ABSTRACT
Human lymphocyte function-associated antigen 3 (hLFA3) has been identified as an important T cell accessory molecule. Rhesus monkeys (Macaca mulatta) have been widely used as animal models for human immune disorders. Due to the species-specificity of immune system, it is necessary to study M. mulatta LFA3 (mmLFA3). In this study, the gene encoding mmLFA3 CD2-binding domain (mmLFA3Sh) was amplified by polymerase chain reaction (PCR) and genetically fused to human IgG1 Fc fragment in pPIC9K to construct the expression plasmid pPIC9K-mmLFA3Sh-Ig. Approximately 3-4 mg mmLFA3Sh-Ig protein was recovered from 1 L of inductive media, and mmLFA3Sh-Ig produced by the P. pastoris can bind to the CD2 positive cells, and suppress the monkey and human lymphocytes proliferation induced by Con A and alloantigen in a dose-dependent manner. These results suggested that mmLFA3Sh-Ig might be used as a novel tool for pathogenesis and experimental immunotherapy of Rhesus monkey immune disorders.
Subject(s)
Animals , Humans , CD58 Antigens , Immunoglobulin G , Lymphocyte Activation , Macaca mulatta , Pichia , Plasmids , Protein Interaction Domains and Motifs , Recombinant Fusion Proteins , T-LymphocytesABSTRACT
With their capability to undergo unlimited self-renewal and to differentiate into all cell types in the body, human embryonic stem cells (hESCs) hold great promise in human cell therapy. However, there are limited tools for easily identifying and isolating live hESC-derived cells. To track hESC-derived neural progenitor cells (NPCs), we applied homologous recombination to knock-in the mCherry gene into the Nestin locus of hESCs. This facilitated the genetic labeling of Nestin positive neural progenitor cells with mCherry. Our reporter system enables the visualization of neural induction from hESCs both in vitro (embryoid bodies) and in vivo (teratomas). This system also permits the identification of different neural subpopulations based on the intensity of our fluorescent reporter. In this context, a high level of mCherry expression showed enrichment for neural progenitors, while lower mCherry corresponded with more committed neural states. Combination of mCherry high expression with cell surface antigen staining enabled further enrichment of hESC-derived NPCs. These mCherry(+) NPCs could be expanded in culture and their differentiation resulted in a down-regulation of mCherry consistent with the loss of Nestin expression. Therefore, we have developed a fluorescent reporter system that can be used to trace neural differentiation events of hESCs.
Subject(s)
Animals , Humans , Mice , Cell Differentiation , Cell Line , Embryonic Stem Cells , Cell Biology , Metabolism , Transplantation , Gene Knock-In Techniques , Genes, Reporter , Homologous Recombination , Luminescent Proteins , Genetics , Mice, SCID , Nestin , Genetics , Neural Stem Cells , Cell Biology , Metabolism , Neurons , Cell Biology , Metabolism , Teratoma , PathologyABSTRACT
As a critical tumor suppressor, p53 is inactivated in human cancer cells by somatic gene mutation or disruption of pathways required for its activation. Therefore, it is critical to elucidate the mechanism underlying p53 activation after genotoxic and cellular stresses. Accumulating evidence has indicated the importance of posttranslational modifications such as acetylation in regulating p53 stability and activity. However, the physiological roles of the eight identified acetylation events in regulating p53 responses remain to be fully understood. By employing homologous recombination, we introduced various combinations of missense mutations (lysine to arginine) into eight acetylation sites of the endogenous p53 gene in human embryonic stem cells (hESCs). By determining the p53 responses to DNA damage in the p53 knock-in mutant hESCs and their derivatives, we demonstrate physiological importance of the acetylation events within the core domain (K120 and K164) and at the C-terminus (K370/372/373/381/382/386) in regulating human p53 responses to DNA damage.
Subject(s)
Humans , Acetylation , Cells, Cultured , DNA Damage , Embryonic Stem Cells , Physiology , Fibroblasts , Physiology , Gene Expression Regulation , Gene Knock-In Techniques , Protein Processing, Post-Translational , Protein Stability , Transcription, Genetic , Tumor Suppressor Protein p53 , PhysiologyABSTRACT
Objective To investigate the effect of percutaneous nephrostolithotomy (PCNL) on patients with different body mass index.Methods The clinical data of 84 patients with kidney stones who were treated with PCNL surgical treatment were enrolled in this study,and they were divided into different groups according to body mass index (BMI),including 16 cases of BMI ＜ 18.5 kg/m2(low BMI group),36 cases of 18.5 kg/m2≤BMI ＜24.0 kg/m2 (normal BMI group),32 cases of BMI≥24.0 kg/m2 (high BMI group),and the curative effect was evaluated.Results Three groups' operation time,multichannel gravel rate,stones clearance,length of hospital stay,blood transfusion rate had no significant difference (P ＞0.05),the incidence of complications in low BMI group [31.25％ (5/16)] was obviously higher than that in normal BMI group [5.56％(2/36)] and high BMI group [9.38％(3/32)],there was significant difference (P ＜ 0.05).Conclusion BMI of PCNL surgery influences on certain effect,low BMI will affect patients with surgery tolerance,cause complications increase,obesity wifl increase the difficulty of the operation.