Toll-like receptor 3 regulates Mx expression in rare minnow Gobiocypris rarus after viral infection

Toll-like receptor 3 (TLR3) plays a key role in activating immune responses during viral infection. To study the genes involved in the regulatory function of TLR3 in the rare minnow Gobiocypris rarus after viral infection, a full-length cDNA of TLR3 (GrTLR3) with a splice variant (GrTLR3s) was identified by homologous cloning and RACE techniques. The antiviral effector molecule Mx gene was cloned and partially sequenced. The mRNA expression levels of GrTLR3, GrTLR3s, and Mx were studied in different tissues before and after virus infection by real-time quantitative RT-PCR. The transcripts of all three genes in liver were significantly increased following GCRV infection (P<0.05). The mRNA levels in liver were upregulated at 24 h post-injection for GrTLR3 and GrTLR3s, and at 12 h for Mx. The upregulated expression levels were several folds for GrTLR3s, tens of folds for GrTLR3, and hundreds of folds for Mx. By semi-quantitative RT-PCR, GrTLR3 and Mx expressed at all the developmental stages, whereas GrTLR3s could only be detected at later developmental stages. Using RNAi and transgenic techniques, GrTLR3 mediated Mx expression but GrTLR3s did not. The time-dependent upregulation of receptor and effector, and the Mx over-expression dependent on TLR3, indicated that GrTLR3 regulated Mx expression in viral infection through a configuration change in rare minnow, and its splice variant did not contribute to the process.

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.

Subcellular localization and characterization of G protein-coupled receptor homolog from lymphocystis disease virus isolated in China

G protein-coupled receptors (GPCRs) constitute a large superfamily involved in various types of signal transduction pathways, and play an important role in coordinating the activation and migration of leukocytes to sites of infection and inflammation. Viral GPCRs, on the other hand, can help the virus to escape from host immune surveillance and contribute to viral pathogenesis. Lymphocystis disease virus isolated in China (LCDV-C) contains a putative homolog of cellular GPCRs, LCDV-C GPCR. In this paper, LCDV-C GPCR was cloned, and the subcellular localization and characterization of GPCR protein were investigated in fish cells. LCDV-C GPCR encoded a 325-amino acid peptide, containing a typical seven-transmembrane domain characteristic of the chemokine receptors and a conserved DRY motif that is usually essential for receptor activation. Transient transfection of GPCR-EGFP in fathead minnow (FHM) cells and epithelioma papulosum cyprini (EPC) cells indicated that LCDV-C GPCR was expressed abundantly in both the cytoplasm and nucleoplasm. Transient overexpression of GPCR in these two cells cannot induce obvious apoptosis. FHM cells stably expressing GPCR showed enhanced cell proliferation and significant anchorage-independent growth. The effects of GPCR protein on external apoptotic stimuli were examined. Few apoptotic bodies were observed in cells expressing GPCR treated with actinomycin D (ActD). Quantitative analysis of apoptotic cells indicated that a considerable decrease in the apoptotic fraction of cells expressing GPCR, compared with. the control cells, was detected after exposure to ActD and cycloheximide. These data suggest that LCDV-C GPCR may inhibit apoptosis as part of its potential mechanism in mediating cellular transformation.

Molecular cloning of growth hormone receptor (GHR) from common carp (Cyprinus carpio L.) and identification of its two forms of mRNA transcripts

The cDNA of growth hormone receptor (GHR) was cloned from the liver of 2-year common carp (Cyprinus carpio L.) by reverse transcription-polymerase chain reaction (RT-PCR) and rapid amplification of cDNA end (RACE). Its open reading frame (ORF) of 1806 nucleotides is translated into a putative peptide of 602 amino acids, including an extracellular ligand-binding domain of 244 amino acids (aa), a single transmembrane domain of 24 aa and an intracellular signal-transduction domain of 334 aa. Sequence analysis indicated that common carp GHR is highly homologous to goldfish (Carassius auratus) GHR at both gene and protein levels. Using a pair of gene-specific primers, a GHR fragment was amplified from the cDNA of 2-year common carp, a 224 bp product was identified in liver and a 321 bp product in other tissues. The sequencing of the products and the partial genomic DNA indicated that the difference in product size was the result of a 97 bp intron that alternatively spliced. In addition, the 321 bp fragment could be amplified from all the tissues of 4-month common carp including liver, demonstrating the occurrence of the alternative splicing of this intron during the development of common carp. Moreover, a semi-quantitative RT-PCR was performed to analyze the expression level of GHR in tissues of 2-year common carp and 4-month common carp. The result revealed that in the tissues of gill, thymus and brain, the expression level of GHR in 2-year common carp was significantly tower than that of 4-month common carp.

Inhibition of progesterone receptor activity in recombinant yeast by soot from fossil fuel combustion emissions and air particulate materials

Numerous environmental pollutants have been detected for estrogenic activity by interacting with the estrogen receptor, but little information is available about their interactions with the progesterone receptor. In this study, emission samples generated by fossil fuel combustion (FFC) and air particulate material (APM) collected from an urban location near a traffic line in a big city of China were evaluated to interact with the human progesterone receptor (hPR) signaling pathway by examining their ability to interact with the activity of hPR expressed in yeast. The results showed that the soot of a petroleum-fired vehicle possessed the most potent anti-progesteronic activity, that of coal-fired stove and diesel fired agrimotor emissions took the second place, and soot samples of coal-fired heating work and electric power station had lesser progesterone inhibition activity. The anti-progesteronic activity of APM was between that of soot from petroleum-fired vehicle and soot from coal-fired establishments and diesel fired agrimotor. Since there was no other large pollution source near the APM sampling sites, the endocrine disrupters were most likely from vehicle emissions, tire attrition and house heating sources. The correlation analysis showed that a strong relationship existed between estrogenic activity and anti-progesteronic activity in emissions of fossil fuel combustion. The discoveries that some environmental pollutants with estrogenic activity can also inhibit OR activity indicate that further studies are required to investigate potential mechanisms for the reported estrogenic activities of these pollutants. (c) 2005 Elsevier B.V. All rights reserved.

大麻受体CB1及CB2在慢性吗啡成瘾大鼠中枢神经系统与免疫系统的分布和表达

吗啡是临床常用的镇痛药物之一，通过模拟内源性抗痛物质脑啡肽的作用，激活中枢神经阿片受体而产生强大的镇痛作用。吗啡属于阿片类生物碱，为阿片受体激动剂，是目前我国主要的毒品成瘾类型之一，对人民生命健康危害极大。目前我国登记在册的吗啡成瘾者约有100万，每年导致的直接经济损失超过1000亿元。因此吗啡成瘾机制的研究以及治疗，是目前神经疾病的研究重点之一。 吗啡成瘾与其结合的受体有关。吗啡除结合阿片受体外，也可能结合大麻素受体，现发现体内有两种大麻素受体的存在：CB1受体和CB2受体。大麻CB1、CB2受体都是G蛋白耦联受体。其中CB1受体主要位于脑、脊髓与外周神经系统中，脑内CB1受体主要分布于基底神经节（黑质、苍白球、外侧纹状体）、海马CA锥体细胞层，小脑和大脑皮层。因此推测大麻CB1受体的功能可能与成瘾、记忆、认知、运动控制的调节有关。而大麻CB2受体主要分布于外周组织，如脾脏边缘区、扁桃体等，它的这种分布可能与免疫抑制作用有关。近来的研究发现大麻CB2受体在中枢神经系统也有分布，目前对其在此分布的功能不明确，推测可能与成瘾、抑郁症等神经类疾病有密切关系。 在药物成瘾导致的精神依赖作用中，奖赏效应是各种药物成瘾的药理学基础。中脑—边缘系统（(mesolimbic dopamine system,MLDS)是药物奖赏效应的神经解剖学基础。目前认为内源性大麻素所起的药理作用与多巴胺能和阿片能的神经传递有密切的关系。因此推断大麻素CB1受体与慢性吗啡成瘾有密切关系，至少是部分参与到慢性吗啡成瘾过程中。 相较于较多的关于大麻CB1受体的研究，有关大麻CB2受体的研究很少。尽管近来证实大麻CB2受体也分布于中枢神经系统，但在慢性吗啡成瘾时，大麻CB2受体表达的改变仍不清楚。在本项目中，我们将对慢性吗啡成瘾动物通过分子生物学、蛋白质化学、免疫组织化学的方法，探讨大麻CB2受体在中枢神经系统的分布和表达，以及大麻CB2受体在吗啡成瘾中可能的作用。 吗啡对免疫系统有抑制作用, 包括抑制淋巴细胞增殖, 减少细胞因子的分泌，减弱自然杀伤细胞（NKC）的细胞毒作用。现已证实激活周围神经系统的CB2受体可诱导IL-4的生成，从而影响阿片μ型受体的转录。此发现提供了内源性大麻系统-阿片系统-免疫系统之间存在相互作用的关系。然而，吗啡吸食是否通过CB2受体从而导致免疫功能的抑制，现在还没有直接证据，在本实验中我们将探讨CB2受体与吗啡成瘾导致免疫功能的改变有关。 实验结果显示（1）应用RT-PCR法，检测到大麻素受体CB1在慢性吗啡成瘾大鼠的皮质和海马处mRNA表达水平与对照组大鼠有明显不同。（2）应用western免疫印迹法，检测到大麻素受体CB1在慢性吗啡成瘾大鼠的皮质，海马和脑干处蛋白表达水平与对照组大鼠有明显不同。在脑干处，虽然mRNA表达水平无变化，但蛋白质的表达水平上升。（3）应用免疫组化检测到大麻素受体CB1在大鼠的皮质，海马，脑干，小脑处都广泛分布。（4）应用RT-PCR法，检测到大麻素受体CB2在慢性吗啡成瘾大鼠的皮质，海马，脑干处mRNA表达水平与对照组大鼠有明显不同。（5）应用western免疫印迹法，检测到大麻素受体CB2在慢性吗啡成瘾大鼠的皮质，海马，脑干蛋白表达水平与对照组大鼠有明显不同。且蛋白质的表达改变趋势与mRNA表达水平的改变相似。（6）应用免疫组化法检测到大麻素受体CB2在大鼠的皮质，海马，脑干，小脑处都广泛分布。但数量明显少于大麻CB1受体。（7）应用直接ELISA法，检测到慢性吗啡成瘾大鼠的血清与对照组大鼠的血清比较，IgM表达下降；IgG表达上升。 实验结果提示大麻受体CB1和CB2 很可能在慢性吗啡成瘾过程起着重要的作用，至少是部分参与到慢性吗啡成瘾的过程中。因为大麻素受体CB1和CB2都属于G 蛋白耦连受体，长期持续使用吗啡，其表达的变化可能会导致cAMP信号通路的上调；提高了腺苷酸环化酶(AC)和蛋白激酶A(PKA)的活性从而激活下游相关基因的表达最终导致成瘾。此外大麻素受体CB1和CB2表达的变化可能与慢性吗啡成瘾后免疫功能的改变有相关性。 通过以上的的实验结果，可以得到以下的结论：（1）我们验证了大麻素受体CB1在慢性吗啡成瘾大鼠的皮质，海马和脑干处mRNA和蛋白质表达水平与对照组大鼠有明显不同，且大麻CB1受体在大鼠中枢神经系统中广泛大量分布，表明大麻素受体CB1很可能在慢性吗啡成瘾过程中起着重要的作用，至少部分参与到慢性吗啡成瘾的过程中。（2）我们第一次证实了大麻素受体CB2在吗啡成瘾大鼠的皮质，海马和脑干处mRNA和蛋白质表达水平与对照组大鼠有明显不同，且大麻CB2受体在大鼠中枢神经系统中少量广泛分布。表明大麻素受体CB2很可能在慢性吗啡成瘾过程中起着重要的作用，至少部分参与到慢性吗啡成瘾的过程中。（3）同时我们发现大麻素受体CB1和CB2在大鼠脑组织中广泛表达，表明内源性大麻系统有可能广泛的参与各种神经疾病，很可能成为治疗的新靶点。（4）最后我们发现慢性吗啡成瘾大鼠血液中IgM表达下降；IgG表达上升，表明慢性吗啡成瘾对机体的免疫功能有广泛的调节作用。慢性吗啡成瘾大鼠血清CB2受体mRNA表达上升。我们证实了大麻受体CB2可能正是把神经系统和免疫系统相联系的一个靶点。

On the chemical synthesis and drug delivery response of folate receptor-activated, polyethylene glycol-functionalized magnetite nanoparticles

We describe here the chemical synthesis and in vitro drug delivery response of polyethylene glycol (PEG)-functionalized magnetite (Fe3O4) nanoparticles, which were activated with a stable ligand, folic acid, and conjugated with an anticancer drug, doxorubicin. The functionalization and conjugation steps in the chemical synthesis were confirmed using Fourier transform infrared spectroscopy. The drug-release behavior of PEG-functionalized and folic acid-doxorubicin-conjugated magnetic nanoparticles was characterized by two stages involving an initial rapid release, followed by a controlled release. (C) 2007 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

One-Step Synthesis of Folic Acid Protected Gold Nanoparticles and Their Receptor-Mediated Intracellular Uptake

We report here a facile method to obtain folic acid (FA)-protected gold nanoparticles (Au NPs) by heating an aqueous solution of HAuCl4/FA in which FA acts as both the reducing and stabilizing agent. The successful formation of FA-protected Au NPs is demonstrated by UV/Vis spectroscopy, transmission electron microscopy (TEM), selected-area electron diffraction (SAED), X-ray photoelectron spectroscopy (XPS), and Fourier transform infrared spectroscopy (FTIR). ne intracellular uptake of these nanoparticles is facilitated by HeLa cells overexpressing the folate reporter, which itself is significantly inhibited by free FA in a competitive assay as quantified by inductively coupled plasma mass spectroscopy (ICP-MS). This simple one-step approach affords a new perspective for creating functional nanomaterials, and the resulting biocompatible, functional Au NPs may find some prospective applications in various biomedical fields.

Folate-Conjugated Micelles and Their Folate-Receptor-Mediated Endocytosis

A folate-conjugated copolymer PEG-PLA-PLL/folate was synthesized and mixed with pure PEG-PLA-PLL and a fluorescent model drug mFITC to prepare folate-conjugated micelles. The distribution of micelles was studied on cancer-cell-bearing mice via frozen slicing. The results show that mFITC is successfully encapsulated into folate(+) and folate(-)micelles; PEG-PLA-PLL micelles the latter can be internalized by both HeLa and CHO cells without selectivity due to their cationic surface charges, while folate(+)micelles exhibit more preferential endocytosis by HeLa cells than by CHO cells. The folate(-)micelles showed retention in both organs and tumors. The folate(+)micelles are a promising active targeting drug delivery system for FR over-expressing cells and they accumulate in tumor beds.

Differential proteomic analysis of neonatal cardiomyocytes in response to beta-adrenergic receptor stimulation

beta-Adrenoceptors(beta-ARs) play a critical role in regulating cardiac functions under both physiological and pathological conditions. To further explore the mechanisms through which beta-ARs perform its actions, proteomic approaches were adopted to study the global protein patterns in cultured neonatal rat cardiomyocytes exposed to isoproterenol (ISO). A modified method, "Mirror Images in One Gel", was used to improve the reproducibility and resolution power of two-dimensional electrophoresis. A 2-DE map with a good reproducibility was obtained in which 1281 70 spots were detected and about 1191 +/- 54 spots were matched, with an average matching rate of 92.9%. Nine proteins with significant changes were identified by using peptide mass fingerprinting(PMF) data obtained via MALDI-MS.

Molecular characterization and effect of RNA interference of retinoid X receptor (RXR) on E75 and chitinase gene expression in Chinese shrimp Fenneropenaeus chinensis

Retinoid X receptor (RXR)/ultraspiracle (USP) is the heterodimeric partner of ecdysteroid receptor and is required for the molting process of arthropods. To better understand the molecular aspects governing the process of molting in shrimp, the full-length cDNA of two RXRs, named as FcRXR-1 and FcRXR-2 were obtained from Chinese shrimp Fenneropenaeus chinensis which were of 1715 and 1700 bp long, revealed a 1315 and 1300 bp open reading frame (ORF) respectively. Quantitative Real time PCR analysis showed a marked tissue-specific difference in the expression of FcRXR transcript, which revealed that the expression of FcRXR Could be regulated in a tissue-specific manner. Moreover, high expression of FcRXR mRNAs was observed in late pre-molt period (D3) and post molt stages (A-B) of shrimp. Among the two isoforms, FcRXR-2 appeared in a considerably high level in all the stages compared to the FcRXR-1. In addition, we examined the temporal expression of two chitinase genes: FcChitinase (FcChi) and FcChitinase-1 (FcChi-1) during the molt cycle of F chinensis. Both the FcChi and FcChi-1 transcripts were detected in all stages of molting, although considerable fluctuations observed through the molt cycle. Injection of double stranded RXR (dsRXR) into juvenile shrimp resulted in a maximum silencing effect at 48 h post injection. We analyzed the expression levels of FcChi, FcChi-1 and the ecdysone inducible gene E75 (FcE75) in samples of dsRXR injected shrimp. Significant reduction in levels of both FcE75, FcChi and FcChi-1 transcripts Occurred in the silenced shrimp. This correlation suggested that RXR might involve in the downstream regulation of E75 and chitinase gene transcription in the ecdysone signaling pathway of decapod crustaceans. (C) 2009 Published by Elsevier Inc.