛋白表达的差异, 从蛋白质水平探讨了维拉帕米心肌保护作用的发生机制. 结果表明, 与假手术组及模型组相比, 维拉帕米给药组心肌组织中有8个蛋白点表达显著上调, 7个蛋白点表达显著下调. 采用质谱(MALDI-TOF-MS)分析结合数据库检索, 共鉴定了其中的15种蛋白质, 可按功能分为如下4类: (1) 能量代谢及线粒体功能相关蛋白; (2) 氧化应激相关蛋白; (3) 细胞骨架蛋白; (4) 其它蛋白. 研究结果表明, 维拉帕米的心肌保护作用与恢复心肌损伤过程中的能量供应及对抗氧化应激等作用有关.     

Metabolomic analysis of normal (C57BL/6J, 129S1/SvImJ) mice by gas chromatography-mass spectrometry: detection of strain and gender differences

Previous studies have shown that the C57 and 129 strains of mice display marked differences in behavioural performance, neuroanatomy, neurochemistry and synaptic plasticity. However, few metabolomic studies of their biofluids have been performed. As part of a series of metabolic phenotyping, the effects of gender and strain upon serum metabolite composition and variation are examined in this study using gas chromatography-mass spectrometry (GC-MS) in normal C57BL/6J and 129S1/SvImJ strains of mice. The 129S1/SvImJ strain is phenotypically distinct from the C57BL/6J strain and characteristic metabotypes are produced for both male and female mice of each strain. These data demonstrate that the C57BL/6J and 129S1/SvImJ strains of mice show a wide range of metabolic differences across glycine, serine and threonine metabolism; valine, leucine and isoleucine biosynthesis; and tricarboxylic acid cycle pathways. Remarkably, the concentration of glyceric acid in the 129S1/SvImJ strain is significantly increased compared to the C57BL/6J mouse strain, reflecting important considerations for studies that use the 129S1/SvImJ mouse as the human d-glycericaciduria model. We infer that a deficiency of d-glycerate kinase would explain such a glyceric acid accumulation in the 129S1/SvImJ strain. More importantly, this differential metabolite level data provide insight into specific metabolic pathways and lay the groundwork for integrated studies of the mouse models.     

Cytokine mRNA expression and serum cortisol evaluation during murine lung inflammation induced by Mycobacterium tuberculosis

A model system was characterized for investigating the potential role of cortisol in MTB induced immunopathology. Serum cortisol levels were evaluated in two mouse strains; C57BL/6 mice develop lung granulomas following acute Mycobacterium tuberculosis infection while A/J mice are deficient in this process. Serum cortisol levels were examined post infection, as well as immunoregulatory mRNA expression in the lung, measured using bioluminescent RT-PCR techniques. Prior to infection, the A/J mice constitutively maintain nearly 75&percent; higher serum cortisol than C57BL/6 mice. Both A/J and C57BL/6 mice exhibited approximately 30&percent; reduction in relative serum cortisol following infection. At no time did serum cortisol levels in the A/J fall below constitutive levels in the non-infected C57BL/6. The overall elevated cortisol in the A/J may affect pulmonary immunoresponsiveness; A/J mice exhibited earlier induction of IL-10 and TNF-alpha than C57BL/6 mice, with a relative lack of IL-2 during late infection. Conversely, the C57BL/6 mice demonstrated higher IL-12(p40) and IL-2 messages at the latter stages of disease than the A/J mice. Both mice demonstrated high IFN-&gama; mRNA. The high constitutive serum cortisol in the A/J mice may therefore contribute to establishment of an environment counter-productive to initiation of protective Th1 cell and granulomatous responses.     

Immunotoxic effects of cis-urocanic acid exposure in C57BL/6N and C3H/HeN mice

Exposure to ultraviolet radiation results in increased levels of intradermal cis-urocanic acid (cUCA) and alters cutaneous immunity by interfering with processing and presentation of antigen by Langerhans cells. Reports on effects of systemic immunotoxicity with 30 day cUCA exposure in laboratory rodents include thymic atrophy, thymic hypocellularity and decreased T-cell-mediated immunity; however, immune effects of single exposure or 5 day cUCA administration, which may better mimic human exposures, are poorly defined. The present study initially evaluated immune effects of single, 5 day, and 4 week cUCA exposure in C57BL/6N mice. Single administration of intradermal cUCA resulted in decreased splenocyte phagocytosis that persisted for 30 days after cUCA exposure. Five day consecutive cUCA exposure decreased numbers of phenotypically mature CD4(+)CD8(-) and CD4(-)CD8(+) (single positive) thymocytes, increased CD4(+)CD8(+) (double positive) immature thymocytes and increased splenocyte proliferation. Prolonged cUCA exposure (4 weeks) caused profound thymic hypocellularity and splenic hypercellularity and increased splenic macrophage chemiluminescence. Because of this apparent sensitivity of C57BL/6N mice to cUCA, thymic hypocellularity was compared between C57BL/6N and C3H/HeN mice dosed with cUCA, and was found to be more pronounced in the C57BL/6N strain. These results are an extension of previous conclusions on immune modulation caused by cUCA in the spleen and thymus. Further, the observed variation in sensitivity between the mouse strains is consistent with known genetic susceptibility of these strains to the immunomodulatory effects of exposure to sunlight.     

Effects of Grape Polyphenols on the Life Span and Neuroinflammatory Alterations Related to Neurodegenerative Parkinson Disease-Like Disturbances in Mice

Functional nutrition is a valuable supplementation to dietary therapy. Functional foods are enriched with biologically active substances. Plant polyphenols attract particular attention due to multiple beneficial properties attributed to their high antioxidant and other biological activities. We assessed the effect of grape polyphenols on the life span of C57BL/6 mice and on behavioral and neuroinflammatory alterations in a transgenic mouse model of Parkinson disease (PD) with overexpression of the A53T-mutant human α-synuclein. C57BL/6 mice were given a dietary supplement containing grape polyphenol concentrate (GPC—1.5 mL/kg/day) with drinking water from the age of 6–8 weeks for life. Transgenic PD mice received GPC beginning at the age of 10 weeks for four months. GPC significantly influenced the cumulative proportion of surviving and substantially augmented the average life span in mice. In the transgenic PD model, the grape polyphenol (GP) diet enhanced memory reconsolidation and diminished memory extinction in a passive avoidance test. Behavioral effects of GP treatment were accompanied by a decrease in α-synuclein accumulation in the frontal cortex and a reduction in the expression of neuroinflammatory markers (IBA1 and CD54) in the frontal cortex and hippocampus. Thus, a GP-rich diet is recommended as promising functional nutrition for aging people and patients with neurodegenerative disorders.     

A mouse serum two-dimensional gel map: application to profiling burn injury and infection

With the importance of mouse as a model to study human diseases and the human and rat plasma/serum two-dimensional (2-D) maps being extensively annotated, this study was aimed at constructing a detailed mouse serum 2-D map. Serum proteins from two different inbred strains of mice (BALB/cJ and C57BL/6J) and mice subjected to two different inflammatory stimuli (20% burn injury and lipopolysaccharide (LPS) injection) were separated on overlapping gels covering pH 3-8 and stained with SYPRO Ruby dye. The tryptic peptides from the resolved spots were analyzed by mass spectrometry, leading to the identification of 38 different gene products. With the exception of major urinary proteins found in abundance in male C57BL/6J mice, little strain difference of the mouse serum 2-D was observed. Many proteins detected in the mouse serum 2-D map were not reported in human or rat serum 2-D maps including epidermal growth factor receptor. Three major murine acute-phase proteins (APPs), haptoglobin, serum amyloid A, and serum amyloid P, were highly induced by both inflammatory stimuli. Image analysis shows that the variations of APPs between these two inflammatory models were not uniform although LPS (100 microg/animal) in general was more effective than 20% burn injury in inducing APPs. Serum amyloid A, much more sensitive to endotoxin than burn injury, may represent a sensitive marker to differentiate these two different inflammatory states.     

Analysis of the mouse proteome. (I) Brain proteins: separation by two-dimensional electrophoresis and identification by mass spectrometry and genetic variation

The total protein of the mouse brain was fractionated into three fractions, supernatant, pellet extract and rest pellet suspension, by a procedure that avoids any loss of groups or classes of proteins. The supernatant proteins were resolved to a maximum by large-gel two-dimensional electrophoresis. Two-dimensional patterns from ten individual mice of the commonly used inbred strain C57BL/6 (species: Mus musculus) were prepared. The master pattern was subjected to densitometry, computer-assisted image analysis and treatment with our spot detection program. The resulting two-dimensional pattern, a standard pattern for mouse brain supernatant proteins, was divided into 40 squares, calibrated, and specified by providing each spot with a number. The complete pattern and each of the 40 squares are shown in our homepage (http://www.charite.de/ humangenetik). The standard pattern comprises 8767 protein spots. To identify the proteins known so far in the brain fraction investigated, a first set of 200 spots was analyzed by matrix-assisted laser desorption/ionization - mass spectrometry (MALDI-MS) after in-gel digestion. By screening protein databases 115 spots were identified; by extending the analysis to selected, genetically variant protein spots, 166 spots (including some spot series) were identified in total. This number was increased to 331 by adding protein spots identified indirectly by a genetic approach. By comparing the two-dimensional patterns from C57BL/6 mice with those of another mouse species (Mus spretus), more than 1000 genetically variant spots were detected. The genetic analysis allowed us to recognize spot families, i.e., protein spots that represent the same protein but that are post-translationally modified. If some members of the family were identified, the whole family was considered as being identified. Spot families were investigated in more detail, and interpreted as the result of protein modification or degradation. Genetic analysis led to the interesting finding that the size of spot families, i.e., the extent of modification or degradation of a protein, can be genetically determined. The investigation presented is a first step towards a systematic analysis of the proteome of the mouse. Proteome analysis was shown to become more efficient, and, at the same time, linked to the genome, by combining protein analytical and genetic methods.     

Serine/threonine-protein phosphatase 1 α levels are paralleling olfactory memory formation in the CD1 mouse

Although olfactory discrimination has already been studied in several mouse strains, data on protein levels linked to olfactory memory are limited. Wild mouse strains Mus musculus musculus, Mus musculus domesticus and CD1 laboratory outbred mice were tested in a conditioned odor preference task and trained to discriminate between two odors, Rose and Lemon, by pairing one odor with a sugar reward. Six hours following the final test, mice were sacrificed and olfactory bulbs (OB) were taken for gel-based proteomics analyses and immunoblotting. OB proteins were extracted, separated by 2-DE and quantified using specific software (Proteomweaver). Odor-trained mice showed a preference for the previously rewarded odor suggesting that conditioned odor preference occurred. In CD1 mice levels, one out of 482 protein spots was significantly increased in odor-trained mice as compared with the control group; it was in-gel digested by trypsin and chymotrypsin and analyzed by tandem mass spectrometry (nano-ESI-LC-MS/MS). The spot was unambiguously identified as serine/threonine-protein phosphatase PP1-α catalytic subunit (PP-1A) and differential levels observed in gel-based proteomic studies were verified by immunoblotting. PP-1A is a key signalling element in synaptic plasticity and memory processes and is herein shown to be paralleling olfactory discrimination representing olfactory memory.     

Ultraviolet-A irradiation to the eye modulates intestinal mucosal functions and properties of mast cells in the mouse

We previously reported that topical irradiation of the eye by ultraviolet-B (UVB) activated hypothalamo-pituitary-adrenal axis (HPA-A) of the mouse to increase 3, 4-dihydroxyphenylalanine (DOPA)-positive melanocytes in the skin by an inducible nitric oxide synthase (iNOS)-dependent mechanism. This work demonstrates that irradiation of the eye by ultraviolet-A (UVA) specifically increased DOPA-positive cells in the mucosa of the jejunum and colon of C57BL/6J mice by some HPA- and iNOS-independent mechanism. UVA-induced increase in DOPA-positive cells in the intestine was inhibited by the administration of hexamethonium or prazosin plus propranolol, blockers for the sympathetic nervous system. UVA irradiation of the eye increased DOPA- and histidine decarboxylase (HDC)-positive cells in the intestinal mucosa of both C57BL/6J and WBB6F1/J mice but not in the mutant strain W/Wv of the latter that lack mast cells. UVA irradiation of the eye suppressed the intestinal peristalsis of control, hypophysectomized or iNOS(-/-) C57BL/6J mice by the mechanism that was inhibited by hexamethonium or prazosin plus propranolol. These observations suggest that UVA irradiation of the eye stimulated the sympathetic nervous system to increase the mucosal DOPA- and HDC-positive mast cells and suppressed the peristalsis of the small intestine of the mouse.