Abstract:Satellite cells, as the major stem cells, are responsible for postnatal skeletal muscle growth, regeneration and maintenance. As a result, satellite cells have great potential as therapeutic agents. Activation of satellite cells in vivo is a key link in the muscle regeneration processes. And their replenishment is necessary to keep the capacity of skeletal muscle to regenerate after recurrence of muscle damage. Cellular and molecular regulation of activation and replenishment for satellite cells issues were reviewed. Hoping through the points of nitric oxide-hepatocyte growth factor (NO-HGF), myostatin and Notch signaling and the niche of satellite cells to overcome the recent obstruction in cell therapy in clinic myopathy, such as Duchenne muscle destrophy.

Abstract:Many apoptosis-related heart diseases such as myocardial infarction, cardiomyopathies, and heart failure severely impair human's health and life. Currently, a key focus for the medical researchers is to find out effective ways to prevent or treat these heart diseases. ARC (apoptosis repressor with caspase recruitment domain) is the first anti-apoptotic protein so far identified to be highly and specifically expressed in the cardiac tissue. ARC could be structurally phosphorylated and involved in various signaling pathways during apoptosis.

Abstract:Toll-like receptors (TLRs), a large family consisting of at least 10 members, are evolutionarily conserved to recognize pathogen-associated molecular patterns (PAMPs). TLRs activation not only initiates innate immunity, but also regulates enhance antigen-specific acquired immunity, and thus associates innate and adaptive immunity. In recent years, studies on the TLRs signaling, especially their negative regulation, rapidly progressed. TLRs signaling pathway and their roles in regulating immune responses against invading pathogens were reviewed.

Abstract:Many surface proteins of Gram-positive bacteria are anchored to the cell wall by the action of sortase enzymes, a recently discovered family of cysteine transpeptidases. As the surface proteins of pathogens are frequently required for virulence, the sortase might be a suitable target for the development of anti-Gram-positive drugs. Recently, the mechanism and active sites of sortase was elucidated by the research of sortaseA(SrtA) in Staphylococcus aureus and a series of SrtA inhibitors have been identified for providing the basis for further development of potent inhibitors.

Abstract:DNA replication and RNA biogenesis happen in the cell nucleus,while protein synthesis occurs in the cytoplasm. Integration of these activities depends on function proteins′ selective transport between the two sub-dimensions. It is a signal mediated process, which needs energy and the participation of soluble factors. By introduction of progress on function protein regulated nuclear translocation, its potential medical application has been explored. With deeper investigation in this field, it will significantly promote the design of anti-virus and gene vector therapy.

Abstract:Six fluorescent fusion protein plasmids were constructed, and the location and interaction of p53 and IκBα (IκBαM, IκBα243N and IκBα244C) in living cells were investigated with cyan (CFP) and red fluorescent protein(DsRed) fusion proteins by fluorescence resonance energy transfer. p53 interacts with the non-ankyrin C terminus PEST domain of IκBα, and the N terminus in p53 plays an important role in the formation of p53·IκBα complex. The change of DsRed/CFP in response to UV-mediated DNA damage was observed in real time and the balance between p53·IκBα complex formation and dissociation was discussed. In addition, the shuttling of p53 and IκBα between cytoplasm and nucleus under LMB stimulation suggested that the relationship among NF-κB, p53, IκBα, Mdm2, and WOX1 is complicated and remains to be determined. IκBα has been an important cross-talk protein between p53 and NF-κB, and this might expand the understanding of these two signaling pathways considerably.

Abstract:Adiponectin is an adipocyte-derived secretory protein. It was found to be associated with insulin resistance, inflammation and arteriosclerosis. To further study the biological function and expression of adiponection in vivo, adipoenctin gene knock-out and LacZ gene knock-in mouse model was constructed. Gene targeting strategy was designed to replace part of exon 2 and exon 3 of adiponectin gene with full length LacZ gene in frame with remaining upstream ATG and signal peptide sequence of exon 2. The targeting vector (Adipo-LacZ-XpPNT) was constructed and verified by restriction enzyme digestion and sequencing. CJ7 ES cells were transfected with targeting vector linearized by NotⅠ digestion, selected in the medium containing both G418 and ganciclovoir. Resistant clones were screened by PCR and further confirmed by Southern blot for correct homologous recombinants. Chimera mice were obtained by routing microinjection of homologous recombined ES cells into blastocysts. After mating, mice heterozygous and further homozygous for adiponectin knockout and LacZ gene knock-in were established. Expression of both endogenous adiponectin and exogenous LacZ gene in mouse tissues and sera were detected by RT-PCR, Northern-blot, Western blot and ELISA. The results show that adiponectin was disrupted at both mRNA and protein levels. LacZ gene is expressed exclusively in adipose tissue of mutant mice. Its expression profile is identical to endogenous adiponection. Unexpectedly, LacZ activity could not be detected in both adipose tissue and serum although LacZ protein can be detected in adipose tissue but not in serum of mutant mice. In conclusion, mice homozygous for adiponectin knockout and LacZ gene knock-in have been successfully constructed. Mutant mice display LacZ expression profile identical to endogenous adiponectin albeit neither LacZ activity nor protein can be detected in serum of mutant mice.

Abstract:The unicellular green alga Haematococcus pluvialis accumulates a highly valuable ketocarotenoid, i.e. astaxanthin up to 4% dry weight under stress conditions. Phytoene synthase is considered to be the first rate limiting enzyme in carotenoid biosynthesis pathway in H. pluvialis. The cDNA and genomic genes of phytoene synthase, i.e. psy from H.pluvialis were cloned and characterized. Result showed that psy had one open reading frame of 1 200 bp encoding a putative polypeptide of 400 amino acids which was interrupted by four introns. Phylogenetic analysis revealed that psy from green algae formed a monophyletic clade, and its closer relationship was higher plants. By using genomic walking approach, an approximate 1 kb 5′ flanking region of psy gene was cloned and a number of putative cis-regulatory elements were revealed. Fusing a 297 bp internal sequence (－297 to －1 bp from the translation initiation codon of psy) with the reporter gene, i.e. lacZ before attempted introducing the construct into the green alga via particle bombardment resulted in lacZ transient expression.

Abstract:Beta2 adrenergic receptor β₂ AR) is one member of G protein coupled receptors (GPCRs), which is a key pharmaceutical target in the treatment of asthma. Evolutionary trace (ET) method was employed to analyze AR sequences and 44 conserved residues were identified. Then molecular dynamics (MD) simulation of the β₂ wild-type receptor, D130N active mutant and D79N inactive mutant were carried out and tried to explore the structural/dynamic features characterizing functionally different states of the receptor, by means of investigating ET identified conserved basic residues in the wild-type receptor and its two mutants. Particularly, it was found that the departing of D130 from R131 of DRY motif and approaching to K149 are highly correlated with the receptor activation, and the movement of helix 2, 4 and 6 upon receptor activation is inferred from the observation that R151 and K270 interact with other residues in the receptor active state on the basis of little change of the side chain orientations. The results might provide further insights into the activating mechanism of β₂ AR mutants, as well as the molecular bases of the diseases induced by the mutations of the receptor.

Abstract:In order to investigate the expression of the glycolysis-associated genes and different proliferation between normal cells and tumor cells in response to hypoxia, and to explore the role of reactive oxygen species (ROS) in mediating metabolism of hepatoma cells and in regulating expression of glycolysis-associated genes and enzyme activity, SMMC-7721 hepatoma cell line and the normal liver cell line LO2 were taken as the investigation objects. The cell suppression ratio and surviving ratio were detected respectively under conditions of simple hypoxia, or 5 mmol/L glucose complicated with hypoxia. The mRNA levels of key enzymes that are involved in regulating glyco-metabolism were detected, including the mRNA levels of pyruvate-kinase, hexokinase, succinic dehydrogenase and isocitric dehydrogenase, and the activity of lactate dehydrognase. The proliferation gene pkb and hypoxia-inducible factor-1 (HIF-1) were detected as well. The results suggest that 1. tumor cell can endure severe hypoxia condition than normal cell, 2. under conditions of hypoxia, the strengthening of the glycolytic pathway is one of the important mechanisms to ensure the rapid proliferation of tumor cells, 3. ROS through HIF-1 mediates the changes of the gene expression of the enzyme of the glycometabolism pathway, and takes part in the regulation of the hypoxia-induced signal transduction. And the use of antioxidant will decrease the ability of tumor cell to endure hypoxia.

Abstract:It is generally believed that sphingomyelin- and cholesterol-enriched microdomains can be isolated as detergent-resistant membranes (DRMs) from plasma membrane and organelle membranes. Here the isolation of microdomains from porcine cardiac sarcoplasmic reticulum (SR) membranes is described. These SR-derived detergent-resistant membranes (SR-DRMs) enriched in sphingomyelin and cholesterol have a low buoyant density. Both ganglioside GM1 and caveolin-3 in the SR, known as a marker protein of caveolae at plasma membrane are present in the SR-DRMs. It was demonstrated that significant amount of SERCAs together with caveolin-3 associates with SR-DRMs, and are fully functional. The results suggested that SR membranes like other biomembranes, such as the plasma membrane, contained microdomains.

Abstract:A thermostable DNA ligase gene was identified, and the biochemical and enzymatic properties of the ligase were characterized from JP2 strain which was enriched from geothermally active sites in Papua New Guinea. The nucleotide and amino acid sequences showed much high identities compared with that of archeabacterium species Sulfolobus solfataricus and Sulfolobus shibatae, especially in the six conserved motif sequences, which are known to be closely related to the key function of ligase. Recombinant JP2 ligase showed high activity in nick-joining reaction. It was the most active when Mn²⁺ present as divalent metal cofactor rather than Mg²⁺ and Ca²⁺. etc.. Assay of thermostability over a range of temperatures showed that at 50～80℃ the enzyme displayed relative high activity. Further thermostability experiment indicated that the activity of JP2 ligase could last for a long time at 80℃ and 85℃, however, at 90℃ and 95℃, it became unstable quickly. An investigation on the acquired thermotolerance of recombinant JP2 ligase was done by applying a chaperonin known as TF55 in thermophile on JP2 ligase reaction. Result showed that TF55 could not help in improving thermostability of ligase at 85℃. The possible reason might be that at 85℃ in vitro, the chaperonin itself was denatured.

Abstract:In order to study the regulation mechanism of Sry in sex determination, the siRNA technique was used to silence the Sry expression in mouse embryos. Two siRNA expression vectors were synthesized, pSilencer4.1/Sry217 and pSilencer 4.1/Sry565. The siRNA expression vectors were injected into gestated mouse through tail vein. The sex was identified by PCR, and the expression level of Sry gene was determined using RT-PCR technique in the male mouse embryos at 11.5 dpc(days post coitum). The time- and dose -dependent effects was studied. The results confirmed that one of the siRNA expression plasmids, pSilencer4.1/Sry565, could inhibit the Sry gene expression significantly. Eighty-five percent inhibition was obtained when injecting 20 μg siRNA vector at 9.5 dpc.

Abstract:The massive β-tricalcium phosphate (β-TCP) scaffold with a central tunnel was impregnated with the sheep mesenchymal stem cells and were continuously perfused with the complete α-MEM medium by a peristaltic pump for 7, 14 and 28 days. Histological study showed that the cells proliferated through the whole scaffolds under dynamic culture. The cells formed a continuous monolayer or multilayer in the first 7 days and some pores in the scaffold packed with cells in 14 days. Most of pores were almost fully filled with cells after 28 days' culture. And the cell coverage were various in various spaces of the scaffold. Unoccupied spaces were found in many macropores and cell death was found in some spaces after 14 days' culture. The CFD was used to simulate the flow conditions within perfused cell-seeded cylindrical scaffolds to give insight into the mechanisms of the cell growth phenomena. Relating the simulation results to perfusion experiments, the average fluid velocity (approximately 0.24～0.53 mm/s) and shear stress (approximately 0.0050～0.023 Pa) were found to correspond to increased cell proliferation within the cell-scaffold constructs.

Abstract:Cortical spreading depression (CSD) is an important disease model for migraine and stroke. Previous studies showed that before the pial artery large dilation which were widely observed in CSD, there is also a small constriction occurring in pial arteres. However, the mechanisms contributing to the regulation of this cerebrovascular response remain unknown. Optical intrinsic signal imaging(OISI) at 550 nm wavelength was used to monitor the responses of the pial arteries during pinprick induced CSD following the application of a K_ATP antagonist glibenclamide (glyb). By applying the glyb, the initial slight constriction (ISC) is obviously reduced, most of the ISC (10 μmol/L: 74.5%; 100 μmol/L: 96.2%) even completely restrained. And the peak of large dilation (LD) in response to the pial arteries was enhanced to (53.8±19.3) % and (59.8±19.6) % of the control with 10 μmol/L and 100 μmol/L glyb, respectively. It can be suggested that K_ATP in nerve cells of pre and post-synaptically plays a lead role in inhibiting CSD-associated hyperemia.