Activity-Induced DNA Breaks Govern the Expression of Neuronal Early-Response Genes.

中翻英：本研究结果，在5-Aza-CdR 与NaB 联合应用下，RASSF1A 基因表达较单独应用5-Aza-CdR 上调，表明了甲基化酶抑制剂5-Aza-CdR 及去乙酰化酶抑制剂NaB 在转录调控作用上的协同作用。

这与Egger 等[24]的研究结果一致。

研究发现，DNA 甲基化和组蛋白乙酰化对基因表达的调控作用有密切联系。

DNA 甲基化可诱导局部组蛋白去乙酰化，使染色质乙酰化水平降低[4]，甲基化CpG 结合蛋白MeCP2 以甲基化依赖的方式结合到染色质上，通过转录抑制结构域与桥蛋白Sin3A 和HDAC 的共同抑制复合体紧密联系[25]Our study shows that the combined administration of 5-Aza-CdR and NaB upregulates the gene expression level of RASSF1A as compared with 5-Aza-CdR alone, demonstrating the synergistic action of methylase inhibitor 5-Aza-CdR and histone deacetylase inhibitor (HDAC) NaB on transcription regulation. Our foundings are also consistent with the study by Egger and colleagues. Numerous studies have found that DNA methylation and histone acetylation correlate closely with gene regulation. DNA methylation can induce local histone deacetylation and therefore reduce the acetylation level of chromatins. Methyl-CpG-binding protein MeCP2 binds to chromatins via a methytation-dependent manner and comes to a close contact with the corepressor complex consisted of HDAC and adaptor protein Sin3A through its transcription repression domain.英翻中A biological indicator for monitoring the effectiveness of a sterilizing, disinfecting, etc., compound or condition is disclosed which comprises a substrate having a surface layer containing functional groups thereon desirably free of silicone linking groups. The functional groups are desirably in the form of a monolayer of a uniformed distribution and of a selected quantity. Various types of microorganisms such as spores and/or etiological agents are covalently bonded to the surface layer functional groups through a crosslinking reagent and thus form a uniform number and distribution of the microorganisms and/or etiological agents. After being subjected to sterilization or other similar treatment, etc., along with various articles such as instruments, the indicator can be cultivated to determine the effectiveness of the sterilization, disinfection, etc. process.本专利公开了一种用于检测杀菌、消毒等化合物有效性的生物指示剂。

1血管紧张素II 对Smad2/3的活化及对Smad3相互作用蛋白质组的影响花芳，周军兰，杨红振，胡卓伟中国医学科学院药物研究所，北京(100050)E-mail （huzhuowei@ ）摘 要： 血管紧张素Ⅱ在心血管纤维化中发挥了十分重要的作用。

AngⅡ作为一种细胞外信号，经过细胞膜AT1受体和细胞内信号传导通道，激活核转录因子进而调控相关基因表达。

了解血管紧张素Ⅱ信号途径中重要的转录因子及其相互作用的辅因子可以从整体的角度更全面认识血管紧张素Ⅱ对心血管系统的生长调节机制。

本研究通过细胞核转录因子微阵列研究发现血管平滑肌细胞中多种核转录因子在血管紧张素Ⅱ的作用下活性发生变化，其中转录因子Smad3/4活性明显增强。

通过对血管紧张素Ⅱ作用前后，Smad3相互作用蛋白的蛋白质组学研究，我们鉴定到两种新的Smad3相互作用蛋白，这对研究Smad3在血管紧张素Ⅱ信号通路中的调控机制提供了新的线索。

关键词：血管紧张素Ⅱ，转录因子，Smad3，相互作用蛋白肾素-血管紧张素－醛固酮系统(RAAS)是机体进化过程中高度保守的内分泌网络调节系统。

血管紧张素Ⅱ(Ang Ⅱ) 是RAAS 的主要效应因子，在维持机体血容量、血流动力学及内环境稳态中发挥主导作用。

研究证明，Ang Ⅱ在许多心血管疾病，包括高血压、心力衰竭和缺血性心脏病的发生、发展过程中发挥了关键作用[1]。

例如，Ang Ⅱ激活AT1受体引起的一系列作用可以引起高血压。

Ang Ⅱ升高血压的作用包括直接收缩血管或通过增加交感神经末端释放去甲肾上腺素，后者收缩血管、增加心肌收缩力和增加心率；直接刺激肾近曲小管增加钠重吸收或通过增加醛固酮分泌引起肾钠水潴留；直接刺激心血管组织引起增生、肥厚或通过促进多种细胞因子的分泌，如炎性细胞因子、促纤维化因子及各种生长因子等，引起心脏和血管细胞的增殖、肥厚[2, 3]。

现已明确，心血管组织重构是高血压和心力衰竭等心血管疾病的核心改变，而心血管组织纤维化是组织重构的最基本病理改变。

gpc6基因GPC6基因是人类基因组中的一个基因，它位于染色体6上。

该基因编码的蛋白质是一种受体蛋白，它在神经细胞中起着重要的作用。

研究表明，GPC6基因与多种疾病的发生和发展有关，包括神经系统疾病、癌症以及心血管疾病等。

本文将重点介绍GPC6基因在神经系统疾病中的作用。

GPC6基因在神经系统发育中起着重要的作用。

研究发现，该基因在胚胎期和幼年期神经细胞的迁移和分化过程中发挥着关键的调控作用。

具体来说，GPC6蛋白质通过与细胞外基质分子相互作用，调控神经细胞的黏附、迁移和定向生长。

这些过程对于神经系统的正常发育至关重要，如果GPC6基因发生突变或异常表达，可能会导致神经系统发育障碍。

GPC6基因与神经退行性疾病密切相关。

多项研究发现，GPC6基因的突变与阿尔茨海默病、帕金森病等神经退行性疾病的发生有关。

这些突变可能导致GPC6蛋白质结构或功能异常，进而影响神经细胞的正常功能和存活。

此外，GPC6基因在神经退行性疾病的早期诊断和治疗中也具有重要意义。

一些研究表明，GPC6基因的表达水平与神经退行性疾病的严重程度和预后密切相关，因此可以作为潜在的诊断和治疗靶点。

GPC6基因还与神经系统肿瘤的发生和发展有关。

研究发现，GPC6基因在一些神经系统肿瘤中呈高表达状态。

这些肿瘤包括神经胶质瘤、神经母细胞瘤等。

进一步的研究表明，GPC6蛋白质通过调控信号通路的活性，参与了肿瘤细胞的增殖、侵袭和转移过程。

这些结果提示，GPC6基因可能是神经系统肿瘤的重要致病基因，并且可以作为潜在的治疗靶点。

在总结上述研究结果的基础上，可以看出GPC6基因在神经系统疾病中具有重要的调控作用。

研究GPC6基因的功能和机制将有助于我们更好地理解神经系统疾病的发生和发展机制，为相关疾病的预防和治疗提供新的思路和方法。

然而，目前对于GPC6基因的研究还存在一些不足之处，例如缺乏大样本的临床研究和动物模型的验证。

因此，今后的研究需要进一步加强，以全面地揭示GPC6基因在神经系统疾病中的作用机制。

RESEARCH ARTICLEDelivery of GDNF by an E1,E3/E4deleted adenoviral vector and driven by a GFAP promoter prevents dopaminergic neuron degeneration in a rat model of Parkinson’s diseaseNA Do Thi 1,P Saillour 1,L Ferrero 2,JF Dedieu 2,J Mallet 1and T Paunio 1,31Laboratoire de Genetique Moleculaire de la Neurotransmission et des Processus Neurodegeneratifs,CNRS,Bat.CERVI,Hopital Pitie-Salpetriere,Paris,France;2Gencell SAS,Vitry sur Seine,France;and 3Department of Molecular Medicine,Biomedicum,Helsinki,FinlandA new adenoviral vector (Ad-GFAP-GDNF)(Ad-¼adenovirus,GFAP ¼glial fibrillary acidic protein,GDNF ¼glial cell line-derived neurotrophic factor)was constructed in which (i)the E1,E3/E4regions of Ad5were deleted and (ii)the GDNF transgene is driven by the GFAP promoter.We verified,in vitro,that the recombinant GDNF was expressed in primary cultures of astrocytes.In vivo,the Ad-GFAP-GDNF was injected into the striatum of rats 1week before provoking striatal 6-OHDA lesion.After 1month,the striatal GDNF levels were 37pg/m g total protein.This quantity was at least 120-fold higher than in nontransduced striatum or after injection of the empty adenoviral vector.At 3months after viral injection,GDNF expression decreased,whereas the viral DNA remained unchanged.Furthermore,around 70%of the dopaminergic (DA)neurons were protected from degeneration up to 3months as compared to about 45%in the control groups.In addition,the ampheta-mine-induced rotational behavior was decreased.The results obtained in this study on DA neuron protection and rotational behavior are similar to those previously reported using vectors with viral promoters.In addition to these results,we established that a high level of GDNF was present in the striatum and that the period of GDNF expression was prolonged after injection of our adenoviral vector.Gene Therapy (2004)11,746–756.doi:10.1038/sj.gt.3302222Published online 15January 2004Keywords:GDNF;Parkinson’s disease;DA neurons;recombinant adenovirusIntroductionParkinson’s disease (PD)is a progressive neurodegen-erative disorder.It is characterized by tremor,bradyki-nesia,rigidity and postural instability that result from a loss of dopaminergic (DA)neurons of the nigrostriatal pathway.The best current therapy of PD is the administration of L -Dopa.However,L -Dopa loses its effectiveness as the disease progresses.Different ther-apeutic approaches are therefore being investigated such as the use of neurotrophic factors,1–4cell/tissue transplantation 5–7and gene transfer of trophic factors using recombinant adenovirus,recombinant adeno-associated virus (AAV)or lentivirus.8,9The ultimate goal of these approaches would be to arrest or to slow down the progressive degenerative process of the disease.Previous research in our laboratory 10,11and in others 12revealed that the progressive degeneration of DA neurons in an adult rat model of PD,13using an E1/E3defective adenovirus (first-generation virus)encoding glial cell line-derived neurotrophic factor (GDNF),under a viral promoter was reduced.The effects obtained in vivo depend on the time and site of administration of recombinant virus.14,15Although these results were encouraging for gene therapy,the first-generation ade-novirus had a toxic effect for transduced cells by inducing the synthesis of viral proteins within these cells.These proteins elicit an immune response in the injected brains,which generates toxicity.It is probable that the toxicity of the E1/E3defective virus is due to the E4region of the type 5adenovirus which is present in the recombinant virus.Yeh et al 16suggested that,in vivo ,a low level of E4expression could be cytotoxic to the recipient cells.In fact,Dedieu et al 17demonstrated that the E1,E3/E4defective adenovirus (third generation)was less toxic for transduced cells than the ‘first-generation’E1/E3defec-tive virus.In addition,the infusion of E1,E3/E4defective virus elicited lower inflammatory responses,improved local tolerance and increased viral DNA persistence in the liver of a lacZ -transgenic mouse.Thus,an E1,E3/E4defective adenovirus represents progression on the path toward preclinical therapy.The aim of our work was to use the E1,E3/E4defective adenovirus to deliver GDNF,a therapeutic gene,into theReceived 17June 2003;accepted 29November 2003;published online 15January 2004Correspondence:J Mallet,Laboratoire de Genetique Moleculaire de la Neurotransmission et des Processus Neurodegeneratifs,Bat.CERVI,Hopital de la Pitie-Salpeˆtriere,83Bd.de l’Hopital,75013Paris,France Gene Therapy (2004)11,746–756&2004Nature Publishing Group All rights reserved 0969-7128/04$/gtbrain of a rat model of PD.The expression of GDNF was targeted to the astrocytes in the lesioned striatum. Astrocytes are the most abundant glial cells in the central nervous system(CNS),and are necessary for the survival of neurons in vivo18and in vitro.19These cells produce and secrete several growth factors,and among them,GDNF and cytokines.20–23In addition,following brain injury,the glialfibrillary acidic protein(GFAP) gene is upregulated in reactive astrocytes.24We therefore used the promoter of the GFAP gene,whose expression in the CNS is restricted to astrocytes,24,25to drive GDNF gene expression.Restriction of GDNF expression to a specific cell type limits the side effects caused by the expression of this gene in surrounding cells,thus facilitating the long-term expression of the transgene. Our results unequivocally showed that the recombinant Ad-GFAP-GDNF,in which the transgene was driven by a glial-specific promoter,prevented DA neurons death after striatal lesions induced by6-OHDA in the rats and improved the drug-induced rotational behavior.As compared to the E1/E3deleted adenovirus used previously in our laboratory,the cytotoxicity in injected animals was much lower.ResultsIn this work we constructed an E1,E3/E4defective recombinant adenovirus encoding rat GDNF under the control of a specific glial promoter,GFAP,by homo-logous recombination in Escherichia coli(see Materials and methods).This defective virus exhibits a large deletion in the E4region,which abrogates the synthesis of all E4-encoded gene products.17The virus was generated in the IGRP2cell line that transcomplements the E4viral function.16In vitro experimentsThe ability of the recombinant Ad-GFAP-GDNF to express GDNF wasfirst tested in primary cultures of astrocytes.The neurotrophic effect of this secreted protein on the survival of DA neurons was performed on mesencephalic cultures.Synthesis of GDNF by various types of cells.To determine whether astrocytes transduced with Ad-GFAP-GDNF express recombinant GDNF,cultivated rat astro-cytes were infected with the recombinant virus as well as an empty control at different doses.Conditioned medium(CM)and cellular pellets were collected at4,6 and8days after infection for analysis by ELISA.The quantity of endogenous GDNF secreted by noninfected astrocytes,or those infected with empty adenovirus was low(120716pg/ml)at all time points tested in CM.The amount of endogenous GDNF was close to the detection limit of the assay(20pg/ml)in the cellular pellet.In astrocytes infected with50viral particles(vp)/cell of Ad-GFAP-GDNF,0.370.05ng/ml of GDNF was secreted in the CM per day.GDNF levels of0.270.04, 0.470.07and0.370.08ng/ml were detected in the cellular pellet at days4,6and8,respectively. However,when astrocytes were infected with Ad-GFAP-GDNF at higher doses(500and103vp/cell), 50–60ng/ml of GDNF was secreted in the CM by105cells per day(Figure1)(P o0.0001for500vp;P¼0.0008for103vp as compared to control).At a dose of5Â103vp/cell,70ng/ml of GDNF was secreted in theCM from day4to day6,and it declined thereafter (Figure1)(P o0.0001as compared to control).In cellular pellets,about4074ng/ml of GDNF was found at thesethree doses at all time points tested.From this result,twodoses of virus,500and5Â103vp/cell,were chosen toinfect the mesencephalic cells.Mesencephalic cells infected with500and5Â103vp/cell of Ad-GFAP-GDNF secreted3079and80720pg/ml of GDNF in the the CM,respectively.In control cultures(cells noninfected by the virus),about35pg/mlof GDNF was found in the CM.In cellular pellets,0.570.1and0.870.12ng/ml ofGDNF were measured in cells infected with500and5Â103vp/cell,respectively,6days after infection.In cellular pellets of control cultures,0.670.09ng/ml ofGDNF was found.These results indicate that recombi-nant GDNF was not effectively synthesized by the mesencephalic cells infected with Ad-GFAP-GDNF.Survival of DA neurons.To test the effect of GDNF onthe survival of neuronal cells,104mesencephalic cellswere plated on a layer of nontransduced astrocytes, astrocytes infected with103vp/cell of Ad-GFAP-GDNFor onto collagen-coated coverslips(control).When plated on transduced astrocytes,457770 tyrosine hydroxylase(TH)-positive neurons were found.This was two-fold lower if plated on noninfected astrocytes(233723).The number of surviving TH-positive neurons was lowest(114718)on collagen-coated coverslips(Figure2).Figure1GDNF levels in cultured astrocytes infected with recombinantvirus.Primary astrocytes were infected with Ad-GFAP-GDNF at differentdoses.d1:500vp/cell;d2:103vp/cell;d3:5Â103vp/cell;C:control, noninfected astrocytes.In all,50–60ng/ml of GDNF was released by105cells per day with both doses d1and d2.At a higher dose(d3),about70ng/ml of GDNF was released by105cells/day until day6after infection.Thenthe quantity of GDNF decreased at day8.*P o0.0001d1versus control atthree times analyzed;**P¼0.008,0.0004,o0.0001d2versus control atthree times analyzed,respectively;***P o0.0001,o0.0001,0.0002d3versus control at three times analyzed,respectively.Degeneration of DA neurons prevented by Ad-GDNFNA Do Thi et al747Gene TherapyIn vivo experimentsThe effect of striatal overexpression of GDNF on the DA neuron survival and motor function in a rat model of PD was investigated by direct in vivo delivery of the transgene,using recombinant Ad-GFAP-GDNF.Body weight.Injection of large doses of recombinant GDNF protein has been found to cause loss of body weight in rats.26No significant differences in weight were observed among the treatment groups over the entire period of experimentation.Thus the quantity of transgenic GDNF detected in the striatum did not affect the body weight of rats.GDNF expression in intact animals.In preliminary experiments ,different doses of virus (107,108,5Â108,109and 3Â109vp/rat)were used to determine an optimal dose both in terms of level of GDNF expression and inflammatory reaction.GDNF expression was assessed by ELISA in striatum obtained from nonlesioned animals that were killed 10days,4,6and 12weeks after vectorinjection.As shown in Table 1,at doses of 108and 5Â108vp/rat,the striatal GDNF content was the highest at all times analyzed as compared to other doses.At a lower dose of the virus (107vp/rat),GDNF levels were 10-fold lower than those measured in rats that had received 108vp of virus,at all time points studied.At doses of 109and 3Â109vp/rat,12–20pg/m g GDNF protein was found (Table 1)in transduced striatum,but a marked,strong inflammatory reaction was observed (data not shown).However,the GDNF protein levels decreased with time at all doses used (Table 1).Analysis with semiquantitative competitive polymer-ase chain reaction (sqc-PCR)showed that the level of viral DNA in injected striatum (108vp/rat)did not change between 10days and 12weeks after viral administration (Figure 3).From these results,we used 108vp/rat in the following experiments.GDNF expression in lesioned rats.The efficacy of adenovirus-mediated GDNF gene transfer was tested on a rat model of PD.13Adult rats were injected stereo-taxically with 108vp of Ad-GFAP-GDNF (G group)into the left striatum as described in Materials and methods.At 1week after viral injection,rats were anesthetized and received stereotaxic injection of 6-OHDA.Striatum and substantia nigra (SN)were dissected out of animals killed at 4,6and 12weeks after viral injection and the GDNF levels in these tissues were determined by ELISA.As shown in Table 2,the GDNF protein levels were 37–40times higher (37.3and 41pg/m g corresponding to 70and 75ng of GDNF per striatum,respectively)in Ad-GFAP-GDNF-transduced striatum at 4and 6weeks as compared to both control groups (OHDA ¼OH group;empty ¼E group),as well as to the noninjected side.The GDNF quantity was decreased at 12weeks (17.2pg/m g corresponds to 35ng of GDNF per striatum)afterviralFigure 2Survival of mesencephalic cells in culture.The survival TH (þ)neurons was two-fold higher on transduced astrocytes (G)than on noninfected cells (A,P ¼0.002)and four-fold higher as compared to control (C,P ¼0.0003).C:neurons on collagen-coated coverslips used as control;A:neurons on noninfected astrocytes;G:neurons on astrocytes infected with Ad-GFAP-GDNF.Table 1GDNF protein levels measured from transduced striatum of intact animals after Ad-GFAP-GDNF injection Doses 107vp 108vp 5Â108vp 109vp 3Â109vp 10days 3.25723579327814.67215734weeks 1.470.227.87223.47215.37316746weeks 2.570.7277834.77522.27220.147212weeks1.370.1214.67626.37312.5731273GDNF protein levels (pg/m g total protein),measured from injected striatum of intact animals (three animals per point),were high at doses of 108and 5Â108vp/rat as compared to other doses of virus.At 12weeks after viral treatment,the quantity of GDNF decreased with all doses used.Values are means 7s.e.m.Figure 3Viral DNA levels of injected striatum,in nonlesioned rats.The relative viral DNA amount in rats injected with 108vp/rat of Ad-GFAP-GDNF was unchanged from day 10to week 12(three animals per point).P ¼0.5,4weeks versus 10days;P ¼0.9,6weeks versus 10days;P ¼0.9,12weeks versus 10days.Degeneration of DA neurons prevented by Ad-GDNFNA Do Thi et al748Gene Therapyinjection.To explain the decline of GDNF levels in Ad-GFAP-GDNF-injected striatum at a later stage,sqc-PCR was performed to determine the relative quantity of the virus at different times after viral injection.As shown in Figure 4,the viral DNA levels were unchanged during 12weeks of experiment,which suggests a downregulation of GFAP promoter in vivo rather than a loss of injected viral DNA.In the SN,the GDNF protein levels were similar in the injected side as compared to the noninjected side and in all groups of animals (Table 2).The viral DNA levels were not detectable in the SN by sqc-PCR.Amphetamine-induced rotation test.The impact of GDNF overexpression on the behavior of the animals was assessed during 12weeks after viral vector injection.As early as 2weeks after the striatal 6-OHDA lesion (3weeks after viral injection),rats injected with Ad-GFAP-GDNF into the striatum began to display reduced amphetamine-induced rotations.As shown in Figure 5,3,4,6and 12weeks after Ad-GFAP-GDNF treatment,rats exhibited a significant reduction inTable 2GDNF protein levels from striatum and SN of lesioned animalsStriatumSubstantia nigraInjected sideNoninjected sideInjected sideNoninjected sideAd-GFAP-GDNF (G group)4weeks *#137.375.10.2970.070.1570.050.2170.096weeks *#14176.50.3470.130.270.060.2370.0612weeks *#117.274.20.2670.060.2670.040.2270.05Empty virus (E group)4weeks 0.1470.020.0970.010.270.040.1570.036weeks 0.0870.010.0770.0030.1270.020.270.0512weeks0.0970.040.0770.0020.1370.020.1470.056-OHDA alone (OH group)4weeks 0.1270.030.1670.030.1670.020.1470.016weeks 0.1470.020.1570.060.1770.020.1370.0112weeks0.1670.010.1270.020.270.050.270.01GDNF protein levels (pg/m g total protein)from striatum and SN measured by ELISA in lesioned animals treated with Ad-GFAP-GDNF (108vp/rat),with empty adenovirus (108vp/rat)or in naive animals that did not receive treatment before inducing 6-OHDA lesion.Seven animals were used per point.GDNF protein levels decreased with time in transduced striatum (G group)and remained unchanged in the injected striatum of E and OH groups.The GDNF levels were similar in the SN (injected and noninjected side)of all groups.Values are means 7s.e.m.*P o 0.0001different from noninjected side;#P o 0.0001G versus E group;P o 0.0001G versus OHgroup.Figure 4Viral DNA levels of injected striatum in lesioned rats at 4,6and 12weeks after viral treatment.The relative quantity of viral DNA was similar during our experiment from 4to 12weeks (five animals per point).P ¼0.9,6weeks versus 4weeks;P ¼0.6,12weeks versus 4weeks.Figure 5Motor performance of the animals using the drug-induced rotation test.Rats were injected with D -amphetamine (2.5mg/kg,i.p.)and their behavior was recorded for 90min.At 3,4,6and 12weeks after viral injection,rats injected with Ad-GFAP-GDNF (G group)exhibited a significant reduction in ipsilateral rotational behavior compared with control groups (OH and E groups).P ¼0.008,G versus OH group (3weeks);P ¼0.2,G versus E group (3weeks);P o 0.0001,G versus E and OH groups (4weeks);P o 0.0001,G versus E and OH group (6weeks);P ¼0.006,G versus E group (12weeks);P ¼0.0002,G versus OH group (12weeks).Degeneration of DA neurons prevented by Ad-GDNF NA Do Thi et al749Gene Therapyipsilateral rotational behavior compared with control groups(OH and E groups)(P o0.0001).In rats injected with empty virus,the rotation score was not significantly different from that of the animals that received6-OHDA alone at3,4and6weeks after viral injection(P¼0.08,0.4and0.3,respectively).Protection of DA neurons in the SN.Survival of DA neurons was analyzed throughout the SN as described in Materials and parison of the percentage of the TH-positive cells in the SN(average results from the three levels analyzed)revealed that about70%of DA neurons remained in the G group as compared to about 45%in the control groups at all three times examined (P o0.0001,0.01and0.0004,4,6and12weeks, respectively)(Figures6,7a and b).This result suggested that a significant protection of DA neurons was found in animals treated with Ad-GFAP-GDNF.Immunoreactivity in the striatum.Following an injec-tion of6-OHDA into the striatum,there is an immediate toxic damage to the DAfibers and axons followed by a rapid degeneration of their terminals.4,27We used NeuN staining to localize the lesion in the striatum(Figure9d), and immunohistochemistry for the TH to assess the extent of denervation induced by intrastriatal6-OHDA lesions(Figure8a and b).The extent of denervation was prominent in the central and dorsal parts of the injected striatum in all animals analyzed.The intensity of TH-positivefiber staining,measured by optical density,in the injected striatum(average results from the three levels analyzed)was similar in both E and OH groups (Figure8b).It was reduced by about70–75%(P o0.0001) in the injected side versus the noninjected side at4weeks and by about80–85%(P o0.0001)at6and12weeks. The TH intensity was slightly higher in animals of G group(þ7%)at4,6and12weeks as compared to controls,but it did not reach statistical significance (P¼0.2)(Figure8a).Abundant TH immunoreactive profiles(dots)of different sizes(Figure8c and f)were observed in the lesion sites of the striatum.Some of these patterns were scattered throughout the parenchyma in animals of G, OH and E groups.However,in animals treated with Ad-GFAP-GDNF(Figure8c),the number of these TH immunoreactive profiles was increased as compared to control animals at all three times analyzed(Figure8f).At higher magnification,we observed TH-positivefibers with numerous axonal varicosities which displayed different intensity of TH staining(Figure8d,compared with Figure8g(control)).In globus pallidus,we observed the TH immunoreactive area,which appears to correspond to the axonal sprouting of TH-positive fibers in animals treated with Ad-GFAP-GDNF vectors (Figure7c).The TH-positivefibers were also observed in the entopeduncular nucleus of these rats(Figure7d). These patterns were not seen in any of the animals of control groups.The GDNF transgene expression in transduced stria-tum was visualized by anti-GDNF antibody.As shown in Figure9a and b,the striatal astrocytes of animals treated with Ad-GFAP-GDNF were stained with GDNF antibody.We also determined the effect of the viruses on the size of the striatum by analyzing the surface of10sections per brain between the coordinates APþ1.7and APþ0.2. The injected striatal size was not modified in G(P¼0.8), E(P¼0.7)and OH(P¼0.4)groups at4weeks as compared to the noninjected side.However,we observed a nonsignificant atrophy,7%as compared to controlate-ral size,of the injected striatum of G(P¼0.5)andEFigure6Rescue of TH immunoreactive neurons in the SN.Significantincrease in the percentage of TH immunoreactive neurons was observed inlesioned rats treated with Ad-GFAP-GDNF(G group)compared with ratsinjected with empty adenovirus(E group)or with lesioned rats(OHgroup).P o0.0001,G versus E group(4weeks);P¼0.001,G versus OHgroup(4weeks);P¼0.01,G versus E group(6weeks);P¼0.003,Gversus OH group(6weeks);P¼0.0004,G versus E group(12weeks);P o0.0001,G versus OH group(12weeks).Figure7TH staining of injected brain.Many TH-positive neuronsremained in the rostral,middle and caudal parts of the SN in animalstreated with Ad-GFAP-GDNF(a),while fewer cells survived in animalslesioned by6-OHDA(b),12weeks after treatment.The presence of TH-positivefibers(asterisk)was seen in globus pallidus(c)and inentopeduncular nucleus(d),4weeks following Ad-GFAP-GDNF injection(injected side¼left side,arrow;right side¼intact side).Scale barrepresents(a,b)250m m and(c,d)150mm.Degeneration of DA neurons prevented by Ad-GDNFNA Do Thi et al750Gene Therapy(P ¼0.6)groups at 6and 12weeks.In the OH group,very mild atrophy was seen (4%as compared to the controlateral side)at 12weeks,but not significant as assessed by one-way analysis of variance (ANOVA),P ¼0.6.Inflammatory response.Injuries to the brain result in a rapid inflammatory reponse that typically involves recruitment and infiltration of different cell populations.Immunohistochemistry using CD4and CD8antibodies allows one to determine the localization of reactive lymphocytes.CD4immunoreactive cells were most numerous at the injection sites of the adenoviral vector with or without transgene,and at the 6-OHDA lesion in all treatment groups (Figure 9e).They were also scatteredthroughout the parenchyma and close to the blood vessels.A few CD8immunoreactive cells were particu-larly concentrated at the injection sites of the adenoviral vector and around the 6-OHDA lesion (Figure 9f).We did not observe more inflammation in G and E groups as compared to the OH group,with both CD4and CD8antibodies (Table 3).Astrocytic response to injury was assessed by using an antibody against GFAP .Glial fibrillaly acidic protein,an intermediate filament protein,is expressed abundantly in astrocytes during development 28of the CNS and in reactive astrocytes (astrogliosis)following CNS in-jury.24,25Reactive astrocytes,characterized by a signifi-cant increase in the GFAP intensity,cellular hypertrophy and increase in the density of GFAPimmunoreactiveFigure 8TH immunostaining of the striatum,4weeks after viral treatment.On the intact side,the TH staining intensity was high throughout the striatum (a,b,right side).After intrastriatal lesion,the TH staining was almost lost at the site of 6-OHDA injection (b,left side).By contrast,Ad-GFAP-GDNF-treated animals had a more lasting TH intensity on the ipsilateral side (a,left side).High-power magnification of boxed area in (b)showed that the axonal terminals in the striatum were degenerated at the lesion site (e,asterisk),whereas some spared terminals remained (arrow).In these GDNF-treated animals,numerous TH-positive axonal profiles (c,dots)and TH immunoreactive fibers with varicosities and sprouting (d,arrow)were seen in the denervated striatum.In the striatum of 6-OHDA lesioned animals,the TH-positive profiles (f)and TH immunoreactive fibers with varicosities (g)were less numerous.Scale bar represents (a,b)200m m and (c–g)50mm.Figure 9Immunostaining of transduced striatum,4weeks after Ad-GFAP-GDNF treatment.At the injected site,a halo of GDNF was seen with GDNF-positive astrocytes (a);at low magnification astrocytes stained with GDNF antibody (b).Numerous cells stained with CD4(e)and CD8(f)at the injected site.In (d)the site of 6-OHDA lesion was stained by NeuN antibody.Inside the lesion,the neurons were degenerated,while around the lesion the nuclei of neurons were stained by NeuN.Reactive astrocytes were stained by GFAP antibody (c)at the injected site of the striatum.Scale bar represents (a)35m m,(b)200m m,(c)50m m,(d)200m m and (e,f)100mm.Degeneration of DA neurons prevented by Ad-GDNF NA Do Thi et al751Gene Therapyprocesses,were detected throughout the ipsilateral striatum.The GFAP staining was particularly intense at the lesion with a dense network of cell bodies and processes in all study groups (Figure 9c).DiscussionThe aim of the present study was to assess the ability of GDNF,expressed by an improved E1,E3/E4defective recombinant adenovirus in which the GDNF gene is driven by a glial-specific promoter,to preserve the integrity of the nigrostriatal DA system (cell bodies,axonal terminals)and the normal motor function after administration of the virus into the striatum before inducing 6-OHDA lesion.Our interest was also to test the GFAP promoter for PD therapy since this promoter was described to direct specifically transgene expression in astrocytes.24,25In our cell cultures,GDNF protein was not synthe-sized by the mesencephalic cells infected with the recombinant GFAP-GDNF adenovirus,whereas this trophic factor was produced and secreted by the transduced astrocytes (Figure 1).Morelli et al 29observed that only cultured neocortical neurons,infected with a recombinant defective adenovirus vector encoding FasL under the control of the neuronal-specific promoter NSE (RAd-NSE-FasL),released the cytotoxic Fas ligand into the culture supernatant.Neurons transduced with a vector under the control of a glial-specific promoter (RAd-GFAP-FasL)were unable to release the FasL cytotoxic activity.Thus,the expression of the transgene was cell-type restricted when the transcription was directed from a glial-or a neuronal-specific promoter in the adenoviral vector.In vivo ,in a rat model of PD,immunohistochemical experiments,performed in the transduced striatum,demonstrated that the expression of the transgene (GDNF)was confined to astrocytes (Figure 9a and b).This observation was supported by the results obtained from ELISA tests (Table 2).After an intrastriatal injection of Ad-GFAP-GDNF,the GDNF protein levels were high in transduced striatum (37–41pg/m g protein from 4to 6weeks;Table 2).At 12weeks the quantity of GDNF protein decreased,whereas the levels of adenoviral DNAremained unchanged from weeks 4to 12(Figure 4).The decline of the transgene expression could result from the host immune responses to the vector in infected cells 30,31or due to the down regulation of the promoter.12In our study,the decline in the GDNF expression was unequi-vocally the result of a downregulation of the GFAP promoter rather than the loss of adenovirus-infected cells,since the quantity of viral DNA in the transduced striatum did not change during the experiment (Figure 4).Using the RSV promoter to drive the expression of the GDNF,Choi-Lundberg et al 32found that GDNF protein and GDNF DNA levels decreased simultaneously from weeks 1to 7.In our study,although the GDNF protein level decreased,it remained relatively high at 12weeks (17pg/m g protein;Table 2).Armentano et al 33and Dedieu et al 17showed that the deleted E1,E3/E4recombinant adenoviruses were unable to sustain a strong and stable transgene expression when under the control of the CMV and RSV promoters.Thus the long-lasting presence of the recombinant GDNF in our experiment cannot be attributed solely to the E1,E3/E4Ad-GFAP-GDNF backbone.The prolongation of the GDNF expression we obtained was probably the consequence of the activity of the GFAP promoter.Despite a downregulation of the GFAP promoter at 12weeks,its remaining activity would still be sufficient to induce the late and high-level expression of the transgene in the astrocytes.In four animals we even observed GDNF expression in the transduced striatum 5months after Ad-GFAP-GDNF injection (unpublished results).In addition,the recombinant E1,E3/E4defective adenovirus used in the present study appeared to be weakly immunogenic in the brain.We did not observe an increased inflammation in the lesioned brains after GFAP-GDNF or empty virus injection as compared to the OHDA-injected animals (Table 3).Moreover,the transduced striatum sizes were not reduced.These results suggest that this is an improvement of the adenoviral vector compared to the first-generation adenovirus used previously by our group.11Another important result was that approximately 70%(as compared to about 45%in the controls)of the nigral DA neurons were still present at 12weeks when the recombinant Ad-GFAP-GDNF had been injected into the striatum,1week before inducing the intrastriatal 6-OHDA lesion.The ratios of protected neurons did not change with time from week 4to week 12after the viral treatment (Figure 6).As the protection of the DA neurons was not complete,it is possible that the recombinant GDNF,synthesized by striatal transduced astrocytes and transported by retrograde axonal transport,was not sufficient in the SN (Table 2).The results obtained in this work are consistent with previous studies by our group 11and others 9,14using Ad/AAV-GDNF injected in the striatum.In addition,these authors 9,11,14found (1)that the intensity of the TH immunoreactivity was increased in the injected striatum,as compared to control 9,11,14and (2)that the axonal sprouting was present in the striatum and the globus pallidus.9In our rats treated with Ad-GFAP-GDNF,although the sprouting was observed in the striatum and the globus pallidus (Figures 7and 8),the intensity of the striatal TH staining was not modified in the injected side.This may be due to the lowTable 3Semiquantitative estimation of the inflammation in injected animals4weeks 6weeks 12weeks GE OH G E OH G E OH 7404040504242636353+524252424242424342++132414141223030212+++050404041303020203++++000000010000Number of animals from G,E and OH groups where the inflammation was produced in the brains from 4to 12weeks after treatment.For each animal,10CD8and CD4-stained sections were examined and scored as described in Materials and methods.First values in each column (G,E,OH)were estimated on CD8-stained sections,and second values (italic)were estimated on CD4-stainedsections.Degeneration of DA neurons prevented by Ad-GDNFNA Do Thi et al752Gene Therapy。

神经科学专业英语（一休十页1）0.3% TritonX-100 solution 0.3% TritonX-1005% goat serum 5%羊血清3.6% Chloral Hydrate 3.6%水和氯醛（1 ml/100 g）acetylate v.[化]加乙酰基于acetylated with 0.25% acetic anhydride in 0.1M triethanolamine (pH 8.0) to prevent non-specific binding of the probes.adaptor protein 衔接蛋白adlibitum 随意地,无限制地,随意,任意administration of 投药adult rats 成鼠affinitypurified 亲和提纯(法)aliquot (整分)部分,可分量,(拉)除得尽allelic 等位的,对偶的 allele等位基因alpha-motoneurons α-运动神经元γ- motoneurons γ-运动神经元幅度,变幅,范围,距离,振幅amplitude n.anatomic a. 解剖的; 解剖学上的 ankle 踝,踝关节anesthesia n.麻醉, 麻痹anesthesia/ anesthetized with 3.6% Chloral Hydrate (1 ml/100 g) ANOV A [analysis of variance] 变异数分析,方差分析anterograde 顺行的BDA anterograde tracingantisense 反义的编程性细胞死亡,脱噬作用apoptosisassay n.化验, 分析; v.化验, 分析astrocyte n.星细胞; 星形胶质细胞avulsion n.扯开, 撕裂, 扯离的部份, 土地的突然转位spinal root avulsion 根性撕脱(神经根撕脱)axon n. (神经的)轴突, 轴突(输出端) distal nerve axotomy 、axonal regeneration axonal transport 轴突转运,轴突运输BBB BBB (Basso, Beattie, Bresnahan) scale scoreBDA tracing technique BDA 追踪技术生物学的 biological actionbiological a.biotinylated biotinylated goat anti-rabbit IgG antibody (1:100 dilution)生物素酰化的山羊抗兔多克隆抗体biotinylated dextran amine 生物素(酰)化的葡聚糖胺（BDA）ions were incubated at 37 C in 1% blocking buffer blocking buffer 封闭缓冲液Then sect(Roche) for 1 hbone morphogenetic proteins 骨形成蛋白,骨形态发生蛋白caudal 尾的centrifuge 离心离心机circuitry n.电路, 线路, 电路学collision tumor 碰撞性瘤computed tomography 计算机(x线)断层摄影术,计算机x线体层摄影术,计算机体层摄影术cortex n.外皮; 皮质 brain cortexcortical somatosensory evoked potential 皮层体感诱发电位cortico- 表示[皮]之意corticospinal tract 皮质脊髓束cranium n.头盖, 头盖骨crashe 粉碎,失败,坠毁,坠落,碰撞暗房darkroom n.days post operation (dpo) 术后几天de novo synthesis 从头合成,重新合成(分)Densitometry analysis 光密度分析derived 衍生的Dexamethasone 地塞米松diameter n.直径, 放大倍数3–4 mm in diameterdiaminobenzidine 二氨基联苯(DAB)n.区别, 分化, 变异, 微分法differentiationdilemma n.困境,进退两难dissect v.解剖, 切细, 仔细分析dissected 切割的dissociated 分裂的(精神病),离解的distal a.末梢的, 远中的distal nerve axotomydistilled water 蒸馏水distributed 分布式的,GDNF mRNAs are widely distributed in a variety of neuronal and non-neuronal tissues of embryos and adultsdopaminergic embryonic ventral midbrain dopaminergic neuronsdorsal 背的,背侧的dorsal column 背柱,脊柱(脊髓)dorsal roots DR背根节dura 硬膜dysfunction n.机能不良, 机能障碍ectopic expression 异位表达edema n 浮肿; 水肿electrode n.电极 reference electrode参考电极,对照电极,参比电极electrophorese 电泳移动elucidate 阐明,解释embryonic a.胚胎的, 像胚胎的endogenous a.内长的; 内的endometrial 子宫内膜的epidemiological 流行病学的epithelium n.上皮, 上皮细胞the nose epithelium 鼻上皮eradicate 根除,消灭escape mechanism 回避机制,逃避机理,(免疫)脱逸机制,逃逸原理esthesioneure 感觉神经元esthesioneure;sensory neuron;SNevoked cortical potential 皮层激发电位exogenous a.外长的; 外生的；外源性的delivery of exogenous neurotrophic factors (NTFs)expression 表达,表情,面容,(面部)表情,压出(法),压榨(法)extensive a.广的, 广泛的, 多方面的 extensive experimentsflaccid a.软弱的, 没气力的, 无活力的, 松弛的 flaccid paralysisfonticulus 囟门forelimb n.前肢 hindlimb n.后肢freezing microtome 冰冻切片机n.频率; [计算机]频率frequencyRanking according to the scoring system described by Basso et al. [26] includes frequency and quality of hindlimb movement as well as forelimb/hindlimb coordination.function recovery 功能恢复弗林蛋白酶,成对碱性氨基酸蛋白酶furingait n.步法, 步态 normal gaitganglia 神经节；神经中枢 dorsal root gangliagastric parietal cells 胃壁细胞gel n.胶化体, 凝胶; v 胶化, 成凝胶状glial cell 神经胶质细胞,胶质细胞guidelines n.指导方针head-holder device 头部支架,头部固定器,头固定器high-altitude 高空的，高高度homogenized a.经过同质加工处理的; 经过均匀加工处理的homozygous deletion 同合型缺失,纯合子缺失horn n.角, 角质, 喇叭; a.角制的; v.用角触, 长角, 干涉hybridization n.杂交, 配种, 杂种培殖In situ hybridization 原位杂交hybridization solution 杂交溶液 prehybridized in a hybridization solution (50% formamide, 10% dextran sulfate, 19 Denhardt’s solution, 0.2 mg/ml Herring sperm DNA, and 10 mM dithiothreitol)immerse v 浸, 陷入, 把……埋入 inimmunohistochemistry Histochemical localization of immunoreactive substances using labeled antibodies as reagents.免疫组化immunoreactivty 免疫试剂immunostain 免疫染色incision n.切成开口, 切割, 雕刻, 切口 midline incisionincubate v.孵化incubation 培育,孵育,潜伏(期),孵化indicate v.显示, 象征, 指示; [计算机]指示inflammation 炎症interval n. 间隔, 距离, 间歇; [计算机]时间间隔at various time intervalsin the caudal segment 尾段intraperitoneally 向腹膜内(地)intraperitoneally injecteintrinsic a.本质的, 原来备有的, 真正的；内源性的（endogenous）investigate v.调查, 研究; [计算机]研究lamina n.薄板, 薄片, 薄层lane (电泳)泳道lesion n.损害, 损伤精神的伤害, 障碍; v.引起……机能障碍locala. 局部的;localize v.停留在一地方; 使地方化;局部化,集中locomotor a.运动的, 移动的, 运转的; n.有运动力之物, 好旅行的人, 移动发动机loss of heterozygosity 杂合子丢失Lysis Buffer thecords were homogenized on ice in a Lysis Buffer containing0.05 M Tris–HCl (pH 7.4, Amresco)malignant 恶性的mechanism 机械装置,机械论,机理mediate a. 居间的, 间接的; v.斡旋, 调停mediated 介导的 mediated by a two-component receptor GFRa-1 and protein tyrosine kinase Retmesangial cell 肾小球膜细胞microinjecte 显微注射microscissors 显微手术剪modified 缓和的,减轻的限制的,改良的,修改的moist chamber 保湿皿motoneurons 运动神经元central nervous system中枢神经系统CNSmuscle twitch 肌肉颤搐nasogastric 鼻胃的neighboring a.附近的; 毗邻的neuroectodermal 神经外胚层的neurologic 神经病学的、神经系统的neurologic dysfunction神经系统技能障碍neurons 神经元neuroplasticity 神经成形术neurotrophic factor 神经元营养因子,神经营养因子neurotrophic factors (NTFs) neurotrophic factors (NTFs)nitrocellulose n. 硝化纤维 nitrocellulose membraneoligonucleotide probe 寡核苷酸探针paraffin-embedded 石蜡包埋的paraformaldehyde p araformaldehyde solution 多聚甲醛parasympathetic 副交感的sympathetic and parasympatheticpartial recovery 部分恢复pathological conditions 病理学的条件pathophysiology 病理生理学perfuse v.灌注rats were perfused with 500 ml of cold phosphate-buffered saline (PBS) peripheral n.周边设备, 外围器具; a.周边的, 周围的, 肤浅的peripheral neurons peripheral nervous system 末梢神经系统,周围神经系统,外周神经系统peroneal nerve 腓神经peroxidase 过氧化物酶,过氧物酶phosphate-bufferedsaline PBS 磷酸盐缓冲生理盐水,磷酸盐缓冲盐水pitfall n.陷阱,缺陷pivotal a.枢轴的; 中枢的placenta 胎盘plasticity n.可塑性, 适应性, 柔软性polyclonal antibody 多克隆抗体posterior a.在后的, 其次的, 后面的;n.后部, 臀部postfix n.后固定This was followed by a postfix for 6–12 hprecipitated 沉淀的,沉(淀)出的,析出的,制备的,精制的前身,前体,先质,产物母体(化合物)precursorpredispose 使易害(病),造成…的因素prehybridize 预杂交premier 第一位的prenatal 产前的,出生前的prescribe 开药方,开处方,建议Prevalence 流行primer n.引发剂Primer premier 5.0 package which was complementary to the rats GDNF gene sequence (33 mer, 50-GCCCTACTTTGTCAC–TCACCAGCCTTCTATTTC-30).probe n.探针, 调查, 探测针; v.用探针测, 详细调查, 使Preparation of probe for in situ hybridizationproducing cells 生成细胞,生产细胞the transplantation ofGDNF-producing cells greatly enhances the survival ofspinal cord motorneuronspromote v.促进, 升迁, 创办prophylactic 预防(性)的,预防剂prophylactic antibiotic 预防性抗生素propriospinal 脊髓固有的regionally projecting propriospinal pathwaysproteinase K 蛋白激酶Kprotein tyrosine kinase PTK蛋白(质)酪氨酸激酶purified 纯化的,精制的,提纯的,净化的,纯净的purify v.纯净, 净化, 去除quench v.淬火；封闭；熄灭, 结束, 冷浸; n.熄灭; 消除rationale 原理.理论reagent n.[化]试剂, 试药; 反应力, 反应物reestablish v.重建, 复兴, 恢复refixe 再固定regenerative a.再生的, 更生的, 更新的regenerative fibers 再生纤维regulation n.规则, 管理, 调整; a 规定的, 正规的report n.报告, 报导, 成绩单;v.报告, 报导, 记录It has been reported that……retrograde 退行的rinse n.清洗, 润丝, 洗刷; v.以清水冲洗, 灌进rodent 侵蚀性,咬的,啮鼠动物rostral 吻的,喙的,嘴的,吻鳞(蛇)rostral and caudal stumps 残枝的头尾端sacrifice 处死SDSpolyacrylamidegel SDSpolyacrylamidegel (SDS-PAGE)sealed 密封的sedimentation n.沉淀, 沉降sequential a.相继的; 连续的sham-operated group 假手术组signal n.信号, 导火线, 动机; v.向……作信号skepticism 多疑癖skull n.头盖骨, 头、颅骨soak n.浸, 湿透, 大雨; v.使上下湿透, 浸, 吸入soake in PBS containing 3% H2O2 for 30 min at room temperature to quench the endogenous peroxidase activitysomatosensory evoked potentials (SEP) 体感诱发电位specificity 特异性,特征,专性,专一性The specificity of antibody for GDNF was confirmed by Western blots using rat spinal cord homogenates.Spinal Cord n.脊髓spinous processes 棘状突,棘突sprout 新芽stain n.污染, 污点, 著色;v. 沾染, 染污, 著色stimulus intensity 刺激强度stromal 间质的,间质状的stump 残肢,残干,树桩,伐根subjected to 遭受substitute n.取代substrate 酶作用物,底物,基质,基层,附着层,基底物质,酶解物sucrose n.蔗糖superficial n 表面, 外表; a.表面的, 肤浅的, 面积的the superficial back muscles and the skin were sutured along the midline supernatant a. 浮在表面的supraspinal 刺突上的,棘上的,脊椎上的,神经索上的 supraspinal neurons,survival n.留住生命, 生存, 残存sympathetic a.有同情心的, 合意的, 赞成的; n.交感神经, 容易感受的人synaptic gene 联会基因synergy 协同作用synthesized 综合tactile sensation 触觉target tissue 靶组织the primary antibody 一抗therapeutic a.治疗的, 治疗学的 therapeutic approachesthermo- 温,热touch sensation 触觉Tracings 描记 The SEP Tracingstransected 横断的transection n.横断; 横切transplantation n.移植, 移民, 移植法；移植术Transverse n.横断物; a.横的traumatic n.外伤药; a.外伤的, 创伤的15–40 traumatic SCI cases ubiquitously ad.无所不在地underwent v.遭遇, 经验ventral a.腹的, 腹部的, 腹侧的; n.腹鳍ventral horn 腹侧端vertebra n.脊椎骨, 椎骨vertebral laminaeviolent a.暴力的, 猛烈的violent injuries 剧烈损伤visible 可见的,看得见的,明显的体外vitro invitrovoltage n.电压, 伏特数warmth sensation 温觉wrist 腕xenograft 奇形嫁接,异种嫁接,异种移植物integration n.整合, 完成, 集成, 集成化intracytoplasmic (细)胞浆内的,细胞质内的,胞浆内的Levodopa 左旋多巴modify v.修饰 Gene-modifiedmuscle n.肌肉, 臂力neurite n.神经突轴突,轴索neurodegenerative 神经变性的neuropathologic lesion 神经病理性损害nigra (拉)黑质optimal a.最佳的, 最理想的outgrowth 向外生长(放),赘疣Parkinson disease Parkinson disease(PD)帕金森plasmid 质粒pluripotent 多向性(的),多能性(的)pluripotent differentiationpluripotent differentiation 间充质细胞,间(充)质的mesenchymal original cells post-transplantation 移植后at 1, 2,4, and 6 weeks post-transplantation. proliferation n.增殖, 分芽繁殖recessive 隐性,劣势的,隐性的,退缩的integration n.整合, 完成, 集成, 集成化intracytoplasmic (细)胞浆内的,细胞质内的,胞浆内的Levodopa 左旋多巴modify v.修饰 Gene-modifiedmuscle n.肌肉, 臂力neurite n.神经突轴突,轴索neurodegenerative 神经变性的neuropathologic lesion 神经病理性损害nigra (拉)黑质optimal a.最佳的, 最理想的outgrowth 向外生长(放),赘疣Parkinson disease Parkinson disease(PD)帕金森plasmid 质粒pluripotent 多向性(的),多能性(的) pluripotent differentiationpluripotent differentiation 间充质细胞,间(充)质的mesenchymal original cellspost-transplantation 移植后at 1, 2,4, and 6 weeks post-transplantation.proliferation n.增殖, 分芽繁殖recessive 隐性,劣势的,隐性的,退缩的recombinant 重组体 recombinant plasmid 重组质粒rejection n.排异反应,拒绝,抵制,排斥,(免疫)排斥现象 immunal rejectionresidual n.残留,残质,后遗症rigidity n.强直rotation n.旋转, 循环; rotation behaviorself-renewal 自我更新surrounding tissues 外围组织integration n.整合, 完成, 集成, 集成化intracytoplasmic (细)胞浆内的,细胞质内的,胞浆内的Levodopa 左旋多巴modify v.修饰 Gene-modifiedmuscle n.肌肉, 臂力neurite n.神经突轴突,轴索neurodegenerative 神经变性的neuropathologic lesion 神经病理性损害nigra (拉)黑质optimal a.最佳的, 最理想的outgrowth 向外生长(放),赘疣Parkinson disease Parkinson disease(PD)帕金森plasmid 质粒pluripotent 多向性(的),多能性(的)pluripotent differentiationpluripotent differentiation 间充质细胞,间(充)质的mesenchymal original cells post-transplantation 移植后at 1, 2,4, and 6 weeks post-transplantation.proliferation n.增殖, 分芽繁殖recessive 隐性,劣势的,隐性的,退缩的recombinant 重组体 recombinant plasmid 重组质粒rejection n.排异反应,拒绝,抵制,排斥,(免疫)排斥现象immunal rejectionresidual n.残留,残质,后遗症rigidity n.强直rotation n.旋转, 循环; rotation behaviorself-renewal 自我更新surrounding tissues外围组织symptoms 症候transgenic 遗传转移,转基因(植物)usage n.用法, 习惯, 待遇symptoms 症候transgenic 遗传转移,转基因(植物)usage n.用法, 习惯, 待遇recombinant 重组体 recombinant plasmid 重组质粒rejection n.排异反应,拒绝,抵制,排斥,(免疫)排斥现象immunal rejectionresidual n.残留,残质,后遗症rigidity n.强直rotation n.旋转, 循环; rotation behaviorself-renewal 自我更新surrounding tissues 外围组织symptoms 症候群transgenic 遗传转移,转基因(植物)usage n.用法, 习惯, 待遇gene cloning 基因克隆CC;cell culture 细胞培养Stem cell transplantation 干细胞移植Screening 筛选、筛查Hippocampus 海马medullary substance 髓质cerebromedullary tube、neural tube 神经管notochord脊索primitive streak 原条 primitive knot、primitive node、protochordal knot 原结gray matter and white matter 灰质和白质myelin sheath髓鞘spinal cord 脊髓posterior root ganglion背根节dorsal columns背柱,脊柱(脊髓)hemisection 半横断 overhemisection 过半横断 transsection 全横断bilateral 两侧的，双边的sensorimotor cortex 感觉运动皮质transferring 转铁蛋白molecular mechanism 分子机制 signal transduction 信号转导 signal pathway 信号通路penicillin 盘尼西林primary culture 原代培养 serial subcultivation 传代培养Januskinase-signal transducer and activator of transcription (JAK-STAT) JAK信号传导子及转录激活子delitescence 潜伏期cortical motor area 皮质运动区datum point、point of reference 基准点acupuncture 针灸针刺oligodendrocyte;oligodendroglial cell 少突胶质细胞 gitter cell、microglia小胶质细胞、astrocyte 星形(胶质)细胞hypothesis 假说localization 定位construction 构建down-regulation 下调 up-regulation 上调verification 验证down-stream 下游 up-stream 上游focus on 病灶关注聚焦neutralization 中和(作用),平衡,抵消,使失效neurotrophic factors(NTFs) 神经营养因子nerve growth factor (NGF)神经生长因子ciliary neurotrophic factor (CNTF) 睫状节神经细胞营养因子brain -derived neurotrophic factor (BDNF) 脑源性神经营养因子platelet-derived growth factor（PDGF）血小板源性生长因子vascular endothelial growth factor (VEGF) 血管内皮(细胞)生长因子glial-derived neurotrophic factor (GDNF) 胶质细胞源性神经营养因子neurotrophin-Ⅲ\Ⅳ\Ⅴ\Ⅵ (NT-3\4\5\6) 神经营养蛋白-3\4\5\6insulin-like growth factor ( IGF - I) 胰岛素样生长因子- Iacid fibroblast growth factor (aFGF) 酸性成纤维生长因子basic fibroblast growth factor（bFGF）碱性成纤维细胞生长因子glial fibrillary acidic protein（GFAP）神经胶质酸性蛋白interleukin-1（IL-1）白介素-1proinflammatory cytokines 致炎(炎症前)细胞因子immune response 免疫应答 immune privilege 免疫赦免innate immunity 先天免疫性、天然免疫acquired immunity 后天免疫proinflammatory 促炎症反应apoptosis 凋亡程序化死亡ERK 细胞外信号调节激酶conjugated to 接合的,结合的,共轭的,共役的,连接的protease 蛋白酶amino acids氨基酸traumatize 使受外伤,使受精神创伤tubulin. 微管素、微管蛋白collagen 胶原 collagen protein 胶原蛋白villus、chorionic 绒毛、绒毛膜的 microvillus（ＭＶ）微绒毛amniotic cavity羊膜腔Varicosity 膨体autophilia 自尊癖,利己狂,自恋heterosexuality 异性性欲、异性恋 homosexuality 同性性欲、同性恋autosomal recessive 常染色体隐性遗传dehydrogenase 脱氢酶,去氢酶encode v. 改为电码; [计算机]编码uterus 子宫homozygous 纯合的,同型结合的plasma 血浆,原浆,原生质profound mental retardation 极重度精神发育迟缓(智商低于20) recessive 隐性,劣势的,隐性的,退缩的syrup 糖浆(剂),糖汁,浆。

Unit 4 Section ADNA Strand Breaks and Chromosomal Abberations Inducedby Ionizing Radiation（1）1.What is the principal target for the biologic effects of radiation on the basis of strong circumstantial evidence?基于详细的证据，辐射生物效应的主要靶点是什么？2. What is the structure of a deoxyribonucleic acid molecule?脱氧核糖核酸分子的结构是什么?DNA Strand BreaksDNA链断裂There is strong circumstantial evidence to indicate that deoxyribonucleic acid (DNA) is the principal target for the biologic effects of radiation, including cell killing mutation, and carcinogenesis. A consideration of the biologic effects of radiation therefore must begin logically with a description of the breaks in DNA caused by charged-particle tracks and by the chemical species produced.有足够多且详细的证据表明脱氧核糖核酸是辐射生物效应的主要的靶，包括细胞杀伤性突变和致癌作用。

因此，从带电粒子和化学产物导致的DNA链断裂来考虑辐射生物效应是较合乎逻辑的。

Persistent activation of p38mitogen-activated protein kinase in a mousemodel of familial amyotrophic lateral sclerosis correlateswith disease progressionM.Tortarolo,a,1P.Veglianese,a,1N.Calvaresi,a A.Botturi,b C.Rossi,cA.Giorgini,c A.Migheli,d and C.Bendotti a,*aLaboratory of Molecular Neurobiology,Department of Neuroscience,Istituto di Ricerche Farmacologiche Mario Negri,Milano,ItalybIstituto Auxologico Italiano,IRCCS Ospedale S.Luca,Milano,Italy cConsorzio Mario Negri Sud,S.Maria Imbaro,Chieti,ItalydLaboratory of Neuropathology,Department of Neuroscience,University of Turin,ItalyReceived 18July 2002;revised 19December 2002;accepted 2January 2003AbstractThe p38mitogen-activated protein kinase (p38MAPK)is activated via phosphorylation in neurones and glial cells by a variety of stimuli including oxidative stress,excitotoxicity,and inflammatory cytokines.Activated p38MAPK can in turn induce phosphorylation of cytoskeletal proteins and activation of cytokines and nitric oxide,thus contributing to neurodegeneration.We investigated the expression and distribution of p38MAPK in the spinal cord of transgenic mice expressing a superoxide dismutase 1mutation (SOD1G93A),a model of familial amyotrophic lateral sclerosis (ALS).Accumulation of p38MAPK was found by immunoblotting in the spinal cord of G93A mice during the progression of disease,but no changes were detected in its mRNA levels.Immunostaining for phosphorylated p38MAPK in lumbar spinal cord sections of SOD1G93A mice at the presymptomatic and early stages of disease showed an increased labeling in motor neurones that colocalized with phosphorylated neurofilaments in vacuolized perikarya and neurites,as detected by confocal microscopy.As the disease progressed,activated p38MAPK also accumulated in hypertrophic astrocytes and reactive microglia,as demonstrated by colocalization with GFAP and CD11b immunostaining,respectively.These data suggest that activation of p38MAPK in motor neurons and then in reactive glial cells may contribute,respectively,to the development and progression of motor neuron pathology in SOD1G93A mice.©2003Elsevier Science (USA).All rights reserved.IntroductionAmyotrophic lateral sclerosis (ALS)is a late-onset neu-rodegenerative disease causing progressive loss of motor neurones in the brain and spinal cord,leading to muscular weakness,paralysis,and early death.The cause of motor neurone death is still unknown;however,the discovery that about one-fifth of the familial forms of ALS are due to mutations in the Cu/Zn superoxide dismutase (SOD1)geneprovided new insight on the disease process (Rosen et al.,1993;Shaw et al.,1998).Familial and sporadic forms have the same clinical and pathological profiles,and may there-fore share common biochemical mechanisms.ALS is re-garded a multifactorial disease which may involve a com-mon cascade of events leading to motor neurone toxicity.Oxidative stress (Ferrante et al.,1997),glutamate-induced excitotoxicity (Rothstein,1995),and inflammatory pro-cesses (McGeer and McGeer,2002)are indicated as poten-tial causes of ALS.These pathological factors may lead to cell death through various intracellular signalling pathways,including the mi-togen activated protein kinase (MAPK)pathways (Mielke and Herdegen,2000).MAPKs are a family of related serine/threonine kinases that integrate various signals directing*Corresponding boratory of Molecular Neurobiology,De-partment of Neuroscience,Mario Negri Institute for Pharmacological Re-search,Via Eritrea 62,20157Milano,Italy.Fax:ϩ39-02-3546277.E-mail address:Bendotti@marionegri.it (C.Bendotti).1Contributed equally to thiswork.Molecular and Cellular Neuroscience 23(2003)180–/locate/ymcne1044-7431/03/$–see front matter ©2003Elsevier Science (USA).All rights reserved.doi:10.1016/S1044-7431(03)00022-8cellular responses to proliferative cues or stressful stimuli.The MAPK family includes extracellular signal-regulated kinases (ERKs),c-Jun-N-terminal kinase or stress activated protein kinase (JNK/SAPK),and p38MAP kinase (p38MAPK).Activation of p38MAPK is often correlated with neuronal degeneration (Horstmann et al.,1998;Skaper and Walsh 1998)and recently it has been shown to occur in neurodegenerative diseases such as Alzheimer ’s and other tau-related diseases (Atzori et al.,2001;Zhu et al.,2000).The p38MAPK is implicated in various functions such as phosphorylation of cytoskeletal proteins and biosynthesis of cytokines and nitric oxide (Mielke and Herdegen,2000;Ono and Han,2000),mechanisms supposed to play a role in the motor neurone degeneration in ALS (Cleveland and Rothstein,2001;Julien,2001;Rowland and Shneider,2001).In order to determine whether p38MAPK pathway is involved in motor neurone degeneration,we examined the expression and distribution of activated p38MAPK in the spinal cord of transgenic mice carrying the human mutant SOD1with the G93A mutation (SOD1G93A),a mouse model of familial ALS (Gurney et al.,1994),during the progression of the disease.We focused our attention on p38MAPK activation not only in motor neurones but also inastrocytes and ing confocal microscopy we showed that p38MAPK was initially activated only in the spinal motor neurones of transgenic mice at the presymp-tomatic stage of the disease and colocalized with phosphor-ylated neuro filaments in the cell bodies and proximal neu-rites.As the disease progressed,the astrocytes and microglial cells also showed a progressive activation of this kinase.Resultsp38MAPK levels progressively increase in the spinal cord of SOD1G93A miceImmunoblot analysis of p38MAPK and its phosphory-lated (activated)form (p38MAPK-P)in spinal cord extracts from control and transgenic mice revealed a single band of about 40kDa corresponding to the band obtained from human monocytes stimulated with fMLP and used as pos-itive control (Fig.1A).An antibody selectively recognizing p38MAPK-P did not reveal any signal in the spinal cord extracts fromnon-Fig.1.Expression of p38MAPK protein in the spinal cord of SOD1G93A mice at early (10weeks,10w)and late (19weeks,19w)stages of the disease in comparison to controls (CTR).(A)Representative immunoblots for phosphospeci fic p38MAPK (P-p38),total p38MAPK (p-38),AKT,and actin.The first lane (ϩ)shows the positive control for P-p38in human monocytes treated with f-MLP (50␮g proteins).Other lanes are tissue homogenates (120␮g protein per lane)from the whole spinal cord.(B)Quantitative analysis of the immunoblots for p38and P-p38MAPK.The optical density was evaluated for each autoradiographic band and the ratios between p38or P-p38to actin were used for statistical analysis,by the nonparametric Kruskal –Wallis test (*P Ͻ0.05).(C)Representative RT-PCR analysis of p38and ␣-actin mRNA from spinal cord of SOD1G93A mice at 10and 19weeks of age,and controls.Histograms of the quantitative analysis of p38MAPK mRNA relative to ␣-actin mRNA show no signi ficant differences among groups.181M.Tortarolo et al./Molecular and Cellular Neuroscience 23(2003)180–192transgenic mice(Fig.1A).By contrast,a moderately intense band was found in spinal cord extracts from19-week-old SOD1G93A mice,while in10-week-old SOD1G93A mice we sometimes observed a very faint specific band which was under the threshold for quantitative detection(Figs.1A and B).In SOD1G93A mice at10and19weeks of age, corresponding to presymptomatic and late stage of the dis-ease respectively,levels of total p38MAPK in the spinal cord were1.5-and2.2-fold higher than those found in nontransgenic littermate mice(Fig.1B).These changes were specific for p38MAPK since no differences were ob-served in AKT,another intracellular kinase involved in the cell signalling cascade,which is also activated by stimuli such as cytokines and stress(Fig.1A).The increase of p38MAPK was not associated with changes in the levels of its mRNA(Fig.1C).Although p38MAPK-P was undetectable by immuno-blotting in spinal cord extracts from control mice,the same antibody showed a specific immunolabeling in spinal cord tissue sections.Specificity of p38MAPK-P immunoreactiv-ity was demonstrated by loss of signal after preadsorbtion of the primary antibody with the immunizing peptide(not shown).Fig.2shows the distribution of p38MAPK-P im-munofluorescence in lumbar spinal cord sections from transgenic SOD1G93A mice at the presymptomatic(8 weeks);early(14weeks),and late(19weeks)stages of the disease,compared to controls and SOD1wild-type(WT) transgenic mice.Control mice had a high level of immuno-fluorescence in the substantia gelatinosa of the dorsal horn. Numerous cells were also moderately labeled in the whole gray matter of the spinal cord,with no signal in the white matter.High-power magnification of motor neurones indi-cated a specific immunoreactivity of p38MAPK-P in the cytosol.Only a few motor neurones showed a prominent immunofluorescence in the nuclei.Interestingly,presymp-tomatic SOD1G93A mice(8weeks)showed a marked in-crease of p38MAPK-P immunofluorescence exclusively in the motor neurones of the ventral horn.The reactivity was mostly in perikarya and proximal neurites.A similar pattern was found in motor neurons of transgenic mice at the symptomatic stage(14weeks)showing a marked vacuol-ization of the cell body.p38MAPK-P immunofluorescence appeared concentrated around vacuoles.At this age,a slight increase of immunofluorescence was also observed in the white matter of SOD1G93A mice.Immunofluorescence of p38MAPK-P in substantia gelatinosa was unchanged at either presymptomatic or early disease-stage SOD1G93A mice(8and14weeks)compared to controls.In SOD1G93A mice with advanced disease(19weeks),p38MAPK-Pflu-orescence was increased in the whole gray matter of the spinal cord including the substantia gelatinosa.The few remaining motor neurones still showed a marked increase in p38MAPK-P immunoreactivity compared to controls.The scattered intense immunofluorescence around the degener-ating,highly vacuolized motor neurones was probably due to neuronal fragments as well as to nonneuronal cells.p38MAPK-P immunostaining was similar in SOD1WT transgenic mice and nontransgenic controls. Colocalization of p38MAPK-P and neurofilaments p38MAPK-P immunoreactivity was examined in relation to phosphorylated and nonphosphorylated neurofilaments labeled with SMI31and SMI32antibodies,respectively. SMI31reacts with a phosphorylated epitope in extensively phosphorylated neurofilament H and,to a lesser extent, with neurofilament M.The distribution of SMI31and p38MAPK-P immunofluorescence in the ventral horn of TgSOD1G93A mice at different stages of the disease and in controls is reported in Fig.3.In control mice SMI31reacted broadly with neuropil,axon terminals,and dendrites,while neuronal cell bodies were totally unstained.In transgenic SOD1G93A mice at different ages,including the presymp-tomatic stage,SMI31staining was found also in large neu-rone cell bodies of ventral horn showing vacuolization. These cells also show increased immunoreactivity for p38MAPK-P which appeared colocalized with phosphory-lated neurofilaments.Double labeling was also found in large vacuolized neurites.As shown in Fig.4,unlike SMI31,SMI32antibody markedly labeled the neuronal cell bodies,dendrites,and some thick axons in the ventral horns.SMI32reacts with a nonphosphorylated epitope in neurofilament H and the staining is masked when the epitope is phosphorylated.In controls,SMI32immunoreactivity was homogeneously dis-tributed in the neuronal perikarya and axons.In transgenic SOD1G93A mice,some neurones showed a nonhomog-enous distribution of SMI32immunoreactivity in their cell bodies with intense immunoreactivity in some regions.In these regions,the immunostaining for p38MAPK-P was either low or absent.On the other hand,highly vacuolized neuronal cell bodies showing high levels of p38MAPK-P immunoreactivity were weakly immunoreactive for SMI32, further indicating lack of colocalization of the two proteins. Axons in the spinal cord sections of transgenic SOD1G93A mice appeared less immunostained for SMI32than controls. Colocalization of Pp38MAPK and glialfibrillary acidic protein(GFAP)immunofluorescenceFig.5shows a high-power magnification of the immu-nostaining for GFAP and p38MAPK-P in the ventral horn of controls and transgenic SOD1G93A mice.In control mice,the astrocytes stained by GFAP antibody showed thin processes originating from a small cell body intensely immunostained.No colocalization was observed with p38MAPK-P.In transgenic SOD1G93A mice at the presymptomatic stage(8weeks)immunostaining for GFAP was not different from that for controls;no colocalization was found with p38MAPK-P.Starting from14weeks of age,GFAP-immunostained astrocytes appeared hypertro-phic with thick processes and showed partial immunoreac-184M.Tortarolo et al./Molecular and Cellular Neuroscience23(2003)180–192tivity for p38MAPK-P.This condition was particularly ev-ident in transgenic SOD1G93A mice at the late stage of disease(19weeks),when the astrocytes markedly increased their volume and appeared strongly labeled with both GFAP and p38MAPK-P antibodies.In many sections from SOD1G93A transgenic mice,hypertrophic double-labeled astrocytes and their processes were located around a degen-erating motor neurone.Colocalization of p38MAPK-P and CD11b-associated immunofluorescenceA strong increase of activated microglia was detected in the gray matter of the spinal cord of SOD1G93A mice at the late stage of disease,as demonstrated by the intense immu-noreactivity of CD11b with the OX42antibody(Fig.6). Microglial cells had round cell bodies and thick short pro-cesses:in these cells,OX42immunostaining almost com-pletely colocalized with p38MAPK-P(Fig.6).At earlier stages,8and14weeks of age,an increase of activated microglia was observed in the ventral horn of SOD1G93A mice compared to controls(data not shown). However,in this case OX42immunostaining was detected only using the TSA amplification system,so we could not analyze the colocalization with p38MAPK-P. DiscussionThe present study reports a progressive accumulation of p38MAPK in the spinal cord of SOD1G93A mice that parallels motor neurone degeneration.Accumulation of the protein was likely due to its reduced degradation rather than to increased synthesis,as we did not detect changes in the levels of its mRNA.It is noteworthy that accumulation of p38MAPK was associated to activation of the kinase,as revealed by the increased p38MAPK-P immunostaining which occurredfirst in the motor neurones,before clinical symptoms became evident,and later in astrocytes and mi-croglial cells.The lack of immunoblotting signal relative to p38MAPK-P at presymptomatic stages in SOD1G93A mice was likely due to the limited effect in motor neurons.At the later stages,when a remarkable activation of p38MAPK occurred also in the reactive astrocytes and microglial cells, the immunoband was clearly revealed.These results strengthen the importance of associating the immunohisto-chemical analysis on tissue sections with the immunoblot-ting from the whole tissue homogenate.We recently reported that at the presymptomatic stage of the disease,in8-week-old and even younger SOD1G93A mice,motor neurones showed remarked subcellular patho-logical alterations such as massive cytoplasm vacuolization and mitochondrial swelling.These morphological changes were never associated with ultrastructural apoptotic features of the nuclei or with nuclear DNA fragmentation,as de-tected by in situ end-labeling(ISEL)technique and acti-vated caspase-3immunostaining even at the later stages of the disease(Bendotti et al.,2001a;Migheli,et al.,1999). Thus,while studies in vitro implicate p38MAPK activation in apoptosis(Kummer et al.,1997),the present in vivo data indicate that an increase of this phosphorylated kinase may precede the motor neurone death,but is not associated with apoptosis.In line with this observation,intracortical injec-tion of the excitotoxin quinolinic acid in rats produced a rapid increase of phosphorylated p38MAPK immunoreac-tivity in the site of lesion,but the dying cell did not show apoptotic morphology nor activation of caspase-3(Ferrer et al.,2001).That activation of p38MAPK may be unrelated to apoptotic changes was also demonstrated in different neu-rodegenerative disease with tau pathology(Atzori et al., 2001).One mainfinding in the present study is the colocaliza-tion of activated p38MAPK with phosphorylated neurofila-ments in the motor neurone cell bodies and proximal axons in SOD1G93A mice.Phosphorylated neurofilaments are normally distributed along the axons or in the terminals. When they become hyperphosphorylated,their transport can be impaired,leading to their accumulation(Bajaj et al., 1999;Miller et al.,2002;Tu et al.,1995).Accumulation of neurofilaments has been reported in motor neurones of pa-tient with ALS as well as in different mouse models of motor neurone degeneration(Migheli et al.,1994;Rouleau et al.,1996;Tu et al.,1996).Slowing of neurofilaments transport was considered an early pathological feature in SOD1mutant mice(Williamson and Cleveland,1999; Zhang et al.,1997).It was reported that glutamate induces neurofilament hyperphosphorylation by members of the MAPK family in cultured neurones(Ackerley et al.,2000).Among theories in the pathogenesis of ALS,glutamate excitotoxicity is considered to play a major role in motor neurone degener-ation.Loss of the astroglial glutamate transporter GLT-1, found in the motor cortex and spinal cord of patients with ALS(Rothstein et al.,1992),may lead to increased extra-cellular concentrations of glutamate with subsequent exci-totoxic degeneration of motor neurones.Although we ob-served a significant decrease of GLT-1in the ventral horn of SOD1G93A mice only at the symptomatic stage of disease (Bendotti et al.,2001b),there is evidence that mutant SOD1 inactivates this glutamate transporter under oxidative con-ditions(Trotti et al.,1999).Thus accumulation of extracel-lular glutamate may cause early activation of MAPK ki-nases,including p38MAPK,leading to neurofilaments hyperphosphorylation.This effect may contribute to the early events in the cascade leading to motor neuron death.p38MAPK is rapidly activated by proinflammatory cy-tokines like IL-1and tumor necrosis factor(TNF)(Lee et al.,2000)and immunoinflammatory factors may be in-volved in motor neuron degeneration(McGeer and McGeer, 2002).An increase of activated microglia and IgG and its receptor for the FC portion(FcgRI)was detected in the ventral horn of transgenic SOD1G93A mice,at the185M.Tortarolo et al./Molecular and Cellular Neuroscience23(2003)180–192presymptomatic stages of the disease(Alexianu et al., 2001).We also observed an increase of activated microglia in the ventral horn of these mice,although the effect was more pronounced at late stages of the ing mi-croarray techniques to examine a series of mRNAs ex-pressed in the spinal cord of SOD1G93A mice.TNF was detected as one of the earliest factors to be induced(Yoshi-hara et al.,2002).An increased TNF production might, therefore,contribute to early and persistent activation of p38MAPK in motor neurones.With the progression of disease,activated p38MAPK accumulates not only in degenerating motor neurones but also in hypertrophic astrocytes and reactive microglia,as demonstrated by colocalization with GFAP and CD11b. TNF and IL-1markedly activate p38MAPK in mouse as-trocytes in vitro(Lee et al.,2000).Activation of p38MAPK in astrocytes is specifically involved in the activation of inducible nitric oxide synthase(iNOS),resulting in sus-tained release of large amounts of nitric oxide(NO)(Da Silva et al.,1997).A series of evidences indicates that the NO may be a causative molecule of motor neurone death in ALS,particularly in mutant SOD1-linked ALS,through the formation of highly reactive molecules such as peroxynitrite (Beckman et al.,1993;Urushitani and Shimahama,2001). Reaction of peroxynitrite with mutant SOD1is reported to produce nitronium-like nitrating species that nitrate the ty-rosine residues of different proteins and accumulate in the cytoplasm of motor neurones with toxic effect(Ferrante et al.,1997).Thus,activation of p38MAPK in reactive astro-cytes and microglia cells by cytokines and NO may in turn activate the production of these inflammatory factors deter-mining a redundant mechanism that contribute to the pro-gression of ALS pathology.Activation of p38MAPK in microglia can also be in-duced by glutamate and this mechanism has been proposed in excitotoxic neuronal death in mixed neuronal–glia spinal cord cultures(Tikka et al.,2001).Minocycline,a tetracy-cline derivative,showed a neuroprotective effect against excitotoxicity in mixed spinal cord cultures,by inhibiting the proliferation and activation of microglia,and by inhib-iting p38MAPK activation(Tikka et al.,2001).Interest-ingly,three recent studies reported that minocycline delayed the onset of disease and prolonged the survival in SOD1 mutant mice(Kriz et al.,2002;Van Den Bosch et al.,2002; Zhu et al.,2002).Although some authors assumed that the drug had a direct effect on the mitochondria inhibiting cytochrome c release(Zhu et al.,2002),other effects of minocycline,such as inhibition of microglia p38MAPK, have also been considered(Kriz et al.,2002;Van Den Bosch et al.,2002).In conclusion,the present study shows an early and persistent activation of p38MAPK in an animal model of motor neurone degeneration.Since activated p38MAPK increases in motor neurones before these cells undergo massive neuropathological alterations and it colocalizes with phosphorylated neurofilaments,we suggest that this mechanism contributes significantly to motor neurone death in mice with SOD1mutations.Activation of p38MAPK in reactive astrocytes and microglia cells at the later stages during disease progression strengthens the possible contrib-utory role of this pathway in the pathological process of ALS.Further experiments aimed at blocking activation of p38MAPK in the spinal cord of SOD1G93A mice are needed to verify these hypotheses,with the hope of achiev-ing some useful therapeutic intervention for ALS. Experimental methodsAnimalsFemale transgenic mice were maintained at a tempera-ture of21Ϯ1°C with relative humidity55Ϯ10%and12h of light.Food(standard pellets)and water were supplied ad libitum.Transgenic mice originally obtained from Jackson Laboratories and expressing a high copy number of mutant human SOD1with a Gly-93-Ala substitution(SOD1G93A) or wild-type human SOD1(SOD1WT)mice were bred and maintained on a C57BL/6mice strain at the Consorzio Mario Negri Sud,S.Maria Imbaro(CH),Italy.Transgenic mice are identified by polymerase chain reaction(PCR) (Rosen et al.,1993).In this study female SOD1G93A mice were killed at8–10,14,and19weeks of age corresponding respectively to presymtomatic,early symptomatic,and late stages of the progression of the motor dysfunction(Bendotti et al.,2001b).Female SOD1WT at19weeks of age and nontransgenic age-matched littermates were used as con-trols.Procedures involving animals and their care were con-ducted in conformity with the institutional guidelines that are in compliance with national(D.L.No.116,G.U.Suppl. 40,Feb.18,1992,Circolare No.8,G.U.,14luglio1994) and international laws and policies(EEC Council Directive 86/609,OJ L358,1DEC.12,1987;NIH Guide for the Care and use of Laboratory Animals,U.S.National Research Council,1996).ImmunoblottingMice(control and SOD1G93A at10and19weeks of age,three tofive mice each group)were killed by decapi-tation and the spinal cord were rapidly removed,frozen in 2-methylbutane atϪ45°C,and stored atϪ80°C until the experiment.Tissues were homogenized in ice-cold lysis buffer(20mM Tris/HCl,pH7.5;10mM EGTA;500mM ␤-glycerophosphate;10mM magnesium chloride;2mM DTT;1mM Na3VO4;1mM PMSF;1%Triton;10mg/mlleupeptin;10mg/ml aprotinin)using a Teflon-on-glass ho-mogenizer and centrifugated at13,000rpm for30min at 4°C.Protein concentrations from supernatant were deter-mined using a Bio-Rad protein assay(Bio-rad).Lysates(50␮g)from human monocytes treated for3min with10Ϫ7M188M.Tortarolo et al./Molecular and Cellular Neuroscience23(2003)180–192N-formylmethionine-leucyl-phenyl-alanine(f-MLP)were loaded as positive controls.Samples were then boiled for3 min in loading buffer(100mM Tris,pH7.5;15%2-mer-captoethanol;4%SDS;15%glycerol;5mM EGTA;5mM EDTA;0.2%bromphenol blue)and100to150␮g proteins/ lane were run on a10%SDS–polyacrylamide gel and trans-ferred to a nitrocellulose membrane(Protran,Scheicher and Schuell).The membrane was incubated in blocking buffer TBST(1ϫTBS;0,1%Tween-20)with5%nonfat milk for 2h at room temperature,followed by overnight incubation at4°C with primary antibodies:rabbit anti-p38MAPK or rabbit anti-p38MAPK phospho-specific(both Calbiochem, 1:1000),in TBST with5%BSA,goat anti-AKT1(Santa Cruz Biotechnology,1:2000)or with mouse anti-actin (Chemicon,10mg/ml)in TBST buffer with5%nonfat dry milk.The blots were then washed in TBST and incubated with secondary antibodies:anti-rabbit,anti-goat,or anti-mouse IgG conjugated to horseradish peroxidase(1:2000) in TBST with5%non-fat dry milk,for1h at room tem-perature.Blots were developed by the ECL technique(Am-ersham Pharmacia Biotech)according to the manufacturer’s instructions.Densitometric analysis of autoradiographic bands was done using a computer-assisted image analysis system(AIS3.0-Imaging Research,Inc.,St.Catherines, Ontario,Canada).RNA extraction and semiquantitative RT-PCRTotal RNA was extracted from frozen spinal cord sam-ples of controls and SOD1G93A mice at10and19weeks of age using the acid guanidium–phenol–chloroform method, previously described(Bendotti et al.2000).Two micro-grams of total RNA was used for cDNA synthesis by MuLV reverse trascriptase(final concentration2.5U/␮l),in the presence of oligo(final concentration2.5␮M),dNTP mix (final concentration1mM each component),RNase inhib-itor(1U/␮l),MgCl2(final concentration5mM)in PCR buffer II1X(Gene Amp RNA PCR Kit,Perkin–Elmer). Incubation was carried out for30min at42°C,followed by 5min at99°C and5min in ice.An aliquot(10/␮l)of each cDNA synthetized in the RT reaction was used for PCR amplification of cDNA encoding for p38MAPK and␣-ac-tin,in the presence of AmpliTaq DNA polymerase(final concentration2.5U/100␮l),MgCl2(final concentration2 mM),PCR buffer II1X(Gene Amp RNA PCR Kit,Perkin–Elmer),and sense and antisense primers(final concentration 0.15␮M each).The sequences of primers for␣-actin are 5Ј-CACACTGTGCCCATCTACGA-3Јand5Ј-CACAG-GATTCCATACCCAAG-3Ј.The sequences of primers for p38MAPK are5Ј-CCGGATCCTGGAAGATGTCGCAG-GAGAG-3Јand5Ј-CCGGATCCCCAGGTGCT-CAGGA-CACCAT-3Ј.In order to verify that amplification of each gene was within the linear range p38MAPK was amplified for25,28,31,34cycles and␣-actin for16,19,22,25 cycles.Quantitative analysis was done by comparing the band of product of28cycles for p38MAPK and of19cycles for␣-actin.Denaturation was reached at94°C for1min, annealing at58°C for1min,and extension at72°C for1min.Each sample was run on1%agarose gel and the opticaldensity of bands was quantified by Image Quant analyzer(Molecular Dynamics).Immunofluorescence in spinal cord sectionsFor immunofluorescence analysis,mice(three nontrans-genic and four SOD1G93A for each age considered andfour19-week-old SOD1WT)were anesthetized with Eq-uithesin(1%phenobarbitol/4%(vol/vol)chloral hydrate,30␮l/10g,ip)and transcardially perfused with20ml saline followed by50ml of sodium phosphate buffered4%para-formaldehyde solution.Spinal cords were rapidly removed,postfixed infixative for2h,transferred to20%sucrosesolution in PBS overnight,then to30%sucrose solutionuntil they sank,andfinally frozen in2-methylbutane at Ϫ45°C.Sections(30␮m)were cut on a cryostat atϪ20°C through the lumbar spinal cord in the transverse plane at the L3–5level.Immunofluorescence of p38MAPK-PSections were incubated in10%normal goat serum,0.1%Triton X-100in PBS for60min and subsequentlywith primary antibody to phosphorylated p38MAPK.(p38MAPK-P,1:500dilution,rabbit polyclonal,Calbio-chem)in0.1%Triton X-100and1%normal goat serumovernight at5°C under constant shaking.They were thenincubated in biotinylated anti-rabbit antibody(1:200dilu-tion,Vector Laboratories)in PBS containing1%normalgoat serum and0.1%Triton X-100for60min at roomtemperature.The secondary biotinylated antibody was re-vealed by TSA kit amplification(Renaissance Direct,Du-pont NEN,NEL705A);the sections were incubated in TNBbuffer(0.1M Tris–HCl,pH7.5/0.15M NaCl/0.5%block-ing reagent)for90min,and then in streptavidin–HRP inTNB(1:50dilution)for30min.After three washes withTNT buffer(0.1M Tris–HCl,pH7.5/0.15M NaCl/0.05%Tween20)the streptavidin–HRP was revealed with tyra-mide conjugated to Cy5(red,1:50dilution)in amplificationdiluent for10min,and then washed with PBS.Processedsections were mounted on slides and coverslipped withFluorsave(Calbiochem).Colocalization with neurofilaments(phosphorylatedSMI31or nonphosphorylated SMI32)Sections prelabeled with p38MAPK-P antibody wereincubated in10%normal goat serum in PBS for60min andkept overnight at5°C in solution containing antibodySMI31or SMI321(1:5000dilution,mouse monoclonal,Sternberg)and3%normal goat serum in PBS.The next day,the sections were incubated in secondary anti-mouse anti-body conjugated to Alexa488(green,1:500dilution,Mo-189M.Tortarolo et al./Molecular and Cellular Neuroscience23(2003)180–192。

大学细胞生物学考试(试卷编号161)1.[单选题]关于病毒的增殖，下列说法错误的是（ ）。

[南京师范大学2008研]A)必须在细胞内进行B)病毒的核酸和病毒蛋白均在宿主细胞内合成C)病毒是否侵染细胞主要取决于病毒表面的识别结构D)存在以RNA为模板反转录为DNA的过程答案:C解析:2.[单选题]细胞内Ca2＋的储备库是（ ）。

[中国科学院大学2016研]A)细胞质B)内质网C)高尔基体D)溶酶体答案:B解析:3.[单选题]Caspase家族的蛋白水解酶能特异的断开（ ）A)半胱氨酸残基后的肽键B)天冬氨酸残基后的肽键C)天冬酰胺残基后的肽键D)丝氨酸残基后的肽键答案:B解析:4.[单选题]下列关于构成生物体的化学元素的叙述中，正确的是（ ）。

[湖南大学2007研]A)生物群落中，碳是以CO2的形式在生产者、消费者和分解者之间传递B)体内血钠含量过低，会引起心肌的自动节律异常，甚至导致心律失常C)落叶与正常叶相比Ca2＋和Mg2＋的含量基本不变D)微量元素B能促进花粉的萌发和花粉管的伸长答案:D解析:5.[单选题]一氧化氮受体是（ ）。

[南开大学2011研]A)G蛋白偶联受体B)鸟苷酸环化酶C)腺苷酸环化酶D)受体酪氨酸激酶（G-cyclase，GC）的激活是NO发挥作用的主要机制。

6.[单选题]负责从内质网到高尔基体物质运输的是（ ）。

A)网格蛋白有被小泡B)COPⅡ有被小泡C)COPⅠ有被小泡D)胞内液泡答案:B解析:A项，网格蛋白有被小泡主要负责蛋白质从高尔基体TGN向质膜和胞内体及溶酶体运输；C项，COPⅠ有被小泡负责将内质网逃逸蛋白质从高尔基体返回内质网等。

7.[单选题]核仁组织者位于中期染色体的（ ）A)主缢痕B)随体C)着丝粒D)次缢痕答案:D解析:8.[单选题]下列细胞器中，不属于细胞内膜系统的是（ ）A)溶酶体B)内质网C)高尔基体D)核糖体答案:D解析:9.[单选题]异染色质时的染色质（ ）。

第一章测试1.下列生物中属于原核生物的一组是（）。

A:绿藻和根瘤菌B:水绵和紫菜C:蓝藻和硝化细菌D:蓝藻和酵母菌答案:C2.在英国引起疯牛病的病原体是（）。

A:立克次体B:支原体C:朊病毒D:RNA病毒答案:C3.以下哪一种描述不属于细胞的基本特征？（）。

A:细胞具有细胞核和线粒B:细胞能够自行增殖C:细胞拥有一套遗传机制D:细胞能对刺激产生反应答案:A4.有关原核细胞与真核细胞的比较，下列说法错误的是（）。

A:真核细胞内有一个较复杂的骨架体系，原核细胞内并没有明显的骨架系统B:现有资料表明真核细胞由原核细胞进化而来，自然界真核细胞的个体数量比原核细胞多C:真核细胞内膜系统分化，内部结构和功能的区域化和专一化，各自行使不同的功能D:真核细胞基因表达有严格的时空关系，并具有多层次的调控答案:B5.由于细菌细胞中没有内膜系统，所以细菌质膜兼有内膜的许多功能，如：具有线粒体的呼吸作用，具有内质网的分泌作用，具有核膜的保护遗传物质的作用。

（）A:对B:错答案:B6.细胞内的生物大分子是指蛋白质、脂类和DNA等。

（）A:对B:错答案:B7.器官的大小主要取决于细胞的数量，与细胞数量成正比，而与细胞大小无关。

（）A:对B:错答案:A第二章测试1.光学显微镜最容易观察到的细胞器或细胞结构是（）。

A:线粒体B:内质网C:细胞核D:微管答案:C2.冰冻蚀刻技术主要用于（）。

A:电子显微镜B:光学显微镜C:原子力显微镜D:激光共聚焦显微镜答案:A3.以下哪些技术一般不用于分离活细胞？（）A:超速离心B:细胞电泳C:差速离心D:流式细胞术答案:AC4.建立分泌单克隆抗体的杂交瘤细胞是通过下列哪种技术构建的（）？A:基因转移B:病毒转化C:核移植D:细胞融合答案:D5.正常细胞培养的培养基中常需加入血清，主要是因为血清中含有（）。

A:生长因子B:氨基酸C:核酸D:维生素答案:A6.适于观察培养瓶中活细胞的显微镜是（）。