Expressed sequence tags from the zhikong scallop (Chlamys farreri): Discovery and annotation of host-defense genes

A high-quality cDNA library was constructed from whole body tissues of the zhikong scallop, Chlamys farreri, challenged by Listonella anguillarum. A total of 5720 clones were sequenced, yielding 5123 expressed sequence tags (ESTs). Among the 3326 unique genes identified, 2289 (69%) genes had no significant (E-value < 1e-5) matches to known sequences in public databases and 194 (6%) matched proteins of unknown functions. The remaining 843 (25%) genes that exhibited homology with genes of known functions, showed broad involvement in metabolic processes (31%), cell structure and motility (20%), gene and protein expression (12%), cell signaling and cell communication (8%), cell division (4%), and notably, 25% of those genes were related to immune function. They included stress response genes, complement-like genes, proteinase and proteinase inhibitors, immune recognition receptors and immune effectors. The EST collection obtained in this study provides a useful resource for gene discovery and especially for the identification of host-defense genes and systems in scallops and other molluscs. (C) 2009 Elsevier Ltd. All rights reserved.

Development of expressed sequence tags from the bay scallop, Argopecten irradians irradians

The bay scallop, Argopecten irradians irradians, introduced from North America, has become one of the most important aquaculture species in China. Inan effort to identify scallop genes involved in host defense, a high-quality cDNA library was constructed from whole body tissues of the bay scallop. A total of 5828 successful sequencing reactions yielded 4995 expressed sequence tags (ESTs) longer than 100 bp. Cluster and assembly analyses of the ESTs identified 637 contigs (consisting of 2853 sequences) and 2142 singletons, totaling 2779 unique sequences. Basic Local Alignment Search Tool (BLAST) analysis showed that the majority (73%) of the unique sequences had no significant homology (E-value >= 0.005) to sequences in GenBank. Among the 748 sequences with significant GenBank matches, 160 (21.4%) were for genes related to metabolism, 131 (17.5%) for cell/organism defense, 124 (16.6%) for gene/protein expression, 83 (11.1%) for cell structure/motility, 70 (9.4%) for cell signaling/communication, 17 (2.3%) for cell division, and 163 (21.8%) matched to genes of unknown functions. The list of host-defense genes included many genes with known and important roles in innate defense such as lectins, defensins, proteases, protease inhibitors, heat shock proteins, antioxidants, and Toll-like receptors. The study provides a significant number of ESTs for gene discovery and candidate genes for studying host defense in scallops and other molluscs.

Microfluidic effects of transporting signaling components in cell coculture chips

A theoretical model has been developed to investigate the microfluidic transport of the signaling chemicals in the cell coculture chips. Using an epidermal growth factor (EGF)-like growth factor as the sample chemical, the effects of velocities and channel geometry were studied for the continuous-flow microchannel bioreactors. It is found that different perfusion velocities must be applied in the parallel channels to facilitate the communication, i.e., transport of the signaling component, between the coculture channels. Such communication occurs in a unidirectional way because the signaling chemicals can only flow from the high velocity area to the low velocity area. Moreover, the effect of the transport of the signaling component between the coculture channels on the growth of the monolayer cells and the multicellular tumor spheroid (MTS) in the continuous-flow coculture environment were simulated using 3D models. The numerical results demonstrated that the concentration gradients will induce the heterogeneous growth of the cells and the MTSs, which should be taken into account in designing the continuous-flow perfusion bioreactor for the cell coculture research.

An ATP-dependent mechanism mediates intercellular calcium signaling in bone cell network under single cell nanoindentation

To investigate the roles of intercellular gap junctions and extracellular ATP diffusion in bone cell calcium signaling propagation in bone tissue, in vitro bone cell networks were constructed by using microcontact printing and self-assembled monolayer technologies. In the network, neighboring cells were interconnected through functional gap junctions. A single cell at the center of the network was mechanically stimulated by using an AFM nanoindenter. Intracellular calcium ([Ca2+](i)) responses of the bone cell network were recorded and analyzed. In the untreated groups, calcium propagation from the stimulated cell to neighboring cells was observed in 40% of the tests. No significant difference was observed in this percentage when the intercellular gap junctions were blocked. This number, however, decreased to 10% in the extracellular ATP-pathway-blocked group. When both the gap junction and ATP pathways were blocked, intercellular calcium waves were abolished. When the intracellular calcium store in ER was depleted, the indented cell can generate calcium transients, but no [Ca2+](i) signal can be propagated to the neighboring cells. No [Ca2+](i) response was detected in the cell network when the extracellular calcium source was removed. These findings identified the biochemical pathways involved in the calcium signaling propagation in bone cell networks. Published by Elsevier Ltd.

Kinetic measurements of cell surface E-selectin/carbohydrate ligand interactions

Selectin/ligand interactions initiate the multistep adhesion and signaling cascades in the recruitment of leukocytes from circulation to inflamed tissues and may also play a role in tumor metastasis. Kinetic properties of these interactions are essential determinants governing blood-borne cells' tethering to and rolling on the vessel wall. Extending our recently developed micropipette method, we have measured the kinetic rates of E-selectin/ligand interactions. Red cells coated with an E-selectin construct were allowed to bind HL-60 or Colo-205 cells bearing carbohydrate ligands. Specific adhesions were observed to occur at isolated points, the frequency of which followed a Poisson distribution. These point attachments were formed at the same rate with both the HL-60 and Colo-205 cells (0.14 +/- 0.04 and 0.13 +/- 0.03 mum(2) s(-1) per unit density of E-selectin, respectively) but dissociated from the former at a rate twice as fast as did from the latter (0.92 +/- 0.23 and 0.44 +/- 0.10 s(-1), respectively). The reverse rates agree well with those measured by the flow chamber. The forward rates are orders of magnitude higher than those of Fc gamma receptors interacting with IgG measured under similar conditions, consistent with the rapid kinetics requirement for the function of E-selectin/ligand binding, which is to capture leukocytes on endothelial surfaces from flow.

Fluid flow induced calcium response in bone cell network

In our previous work, bone cell networks with controlled spacing and functional intercellular gap junctions had been successfully established by using microcontact printing and self assembled monolayers technologies [Guo, X. E., E. Takai, X. Jiang, Q. Xu, G. M. Whitesides, J. T. Yardley, C. T. Hung, E. M. Chow, T. Hantschel, and K. D. Costa. Mol. Cell. Biomech. 3:95-107, 2006]. The present study investigated the calcium response and the underlying signaling pathways in patterned bone cell networks exposed to a steady fluid flow. The glass slides with cell networks were separated into eight groups for treatment with specific pharmacological agents that inhibit pathways significant in bone cell calcium signaling. The calcium transients of the network were recorded and quantitatively evaluated with a set of network parameters. The results showed that 18 alpha-GA (gap junction blocker), suramin (ATP inhibitor), and thapsigargin (depleting intracellular calcium stores) significantly reduced the occurrence of multiple calcium peaks, which were visually obvious in the untreated group. The number of responsive peaks also decreased slightly yet significantly when either the COX-2/PGE(2) or the NOS/nitric oxide pathway was disrupted. Different from all other groups, cells treated with 18 alpha-GA maintained a high concentration of intracellular calcium following the first peak. In the absence of calcium in the culture medium, the intracellular calcium concentration decreased slowly with fluid flow without any calcium transients observed. These findings have identified important factors in the flow mediated calcium signaling of bone cells within a patterned network.

Identification of genes differentially expressed in wild type and Purkinje cell degeneration mice

Purkinje cell degeneration (pcd) mice are characterized by death of virtually all cerebellar Purkinje cells by postnatal day 30. In this study, we used DNA microarray analysis to investigate differences in gene expression between the brains of wild type and pcd mice on postnatal day 20, before the appearance of clear-cut phenotypic abnormalities. We identified 300 differentially expressed genes, most of which were involved in metabolic and physiological processes. Among the differentially expressed genes were several calcium binding proteins including calbindin -28k, paravalbumin, matrix gamma-carboxygluta mate protein and synaptotagamins 1 and 13, suggesting the involvement of abnormal Ca2+ signaling in the pcd phenotype.

Comparative Analysis of Five Different Homologous Feeder Cell Lines in the Ability to Support Rhesus Embryonic Stem Cells

我们以前的研究建立了五株猕猴饲养层细胞系来支持猕猴胚胎干细胞(rESCs)的生长:一岁猴耳皮肤成纤维细胞(MESFs)、两岁猴输卵管成纤维细胞(MOFs)、成年猴卵泡颗粒成纤维样细胞(MFGs)、成年猴卵泡颗粒上皮样细胞(MFGEs),以及MESFs的克隆成纤维细胞(CMESFs).我们发现MESFs、CMESFs、MOFs和MFGs,而不足MFGEs支持猕猴胚胎干细胞(rESCs,rhesus embryonic stem cells)的生长.通过半定量PCR的方法,我们在支持性的饲养层细胞中检测到了一些基因的高表达.在本研究中,我们运用Affymetrix公司的GeneChip Rhesus Macaque Genome Array芯片来研究这五株同源饲养层的表达谱,希望发现哪些细胞因子和信号通路在维持rESCs中起到重要作用.结果表明,除MFGE外,包括GREM2、bFGF,、KITLG,、DKK3、GREM1、AREG、SERPINF1和LTBF1等八个基因的mRNA在支持性的饲养层细胞中高表达.本研究结果提示,很多信号通路在支持rESCs的未分化生长和多潜能性方面可能起到了冗余的作用.

Involvement of Fas/FasL system in apoptotic signaling in testicular germ cells of male Wistar rats injected i.v. with microcystins

Previous studies have shown that gonads were the second target organ of microcystins (MCs), and that MCs exposure exerted obvious toxic effects on male reproductive system of mammals. However, relevant molecular evidences are still lacking. Fas-signaling pathway plays a key role in toxicant-induced germ cell apoptosis. This study was to evaluate the responses of Fas/FasL system related genes and proteins in testes of rats injected intravenously with MCs. Enhanced apoptosis of germ cells in the testes of MCs-treated rats was detected by the terminal deoxynucleotidyl transferase-mediated deoxy-UTP nick end labeling (TUNEL) associated with up-regulation of the Fas/FasL system. Both Fas and FasL protein expression were induced evidently from I h post-injection, and this high expression level maintained throughout the experiment. In addition, the activation of caspase-8 and caspase-3 protein was also observed, which were indicators of apoptosis. These results suggested the likely involvement of Fas/FasL system in the MCs-induced germ cell apoptosis. It is also suggested that MCs can cause damage to Sertoli cells directly. (C) 2009 Elsevier Ltd. All rights reserved.