Enhanced contextual fear memory in central serotonin-deficient mice

Central serotonin (5-HT) dysregulation contributes to the susceptibility for mental disorders, including depression, anxiety, and posttraumatic stress disorder, and learning and memory deficits. We report that the formation of hippocampus-dependent spatia

海马突触可塑性与异常记忆

5-羟色胺(5-HT)是中枢神经系统内非常重要的神经递质，广泛参与各种行为和生理过程。5-羟色胺功能低下可导致多种精神类疾病尤其是焦虑、抑郁和创伤后应激障碍等，而这些疾病都伴有学习和记忆的障碍；海马是参与学习记忆的重要脑区。海马接受5-HT神经元的直接投射且富含5-HT受体，因而海马也可以通过5-HT系统调控焦虑、抑郁及学习记忆。海马突触可塑性是学习记忆的细胞分子机制，是学习记忆的基础。我们条件性敲除转录因子Lmx1b得到中枢5-HT缺失小鼠，利用该小鼠进行中枢神经系统5-HT功能的研究。我们发现该小鼠的脑结构和运动能力正常；水迷宫空间学习能力正常，但空间记忆受损；焦虑水平降低，但是环境恐惧学习和记忆能力增强，增强的恐惧记忆能被外源给予的5-HT逆转；在中枢5-HT缺失小鼠中，应激对海马可塑性的作用即损伤LTP易化LTD消失，外源给予5-HT可以恢复应激的效果。这些结果提示应激导致海马LTP损伤可能是保护机制，缺乏这种保护机制可能导致恐惧记忆相关的创伤后应激障碍(PTSD)的易感。成瘾的核心特征是对药物的强迫性渴求和复吸。成瘾与学习记忆有很多共同的脑区和分子通路，它可能通过篡夺正常生理神经通路而产生比正常生理反应更强烈的可塑性，形成有害的异常记忆。以前的报道证实海马的兴奋性突触可塑性在成瘾过程中的适应性改变可能是成瘾的机制；但是成瘾涉及复杂的生物机制，因而不可能仅是兴奋性突触可塑性的贡献。我们研究了5-HT系统和抑制性系统（主要是GABA能系统）在成瘾中的贡献。利用中枢5-HT缺失小鼠，我们发现5-HT缺失小鼠的吗啡显著地易化了5-HT CKO的海马LTP，同时也导致成瘾行为持续不消退；5-HT和5-HT1a受体激动剂能逆转此现象。这提示毒品成瘾可能导致中枢5-HT缺失，进而增强海马LTP，使毒品相关记忆牢固不消退。GABA能系统是中枢神经系统最重要的抑制性系统，我们研究发现一次吗啡对内源性大麻受体（CB1R）依赖的抑制性突触的长时程抑制（Inhibitory long-term depression，I-LTD）没有影响，成瘾后I-LTD抑制，而吗啡成瘾后戒断导致了内源性大麻受体(CB1R)和L-型钙通道(LTCC)依赖的GABA能LTD (I-LTD)，使I-LTD增大了一倍，提示在吗啡成瘾阶段过程中，有组合突触可塑性发生，进而增强了突触可塑性的调控范围。 本论文是对中枢5-HT系统对海马兴奋性突触可塑性在焦虑、应激、成瘾等异常记忆中的调节作用以及海马抑制性系统在成瘾和戒断中的贡献进行研究，表明恐惧记忆和毒品成瘾记忆存在许多共同的细胞分子机理，对今后治疗焦虑、创伤后应激障碍和成瘾提供了新的思路。

Dissection and localization of the immunostimulating domain of Edwardsiella tarda FliC

Bacterial flagellin is known to induce potent immune response in vertebrate systems via the toll-like receptor (TLR) 5. As a result, flagellin has been studied extensively as a vaccine adjuvant. In a previous study, we examined the vaccine and adjuvant potentials of the flagellin (FliC) of the fish pathogen Edwardsiella tarda. We found that E. tarda FliC induced low protective immunity by itself but could function as a molecular adjuvant and potentiate the specific immune response induced by the E. tarda antigen Eta6. Since FliC is a large protein and organized into distinct structural domains, we wondered whether the immunostimulating effect observed with the full-length protein could be localized to a certain region. To investigate this question, we in the present study dissected the FliC protein into several segments according to its structural features: (i) N163, which consists of the conserved N-terminal 163 residues of FliC; (ii) M160, which consists of the variable middle 160 residues; (iii) C94, which consists of the conserved C-terminal 94 residues; (iv) NC257, which is an artificial fusion of N163 and C94. To examine the adjuvanticity of the FliC fragments, DNA vaccine plasmids expressing FliC fragments in fusion with Eta6 were constructed and used to immunize Japanese flounder. The results showed that N163 produced the best adjuvant effect, which, in respect to improvement in the relative percent survival of the vaccinated fish, was comparable to that of the full-length FliC. None of the other FliC fragments exhibited apparent immunopotentiating effect. Further analysis showed that N163 enhanced the production of serum specific antibodies and, like full-length FliC, significantly upregulated the expression of the genes that are possibly involved in innate and adaptive immunity. These results indicate that N163 is the immunodominant region of FliC and suggest that E. tarda FliC may induce immune responses in Japanese flounder via mechanisms alternative to that involving TLR5. (C) 2010 Elsevier Ltd. All rights reserved.

双壳贝类幼虫变态诱导及其机理研究

变态过程是双壳贝类由幼虫向成体转变的一个必不缺少的发育阶段。研究双壳贝类幼虫的变态过程及其机理，对于阐明它们的种群数量变动，促进重要经济双壳贝类增养殖的发展有重要的理论和实践意义。本论文除了用化学物质对几种双壳贝类(海湾扇贝、墨西哥湾扇贝和硬壳蛤)幼虫的变态进行诱导外，主要以激素和神经递质的作用方式为基础，通过直接测定双壳贝类(以海湾扇贝为代表)幼虫体内激素和神经递质、第二信使cAMP等生化物质含量的变化来研究双壳贝类幼虫变态过程中的信息传递途径，从分子生物学和神经生物学角度阐明双壳贝类幼虫变态机理。主要结果如下：1．通过参考国内外大量文献的基础上，较为系统地评述了近二十年来海洋无脊椎动物幼虫附着变态研究的一些进展情况，主要包括诱导因子、附着变态机理模型、人工诱导物的应用和延迟变态四个方面。到目前为止，人们已经发现了许多海洋无脊椎动物幼虫附着变态的诱导物质，主要分为天然诱导物和人工诱导物两大类，一些人工诱导物如GABA、肾上腺素和去甲肾上腺素已经在经济贝类苗种生产中得到应用。幼虫附着变态机理模型主要有长牡蛎(Crassostrea gigas)幼虫附着变态的双调控模型、红鲍(Haliotis rufescens)幼虫附着变态的上行调节模型以及多毛类Phragmatopoma california幼虫附着变态的脂肪酸调控模型。本论文还评述了海洋无脊椎动物幼虫发生延迟变态的原因以及延迟变态对海洋无脊椎动物造成的影响，并提出了解决的方法和今后研究的重点问题。2．在室内用氯化乙酰胆碱、ATP和CaCl_2 3种化学物质对海湾扇贝幼虫的变态进行了诱导实验。结果表明，虽然在个别浓度和处理时间氯化乙酰胆碱和ATP有诱导作用，但总体诱导效果不显著。而10×10~(-3)～40×10~(-3M的CaCl_2在处理12～24h后诱导效果较显著，其诱导效果对处理时间的依赖性较显著，在浓度为40×10~(-3)M和处理时间为24h时诱导效果最好，与对照组相比，变态率提高23.18％。3种诱导物对幼虫死亡率均有显著影响，并且死亡率对浓度和处理时间均有显著的依赖性，浓度越高，处理时间越长，死亡率越高。3．用KCl、肾上腺素、去甲肾上腺素和氯化胆碱进行了墨西哥湾扇贝(Argopectenirradians concentricus Say)幼虫变态的诱导作用实验。结果表明，KCl、肾上腺素、去甲肾上腺素和氯化胆碱对墨西哥湾扇贝幼虫变态均有显著诱导作用。KCl在处理时间为12h～48h范围内均有诱导作用；13.42×10~(-3)M和20.13×10~(-3)M的KCl诱导效果较好，变态率平均提高10％以上。1．O×10~(-6)M～50×10~(-6)M的肾上腺素在处理时间为lh～12h较适宜，此时变态率均提高10％以上。1.0×10~(-6)M～50×10~(-6)M的去甲肾上腺素在处理时间为1h～24h都较适宜，变态率平均均提高10％以上，最高可提高31．07％。0.01×10~(-4)M～1.O×10~(-4)M的氯化胆碱在处理时间为12h～48h时诱导效果均较好，它们之间的平均变态提高率并没有显著差别，均在12％～13％之间。10×10~(-4)M的氯化胆碱在处理时间为12h时诱导效果较明显，变态率可以提高19.14％，超过12h，变态率明显下降，100×10~(-4)M的氯化胆碱明显产生毒害作用，幼虫变态率均为零，而幼虫的死亡率均为100％。4．用KCl、肾上腺素、去甲肾上腺素、L-DOPA、5-羟色胺(5-hydroxytryptamine，Serotonin，5-HT)和GABA(γ-氨基丁酸)进行了不同浓度不同处理时间对硬壳蛤(Mercenaria mercenaria L.)幼虫变态诱导实验。结果表明，KCl、肾上腺素、去甲肾上腺素、L-DOPA和5-羟色胺对硬壳蛤幼虫的变态均有诱导作用，而GABA的诱导 作用不显著。KCl的最佳诱导浓度随处理时间不同而有所不同。当处理时间为1～24h时，KCl的最佳诱导浓度为33.56×10~(-3)M，此时幼虫变态率均提高24％以上，当处理时间为48h时，KCl的最佳诱导浓度为20.13～26.85×10~(-3)M，处理时间为72h时，最佳诱导浓度为13.42×10~(-3)M。肾上腺素和去甲肾上腺素的诱导作用与浓度和处理时间均有关。肾上腺素的最佳处理浓度为100×10~(-6)M，最佳处理时间均为8h，此时幼虫变态率提高最大，为36.97％。当去甲肾上腺素的诱导浓度为100×10~(-6)M，处理时间为8h～16h时，幼虫变态提高率较高，均大于18％，死亡提高率均低于30％，当去甲肾上腺索诱导浓度为500×10~(-6)M时，虽然在8h～16h的处理时间范围内，幼虫变态提高率也较高，均大于18％，但当处理时间超过8h，在16～48h范围内，幼 虫死亡提高率明显升高，均大于50％。L-DOPA的适宜诱导浓度为10×10~(-6)M～50×10~(-6)M，适宜处理时间为8～24h，此时幼虫变态率均提高30％以上，最高可提高79.43％。5-羟色胺的诱导作用较强，其适宜诱导浓度为100×10~(-6)M—1000×10~(-6)M，适宜处理时间为0.5～24h，此时幼虫变态率提高均在30％以上，当处理时间为8h时，最佳诱导浓度为1000×10~(-6)M，此时幼虫变态率提高57.5％，当处理时间为24h时，最佳诱导浓度为100×10~(-6)M，此时幼虫变态率提高69.29％。GABA的诱导作用较弱，最佳诱导浓度随处理时间的不同而有所不同。处理时间为24h和48h时，最佳诱导浓度为0.1×10~(-6)M；处理时间为0.5～16h时，最佳诱导浓度为100×10~(-6)M。5．KCl、肾上腺素、去甲肾上腺索、L-DOPA、5-羟色胺、GABA、茶碱和咖啡因8种诱导物对不同发育阶段海湾扇贝幼虫变态的诱导作用是不同的。13．42×10~(-3)M和20.13×10~(-3)M的KCl对第12天幼虫的变态有抑制作用，变态提高率为负值；之后当幼虫发育至第13和14天时，两浓度的KCl能够明显诱导幼虫变态，变态提高率均高于20％，而对于第16天的幼虫诱导作用有所减弱，变态提高率有所降低；26.85×10~(-3)M的KCl对第12和13天幼虫的变态均有抑制作用，变态提高率为负值，对第14和16天幼虫的变态却有明显的持续的诱导作用，变态提高率分别为22.98％和37.5％。神经递质肾上腺素、去甲肾上腺素、L-DOPA、5-羟色胺和GABA的诱导作用规律基本相似，即对第13天海湾扇贝幼虫的变态有明显的抑制作用，变态提高率均为负值，而对第14天幼虫的诱导作用较显著。茶碱和咖啡因作为影响细胞内cAMP的物质，它们的诱导作用规律与神经递质有所不同。它们对第13天海湾扇贝幼虫变态的诱导效果最好。6．测定了不同发育阶段及人工诱导后海湾扇贝幼虫体内去甲肾上腺素、多巴胺和5-羟色胺含量的变化规律。结果表明，海湾扇贝幼虫体内去甲肾上腺索含量在变态前和变态后没有明显变化，变态前为2352(pg／mg湿重)，变态后为2770(pg／mg湿重)。多巴胺和5-羟色胺含量在变态前随幼虫的发育而增加，变态前(第13天)急剧增加，第13天的幼虫比第12天的幼虫分别增加了2.8倍和5.7倍，变态后急剧下降，变态后幼苗比第13天的幼虫分别降低了25.1倍和16.4倍。海湾扇贝幼虫体内DA：NE比和5-HT：NE比在变态前和变态后变化比较剧烈。DA：NE比和5-HT：NE比在变态前(第13天)急剧增加，第13天的幼虫比第12天的幼虫增加了3.O倍(DA：NE比)和5.0倍(5-HT:NE比)；变态后急剧降低，变态后幼苗比第13天的幼虫降低了29.8倍(DA：NE比)和19.5倍(5-HT：NE比)。海湾扇贝幼虫经KCl和氯化钙诱导24h后，体内去甲肾上腺素、多巴胺和5-羟色胺以及DA：NE比和5-HT：NE比均有所降低。本实验的结果表明，多巴胺和5-羟色胺可能启动了海湾扇贝幼虫的变态过程。7．茶碱和咖啡因对墨西哥湾扇贝幼虫的变态均有明显诱导作用。它们的诱导作用均对浓度的依赖性较强，对处理时间的依赖性较弱。10×10~(-4)M的茶碱诱导效果最好，平均变态提高率达33％，其次为1.0×10~(-4)M和100×10~(-4)M的茶碱，平均变态提高率分别为23.15％和21.97％。处理时间对茶碱诱导效果影响不显著，在1～24h范围内，平均变态提高率在19.07～26.1％之间变动。10×10~(-4)M的咖啡因诱导效果最佳，4个处理时间的平均变态提高率为36.01％，其次为100×10~(-4)M，平均变态提高率为26.43％。处理时间对茶碱的诱导效果影响不大，在1～24h范围内，平均变态提高率在19.65～22.02％之间变动。8．采用直接测定cAMP的方法来研究cAMP是否参与了海湾扇贝幼虫的变态过程。结果表明，cAMP参与了海湾扇贝幼虫的变态过程。海湾扇贝幼虫体内cAMP含量随着发育阶段的不同而有所变化。在D形幼虫期最低，为73 pmol／(mg蛋白质)；当到达壳顶期幼虫时cAMP含量明显增加，比D形幼虫期提高了12.7倍。从壳顶期幼虫到眼点幼虫(100％，第13天)cAMP含量增加速度较慢，各发育阶段分别比前一发育阶段增加了0.4倍、0.3倍和0.2倍。但当幼虫变态后，体内cAMP含量又急剧增加，幼苗体内cAMP含量比眼点幼虫(100％，第13天)增加了6.1倍。当用KCl、肾上腺索和L-DOPA诱导后，幼虫体内cAMP含量明显增加，分别比对照组提高了7.8倍、1.5倍和10.7倍，说明cAMP参与了这3种诱导物诱导海湾扇贝幼虫变态的过程。9．在前面实验结果和参考有关文献的基础上，初步提出了以海湾扇贝为代表的双壳贝类幼虫变态机理模型：幼虫变态分为两个过程：启动过程和后续过程。当幼虫发育到一定阶段，在外界刺激因子的作用下，体内分泌多巴胺和5-羟色胺，多巴胺和5-羟色胺通过某种信号转导途径(如以DG和IP_3为第二信使)启动变态过程，变态过程启动后，又激活以cAMP为第二信使的信号转导途径(暂时称为后续过程)，两者共同完成了幼虫的变态过程。

Cflec-5, a pattern recognition receptor in scallop Chlamys farreri agglutinating yeast Pichia pastoris

C-type lectins are a superfamily of carbohydrate-recognition proteins which play crucial roles as pattern recognition receptors (PRRs) in the innate immunity. In this study, the full-length cDNA of a C-type lectin was cloned from scallop Chlamys farreri (designated as Cflec-5) by expression sequence tag (EST) analysis and rapid amplification of cDNA ends (RACE) approach The full-length cDNA of Cflec-5 was of 1412 bp. The open reading frame encoded a polypeptide of 153 amino acids, including a signal sequence and a conserved carbohydrate-recognition domain with the EPN motif determining the mannose-binding specificity The deduced amino acid sequence of Cflec-5 showed high similarity to members of C-type lectin superfamily. The quantitative real-time PCR was performed to investigate the tissue distribution of Cflec-5 mRNA and its temporal expression profiles in hemocytes post pathogen-associated molecular patterns (PAMPs) stimulation. In healthy scallops, the Cflec-5 mRNA was mainly detected in gill and mantle, and marginally in other tissues The mRNA expression of Cflec-5 could be significantly induced by lipopolysaccharide (LPS) and glucan stimulation and reached the maximum level at 6 h and 12 h, respectively But its expression level did not change significantly during peptidoglycan (PGN) stimulation The function of Cflec-5 was investigated by recombination and expression of the cDNA fragment encoding its mature peptide in Escherichia coli Rosetta Gami (DE3) The recombinant Cflec-5 agglutinated Pichia pastoris in a calcium-independent way The agglutinating activity could be inhibited by D-mannose. LPS and glucan, but not by D-galactose or PGN. These results collectively suggested that Cflec-5 was involved in the innate Immune response of scallops and might contribute to nonself-recognition through its interaction with various PAMPs (C) 2010 Elsevier Ltd All rights reserved

Attention deficit/hyperactivity disorder children with a 7-repeat allele of the dopamine receptor D4 gene have extreme behavior but normal performance on critical neuropsychological tests of attention

An association of the dopamine receptor D4 (DRD4) gene located on chromosome 11p15.5 and attention deficit/hyperactivity disorder (ADHD) has been demonstrated and replicated by multiple investigators. A specific allele [the 7-repeat of a 48-bp variable number of tandem repeats (VNTR) in exon 3] has been proposed as an etiological factor in attentional deficits manifested in some children diagnosed with this disorder. In the current study, we evaluated ADHD subgroups defined by the presence or absence of the 7-repeat allele of the DRD4 gene, using neuropsychological tests with reaction time measures designed to probe attentional networks with neuroanatomical foci in D4-rich brain regions. Despite the same severity of symptoms on parent and teacher ratings for the ADHD subgroups, the average reaction times of the 7-present subgroup showed normal speed and variability of response whereas the average reaction times of the 7-absent subgroup showed the expected abnormalities (slow and variable responses). This was opposite the primary prediction of the study. The 7-present subgroup seemed to be free of some of the neuropsychological abnormalities thought to characterize ADHD.

Enhancement of GABAA Receptor-Mediated Inhibitory Postsynaptic Currents Induced by "Partial Oxygen-Glucose Deprivation

下载PDF阅读器"氧糖剥夺"模型作为研究脑缺血的离体模型被广泛使用,该模型模拟了局灶性脑缺血的主要病理变化.然而在缺血病灶核心区与正常脑组织之间称为缺血半暗带的区域,脑血流也有程度不一的降低.为了模拟这种病理变化,发展了一种"不完全氧糖剥夺"的离体脑片模型,该模型满足两个条件,灌流液里氧气部分剥夺而葡萄糖含量降低;"氧糖剥夺"可以导致谷氨酸介导的兴奋性毒性,从而引起神经细胞的坏死.而A型γ-氨基丁酸受体(GABAAR)介导的神经元抑制性活动可以对抗谷氨酸引起的兴奋性毒性,因此近年来引起广泛的研究兴趣.而谷氨酸受体和γ-氨基丁酸受体功能在缺血半暗带是否有改变尚不得而知.因此本文采用海马脑片全细胞膜片钳的记录方法,研究"不完全氧糖剥夺"对海马CA1区神经元的A型γ-氨基丁酸受体介导的抑制性突触后膜电流(IPSCs)的影响.研究发现"不完全氧糖剥夺"使GABAAR介导的IPSCs的峰值增加而衰减时程延长.进一步研究发现该电流的峰值增加是由于GABAAR-氯离子通道的电导增加所致,而与氯离子的反转电位变化无关.这些发现提示在脑缺血的缺血半暗带区域GABAAR介导的神经元抑制性活动可能是增强的,这可能是神经元面对缺血状态产生自我保护的一种内稳态机制.

N-methyl-D-aspartate receptor-dependent long-term potentiation in CA1 region affects synaptic expression of glutamate receptor subunits and associated proteins in the whole hippocampus

Long term potentiation in hippocampus, evoked by high-frequency stimulation, is mediated by two major glutamate receptor subtypes, alpha-amino-3-hydroxyl-5-methyl-4-isoxazole propionate receptors and N-methyl-D-aspartate receptors. Receptor subunit compos

Expression and regulation of orphan receptor TR2 mRNA in germ cells of cryptorchid testis in rat and rhesus monkey

Expression and cellular localization of orphan receptor TR2 mRNA in relation to germ cell apoptosis in cryptorchid testes of rat and rhesus monkey have been studied by using in situ hybridization and in situ 3'-end labeling of DNA fragments (TUNEL). The results show that: (i) TR2 mRNA is specifically expressed in the germ cells, mainly in the spermatocytes, round and elongated spermatids. The expression level of TR2 mRNA varies with the seminiferous cycle, (ii) In the rat cryptorchid testes on days 3 and 5 after the surgery, the germ cells began to undergo apoptosis with no evident decrease in TR2 mRNA level. On day 7.5, however, most germ cells underwent apoptosis, while the expression level of TR2 mRNA declined markedly, and TR2 mRNA was rarely expressed on day 10 thereafter. (iii) On days 15 and 20 of the cryptorchid testes of rhesus monkey, TR2 mRNA was only expressed in a few of primary spermatocytes and the mRNA was almost undetectable on days 30, 45, 60. These results suggest that TR2 mRNA probably plays an important role in spermatogenesis and germ cell apoptosis.

Characterization of C-C chemokine receptor subfamily in teleost fish

Chemokines and their receptors play important roles in nervous and immune systems. Little information, however, exists concerning this gene family in teleost fish. In the present Study, 17 C-C chemokine receptors genes were identified from Danio rerio, 9 from Gasterosteus aculeatus, 10 from Oryzius latipes, 8 from Takifugu rubripes and 5 from Tetraodon nigroviridis. Phylogenetic analysis showed that the orthologs to mammalian CCR6, 7, 8, 9 and CCRL1 receptors were evident in zebrafish, but the clear orthologs to mammalian CCR1, 2, 3, 4, 5 and 10 were not found in zebrafish. The gene structure of zebrafish CCR (zfCCR) was further analyzed. The open reading frame of zfCCR3-1, zfCCR3-3, zfCCR6-1, zfCCR6-2, zfCCR8-2 contain one exon, and two exons were identified for zfCCR2-1, zfCCR2-2, zfCCR4 and zfCCRLI-1, three exons for zfCCR3-2, zfCCR5 and zfCCR7, four exons for zfCCR8-1 and zfCCR9-1. The expression analyses showed that in zebrafish, most C-C chemokine receptor genes Were expressed in fertilized eggs and oocytes, and all the receptor genes were expressed in larval stages. The zfCCR2-2, zfCCR3-1, zfCCR4 and zfCCR6-2 genes were expressed in all normal organs examined, whereas not for zfCCR2-1, zfCCR3-3, zfCCR6-1, zfCCR8-1, zfCCR9-2 and zfCCRL1-2. The expression of zfCCR3-2, zfCCR5, zfCCR7, zfCCR9-1 and zfCCRLI-1 were detected in the majority organs. and zfCCR8-2 and zfCCR8-3 detected only in brain. The differential expression pattern of different paralogues in organs may indicate their difference in function, which requires further investigation. (C) 2008 Elsevier Ltd. All rights reserved.

Staphylococcus aureus lipoproteins trigger human corneal epithelial innate response through toll-like receptor-2

Bacterial lipoproteins (LP) are a family of cell wall components found in a wide variety of bacteria. In this study, we characterized the response of HUCL, a telomerase-immortalized human corneal epithelial cell (HCEC) line, to LP isolated from Staphylococcus (S) aureus. S. aureus LP (saLP) prepared by Triton X-114 extraction stimulated the activation of NF-kappa B, JNK, and P38 signaling pathways in HUCL cells. The extracts failed to stimulate NF-kappa B activation in HUCL cells after lipoprotein lipase treatment and in cell lines expressing TLR4 or TLR9, but not TLR2, indicating lipoprotein nature of the extracts. saLP induced the up-regulation of a variety of inflammatory cytokines and chemokines (IL-6, IL-8, ICAM-1). antimicrobial molecules (hBD-2, LL-37, and iNOS), and homeostasis genes (Mn-SOD) at both the mRNA level and protein level. Similar inflammatory response to saLP was also observed in primarily cultured HCECs using the production of IL-6 as readout. Moreover, TLR2 neutralizing antibody blocked the saLP-induced secretion of IL-6, IL-8 and hBD2 in HUCL cells. Our findings suggest that saLP activates TLR2 and triggers innate immune response in the cornea to S. aureus infection via production of proinflammatory cytokines and defense molecules. (C) 2007 Elsevier Ltd. All rights reserved.

Molecular identification and expression analysis of tumor necrosis factor receptor-associated factor 2 in grass carp Ctenopharyngodon idella

Tumor necrosis factor receptor-associated factor 2 (TRAF2) is a crucial component of almost the entire tumor necrosis factor receptor superfamily signaling pathway. In the present study, a TRAF2 gene has been cloned from grass carp (Ctenopharyngodon idella) by reverse transcription-polymerase chain reaction and rapid amplification of cDNA ends. The full-length cDNA is 3162 bp, including a 60 bp 5' untranslated region (UTR), a 1611 bp open reading frame, and a 1491 bp 3' UTR. The polyadenylation signal (AATAAA) and the mRNA instability motifs (ATTTTA, ATTTA) were followed by a poly(A) tail in the 3' UTR. No signal peptide or transmembrane region has been found in the putative amino acids of grass carp TRAF2 (gcTRAF2). Phylogenetic tree analysis clearly showed that gcTRAF2 is nearest to the TRAF2 gene of goldfish. The identity of gcTRAF2 with its homologs in other vertebrates ranges from 56% to 97%. It is characterized by one RING-type signature at the N-terminus, one zinc finger in the middle part, and one conserved TRAF domain consisting of a C-proximal (TRAF-C) subdomain and a N-proximal (TRAF-N) subdomain. The identity of TRAF-C among all TRAF2 homologs in vertebrates varies from 78% to 97%, whereas the identity of TRAF-N ranges from 56% to 100%. The recombinant gcTRAF2 has been expressed in Escherichia coli using pET-32a expression vector. The rabbit anti-gcTRAF2 polyclonal antibody was obtained. The expression of gcTRAF2 in different organs was examined by real-time quantitative polymerase chain reaction and Western blot analysis. It was widely distributed in heart, head kidney, thymus, brain, gill, liver, spleen, and trunk kidney. This is the first report of a TRAF2 homolog molecule in fish.

Androgen receptor targets NFKB and TSPI to suppress prostate tumor growth in vivo

The androgen role in the maintenance of prostate epithelium is subject to conflicting opinions. While androgen ablation drives the regression of normal and cancerous prostate, testosterone may cause both proliferation and apoptosis. Several investigators note decreased proliferation and stronger response to chemotherapy of the prostate cancer cells stably expressing androgen receptor (AR), however no mechanistic explanation was offered. In this paper we demonstrate in vivo anti-tumor effect of the AR on prostate cancer growth and identify its molecular mediators. We analyzed the effect of AR on the tumorigenicity of prostate cancer cells. Unexpectedly, the AR-expressing cells formed tumors in male mice at a much lower rate than the AR-negative controls. Moreover, the AR-expressing tumors showed decreased vascularity and massive apoptosis. AR expression lowered the angiogenic potential of cancer cells, by increasing secretion of an anti-angiogenic protein, thrombospondin-1. AR activation caused a decrease in RelA, a subunit of the pro-survival transcription factor NF kappa B, reduced its nuclear localization and transcriptional activity. This, in turn, diminished the expression of its anti-apoptotic targets, Bcl-2 and IL-6. Increased apoptosis within AR-expressing tumors was likely due to the NF kappa B suppression, since it was restricted to the cells lacking nuclear (active) NF kappa B. Thus we for the first time identified combined decrease of NF kappa B and increased TSP1 as molecular events underlying the AR anti-tumor activity in vivo. Our data indicate that intermittent androgen ablation is preferable to continuous withdrawal, a standard treatment for early-stage prostate cancer. (C) 2007 Wiley-Liss, Inc.