Construction and characterization of a bacterial artificial chromosome library of marine macroalga Porphyra yezoensis (Rhodophyta)

A large-DNA-fragment library is necessary for research into the Porphyra genome. In this study, a bacterial artificial chromosome (BAC) library of Porphyra yezoensis was constructed and characterized. The library contains 54,144 BAC clones with an average insert size of about 65 kb and fewer than 0.7% of clones without large inserts. Therefore, its capacity is more than 6.6 P. yezoensis genome equivalents, and the probability of recovering any nuclear DNA sequence from the library is higher than 99%. The library shows good fidelity and stability. A putative trehalose-6-phosphate synthase (TPS) gene was successfully screened out from the library. The above results show that the library is useful for gene cloning and genomic research in P. yezoensis.

Proteomic analysis of differentially expressed proteins in lymphoid organ of Fenneropenaeus chinensis response to Vibrio anguillarum stimulation

To gain an insight into the function of shrimp lymphoid organ at protein level, we analyzed the proteome of lymphoid organ in healthy Chinese shrimp Fenneropenaeus chinensis (F. chinensis) through two-dimensional gel electrophoresis (2-DE) based proteomic approach. A total of 95 spots representing 75 protein entries were identified by liquid chromatography tandem mass spectrometry (LC-MS/MS) with both online and in-house database. According to Gene Ontology (GO) annotation of biological process, the identified proteins were classified into 13 categories. Among them, approximately 36% of proteins related to cytoskeleton are noticeable. Then, a comparative proteomic approach was employed to investigate the differentially expressed proteins in lymphoid organ of Vibrio anguillarum-challenged F. chinensis. At 24 h post-injection (hpi), 17 differentially expressed protein spots were successfully identified, including 4 up-regulated protein spots (represent 4 proteins: cathepsin L protein similar to squid CG16901-PC, protein kinase C and protein similar to T-complex Chaperonin 5 CG8439-PA), and 13 down-regulated protein spots (represent 9 proteins: actin, beta-actin, cytoplasmic actin CyII, alpha tubulin, beta tubulin, protein similar to proteasome delta, vacuolar ATP synthase subunit B, elongation factor 2, carboxypeptidase B). These data may help us to understand the function of lymphoid organ and the molecular immune mechanism of shrimp responsive to pathogen infection. (C) 2010 Elsevier Ltd. All rights reserved.

Discovery of the genes in response to white spot syndrome virus (WSSV) infection in Fenneropenaeus chinensis through cDNA microarray

We used microarray technology to study differentially expressed genes in white spot syndrome virus (WSSV)-infected shrimp. A total of 3136 cDNA targets, including 1578 unique genes from a cephalothorax cDNA library and 1536 cDNA clones from reverse and forward suppression subtractive hybridization (SSH) libraries of Fenneropenaeus chinensis, plus 14 negative and 8 blank control clones, were spotted onto a 18 x 18 mm area of NH2-modified glass slides. Gene expression patterns in the cephalothorax of shrimp at 6 h after WSSV injection and moribund shrimp naturally infected by WSSV were analyzed. A total of 105 elements on the arrays showed a similar regulation pattern in artificially infected shrimp and naturally infected moribund shrimp; parts of the results were confirmed by semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR). The up-regulated expression of immune-related genes, including heat shock proteins (HSP70 and HSP90), trehalose-phosphate synthase (TPS), ubiquitin C, and so forth, were observed when shrimp were challenged with WSSV. Genes including myosin LC2, ATP synthase A chain, and arginine kinase were found to be down-regulated after WSSV infection. The expression of housekeeping genes such as actin, elongation factor, and tubulin is not stable, and so these genes are not suitable as internal standards for semiquantitative RT-PCR when shrimp are challenged by WSSV. As a substitute, we found that triosephosphate isomerase (TPI) was an ideal candidate of interstandards in this situation.

南大洋印度洋扇形区南极绕极流和南极沿岸流的相互作用研究

本文利用数值方法对南大洋印度洋扇形区南极绕极流（ACC）和南极沿岸流的相互作用进行了研究，重点研究了凯尔盖朗海台区ACC的非纬向性特征及其季节变化，对其非纬向性的形成机制进行了理论探讨。基于OPYC模式，我们建立了一个冰－海耦合的南大洋等密面模式，模式中全面考虑了热力学和动力学过程，采用月均的ECMWF资料作为强迫场，海－气间的热量通量和淡水通量均根据实测的大气资料计算。模式的计算域包括了整个南大洋，利用变网格技术在所关心的重点区域进行了网格加密。模拟出的南大洋环流和海冰与已有的实测结果符合良好。本文计算的德雷克海峡处ACC的流量为145Sv，较FRAM的结果（200Sv）更加接近实测值（134Sv）。对模拟结果的分析表明，ACC与沿岸流的相互作用可呈现出不同的方式。在印度洋扇形区，ACC与沿岸流非常接近，由于岸线和地形的变化在ACC和沿岸流之间形成一系列的涡旋，这里是ACC与沿岸流直接发生相互作用的地区。这种相互作用与凯尔盖朗海台区ACC的带状分布和非纬向性特征有关。ACC流经凯尔盖朗海台时，分别从海台的北部、中部和南部经过，其南部的一个分支贴近南极大陆沿岸，它与西向的陆坡流之间的相互作用构成了印度洋扇形区ACC与沿岸流相互作用的主要部分。模拟结果显示，凯尔盖朗海台以北ACC的变化以年周期为主，海台以南的变化以半年周期为主，其时间变化规律与这里风应力的变化规律是一致的。理论分析表明，凯尔盖朗海台区ACC的非纬向性主要是由风应力和海底地形的变化造成的，它通过该海台时的南北路径的分叉和转向与该海区风应力与水深之比的旋度的分布相吻合。

靶向斑马鱼VEGF的shRNA表达载体的构建及其对斑马鱼胚胎血管发育的影响

血管内皮生长因子(vascular endothelial growth factor, VEGF)是一种多功能的细胞因子，其主要作用是促进血管内皮细胞增殖和增加血管通透性，是肿瘤及正常组织血管生成的中心调控因素，以VEGF为靶点的肿瘤血管靶向性治疗成为近几年肿瘤治疗的新途径。RNAi是近年来新发展的一项反向遗传学技术，是一种研究基因功能的有力工具。斑马鱼作为一种重要的模式生物，被广泛用于胚胎的分子发育机制、疾病模型的构建以及药物筛选等研究中。然而在斑马鱼中运用RNAi技术进行基因功能研究是一个相对较新的领域，研究资料较少，并且目前进行的斑马鱼RNAi实验中，siRNA大都是通过化学方法或体外转录合成的。体外合成的siRNA在进入体内后会被降解而无法达到持久阻抑基因表达的目的。因此本研究旨在探讨VEGF特异性siRNA表达载体对斑马鱼VEGF基因的沉默作用，通过分析表型及相关细胞因子的变化，阐明VEGF对斑马鱼胚胎血管生成的影响及作用机制。 研究通过计算机辅助设计软件，针对斑马鱼VEGF mRNA不同位点设计合成了4段含siRNA特异序列的DNA单链，经退火，克隆入pSilencer 4.1-CMV neo载体CMV启动子下游，构建了重组质粒pS1-VEGF、pS2-VEGF、pS3-VEGF及pS4-VEGF。 通过显微注射的方法将载体导入1-2细胞期斑马鱼体内，于胚胎发育的48 h采用RT-PCR的方法检测VEGF基因的表达量，研究不同干扰序列对VEGF基因表达的干涉作用。结果显示，针对不同位点的表达载体对VEGF基因表达的抑制效率有显著差异。它们对VEGF mRNA的抑制率分别为80.5%，42.8%，12.5%，40.7%。通过筛选我们得到了一条具有高效抑制作用的载体pS1-VEGF，该载体的相应序列靶向斑马鱼两个主要异构体VEGF165和VEGF121的共有外显子序列。 形态学检测结果显示，注射了pS1-VEGF的胚胎出现了心包膜水肿、血流速度减慢、循环红细胞堆积等症状。定量碱性磷酸酶染色显示，注射pS1-VEGF能够抑制斑马鱼胚胎新生血管的形成，当注射剂量为0.4 ng时，血管生成的抑制率为31.8%。NBT/BCIP血管染色显示，注射该载体后72 h，50%的斑马鱼肠下静脉、节间血管以及其它血管的发育受到不同程度的抑制。随着注射剂量的加大，血管发育受抑制的情况也随之加重，当注射剂量为1 ng时，只有心脏、头部及卵黄有血液循环。对干扰效果的特异性进行了研究，结果表明pS1-VEGF对斑马鱼内源基因胸苷酸合成酶（thymidylate synthase, TS）基因的表达没有明显的抑制作用。针对TS基因的shRNA表达载体及与斑马鱼没有同源性的对照载体对VEGF基因表达也没有明显的抑制作用。浓度梯度实验表明在0-1.2 ng的范围内干扰效果具有剂量依赖性。 以胚胎整体原位杂交的方法检测质粒对VEGF基因受体NRP1基因表达的影响，发现VEGF特异性shRNA表达载体能够引起NRP1基因表达的降低，说明斑马鱼中VEGF所介导的血管生成作用至少在部分上是依赖于NRP通路所调节的。 本研究工作为进一步研究斑马鱼基因功能、VEGF调控网络提供了一个快速、有效的手段，为阐明斑马鱼的血管生成机制提供了新的资料，为采用RNAi技术，以VEGF为靶点，以斑马鱼为模型对肿瘤进行基因治疗研究奠定了基础。

胸苷酸合成酶和二氢叶酸还原酶mRNA亲和肽的发现及mRNA体外展示技术的优化

胸苷酸合成酶（thymidylate synthase，简称TS）和二氢叶酸还原酶（dihydrofolate reductase, 简称DHFR）都是叶酸依赖性酶，在维持DNA合成和DNA修复上发挥关键作用，并且多年来一直是肿瘤研究和化疗的重要靶点。我们前期的研究发现，TS和DHFR在翻译水平上存在负反馈调控机制。人TS和DHFR可以与其自身的mRNA结合，从而抑制mRNA的表达，化疗药物可以与TS或者DHFR相互作用，形成的复合物不能与TS mRNA结合， 使负反馈机制丧失。因此深入研究TS和DHFR的翻译调控机理，对阐明肿瘤抗药性机制，对发现新的抗肿瘤药物和肿瘤的治疗都具有十分重要的意义。 本论文利用mRNA体外展示技术，构建多肽库（约10万亿种多肽分子），利用多种实验手段将mRNA体外展示技术进行优化，提高了多肽库融合肽的产量，提高了mRNA体外展示技术筛选的特异性。将TS mRNA分子上的顺式因子TS30 RNA固定于磁珠上，将融合肽库与顺式因子作用，经过6轮循环，由多肽库中获得了与TS mRNA高度亲和的多肽序列，体外结合实验证明亲和肽可以与TS全长mRNA结合，体外翻译实验证明多肽可以抑制TS mRNA的翻译。并且利用phage display技术由噬菌体肽库（12个氨基酸随机肽库）经过四轮筛选，分别筛选到TS和DHFR的亲和肽，凝胶阻滞实验证明它们分别能与TS和DHFR mRNA结合。 本论文利用的展示技术可以广泛应用于特异靶点的蛋白质筛选，并且本论文筛选到的TS和DHFR亲和肽可以作为TS和DHFR的抑制剂，从而为获得新型的抗肿瘤药物奠定基础。

Comparative researches on effects of sodium dodecylbenzene sulfonate and sodium dodecyl sulfate upon Lateolabrax japonicus biomarker system

Fish Lateolabrax japonicus were exposed to anion surfactant sodium dodecylbenzene sulfonate (SDBS) and sodium dodecyl sulfate (SDS) at 1 mg/l, respectively, for 6, 12 and 18 d, with one control group. Liver antioxidant enzymes, including superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), reduced glutathione (GSH) and glutathione S-transferase (GST) were determined; brain acetylcholinesterase (AChE) and liver inducible nitric oxide synthase (NOS) activities were also measured. The results of the study indicated that these parameters made different, sometimes, adverse responses to SDBS and SDS exposure, such as the activity of NOS can be inhibited by SDBS and induced by SDS, the different physico-chemical characteristics of SDBS and SDS should be responsible for their effects on enzyme activities. (c) 2005 Elsevier B.V. All rights reserved.

Modulatory effects of ammonia-N on the immune system of Penaeus japonicus to virulence of white spot syndrome virus

To study response to white spot syndrome virus (WSSV) under ammonia stress, Penaeus japonicus were exposed to 5 mg l(-1) ammonia-N and challenged orally with WSSV (NW). Controls consisted of an ammonia-N-exposed control group (N), a WSSV-challenged positive control group (W), and an untreated control group (control). Immune parameters measured were total haemocyte count (THC), haemocyte phagocytosis, plasma protein content and haemolymph enzymatic activities for prophenoloxidase (proPO), alkaline phosphatase (ALP), and nitric oxide synthase (NOS). THC and plasma protein had downward trends with time in all treatment groups (NW, N, and W) in contrast to the untreated control group (control). The percentage phagocytosis, NOS activity, and ALP and proPO activity of W and NW decreased initially then increased from 6 to 78 h (except for NOS and ALP, from 6 to 54 h) before declining thereafter until the end of the experiment. Compared with untreated controls (control), there was a downward trend for all measured parameters in the treatment groups (N, NW, and W), but the degree was W > NW > N. WSSV was detected at 78 h postchallenge in both W and NW. In conclusion, 5 mg l(-1) ammonia-N reduced the immunocompetence of P japonicus and may have decreased the virulence of WSSV (C) 2004 Elsevier B.V. All rights reserved.

Response of integrated biomarkers of fish (Lateolabrax japonicus) exposed to benzo[a]pyrene and sodium dodecylbenzene sulfonate

Fish Lateolabrax japonicus were exposed to 0.1 and 1 mg/L of anion surfactant sodium dodecylbenzene sulfonate (SDBS) and to 2 and 20 mu g/L of benzo[a]pyrene (B[a]P) for 6, 12, and 18 days, with control and solvent control groups. Liver antioxidant enzymes, including superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), reduced glutathione (GSH), and glutathione S-transferase (GST), were determined; brain acetyleholinesterase (AChE) and liver inducible nitric oxide synthase (iNOS) activities were also measured. The results indicated that (1) L. japonicus avoided oxidative damage through antioxidant systems; (2) SOD, GPx, and GSH were induced, and GST was inhibited and then induced by B[a]P exposure; and (3) CAT, GPx, and AChE were induced while NOS was inhibited, and GST was induced and then inhibited by SDBS stress in experimental period. (c) 2005 Elsevier Inc. All rights reserved.

Effects of forage composition and growing season on methane emission from sheep in the Inner Mongolia steppe of China

Understanding the effects of dietary composition on methane (CH4) production of sheep can help us to understand grassland degradation resulting in an increase of CH4 emission from ruminant livestock and its resulting significance affecting CH4 source/sink in the grazing ecosystem. The objective of this study was to investigate the effect of forage composition in the diet of sheep in July and August on CH4 production by sheep in the Inner Mongolia steppe. The four diet treatments were: (1) Leymus chinensis and Cleistogenes squarrosa (LC), (2) Leymus chinensis, Cleistogenes squarrosa and concentrate supplementation (LCC), (3) Artemisia frigida and Cleistogenes squarrosa (AC), and (4) Artemisia frigida, Cleistogenes squarrosa and concentrate supplementation (ACC). CH4 production was significantly lower in July than in August (31.4 and 36.2 g per sheep-unit per day, respectively). The daily average CH4 production per unit of digestive dry matter (DM), organic matter (OM) and neutral detergent fiber (NDF) increased by 10.9, 11.2 and 42.1% for the AC diet compared with the LC diet, respectively. Although concentrate supplementation in both the AC and LC diets increased total CH4 production per sheep per day, it improved sheep productivity and decreased CH4 production by 14.8, 12.5 and 14.8% per unit of DM, OM and NDF digested by the sheep, respectively. Our results suggested that in degraded grassland CH4 emission from sheep was increased and concentrate supplementation increased diet use efficiency. Sheep-grazing ecosystem seems to be a source of CH4 when the stocking rate is over 0.5 sheep-units ha(-1) during the growing season in the Inner Mongolia steppe.

阿片药物依赖的神经机制---内侧前额叶与药物的心理依赖

Mental dependence, characterized by craving and impulsive seeking behavior, is the matter of intensive study in the field of drug addiction. The mesolimbic dopamine system has been suggested to play an important role in rewarding of drugs and relapse. Although chronic drug use can induce neuroadaptations of the mesolimbic system and changes of drug reinforcement, these mechanisms cannot fully account for the craving and the compulsive drug-using behavior of addicts. Acknowledging the reinforcement effects of drugs, most previous studies have studied the impact of environmental cues and conditioned learning on addiction behavior, often using established classical or operant conditioning model. These studies, however, paid little attention to the role of cognitive control and emotion in addiction. These mental factors that are believed to have an important influence on conditioned learning. The medial prefrontal cortex (mPFC) has close anatomic and functional connections with the mesolimbic dopamine system. A number of the cognitive neurological studies demonstrate that mPFC is involved in motivation, emotional regulation, monitoring of responses and other executive functions. Thus we speculated that the function of abnormality in mPFC following chronic drug use would cause related to the abnormal behavior in addicts including impulse and emotional changes. In the present study of a series of experiments, we used functional magnetic resonance imaging to examine the hemodynamic response of the mPFC and related circuits to various cognitive and emotional stimuli in heroin addicts and to explore the underlying dopamine neuromechnism by microinjection of tool drugs into the mPFC in laboratory animals. In the first experiment, we found that heroin patients, relative to the normal controls, took a much shorter time and committed more errors in completing the more demanding of cognitive regulation in the reverse condition of the task, while the neural activity in anterior cingulate cortex (ACC) was attenuated. In the second experiment, the scores of the heroin patients in self-rating depression scale (SDS) and Self-rating anxiety scale (SAS) were significantly higher than the normal controls and they rated the negative pictures more aversive than the normal controls. Being congruent with the behavioral results, hemodynamic response to negative pictures showed significant difference between the two groups in bilateral ventral mPFC (VMPFC), amygdala, and right thalamus. The VMPFC of patients showed increased activation than normal controls, whereas activation in the amygdala of patients was weaker than that in normal subjects. Our third experiment showed that microinjection of D1 receptor agonist SKF38393 into the mPFC of rats decreased hyperactivity, which was induced by morphine injection, in contrast, D1 receptor antagonist SCH23390 increased the hyperactivity, These findings suggest: (1) The behavior and neural activity in ACC of addicts changed in chronic drug users. Their impulsive behavior might result from the abnormal neural activity in the mPFC especially the ACC. (2) Heroine patients were more depress and anxiety than normal controls. The dysfunction of the mPFC---amygdala circuit of heroine addicts might be related to the abnormal emotion response. (3) Dopamine in the mPFC has an inhibitory effect on morphine induced behavior. The hyperactivity induced by chronic morphine was reduced by dopamine increase with D1 receptor agonist, confirm the first experiment that the neuroadaption of mPFC system induced by chronic morphine administration appears to be the substrate the impulse behavior of drug users.

态度结构一致性对态度-行为关系的影响：个体与群体水平的研究

Social psychologists have long been interested in understanding the conditions under which attitudes influence behaviors, and they have formed two relatively independent theoretical perspectives in the attitude-behavior domain. One perspective, which we adopt in the present paper, focuses on investigating the effect of attitude strength on the attitude-behavior relationship. Specifically, the present research investigates the role of structural consistency, a dimension of attitude strength, on the attitude-behavior relationship. Structural consistency has typically been defined as the extent to which one aspect of individual’s attitude is consistent with either his or her overall attitude toward the object or the other aspect of the attitude, including affective-cognitive consistency (ACC), evaluative-cognitive consistency (ECC) and evaluative-affective consistency (EAC). The ECC and EAC can be integrated into the attitude base. Despite the evidence that structural consistency is associated with attitude-behavior relationship, there are some limitations in previous studies: (1) attitude base and ACC have been rarely studied together ;( 2) researchers often used the term behavior in a broad sense to encompass behavioral intentions as well as actual behaviors ;( 3) the effects were all investigated at individual level. The present research tried to explore the effects of ACC and attitude base on attitude-behavioral intention-actual behavior relationships at individual and group levels in different behavior areas including shopping, supportive behavior towards officials, and offensive behavior towards governors and central and local governments. The roles of affective and cognitive components of attitudes on general attitudes and following behaviors were also examined in this research. The results indicated that: A. At individual and group levels, the effect of structural consistency on attitude –intention relation is as the same as its effect on attitude-behavior relation, but it varies in different areas. On the other hand, the effect of structural consistency on explicit attitude-behavior relation is different from its effect on implicit attitude-behavior relation. B. The cognitive components of the individuals and groups’ implicit attitudes towards officials, the implicit and explicit attitudes towards governors, and the public’s attitudes towards central and local governments exert the stronger influence on general attitudes. Similarly, the cognitive components also exert the stronger influence on groups’ supportive behaviors towards officials and offensive behaviors towards governors and governments. C. Both group attitude and emotion can influence the group offensive behavior towards governors. The group attitude exerts its influence through the mediating effects of behavior intention and group emotion. The group emotion which is the core factor can exert the direct influence on group offensive behavior towards governors. D. At individual and group levels, the implicit and explicit measures should be used together to collect the attitude data, because the implicit and explicit attitudes can both predict behaviors under most conditions. It is the best implicit attitude measure to use the experts’ evaluations of listed words. And the emotion should be measured by the experts’ evaluations of listed words if the emotion data is used to predict the following behavior.

Influence of different neuroprotective drugs on dopamine neurotoxicity induced by 3,4-methylenedioxymethamphetamine and MPTP in mice

Introduction: Parkinson‟s disease (PD) is characterized by a chronic progressive loss of nigrostriatal dopaminergic neurons that is associated with chronic neuroinflammation. Current treatments for PD can significantly improve symptoms but do not cure the disease or slow its progression. An approach used in existing therapies is based on the inhibition of monoamine oxidase (MAO), enzyme involved in the metabolic degradation of dopamine. Although, preclinical studies showed that MAO-B inhibitors have neuroprotective activity in cellular and animal models of PD, clinical trials did not completely confirm this result. Therefore a large number of new molecules, with more potent MAO-B inhibitory activity and a possible neuroprotective effect, have been proposed to replace the pre-existing MAO-B inhibitors. The profile of the recent MAO inhibitor, SZV558, appears to be particularly interesting because of its pharmacodynamic, favorable for disease-modifying properties and its irreversible MAO-B enzyme bind. The enhancement of adult neurogenesis could be of great clinical interest in the management of neurodegenerative disorders. In line with this, the metformin, a well-known antidiabetic drug, has recently been proposed to promote neurogenesis and to have a neuroprotective effect on the neurodegenerative processes induced by the dopaminergic neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) in a mice PD model. Although, PD has multiple origins, one hypothesis is that amphetamine-related drugs may be part of the wide array of factors leading to the dopaminergic neuron degeneration that causes the disease. These hypothesis are supported by different results that showed a persistent, long-term dopaminergic toxicity induced by 3,4-methylenedioxymethamphetamine (MDMA) in mice. Moreover, the MDMA, altering the dopaminergic transmission, may affect neurogenesis and synaptogenesis. On these basis, considering that the young brain is particularly sensitive to drug-induced neurotoxicity, the consumption of MDMA during the adolescence might increase the vulnerability of dopaminergic neurons. However, the use of amphetamine-related drugs by adolescent and young people is often combined with caffeinated energy drinks in order to amplify their stimulant actions. Although caffeine use is safe, the combined treatment of caffeine and MDMA increases not only the DA release but also the microglia and astroglia activation. Aims: During my Ph.D. I studied the influence of neuroprotective drugs, such as MAO inhibitors and metformin, or substances, such as caffeine, on the neurodegenerative effects of two dopaminergic toxins, MDMA and MPTP, in mice. 1. In the first phase of my study, I evaluated the neuroprotective activity of the new MAO-B inhibitor SZV558, compared with well-known rasagiline, in a chronic mouse model of MPTP plus probenecid (MPTPp), which induces a progressive loss of nigrostriatal dopaminergic neurons. 2. Previous results showed that when MDMA is associated with caffeine, a more pronounced degeneration in adolescent compared with adult mice was observed. To better clarify the molecular mechanism at the base of the different neurotoxic effect of this drug association at different ages, I evaluated the neuronal nitric oxide synthase (nNOS) expression, which plays a critical role in the integration of dopaminergic and glutamatergic transmissions, in the CPu of adolescent or adult mice treated with MDMA, alone or in combination with caffeine. 3. Finally, I investigated the neuroprotective effect of metformin against dopaminergic neurotoxicity induced by MDMA in the CPu and SNc of adult mice. Conclusions: These results demonstrated that the dopaminergic neurodegenerative process may be induced or conditioned by environment stressors or substances which influence, through different ways, the development of neurodegenerative mechanisms. In the present study I evaluated the effects of 3 substances, known as potentially neuroprotective, in combination with two different neurotoxins that affect the nigrostriatal dopaminergic system. The SZV558 MAO-B inhibitor and the metformin protected the nigrostriatal pathway, usually affected in PD, by MPTP- and MDMA- induced neurotoxicity, respectively. On the other hand, caffeine, administrated with MDMA, showed a neurotoxic potential depending on the age of consumers, confirming the vulnerability of adolescent brain to consumption of drug and substances that affected the dopaminergic system. In conclusion, the study of neurodegenerative processes may be relevant to understand the human pharmacology, the origin and development of neurodegenerative disease and to predict the neurotoxic effect of drug abuse.

Phenotype consequences of myophosphorylase dysfunction: insights from the McArdle mouse model

McArdle disease, caused by inherited deficiency of the enzyme muscle glycogen phosphorylase (GP-MM), is arguably the paradigm of exercise intolerance. The recent knock-in (p.R50X/p.R50X) mouse disease model allows an investigation of the phenotypic consequences of muscle glycogen unavailability and the physiopathology of exercise intolerance. We analysed, in 2-month-old mice [wild-type (wt/wt), heterozygous (p.R50X/wt) and p.R50X/p.R50X)], maximal endurance exercise capacity and the molecular consequences of an absence of GP-MM in the main glycogen metabolism regulatory enzymes: glycogen synthase, glycogen branching enzyme and glycogen debranching enzyme, as well as glycogen content in slow-twitch (soleus), intermediate (gastrocnemius) and glycolytic/fast-twitch (extensor digitorum longus; EDL) muscles.

Muscle signaling in exercise intolerance: Insights from the McArdle mouse model

We recently generated a knock-in mouse model (PYGM p.R50X/p.R50X) of McArdle disease (myophosphorylase deficiency). One mechanistic approach to unveil the molecular alterations caused by myophosphorylase deficiency, which is arguably the paradigm of 'exercise intolerance', is to compare the skeletal-muscle tissue of McArdle, heterozygous, and healthy (wild type (wt)) mice. We analyzed in quadriceps muscle of p.R50X/p.R50X (n=4), p.R50X/wt (n=6) and wt/wt mice (n=5) (all male, 8 wk-old) molecular markers of energy-sensing pathways, oxidative phosphorylation (OXPHOS) and autophagy/proteasome systems, oxidative damage and sarcoplamic reticulum (SR) Ca handling. We found a significant group effect for total AMPK (tAMPK) and ratio of phosphorylated (pAMPK)/tAMPK (P=0.012 and 0.033), with higher mean values in p.R50X/p.R50X mice vs. the other two groups. The absence of massive accumulation of ubiquitinated proteins, autophagosomes or lysosomes in p.R50X/p.R50X mice suggested no major alterations in autophagy/proteasome systems. Citrate synthase activity was lower in p.R50X/p.R50X mice vs. the other two groups (P=0.036) but no statistical effect existed for respiratory chain complexes. We found higher levels of 4-hydroxy-2-nonenal-modified proteins in p.R50X/p.R50X and p.R50X/wt mice compared with the wt/wt group (P=0.011). Sarco(endo)plasmic reticulum ATPase 1 (SERCA1) levels detected at 110kDa tended to be higher in p.R50X/p.R50X and p.R50X/wt mice compared with wt/wt animals (P=0.076), but their enzyme activity was normal. We also found an accumulation of phosphorylated SERCA1 in p.R50X/p.R50X animals. Myophosphorylase deficiency causes alterations in sensory energetic pathways together with some evidence of oxidative damage and alterations in Ca handling but with no major alterations in OXPHOS capacity or autophagy/ubiquitination pathways, which suggests that the muscle tissue of patients is likely to adapt overall favorably to exercise training interventions.