Biomedical subjects

Siming Li

Publications and source records attributed to Siming Li.

11 recordsLinked to original sources

PubMed2024

Identification and expression analysis of calcium-dependent protein kinase family in oat (Avena sativa L.) and their functions in response to saline-alkali stresses.

Calcium-dependent protein kinases (CDPKs) serve as calcium ion sensors and play crucial roles in all aspects of plant life cycle. While CDPK gene family has been extensively studied in various plants, there is limited information available for CDPK members in oat, an important cereal crop worldwide. Totally, 60 AsCDPK genes were identified in oat genome and were classified into four subfamilies based on their phylogenetic relationship. The members within each subfamily shared similar gene structure and conserved motifs. Collinearity analysis revealed that AsCDPK gene amplification was attributed to segmental duplication events and underwent strong purifying selection. AsCDPK promoters were predicted to contain cis-acting elements associated with hormones, biotic and abiotic stresses. AsCDPK gene expressions were induced by different salt stresses, exhibiting stress-specific under different salt treatments. Moreover, overexpression of AsCDPK26 gene enhanced salt resistance in C. reinhardtii, a single-cell photoautotrophic model plants. Further analysis revealed a significant correlation between AsCDPK26 and Na+/H+ antiporter 1 (p<0.05), suggesting that AsCDPK26 may interact with ion transporter to modulate salt resistance. These results not only provide valuable insights into AsCDPK genes in response to different salt stresses, but also lay the foundation to mine novel candidates for improving salt tolerance in oat and other crops.

Chlamydomonas reinhardtii↗

PubMed2005

Towards a proteome-scale map of the human protein-protein interaction network.

Systematic mapping of protein-protein interactions, or 'interactome' mapping, was initiated in model organisms, starting with defined biological processes and then expanding to the scale of the proteome. Although far from complete, such maps have revealed global topological and dynamic features of interactome networks that relate to known biological properties, suggesting that a human interactome map will provide insight into development and disease mechanisms at a systems level. Here we describe an initial version of a proteome-scale map of human binary protein-protein interactions. Using a stringent, high-throughput yeast two-hybrid system, we tested pairwise interactions among the products of approximately 8,100 currently available Gateway-cloned open reading frames and detected approximately 2,800 interactions. This data set, called CCSB-HI1, has a verification rate of approximately 78% as revealed by an independent co-affinity purification assay, and correlates significantly with other biological attributes. The CCSB-HI1 data set increases by approximately 70% the set of available binary interactions within the tested space and reveals more than 300 new connections to over 100 disease-associated proteins. This work represents an important step towards a systematic and comprehensive human interactome project.

Cloning, Molecular↗

PubMed2005

Hyperlipidemic effects of dietary saturated fats mediated through PGC-1beta coactivation of SREBP.

The PGC-1 family of coactivators stimulates the activity of certain transcription factors and nuclear receptors. Transcription factors in the sterol responsive element binding protein (SREBP) family are key regulators of the lipogenic genes in the liver. We show here that high-fat feeding, which induces hyperlipidemia and atherogenesis, stimulates the expression of both PGC-1beta and SREBP1c and 1a in liver. PGC-1beta coactivates the SREBP transcription factor family and stimulates lipogenic gene expression. Further, PGC-1beta is required for SREBP-mediated lipogenic gene expression. However, unlike SREBP itself, PGC-1beta reduces fat accumulation in the liver while greatly increasing circulating triglycerides and cholesterol in VLDL particles. The stimulation of lipoprotein transport upon PGC-1beta expression is likely due to the simultaneous coactivation of the liver X receptor, LXRalpha, a nuclear hormone receptor with known roles in hepatic lipid transport. These data suggest a mechanism through which dietary saturated fats can stimulate hyperlipidemia and atherogenesis.

Animals↗

PubMed2004

Systematic interactome mapping and genetic perturbation analysis of a C. elegans TGF-beta signaling network.

To initiate a system-level analysis of C. elegans DAF-7/TGF-beta signaling, we combined interactome mapping with single and double genetic perturbations. Yeast two-hybrid (Y2H) screens starting with known DAF-7/TGF-beta pathway components defined a network of 71 interactions among 59 proteins. Coaffinity purification (co-AP) assays in mammalian cells confirmed the overall quality of this network. Systematic perturbations of the network using RNAi, both in wild-type and daf-7/TGF-beta pathway mutant animals, identified nine DAF-7/TGF-beta signaling modifiers, seven of which are conserved in humans. We show that one of these has functional homology to human SNO/SKI oncoproteins and that mutations at the corresponding genetic locus daf-5 confer defects in DAF-7/TGF-beta signaling. Our results reveal substantial molecular complexity in DAF-7/TGF-beta signal transduction. Integrating interactome maps with systematic genetic perturbations may be useful for developing a systems biology approach to this and other signaling modules.

Animals↗

PubMed2004

A map of the interactome network of the metazoan C. elegans.

To initiate studies on how protein-protein interaction (or "interactome") networks relate to multicellular functions, we have mapped a large fraction of the Caenorhabditis elegans interactome network. Starting with a subset of metazoan-specific proteins, more than 4000 interactions were identified from high-throughput, yeast two-hybrid (HT=Y2H) screens. Independent coaffinity purification assays experimentally validated the overall quality of this Y2H data set. Together with already described Y2H interactions and interologs predicted in silico, the current version of the Worm Interactome (WI5) map contains approximately 5500 interactions. Topological and biological features of this interactome network, as well as its integration with phenome and transcriptome data sets, lead to numerous biological hypotheses.

Animals↗

PubMed2004

Increasing specificity in high-throughput yeast two-hybrid experiments.

Since its inception, the yeast two-hybrid (Y2H) system has proven to be an efficient system to identify novel protein-protein interactions. However, Y2H screens are sometimes criticized for generating high rates of false-positives. Minimizing false-positive interactions is especially important in proteome wide high-throughput (HT) Y2H. Here, we summarize various approaches that reduce false-positives in HT-Y2H projects. We evaluated the potential of examining putative positives after removing the prey encoding plasmid by negative selection. We found that this method reliably identifies false-positives caused by spontaneous conversion of baits into auto-activators and provides significant time-savings in HT screens. In addition, we present a method to eliminate an important source of false-positives: contaminating prey plasmids. Y2H interactors can be wrongly identified due to the presence of two or more different plasmids in the cells of a single yeast colony. Of these independent plasmids, only one encodes a genuine interactor. Contaminating plasmids are eliminated by extended culture of yeast cells under positive selection for the interaction, allowing the identification of the true interaction partner.

Animals↗

PubMed2004

Human ORFeome version 1.1: a platform for reverse proteomics.

The advent of systems biology necessitates the cloning of nearly entire sets of protein-encoding open reading frames (ORFs), or ORFeomes, to allow functional studies of the corresponding proteomes. Here, we describe the generation of a first version of the human ORFeome using a newly improved Gateway recombinational cloning approach. Using the Mammalian Gene Collection (MGC) resource as a starting point, we report the successful cloning of 8076 human ORFs, representing at least 7263 human genes, as mini-pools of PCR-amplified products. These were assembled into the human ORFeome version 1.1 (hORFeome v1.1) collection. After assessing the overall quality of this version, we describe the use of hORFeome v1.1 for heterologous protein expression in two different expression systems at proteome scale. The hORFeome v1.1 represents a central resource for the cloning of large sets of human ORFs in various settings for functional proteomics of many types, and will serve as the foundation for subsequent improved versions of the human ORFeome.

Cloning, Molecular↗

PubMed2003

C. elegans ORFeome version 1.1: experimental verification of the genome annotation and resource for proteome-scale protein expression.

To verify the genome annotation and to create a resource to functionally characterize the proteome, we attempted to Gateway-clone all predicted protein-encoding open reading frames (ORFs), or the 'ORFeome,' of Caenorhabditis elegans. We successfully cloned approximately 12,000 ORFs (ORFeome 1.1), of which roughly 4,000 correspond to genes that are untouched by any cDNA or expressed-sequence tag (EST). More than 50% of predicted genes needed corrections in their intron-exon structures. Notably, approximately 11,000 C. elegans proteins can now be expressed under many conditions and characterized using various high-throughput strategies, including large-scale interactome mapping. We suggest that similar ORFeome projects will be valuable for other organisms, including humans.

Alternative Splicing↗

PubMed2002

Biologic characteristics of fibroblast cells cultured from the knee ligaments.

OBJECTIVE: To culture fibroblast cells from the knee ligaments and to study the biological characteristics of these cells. METHODS: Cells of the anterior cruciate ligament (ACL) and the medial collateral ligament (MCL) from New Zealand white rabbit were cultured in vitro. Cellular growth and expression of the collagen were analyzed. Moreover, an in vitro wound closure model was established and the healing of the ACL and the MCL cells was compared. RESULTS: Maximal growth for all these cells were obtained with Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, but RPMI 1640 and Ham's F12 media were not suitable to maintain these cells. Morphology of both ACL and MCL cells from New Zealand white rabbit was alike in vitro, but the MCL cells grew faster than the ACL cells. Both cell types produced similar amount of collagen in culture, but the ratio of collage type I to type III produced by ACL cells was higher than that produced by MCL cells. Wound closure assay showed that at 36 hours after injury, cell-free zones created in the ACL cultures were occupied partially by the ACL cells; in contrast, the wounded zone in the MCL cultures was almost completely covered by the cells. CONCLUSIONS: Although the ACL cells and the MCL cells from New Zealand white rabbit show similar appearance in morphology in culture, the cellular growth and the biochemical synthesis of collagen as well as the healing in vitro were significantly different. These differences in intrinsic properties of the two types of cells in vitro might contribute to the differential healing potentials of these ligaments in vivo.

Animals↗

PubMedDate not supplied

A transgenic analysis of mouse lactate dehydrogenase C promoter activity in the testis.

Transcription of the mouse testis-specific lactate dehydrogenase c (mldhc) gene is limited to cells of the germinal epithelium. Cloning and analysis of the mldhc promoter revealed that a 100-bp core promoter was able to regulate testis-specific transcription in vitro and in transgenic mice. Surprisingly, expression of the reporter in transgenic testes was limited to pachytene spermatocytes, whereas native LDH-C(4) was detected in pachytene and all later germ cells. To locate additional regulatory sequence that could recapitulate the native LDH-C(4) distribution pattern, we investigated the contribution of 5' and 3' flanking sequences to the regulation of LDH-C(4) expression. We found that transcription factor YY1 binds to the mldhc promoter, that the mldhc 3' untranslated sequence does not permit a postmeiotic expression of a beta-galactosidase reporter in transgenic mice, and that native mldhc mRNA is predominately meiotic, with only a low level of postmeiotic distribution. Our results suggest that the high level of LDH-C(4) in postmeiotic cells results from mRNA and protein stability.

3' Flanking Region↗

PubMedDate not supplied

Heat-induced apoptosis of mouse meiotic cells is suppressed by ectopic expression of testis-specific calpastatin.

Calpastatin is a naturally occurring inhibitor of calpain, a protease involved in apoptotic cell death. A testis-specific isoform of calpastatin (tCAST) has been identified that is transcribed in haploid germ cells but not in spermatocytes. To investigate the possible function(s) of tCAST, we tested the hypothesis that the ectopic expression of calpastatin in spermatocytes would suppress the death of these cells in response to an apoptosis-inducing stimulus in vivo. To this end, the 5'-flanking region of the mouse ldhc gene was linked to tCAST, and transgenic mice were generated. Immunohistochemical analysis revealed that, in contrast to control sections in which the signal for tCAST was seen in round spermatids, intense staining was visualized in pachytene spermatocytes in the transgenic animals, indicating that the strategy we used to generate the transgenic animals resulted in the ectopic expression of tCAST in spermatocytes. We then tested the effect of a short period of heating on germ cell apoptosis in the testes of wild-type and transgenic mice. Pachytene spermatocytes were the major germ cell type seen to undergo apoptosis after heat treatment. There were no differences in the number of apoptotic germ cells per seminiferous tubule between wild-type and tCAST transgenic control mice; thus, there was no apparent effect of the transgene on normal apoptosis. Heating resulted in increased numbers of TUNEL-positive germ cells in both wild-type and tCAST transgenic mice, as well as increased testicular DNA fragmentation. Heating the tCAST transgenic mouse testes resulted in significantly fewer apoptotic cells per seminiferous tubule than in wild-type mice at both 8 and 24 hours after treatment. Thus, as hypothesized, the ectopic expression of tCAST in pachytene spermatocytes suppressed germ cell apoptosis.

Animals↗