Pharmacological and immunocytochemical investigation of the role of catecholamines on larval metamorphosis by beta-adrenergic-like receptor in the bivalve Meretrix meretrix

Catecholamines regulate several physiological processes in mollusks. Many pharmacological experiments have been conducted to determine the effects of adrenergic agonist and antagonist of catecholamine receptors on Meretrix meretrix metamorphosis. Results showed that adrenaline (AD) and noradrenaline (NA) had substantial effects (p < 0.05) on larval metamorphosis at concentrations ranging from 10 mu M to 100 mu M. 10 mu M beta-adrenergic receptor (AR) agonist isoproterenol showed the same inducement effect as that of NA and AD on metamorphosis, whereas the alpha-AR agonist phenylephrine had no significant effect at concentrations between 0.1 mu M and 100 mu M concentrations (p > 0.05). Furthermore, I mu M beta-AR antagonist propanolol, but not alpha-AR antagonist prazosin, depressed the larval metamorphosis induced by NA or AD. By immunocytochemistry, two cell bodies of beta-adrenergic-like receptor, C/A1, C/A2, were observed in the cerebral/apical ganglion of competent larvae. In addition, there were other immunoreactive dots near C/A1 and C/A2. The results of pharmacology and immunocytochemistry suggests that beta-adrenergic-like receptor located in the larval CNS, might play a considerable role in the larval metamorphosis of M meretrix by AD or NA. (c) 2006 Elsevier B.V. All rights reserved.

栉孔扇贝(Chlamys farreri)Toll样受体及其信号传递相关基因的克隆与表达

扇贝是我国海水养殖的重要品种，但自1994年以来，养殖扇贝陆续爆发的大规模死亡，不但造成了巨大的经济损失，而且直接威胁到现有产业的生存和发展。引起扇贝大规模死亡原因是多方面的，其主要原因是养殖环境恶化、扇贝种质衰退和抗病力下降。因此，深入研究扇贝免疫防御机制，探讨提高机体抗病力的有效途径和方法，改良种质和培育抗病品系，无疑是解决目前困扰扇贝养殖业健康可持续发展的必经之路。 Toll样受体（TLRs）家族是新近发现的模式识别受体（PRRs），参与识别病原体相关的分子模式（PAMPs），在天然免疫系统中起着非常重要的作用。哺乳动物中Toll样受体信号通路还参与诱导树枝状细胞成熟、参与免疫耐受、参与凋亡发生发展、介导非感染性因素的识别等，被视为联系天然免疫和获得性免疫的桥梁。同时果蝇的Toll信号通路也是不具备获得性免疫的果蝇赖以抵御病毒、细菌和真菌感染，介导天然免疫反应的重要信号通路。 本研究采用大规模EST测序方法，结合Genome Walker库的构建和cDNA末端快速扩增技术，从栉孔扇贝克隆得到CfToll-1、CfMyd88、CfTRAF6和CfCactus这四个Toll样受体信号通路基因的全长cDNA，同时用荧光实时定量PCR技术检测了这些基因的组织分布及在脂多糖（LPS）和肽聚糖（PGN）刺激下的表达规律。 栉孔扇贝Toll样受体（CfToll-1）的cDNA序列全长4308 bp，包含5’非翻译区（UTR）211 bp，3597 bp的开放阅读框，500 bp的3’UTR，最后为18个腺嘌呤的ploy A 尾巴。开放阅读框编码1198个氨基酸的多肽，该多肽的估计分子量为137.41kd，估计的等电点为5.62，该多肽有信号肽，具有一个预测的跨膜区，因此是一种跨膜蛋白。经BLAST比对，CfToll-1基因与节肢动物多种Toll蛋白高度的相似性。SMART（Simple Modular Architecture Research Tool）软件分析，CfToll-1包含典型的Toll样受体的结构：富含亮氨酸的重复序列的胞外区（leucine-rich repeats, LRR），一段跨膜结构域，以及胞内区的TIR结构域（Toll/IL-1 receptor homologous region）。利用Real-time RT-PCR发现CfToll-1mRNA在扇贝体内普遍存在于血细胞、肌肉、外套膜、心、性腺和鳃组织中。利用体外培养的原代血细胞系研究不同浓度LPS刺激后CfToll-1的表达变化，结果显示低剂量（100ng.mL-1 ）LPS 使CfToll-1 mRNA表达量减小，该变化在1.5h、3h 和9h组差异显著，虽然在6h组表达量稍有恢复，但尚未达到对照水平；用1μg.mL-1LPS处理细胞时， 6h组CfToll-1表达量明显上调，约为对照水平的2倍。证实细菌结构脂多糖对CfToll-1基因的表达有影响，且这种影响有剂量依赖效应。 栉孔扇贝Myd88同源基因（CfMyd88）的cDNA序列全长1554bp，包含5’UTR 427 bp，1101bp的开放阅读框，最后为18个腺嘌呤的ploy A 尾。CfMyd88的开放阅读框可编码367个氨基酸的多肽，该多肽的估计分子量为42.37kD，估计的等电点为5.71。利用SMART程序分析发现CfMyd88编码了Death和TIR结构域, 这两个结构域是Myd88特征结构。BLAST程序发现扇贝的序列与数据库哺乳动物的Myd88基因高度同源。原代培养的扇贝血细胞在受到PGN刺激后，CfMyd88 mRNA表达在1.5小时开始下调，直到9小时下调至对照表达量的1/10，证实肽聚糖结构对CfMyd88基因的表达有影响。 栉孔扇贝TRAF6同源基因（CfTRAF6）的cDNA序列全长2510bp，包含5’UTR 337 bp，1965bp的开放阅读框，3’UTR 208bp，最后为21 个腺嘌呤的ploy A 尾巴。CfTRAF6开放阅读框编码655个氨基酸的多肽，该多肽的估计分子量为74.09kD，估计的等电点为6.01。InterPro Scan在线分析发现CfTRAF6有典型的TRAF蛋白家族的特征结构，包括的一个指环结构，两个锌指结构，一个MATH （the meprin and TRAF homology）结构域以及Coiled-coil区域。CfTRAF6的序列与数据库多物种的TRAF6高度同源，同源性最高的是乌贼序列（Identity＝68）和鼠类（Identity＝45%）。利用Real-time RT-PCR，发现CfTRAF6在各组织普遍存在，在性腺中的表达最高。原代培养的扇贝血细胞在受到不同浓度PGN刺激后，与CfMyd88的情况一样，CfTRAF6的表达量变化减少，且这种变化随剂量的增加更加明显。 栉孔扇贝Cactus同源基因（CfCactus）的cDNA序列全长2488bp，包含5’UTR 181 bp，840bp的开放阅读框， 3’UTR 1467bp，最后为19个腺嘌呤的ploy A 尾巴。CfCactus的开放阅读框编码279个氨基酸的多肽，该多肽的估计分子量为31.37 kD；估计的等电点为4.74，与果蝇的Cactus基因的等电点相近（4.5）。利用SMART程序分析发现CfCactus主要编码了ANK结构域（ankyrin repeats）。Cactus基因为哺乳动物NF-κB抑制蛋白IκB的同源分子，BLAST 程序发现扇贝的序列与数据库多物种的Cactus或IκB基因高度同源。同源性最高的是太平洋牡蛎（Identity=35%）和圆尾鲎（Identities = 44%）。对CfTCactus mRNA在扇贝的血细胞、性腺、 肠的组织表达进行分析，并同时与CfTRAF6和CfMyd88的表达量进行了对比，发现CfCactus的表达水平明显高于这两个基因，而且CfTRAF6的基因表达量也高于CfMyd88，表现出级联放大效应。正常情况下，三个基因在性腺的表达量最高，推测这条通路可能和发育等功能密切相关。 通过本研究我们首次在双壳类软体动物找得到与果蝇Toll蛋白家族高度同源的CfToll-1基因，同时发现其他三个在Toll样受体信号传递过程中起重要作用的基因，其中包括在软体动物中获得的第一个Toll样受体的接头分子－CfMyd88基因，该结果直接证明软体动物具有与哺乳动物和节肢动物高度类似Myd88依赖的Toll样受体信号通路。同时通过这些基因组织分布的研究以及细菌结构LPS和PGN对这条通路上基因表达的影响，证明扇贝Toll信号通路可能与在果蝇中一样，参与扇贝的发育和免疫防御等多种功能。

STAT1 – główny czynnik regulatorowy w zapaleniu i dysfunkcji naczyniowej

Wydział Biologii: Instytut Biologii Molekularnej i Biotechnologii

Prolactin receptor signaling is essential for perinatal brown adipocyte function: a role for insulin-like growth factor-2.

BACKGROUND: The lactogenic hormones prolactin (PRL) and placental lactogens (PL) play central roles in reproduction and mammary development. Their actions are mediated via binding to PRL receptor (PRLR), highly expressed in brown adipose tissue (BAT), yet their impact on adipocyte function and metabolism remains unclear. METHODOLOGY/PRINCIPAL FINDINGS: PRLR knockout (KO) newborn mice were phenotypically characterized in terms of thermoregulation and their BAT differentiation assayed for gene expression studies. Derived brown preadipocyte cell lines were established to evaluate the molecular mechanisms involved in PRL signaling on BAT function. Here, we report that newborn mice lacking PRLR have hypotrophic BAT depots that express low levels of adipocyte nuclear receptor PPARgamma2, its coactivator PGC-1alpha, uncoupling protein 1 (UCP1) and the beta3 adrenoceptor, reducing mouse viability during cold challenge. Immortalized PRLR KO preadipocytes fail to undergo differentiation into mature adipocytes, a defect reversed by reintroduction of PRLR. That the effects of the lactogens in BAT are at least partly mediated by Insulin-like Growth Factor-2 (IGF-2) is supported by: i) a striking reduction in BAT IGF-2 expression in PRLR KO mice and in PRLR-deficient preadipocytes; ii) induction of cellular IGF-2 expression by PRL through JAK2/STAT5 pathway activation; and iii) reversal of defective differentiation in PRLR KO cells by exogenous IGF-2. CONCLUSIONS: Our findings demonstrate that the lactogens act in concert with IGF-2 to control brown adipocyte differentiation and growth. Given the prominent role of brown adipose tissue during the perinatal period, our results identified prolactin receptor signaling as a major player and a potential therapeutic target in protecting newborn mammals against hypothermia.

Nutritional Control of L1 Arrest and Recovery in Caenorhabditis elegans by Insulin-like Peptides and Signaling

Animals must coordinate development with fluctuating nutrient availability. Nutrient availability governs post-embryonic development in Caenorhabditis elegans: larvae that hatch in the absence of food do not initiate post-embryonic development but enter "L1 arrest" (or "L1 diapause") and can survive starvation for weeks, while rapidly resume normal development once get fed. Insulin-like signaling (IIS) has been shown to be a key regulator of L1 arrest and recovery. However, the C. elegans genome encodes 40 insulin-like peptides (ILPs), and it is unknown which peptides participate in nutritional control of L1 arrest and recovery. Work in other contexts has identified putative receptor agonists and antagonists, but the extent of specificity versus redundancy is unclear beyond this distinction.

We measured mRNA expression dynamics with high temporal resolution for all 40 insulin-like genes during entry into and recovery from L1 arrest. Nutrient availability influences expression of the majority of insulin-like genes, with variable dynamics suggesting complex regulation. We identified 13 candidate agonists and 8 candidate antagonists based on expression in response to nutrient availability. We selected ten candidate agonists (daf-28, ins-3, ins-4, ins-5, ins-6, ins-7, ins-9, ins-26, ins-33 and ins-35) for further characterization in L1 stage larvae. We used destabilized reporter genes to determine spatial expression patterns. Expression of candidate agonists was largely overlapping in L1 stage larvae, suggesting a role of the intestine, chemosensory neurons ASI and ASJ, and the interneuron PVT in systemic control of L1 development. Transcriptional regulation of candidate agonists was most significant in the intestine, as if nutrient uptake was a more important influence on transcription than sensory perception. Scanning in the 5' upstream promoter region of these 40 ILPs, We found that transcription factor PQM-1 and GATA putative binding sites are depleted in the promoter region of antagonists. A novel motif was also found to be over-represented in ILPs.

Phenotypic analysis of single and compound deletion mutants did not reveal effects on L1 recovery/developmental dynamics, though simultaneous disruption of ins-4 and daf-28 extended survival of L1 arrest without enhancing thermal tolerance, while overexpression of ins-4, ins-6 or daf-28 shortened L1 survival. Simultaneous disruption of several ILPs showed a temperature independent, transient dauer phenotype. These results revealed the relative redundancy and specificity among agonistic ILPs.

TGF- 46; and steroid hormone (SH) signaling have been reported to control the dauer formation along with IIS. Our preliminary results suggest they may also mediate the IIS control of L1 arrest and recovery, as the expression of several key components of TGF- 46; and SH signaling pathway genes are negatively regulated by DAF-16, and loss-of-function of these genes partially represses daf-16 null phenotype in L1 arrest, and causes a retardation in L1 development.

In summary, my dissertation study focused on the IIS, characterized the dynamics and sites of ILPs expression in response to nutrient availability, revealed the function of specific agonistic ILPs in L1 arrest, and suggested potential cross-regulation among IIS, TGF- 46; signaling and SH signaling in controlling L1 arrest and recovery. These findings provide insights into how post-embryonic development is governed by insulin-like signaling and nutrient availability.

Sensitive and precise quantification of insulin-like mRNA expression in Caenorhabditis elegans.

Insulin-like signaling regulates developmental arrest, stress resistance and lifespan in the nematode Caenorhabditis elegans. However, the genome encodes 40 insulin-like peptides, and the regulation and function of individual peptides is largely uncharacterized. We used the nCounter platform to measure mRNA expression of all 40 insulin-like peptides as well as the insulin-like receptor daf-2, its transcriptional effector daf-16, and the daf-16 target gene sod-3. We validated the platform using 53 RNA samples previously characterized by high density oligonucleotide microarray analysis. For this set of genes and the standard nCounter protocol, sensitivity and precision were comparable between the two platforms. We optimized conditions of the nCounter assay by varying the mass of total RNA used for hybridization, thereby increasing sensitivity up to 50-fold and reducing the median coefficient of variation as much as 4-fold. We used deletion mutants to demonstrate specificity of the assay, and we used optimized conditions to assay insulin-like gene expression throughout the C. elegans life cycle. We detected expression for nearly all insulin-like genes and find that they are expressed in a variety of distinct patterns suggesting complexity of regulation and specificity of function. We identified insulin-like genes that are specifically expressed during developmental arrest, larval development, adulthood and embryogenesis. These results demonstrate that the nCounter platform provides a powerful approach to analyzing insulin-like gene expression dynamics, and they suggest hypotheses about the function of individual insulin-like genes.

Achiral, selective CCK2 receptor antagonists based on a 1,3,5-benzotriazepine-2,4-dione template

Novel, achiral 1H-1,3,5-benzotriazepine-2,4(3H,5H)-diones have been prepared and structurally characterized. These compounds are potent CCK2 receptor antagonists that display a high degree of selectivity over CCK1 receptors.

N-terminal His(7)-modification of glucagon-like peptide-1(7-36) amide generates dipeptidyl peptidase IV-stable analogues with potent antihyperglycaemic activity

Glucagon-like peptide-1(7-36)amide (GLP-1) possesses several unique and beneficial effects for the potential treatment of type 2 diabetes. However, the rapid in-activation of GLP-1 by dipeptidyl peptidase IV (DPP IV) results in a short half-life in vivo (less than 2 min) hindering therapeutic development. In the present study, a novel His(7)-modified analogue of GLP-1, N-pyroglutamyl-GLP-1, as well as N-acetyl-GLP-1 were synthesised and tested for DPP IV stability and biological activity. Incubation of GLP-1 with either DPP IV or human plasma resulted in rapid degradation of native GLP-1 to GLP-1 (9-36),amide, while N-acetyl-GLP-1 and N-pyroglutamyl-GLP-1 were completely resistant to degradation. N-acetyl-GLP-1 and N-pyroglutamyl-GLP-1 bound to the GLP-1 receptor but had reduced affinities (IC50 values 32(.)9 and 6(.)7 nM, respectively) compared with native GLP-1 (IC50 0(.)37 nM). Similarly, both analogues stimulated cAMP production with EC50 values of 16(.)3 and 27 nM respectively compared with GLP-1 (EC50 4(.)7 nM). However, N-acetyl-GLP-1 and N-pyroglutamyl-GLP-1 exhibited potent insulinotropic activity in vitro at 5(.)6 mM glucose (P

Novel dipeptidyl peptidase IV resistant analogues of glucagon-like peptide-1(7-36)amide have preserved biological activities in vitro conferring improved glucose-lowering action in vivo

Although the incretin hormone glucagon-like peptide-1 (GLP-1) is a potent stimulator of insulin release, its rapid degradation in vivo by the enzyme dipeptidyl peptidase IV (DPP IV) greatly limits its potential for treatment of type 2 diabetes. Here, we report two novel Ala(8)-substituted analogues of GLP-1, (Abu(8))GLP-1 and (Val(8) GLP-1 which were completely resistant to inactivation by DPP IV or human plasma. (Abu(8))GLP-1 and (Val(8))GLP-1 exhibited moderate affinities (IC50: 4.76 and 81.1 nM, respectively) for the human GLP-1 receptor compared with native GLP-1 (IC50: 0.37 nM). (Abu(8))GLP-1 and (Val(8))GLP-1 dose-dependently stimulated cAMP in insulin-secreting BRIN BD11 cells with reduced potency compared with native GLP-1 (1.5- and 3.5-fold, respectively). Consistent with other mechanisms of action, the analogues showed similar, or in the case of (Val(8))GLP-1 slightly impaired insulin releasing activity in BRIN BD11 cells. Using adult obese (ob/ob) mice, (Abu(8))GLP-1 had similar glucose-lowering potency to native GLP-1 whereas the action of (Val(8))GLP-1 was enhanced by 37%. The in vivo insulin-releasing activities were similar. These data indicate that substitution of Ala(8) in GLP-1 with Abu or Val confers resistance to DPP IV inactivation and that (Val(8))GLP-1 is a particularly potent N-terminally modified GLP-1 analogue of possible use in type 2 diabetes.

Differential expression of components of the cardiomyocyte adrenomedullin/intermedin receptor system following blood pressure reduction in nitric oxide-deficient hypertension.

Adrenomedullin (AM) and intermedin (IMD; adrenomedulln-2) are vasodilator peptides related to calcitonin gene-related peptide (CGRP). The actions of these peptides are mediated by the calcitonin receptor-like receptor (CLR) in association with one of three receptor activity-modifying proteins. CGRP is selective for CLR/receptor activity modifying protein (RAMP)1, AM for CLR/RAMP2 and -3, and IMD acts at both CGRP and AM receptors. In a model of pressure overload induced by inhibition of nitric-oxide synthase, up-regulation of AM was observed previously in cardiomyocytes demonstrating a hypertrophic phenotype. The current objective was to examine the effects of blood pressure reduction on cardiomyocyte expression of AM and IMD and their receptor components. Nomega-nitro-L-arginine methyl ester (L-NAME) (35 mg/kg/day) was administered to rats for 8 weeks, with or without concurrent administration of hydralazine (50 mg/kg/day) and hydrochlorothiazide (7.5 mg/kg/day). In left ventricular cardiomyocytes from L-NAME-treated rats, increases (-fold) in mRNA expression were 1.6 (preproAM), 8.4 (preproIMD), 3.4 (CLR), 4.1 (RAMP1), 2.8 (RAMP2), and 4.4 (RAMP3). Hydralazine/hydrochlorothiazide normalized systolic blood pressure (BP) and abolished mRNA up-regulation of hypertrophic markers sk-alpha-actin and BNP and of preproAM, CLR, RAMP2, and RAMP3 but did not normalize cardiomyocyte width nor preproIMD or RAMP1 mRNA expression. The robust increase in IMD expression indicates an important role for this peptide in the cardiac pathology of this model but, unlike AM, IMD is not associated with pressure overload upon the myocardium. The concordance of IMD and RAMP1 up-regulation indicates a CGRP-type receptor action; considering also a lack of response to BP reduction, IMD may, like CGRP, have an anti-ischemic function.