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.

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.

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

Ecdysteroid-responsive genes: E75 and Retinoid X Receptor (RXR), essential molt-regulating genes in the shrimp, Fenneropenaeus chinensis

本论文主要研究两种重要的调节蜕皮过程的基因—蜕皮激素效应基因E75和RXR在中国明对虾蜕皮中的作用。利用RT-PCR和RACE技术获得了编码FcE75和FcRXR的全长cDNA序列。FcRXR包含7个内含子，在对虾中存在不同的异形体，命名为RXR-1和RXR-2。应用荧光实时定量PCR分析表明FcE75和FcRXR基因在中国明对虾蜕皮前期（D3）其转录表达量明显上调。另外，FcE75和FcRXR基因在不同组织中的转录表达存在明显的差异。利用FcE75和FcRXR基因的双链RNA注射对虾能有效降低FcE75和FcRXR的表达水平。FcE75和FcRXR的体内沉默完全抑制了对虾的蜕皮过程，并且引起对虾的死亡。对不能正常蜕皮个体进行观察的结果表明，FcE75沉默的对虾，其上皮的收缩、新的刚毛及新表皮的形成均收到限制。在FcE75双链RNA沉默后的对虾中，我们检测了与蜕皮相关的一些效应因子，如chitinase等的转录，发现这些效应因子的转录明显受到抑制，说明FcE75和FcRXR在蜕皮过程中起到非常重要的作用。本论文首次阐明了这些基因在十足目甲壳动物蜕皮过程中的功能。