PTP1B抑制剂的制备与构效关系研究

蛋白酪氨酸磷酸酶1B（protein tyrosine phosphatase, PTP1B）是蛋白酪氨酸磷酸酶（protein tyrosine phosphatases, PTPs）家族中的一个经典的非受体型酪氨酸磷酸酶，在胰岛素信号通路中起着重要的负调控作用，是目前公认的一个新颖的糖尿病和肥胖症治疗靶点。寻找PTP1B的高活性抑制剂对糖尿病和肥胖症治疗有着重要的应用前景。 双-(2,3-二溴-4,5-二羟基苯基)-甲烷（BDDPM）是从松节藻醇提物中分离鉴定出的溴酚类化合物，体外活性筛选发现，它具有极强的蛋白酪氨酸磷酸酶1B(PTP1B)抑制活性(IC50=2.4μmol/L)。采用高脂饮食-链脲佐菌素诱导的大鼠模型（STZ-DM）对富含BDDPM的松节藻醇提物进行动物实验，发现中、高剂量组同样表现出惊人的活性，降糖效果优于阳性对照临床药物文迪雅，并呈剂量依赖性。于是拟采用STZ-DM大鼠模型对单一组分BDDPM进行药理、药效学等体内降糖活性研究，但体内动物实验需要30g以上BDDPM，所以首先要解决药源的问题。本文尝试从天然海藻提取分离和化学合成两种途径来解决BDDPM制备的问题。 首先本文尝试从松节藻中提取分离BDDPM的制备方法。通过正相硅胶色谱、凝胶Sephadex LH-20色谱和重结晶等纯化手段分离纯化目标化合物BDDPM，并借助IR，MS和NMR等技术确定了其化学结构。最终从常温风干的50kg松节藻干样品中分离得到7.8g BDDPM。由于松节藻藻体构成复杂，给分离纯化BDDPM带来极大困难，致使分离纯化过程耗费大量时间和金钱；并且原材料松节藻的采集也易受季节和原料短缺等自然因素的影响。所以，从天然海藻中分离纯化的方法不适宜用于BDDPM的制备。 本文的重点是对BDDPM（4e）的化学合成途径进行研究。本文通过5步合成法（Friedel-Craftz酰基化反应、苯环逐级溴代、羰基还原、羟基脱保护）成功地合成了BDDPM，合成总产率为23.6%。同时本文采用上述合成路线获得了四个系列共计20个溴酚系列衍生物（4e为目标产物BDDPM，其余19个为溴酚系列衍生物），其中10个为新化合物。合成的20个化合物经1H NMR、13C NMR、MSEI和IR进行了结构鉴定。合成的20个化合物在体外活性筛选中均表现出不同程度的PTP1B抑制活性，其中合成的目标产物4e具有与天然分离纯化获得的BDDPM同等效率的PTP1B抑制作用。 另外，通过比较四个系列化合物PTP1B抑制活性间的差异，对此类溴酚化合物的构效关系作了初步分析，结果表明：1．羰基官能团的存在会明显降低此类化合物的PTP1B抑制率；2．化合物中的羟基官能团被甲氧基保护后，PTP1B抑制活性会得到一定程度的加强；3．化合物苯环上溴原子取代基数目增多时，其PTP1B抑制率也会随之增强。但是，筛选结果中也有少部分化合物的PTP1B抑制作用与上述规则相违背。因此，本文总结的初步构效关系还需要进一步的实验研究加以验证。 最后本文通过化学合成的方法，经过5步反应成功地制备出了30g BDDPM，为后续的药理、药效学研究奠定了基础。

Characterization of eastern oyster (Crassostrea virginica Gmelin) chromosomes by fluorescence in situ hybridization with bacteriophage P1 clones

Chromosome identification is an essential step in genomic research, which so far has not been possible in oysters. We tested bacteriophage P1 clones for chromosomal identification in the eastern oyster Crassostrea virginica, using fluorescence in situ hybridization (FISH). P1 clones were labeled with digoxigenin-11-dUTP using nick translation. Hybridization was detected with fluorescein-isothiocyanate-labeled anti-digoxigenin antibodies and amplified with 2 layers of antibodies. Nine of the 21 P1 clones tested produced clear and consistent FISH signals when Cot-1 DNA was used as a blocking agent against repetitive sequences. Karyotypic analysis and cohybridization positively assigned the 9 P1 clones to 7 chromosomes. The remaining 3 chromosomes can be separated by size and arm ratio. Five of the 9 P1 clones were sequenced at both ends, providing sequence-tagged sites that can be used to integrate linkage and cytogenetic maps. One sequence is part of the bone morphogenetic protein type 1b receptor, a member of the transforming growth factor superfamily, and mapped to the telomeric region of the long arm of chromosome 2. This study shows that large-insert clones such as P1 are useful as chromosome-specific FISH probes and for gene mapping in oysters.

Total synthesis of bis-(2,3-dibromo-4,5-dihydroxyphenyl)-methane as potent PTP1B inhibitor

Protein tyrosine phosphatase 1B (PTP1B) plays an important role as a negative regulator and has been proved to be an effective target for the treatment of type 2 diabetes mellitus. Bis-(2,3-dibromo-4,5-dihydroxyphenyl)-methane 7 was first reported as a natural bromophenol with significant inhibition against PTP1B which was isolated from red algae Rhodomela confervoides. Intrigued by its astonishing activity (IC50 = 2.4 mu mol/L), compound 7 was synthesized with the overall yield of 24% and evaluated for its PTP1B inhibitory activity compared with natural compound. (C) 2008 Li Jun Han. Published by Elsevier B.V. on behalf of Chinese Chemical Society. All rights reserved.

Inhibition of bromophenols against PTP1B and anti-hyperglycemic effect of Rhodomela confervoides extract in diabetic rats

Protein tyrosine phosphatase 1B (PTP1B) plays an important role as a negative regulator in insulin signaling pathways. PTP1B is an effective target for the treatment of type 2 diabetes mellitus. Four bromophenol derivatives from red algae Rhodomela confervoides, 2,2',3,3'-tetrabromo-4,4',5,5'-tetra-hydroxydiphenyl methane (1), 3-bormo-4,5-bis(2,3-dibromo-4,5-dihydroxybenzyl) pyrocatechol (2), bis(2,3-dibromo-4,5-dihydroxybenzyl) ether (3) and 2,2',3-tribromo-3',4,4',5-tetrahydroxy-6'-ethyloxy-methyldiphenylmethane (4) showed significant inhibitory activity against PTP1B (IC50 were 2.4, 1.7, 1.5 and 0.84 mu mol/L, respectively) as potential therapeutical agents for the treatment of type 2 diabetes mellitus. The anti-hyperglycemic effects of the ethanol extracts from R. confervoides on streptozotocin-induced diabetes (STZ-diabetes) in male Wistar rats fed with high fat diet were investigated. The STZ-diabetic rats treated with medium-dose and high-dose alga extracts showed remarkable reductions in fasting blood glucose (FBG) as compared with the STZ-diabetic control. The results indicate that the in vivo anti-hyperglycemic activity of the R. confervoides extracts can be partially attributed to the inhibitory actions against PTP1B of the bromophenol derivatives and that may be of clinical importance in improving the management of type 2 diabetes mellitus.

氢氧化锂存在下（焦）脱镁叶绿酸-a甲酯的空气重排反应

在氢氧化锂存在下，脱镁叶绿酸-a甲酯（1a）发生空气氧化和重排反应，经盐酸酸化和重氮甲烷甲基化，得到由紫红素-7三甲酯（2）、紫红素-18甲酯（3）、卟吩-P6三甲酯（4）、地质卟啉衍生物（5）和3-环氧乙基-3-去乙烯基紫红素-18甲酯（6）所组成的混合物．用相同的方法处理焦脱镁叶绿酸-a甲酯（1b），则分离出13^2-氧代焦脱镁叶绿酸-a甲酯（7）、15-甲酰基紫红素-5二甲酯（8）、紫红素-18甲酯（3）和3-环氧乙基-3-去乙烯基紫红素-18甲酯（6）-所得新叶绿素衍生物5，6和8的化学结构均经UV，IR，^1H NMR及元素分析得以证实，并对相应的反应提出可能的反应机理．

国产6种点地梅属植物的核型及其系统学意义

报道了点地梅属6种12个居群的染色体数目和核型。它们的染色体数目（2n）、核型公式（KF）、染色体相对长度组成（CRL）、核型不对称性系数（A. sK％）和核型类型（KT）分别为：点地梅在南京的2个居群都是2n=2x=20，KF=18m+2sm, CRL=2L+6M_2+10M_1+2S，核型KT属于1A型，但核型不对称性系数As. K％分别是55.04％和56.71％；北点地梅在内蒙古锡林郭勒和白音锡勒居群的2n=2x=20，KF分别为16m+2sm+2st+1b和16m+2sm+2st+4b，染色体相对长度组成分别是14M_2+4M_1+2S+1b和12M_2+6M_1+2S+4b，核型不对称性系数As. K％分别是57.65％和58.86％，属2A型和2B型；高原点地梅在青海玛沁县昌马河居群和兴海县温泉居群都是2n=2x=20，KF=10m+8sm+2st。但相对长度组成分别为12M_2+8M_1和4L+6M_2+6M_1+4S, As. K％分别是60.35％和62.57％，属2A型和2B型；雅江点地梅在青海玛多县巴颜喀拉山居群和大通县达坡山居群分别为2n=4x=40和20n=6x=60，KF=36m+2sm+2st和KF=46m+10sm+4st+2b, CRL=2l+20M_2+14M_1+4S和GRL=6L+30M_2+16M_1+8S+2b, As.K％分别为55.62％和58.31％，核型均属于2B；巴颜喀拉山北坡的鳞叶点地梅2n=40、60、80。西藏点地梅青湖居群中有2种细胞型：①2n=2x=24, KF=12m+6sm+6st+4b, CRL=6L+2M_2+8M_1+4S+4b, As. K％＝65.74％，核型属2B；②2n=2x=22,KF=14m+4sm(2SAT)+4st, CRL=4L+6M_2+8M_1(2SAT)+4S,As. K％=63.40％，核型也属于2B。西宁西山湾居群也有2种细胞型；①2n=3x=36, KF=36m,CRL=4L+12M_2+20M_1, As. K％=54.82％，核型属1A；②2n=3x=33,KF=33m, CRL=3L+12M_2+18M_1,As. K％=52.11％，核型属1A。点地梅属的染色体原始基数可能是x=10，在种间或种内观察到有3种核型变化：染色体非整倍性变化、多倍化和核型不对称性变化。将2倍体居群的核型和不对称性进行比较，可以看出点地梅是较对称的核型。因此，在研究的种中应是比较原始的类群。北点地梅的核型不对称性和进化程度高于点地梅而低于高原点地梅和西藏点地梅。染色体多倍化的雅江点地梅、鳞叶点地梅和西藏点地梅等在核型上也许是最进化的类群。

巨穗小麦种质小穗数的染色体定位研究

巨穗小麦新种质材料是一具有茎秆粗壮、叶片短宽直立、穗大、粒大、结实率高等特点的种质资源。应用单体分析和双端体分析方法对“241”材料进行遗传学研究，结果表明，小麦新种质材料“241”的3A、1B、2B和6B染色体上具有控制小穗数的隐性基因，其中3A、1B和2B染色体上的基因表现为强效，6B染色体上的基因表现为弱效。通过双端体分析进一步将控制小穗数的基因定位到3AS、1BL和6BS上，其中3AS、6BS上可能具有控制“241”小穗数的新基因。控制“241”穗粒数和控制小穗数的基因可能存在一定的连锁关系。

雅江点地梅（报春花科）3个居群的核型和倍性变化

首次报道了雅江点地梅(Androsace yargongensis) 3 个居群的染色体数目和核型, 对倍性也进行研究。3个居群的染色体数目(2n) , 核型公式(KF) , 染色体相对长度组成(C.RL ), 核型不对称系数(A s. K% ) 和核型类型(KT) 分别为: 野牛沟居群2n= 40; KF = 36 m(1SA T) + 2SM + 2ST + 2b, C. RL = 2L + 14M 2+ 22M 1+ 2S+ 2bS,As.K= 54. 75% , KT = 2A;巴颜喀拉山居群2n= 40, KF = 36 m + 2 sm + 2 st+ 1b, C.RL = 4L + 16M 2+ 18M 1+ 2S+1bS,As.K= 56. 31% , KT = 2B; 达坂山居群2n= 60, KF= 40 m + 14 sm + 6 st, C.RL = 4L +24M 2+ 26M 1+ 6S,As.K= 59. 56% , KT = 2B。根据3 个居群的染色体和核型不对称性与居群所在地的地理位置, 认为雅江点地梅核型和倍性的演化与高海拔生态环境和寒冷、干旱的气候加剧有密切关系。

溶液介质条件和腐殖影响下REE在水/粘土界面分配和分馏的实验研究

过去几十年，由于REE具有重要的物源和过程示踪的地球化学意义，又与核放射性元素（钢系元素）的地球化学结构类似，因此，稀土元素的表生地球化学分配和行为研究便成为微量元素地球化学研究的一个重要部分。目前"通过水体悬浮物吸附态REE组成变化分析研究水／微粒界面作用REE分异现象．的工作不少，但至今进行的胶体或其他微粒吸附REE的实验研究不多，且已有的实验未能对溶液介质条件（如pH、离子强度、阴离子、固／液比），尤其是天然有机物的控制机理进行系统研究，对所观察到的水体中REE及其他微量元素分布变化多样性的解释仍缺乏实验依据。本文用结晶良好且粘土矿物含量高的苏州高岭土、美国粘土协会高岭土（Kga-1b）和蒙脱土（Wwy-2）作为吸附剂，采用系列吸附实验定量研究了不同理化条件（如pH值、离子强度、固／液比）下，受溶液阴离子（Cl-、C1O4-、SO42-、HCO3-）和Fluka胡敏酸（HA）影响，REE在粘土／水界面的分配和分馏，并讨论了HA和粘土的相互作用及胡敏酸存在与不存在时的REE形态分布。得到以下几点重要认识，为合理解释地表水体中既E和其他微量元素分布变化的多样性提供了实验依据：1、REE在苏州高岭土／水界面的重现性实验结果为：稀土配分系数D的相对标准偏差最大值为Eu8.4％，其他大多介于5．0％和6．6％间。而稀土吸附率Rd的相对标准偏差最大值为D2.2％，其他大多＜2％。较小误差表明本次研究所用实验方法是可行的。2、REE在苏州高岭土／水界面的动力学实验结果表明：短时间内（几分钟）稀土快速吸附在高岭土：接着因为粘土的层状结构，在20h内粘土层间金属与REE发生交换，稀土分配系数变化较大；20h以后能达到稳定的吸附／解吸平衡。因此本次研究采用的平衡时间为24h。3、pH分别为4.5和6.5时REE在苏州高岭土冰界面的分配能用Langmuir吸附等温线模拟和MINEQL＋软件表达。与静电吸附相对应，pH值越高REE最大吸附量越大。同时REE浓度的差异造成了REE分馏，总的趋势是REE含量越高，分馏越不明显。4、近中性（pH＝6.5）条件下不同阴离子的存在对REE在苏州高岭土／水界面分配和分馏的影响表明：随阴离子（Cl-、ClO4-、SO42-）含量升高REE吸附率降低，其中SO42-对REE吸附的影响最大，说明Na＋质量效应和阴离子配合的影响；同时由于不同阴离子与轻重R陇的络合差异所致，阴离子含量越高，轻重稀土的分馏越明显（La／Yb＝0.14-0.96），一般为阴离子含量的增加使得重稀土更多的被吸附，其中C1-和SO42+的影响最为明显。HCO3-虽然与REE有较强配合，但可能由于我们的HCO3-实验浓度低（＜0.0O25mol／L），在我们的实验结果中其对REE吸附和分馏的影响较小。5、由于不同pH和介质条件下，REE的络合形态分布不同，它们可以影响其在水／粒界面的分配。应用MINEQL＋模型，考虑REE的氢氧化物、碳酸盐和腐殖酸的影响，研究了REE的形态分布，结果表明fIA的存在对REE形态有很大影响：在HA不存在时，pH7-8间REECO3＋为主要的REEs形态，在更低和更高pH值，REE主要存在形式分别为REE加和REE（CO3）2-；而当HA存在时，在1）H值3-9，REEHA成为主要形态，在低pH（＜3）和高pH（＞9）时REE3+和REE（CO3）2-分别为主要形态。6、在较宽的pH范围HA能吸附在粘土上意味着在大多数含HA的天然水体中，粘土表面被HA覆盖。随H增加粘土对HA的吸附降低，反应了配位体交换或表面络合反应引起的专属吸附，其他如疏水性、、腐殖物质的溶解和HA的结构变化可能也影响了吸附。HA含量、矿物表面积和离子强度等理化条件对HA吸附会产生影响，从而影响HA对粘土表面的覆盖和接下来的粘土对REE的吸附。7、溶液介质条件对REE在粘土（Kga-1b和SWy-2）／水界面分配和分馏的影响表明：主要与静电相互作用、离子交换反应相对应，除低PH外，REE在高岭土上的吸附表现出弱的pH依赖性；而随pH增加REE在蒙脱土上的吸附呈下降趋势，显示交换反应为吸附过程的主要因素。在两种粘土中REE吸附均为随离子强度增加而降低，反应了Na质量效应。8、腐殖物质在粘土表面的吸附改变了吸附剂的属性。HA存在时REE在固液界面的分配反应了REEHA在固液界面的分配和HA在高岭土或蒙脱土／水界面上的分配，其他如静电吸附等机制也影响了吸附：在高岭土中，HA会增加低pH（＜4）吸附，随pH增加（＞5）HA会降低吸附。与此不同，在整个PH范围（3-10），HA的存在明显降低了REE在蒙脱土上的吸附。REE吸附在高岭土上随队含量增加是先增加后降低。而蒙脱土实验中，在低HA含量（＜5mg／L）处，RE阮吸附率与HA含量增加呈线性降低至几个百分比，而在高队含量处，REEs吸，附率无明显变化。这些清楚地说明水体环境中有机物的存在通常降低微量金属吸，附在微粒上，增加微量金属在水体中的溶解量，从而促进微量金属在水体环境中的长距离迁移。9、HA存在和不存在时，吸附／解吸过程中的REE分馏随pH或离子强度的变化都不明显，但由于REE系列与腐殖物质和矿物络合的差异，会随队含量的变化发生明显变化。通常溶液中高队含量增加了LREE在高岭土／蒙脱土上的吸附。这些结果是在HA存在时，微粒相上产生LREE富集的一个实验证实，也和大多数天然水体中的REEs分馏相一致。

Genômica e proteômica de Anaplasma marginale: contribuições para o controle da riquétsia.

A anaplasmose bovina é causada pela riquétsia intra-eritrocítica Anaplasma marginale, responsável por importantes prejuízos econômicos, por causa da alta morbidade e mortalidade em rebanhos bovinos suscetíveis. A vacinação tem sido uma forma econômica e eficiente de controlar a enfermidade. No entanto, os métodos de imunização tradicionais apresentam efeitos adversos em algumas categorias de animais. Nas últimas décadas, os estudos sobre imunização contra Anaplasma concentraram-se nas proteínas de superfície MSP1a, 1b, 2, 3, 4 e 5. No entanto, até o momento, os resultados foram pouco promissores, apontando a necessidade de ampliar o conhecimento sobre o rol das proteínas de membrana da riquétsia e das relações estruturais entre elas. Nesse contexto, os estudos do genoma e do proteoma da riquétsia têm contribuído com essa finalidade. Pela análise genômica, 14 genes para novas proteínas de membrana externa foram identificados (omp 1-14), dentre os quais, omp2, 3 e 6 não são transcritos. Esses genes ostraram-se altamente conservados entre isolados da riquétsia. As proteínas OMP4, 7, 10 e 14 foram reconhecidas por soros de bovinos imunizados com membrana de A. marginale, mostrando potencial para desenvolvimento de imunógenos. Além disso, mediante análise proteômica, foi possível detectar novas proteínas de membrana, negligenciadas pela anotação genômica. Dentre elas estão AM097 - conjugal transfer protein, AM956 - PepA citosol amino peptidase, AM254 - fator de elongação Tu e quatro proteínas de função desconhecida: AM127, 197, 387 e 854, as quais também foram reconhecidas por soros de bovinos imunizados com membrana de A. marginale.

Orientações práticas para a fertirrigação do mamoeiro.

Os métodos de irrigação mais recomendados para a cultura do mamoeiro têm sido os métodos pressurizados, isto é, a irrigação por aspersão e localizada. Dentre os sistemas de irrigação por aspersão, os sistemas autopropelidos (Figura 1a) e os pivôs centrais (Figura 1b) têm sido os mais utilizados. Em se tratando de sistemas de irrigação localizada, a fertirrigação via microaspersão deve levar em conta a distribuição de água pelo microaspersor, que segue um padrão conforme a Figura 2, onde a maior quantidade de água cai próximo do emissor reduzindo-se à medida em que se afasta deste. A concentração de íons da água de irrigação é uniforme, isto é, apresenta pequena variação na área molhada na superfície do solo, consequentemente, a distribuição do fertilizante é desuniforme, ou seja, a região mais próxima do emissor recebe maior quantidade de fertilizante comparada às regiões mais afastadas do emissor.

3'-UTR SIRF: A database for identifying clusters of short interspersed repeats in 3' untranslated regions

BACKGROUND:Short (~5 nucleotides) interspersed repeats regulate several aspects of post-transcriptional gene expression. Previously we developed an algorithm (REPFIND) that assigns P-values to all repeated motifs in a given nucleic acid sequence and reliably identifies clusters of short CAC-containing motifs required for mRNA localization in Xenopus oocytes.DESCRIPTION:In order to facilitate the identification of genes possessing clusters of repeats that regulate post-transcriptional aspects of gene expression in mammalian genes, we used REPFIND to create a database of all repeated motifs in the 3' untranslated regions (UTR) of genes from the Mammalian Gene Collection (MGC). The MGC database includes seven vertebrate species: human, cow, rat, mouse and three non-mammalian vertebrate species. A web-based application was developed to search this database of repeated motifs to generate species-specific lists of genes containing specific classes of repeats in their 3'-UTRs. This computational tool is called 3'-UTR SIRF (Short Interspersed Repeat Finder), and it reveals that hundreds of human genes contain an abundance of short CAC-rich and CAG-rich repeats in their 3'-UTRs that are similar to those found in mRNAs localized to the neurites of neurons. We tested four candidate mRNAs for localization in rat hippocampal neurons by in situ hybridization. Our results show that two candidate CAC-rich (Syntaxin 1B and Tubulin beta4) and two candidate CAG-rich (Sec61alpha and Syntaxin 1A) mRNAs are localized to distal neurites, whereas two control mRNAs lacking repeated motifs in their 3'-UTR remain primarily in the cell body.CONCLUSION:Computational data generated with 3'-UTR SIRF indicate that hundreds of mammalian genes have an abundance of short CA-containing motifs that may direct mRNA localization in neurons. In situ hybridization shows that four candidate mRNAs are localized to distal neurites of cultured hippocampal neurons. These data suggest that short CA-containing motifs may be part of a widely utilized genetic code that regulates mRNA localization in vertebrate cells. The use of 3'-UTR SIRF to search for new classes of motifs that regulate other aspects of gene expression should yield important information in future studies addressing cis-regulatory information located in 3'-UTRs.

Novel insights into the role of the GABAB receptor in depression and anxiety

The GABAB receptor is a functional heterodimer comprising the GABAB1 and GABAB2 subunits, with the GABAB1 subunit displaying two major isoforms, GABAB(1a) and GABAB(1b). Preclinical findings have strongly implicated the GABAB receptor in stress-related psychiatric disorders, however, the precise contribution of the GABAB receptor in depression and anxiety disorders remains unknown. Emerging data suggest that the interaction between adverse environmental conditions, such as early life stress, and a specific genetic composition can increase the risk to develop psychiatric disorders in adulthood. This thesis investigated the role of the GABAB receptor alone or in combination with early-life stress (maternal separation), in modulating antidepressant like and anxiety-related behaviours. Pharmacological blockade of the GABAB receptor with CGP52432 had antidepressant-like behavioural effects. Moreover, mice lacking the GABAB(1b) receptor subunit isoform exhibited antidepressant-like behaviours in adulthood but anxiety-like behaviour in early-life. In response to maternal separation, GABAB(1a)-/- mice exhibited early-life stress-induced anhedonia, a core symptom of depression, while GABAB(1b)-/- mice exhibited a more resilient phenotype. Moreover, when compared with wildtype or GABAB(1a)-/- mice, GABAB(1b)-/- mice that underwent maternal separation exhibited enhanced stressinduced neuronal activation in the hippocampus and in the nucleus accumbens (NAcc), a critical area for anhedonia thus suggesting that enhanced stress-induced neuronal activation in the hippocampus and NAcc in GABAB(1b)-/- mice may be important for their antidepressant-like phenotype and their resilience to stress-induced anhedonia. Pharmacological blockade of GABAB receptor and GABAB(1b) receptor subunit isoform loss of function increased adult hippocampal cell proliferation, thus suggesting that increased hippocampal neurogenesis could be a potential mechanism for the antidepressant-like effects of GABAB receptor antagonists and GABAB(1b) receptor subunit isoform disruption. Finally, this thesis investigated whether the expression of several genes involved in hippocampal neurogenesis or the antidepressant response were altered in the mouse hippocampus following chronic treatment with a GABAB receptor antagonist.

Novel roles for the GABAB receptor in anxiety and cocaine addiction

The GABAB receptor has been postulated as a possible drug target in the treatment of anxiety disorders and cocaine addiction. Indeed, a wealth of preclinical data is emerging that has shown that mice lacking functional GABAB receptors display a highly anxious behaviour across a range of behavioural models of anxiety. Additionally, novel compounds that act by altering the allosteric conformation of the GABAB receptor to a more active state; the GABAB receptor positive modulators, have been repeatedly demonstrated to have anxiolytic effects in animals. In addition to being a putative anxiolytic drug target, the GABAB receptor has been identified as a novel target for antiaddictive therapies. Indeed GABAB receptor positive modulators have been demonstrated to have anti-addictive properties across a broad variety of behavioural paradigms. Despite these findings, several gaps in our knowledge of the role played by the GABAB receptor in both anxiety and drug abuse disorder exist. The aim of this thesis was to use preclinical animal models in an effort to further probe the role played by the GABAB receptor in anxiety and addiction. Our studies initially examined the role played by the GABAB receptor in the neurodevelopmental processes underpinning of anxiety. Our studies demonstrated that treating mouse pups in early life with the GABAB receptor agonist baclofen produced an anxious phenotype in adult life, whereas treatment with the GABAB receptor antagonist CGP52432 produced no effects on adult behaviour. Further to this, we examined whether the anxious behaviour induced by early life blockade of the serotonin reuptake transporter was dependant on alterations in GABAB receptor function. Our studies however revealed no effect of early life selective serotonin reuptake inhibitor treatment on adult life baclofen sensitivity. The next issue addressed in this thesis is the characterization of the effects of a GABAB receptor positive modulator and a GABAB receptor antagonist in a behavioural model of conditioned fear behaviour. These novel classes of GABAB receptor ligands have been considerably less well characterized in this facet of preclinical anxiety behaviour than in terms of innate anxiety behaviour. Our study however revealed that the GABAB receptor positive modulator GS39783 and the GABAB receptor antagonist CGP52432 were without effect on the acquisition, expression or extinction of conditioned fear in our model. The next element of this thesis dealt with the characterization of a novel mouse model, the GABAB(2)- S892A mouse. This mouse has been engineered to express a form of the GABAB(2) receptor subunit wherein the function determining serine phosphorylation site cannot be phosphorylated. We initially tested this mouse in terms of its GABAB receptor function in adult life, followed by testing it in a battery of tests of unconditioned and learned anxiety behaviour. We also examined the behavioural and molecular responses of the GABAB(2)-S892A mouse to cocaine. All of our studies appear to show that the GABAB(2)-S892A mouse is indistinguishable from wildtype controls. The final aim of the thesis was to investigate the behavioural and molecular sensitivity of the GABAB(1) subunit isoform null mice, the GABAB(1a) -/- and GABAB(1b) -/- mice to cocaine. Our studies revealed that these mice display differing behavioural responses to cocaine, with the GABAB(1a) -/- mouse displaying a hypersensitivity to the acute locomotor effects of cocaine, while the GABAB(1b) -/- displayed blunted locomotor sensitisation to cocaine.

Hybrid transgenic mice reveal in vivo specificity of G protein-coupled receptor kinases in the heart.

G protein-coupled receptor kinases (GRKs) phosphorylate activated G protein-coupled receptors, including alpha(1B)-adrenergic receptors (ARs), resulting in desensitization. In vivo analysis of GRK substrate selectivity has been limited. Therefore, we generated hybrid transgenic mice with myocardium-targeted overexpression of 1 of 3 GRKs expressed in the heart (GRK2 [commonly known as the beta-AR kinase 1], GRK3, or GRK5) with concomitant cardiac expression of a constitutively activated mutant (CAM) or wild-type alpha(1B)AR. Transgenic mice with cardiac CAMalpha(1B)AR overexpression had enhanced myocardial alpha(1)AR signaling and elevated heart-to-body weight ratios with ventricular atrial natriuretic factor expression denoting myocardial hypertrophy. Transgenic mouse hearts overexpressing only GRK2, GRK3, or GRK5 had no hypertrophy. In hybrid transgenic mice, enhanced in vivo signaling through CAMalpha(1B)ARs, as measured by myocardial diacylglycerol content, was attenuated by concomitant overexpression of GRK3 but not GRK2 or GRK5. CAMalpha(1B)AR-induced hypertrophy and ventricular atrial natriuretic factor expression were significantly attenuated with either concurrent GRK3 or GRK5 overexpression. Similar GRK selectivity was seen in hybrid transgenic mice with wild-type alpha(1B)AR overexpression concurrently with a GRK. GRK2 overexpression was without effect on any in vivo CAM or wild-type alpha(1B)AR cardiac phenotype, which is in contrast to previously reported in vitro findings. Furthermore, endogenous myocardial alpha(1)AR mitogen-activated protein kinase signaling in single-GRK transgenic mice also exhibited selectivity, as GRK3 and GRK5 desensitized in vivo alpha(1)AR mitogen-activated protein kinase responses that were unaffected by GRK2 overexpression. Thus, these results demonstrate that GRKs differentially interact with alpha(1B)ARs in vivo such that GRK3 desensitizes all alpha(1B)AR signaling, whereas GRK5 has partial effects and, most interestingly, GRK2 has no effect on in vivo alpha(1B)AR signaling in the heart.