Abstract:A novel receptor family, the Mas-related G protein-coupled receptors (Mrgs, also called Sensory neuron-specific G protein-coupled receptors or SNSRs), was found in 2001. Interestingly, the mRNAs of Mrgs are expressed predominantly, if not exclusively, in non-overlapping subsets of nonpeptidergic neurons in dorsal root (DRG) and trigeminal ganglia (TG). This specific expression pattern implies significance in physiological and pharmaceutical sciences of pain processing. Study of Mrgs is a new field and will help us to understand the mechanism of pain and nociceptor development. It would also contribute to develop a new analgesic with less central side-effects. The present article summarizes the research progress about Mrgs and provides useful information, such as classification, distribution, expressing regulation and the possible functions in neural circuit and nociception.

Abstract:Mucins are heavily O-glycosylated glycoproteins found in mucous secretions and as transmembrane glycoproteins of the cell surface with the glycan exposed to the external environment. In mucins, O-glycans are covalently α-linked via an N-acetylgalactosamine(GalNAc) moiety to serine or threonine, and the structures are named mucin-type O-glycans. Mucin-type O-glycans are initiated by UDP-GalNAc∶ polypeptide N-acetylgalactosaminyltransferases, which enzymatic mechanism and structural features have been a hot topic of glycosyltransferases research. Mucin-type O-glycans of cancer cells are often changed, both in structure and in quantity, developing several cancer-associated glycans, such as T and Tn antigens. These structural changes can alter the function of the cancer cells, and its antigenic and adhesive properties, as well as its potential to invade and metastasize. These cancer-associated glycans can be exploited to tumor diagnosis, and in the development of anti-tumor drug or vaccine.

Abstract:Terahertz(THz) radiation, a newly developed coherent far infrared source, has been comprehensively applied in investigation on the structure and dynamics characteristic of biological macromolecules. Combining the characteristic of THz spectroscopy, a general review of its application in protein, carbohydrate, DNA, and bio-molecule in liquid water was provided. The problem and the prospect of the THz spectroscopy's application at this stage are also discussed.

Abstract:Mammary stem cells (MaSCs) is an ideal model for studies of organogenesis, cell proliferation, differentiation, survival and apoptosis. Recent researches showed that many adult stem cell surface markers belonged to cell adhesion molecule (CAM) family. Therefore, analysis of relationship between mammary stem/progenitor cells and the expression pattern of CAM have directive meanings for identifying embryonic mammary stem/progenitor cells and understanding their properties. Here, cells in E14 mouse mammary anlagen were purified using the adult mouse mammary epithelial stem cell (MaESC) markers of CD24 and CD49f. It was found that CD24 and CD49f double-positive cells contain two distinct cell populations: CD24^hiCD49f⁺ and CD24^medCD49f⁺, and their percentages in total mammary anlagen cells were 16% and 47%, respectively. In the following monolayer culture and in vivo transplantation tests, it was found that the CD24^medCD49f⁺ cells could attach plate and regenerate mammary ductal units; correspondingly, CD24^hiCD49f⁺ cells did not possess of these capabilities. These results indicate that the two mammary anlagen cell populations represent different cell types, and CD24^medCD49f⁺ population may contain the self-renewal mammary anlagen stem/progenitor cells. Afterwards, the differences in the expressions of 19 mammary-related CAM transcripts in the two cell populations were validated by quantitative real-time PCR analysis. The data show that the CAM gene expressions of mammary repopulating-CD24^medCD49f^{+ cells are dramatically different from that of CD24hiCD49f+ cell population. The expressions of several adult stem cell markers in CD24medCD49f+ cell population are remarkable higher than those in CD24hiCD49f+ cell population.}

Abstract:Members of the serum and glucocorticoid-regulated kinase (SGK) family are important mediators of growth factor and hormone signaling. To investigate the biological function of SGK family member SGK2α, eukaryotic expression plasmid pEGFP-N1-SGK2α was constructed and introduced into HEK293 cells by transient transfection. The subcellular localization of SGK2α-GFP fusion protein was preferentially localized in the cytoplasm by laser scanning confocal microscope. The interaction of SGK2α and glycogen synthase kinase 3β (GSK3β) was confirmed by coimmunoprecipitation experiment. Stable BEL7402 cell line expressing SGK2α proteins was generated by PCDNA6-V5-HisB-SGK2α plasmid transfection. The growth of BEL7402 cells was suppressed and the cell doubling time was prolonged after SGK2α gene transfection by cell proliferation experiment. Compared with the control cells, the tumorigenic capacity of BEL7402 cells was clearly decreased after SGK2α gene expression by tumorigenecity assay. Overexpression of SGK2α decreased the expression of β-catenin and Cyclin D1 but not affected the expression of GSK3β in BEL7402 cells by Western blotting. These results suggest that overexpression of exogenous SGK2α protein might inhibit tumor cell growth both in vitro and in vivo by decreasing the expression of Wnt/β-catenin signal pathway molecules.

Abstract:This research aimed to construct a new combination gene vector: pcDNA3.1(-)VEGF-siRNA/　yCDglyTK, study its expression quality and lethal effet in human gastric cancer cell line SGC7901. First, RNA interference (RNAi) targeting vascular endothelial growth factor(VEGF) was applied to construct interfering plasmid pGenesil-VEGF-siRNA. Then, the siRNA expression cassette (including U6 promotor ) was amplified by PCR and subcloned into pcDNA3.1(-)CV-yCDglyTK to build a new combination gene plasmid: pcDNA3.1(-)VEGF-siRNA/yCDglyTK. The recombinant plasmid was identified by restriction enzyme digestion and gene sequencing. All of the three plasmids were delivered into SGC7901 cells using calcium phosphate nanoparticles (CPNPs). Expressions of yCDglyTK and VEGF were detected by RT-PCR and Western-blot. MTT assays were applied to determine the cytotoxic effect of plasmids in the presence of 5-FC. Restriction enzyme digestion and gene sequencing confirmed the combination gene vector pcDNA3.1(-)VEGF-siRNA/yCDglyTK was constructed successfully. RT-PCR, Western-blot showed expression of yCDglyTK and inhibition of VEGF in SGC7901 cells transfected with the combined gene plasmid, which were the most sensitive to 5-FC in the MTT assays. The combination gene vector pcDNA3.1(-)VEGF-siRNA/yCDglyTK was constructed successfully. It was tentatively confirmed that RNAi targeting VEGF could synergize with suicide gene therapy.

Abstract:In order to investigate whether excitatory neurotransmission system takes roles in tau phosphorylation caused by cold water stress, mice were treated with cold water stress (CWS), which were forced to swim at 4℃ for 5 min. The tau phosphorylation in mice brains was analyzed by immunobloting and immunohistochemistry with c-fos and phosphorylation-dependent tau antibodies. To evaluate the imbalance of excitatory or inhibitory neurotransmitters system, HPLC was used to detect amino acid neurotransmitters in brain after CWS. And the phosphorylated tau in brains of CWS mice, which were pre-treated with different antagonists for excitatory amino acid receptors and L-type calcium channel, was analyzed. The phosphorylated tau in hippocampus was significantly increased accompanied by an increase of c-fos expression at 1 h after CWS. HPLC showed that the content of all detected excitatory and inhibitory amino acid neurotransmitters appeared an acute increase then decrease pattern. At 15 min after CWS, aspartate and glycine appeared a significant increase, and aspartate, glutamate, taurine and GABA significantly decreased at 1 h. When the animals were pre-treated with NMDA receptor antagonist MK-801 (5 mg/kg) and AMPA receptor antagonist DNQX (0.5, 5 mg/kg), tau phosphorylation caused by CWS were significantly suppressed. Whereas, metabolic glutamate receptor antagonist, MAP-4, had no significant effect on tau phosphorylation. In addition, L-type calcium channel blocker, nimodipine (0.05, 0.5 mg/kg), also could inhibit CWS caused tau phosphorylation. These results indicated that CWS affects tau phosphorylation by mediating excitatory neurotransmission system through ionic excitatory amino acid receptors. The activation of excitatory neurotransmission system takes roles in CWS induced tau phosphorylation in hippocampus.

Abstract:Availability of high-throughput protein-protein interaction and domain-domain interaction information make it possible to study human structural interaction network, and uncover the inner relationship between structure and function of proteins in proteomics area．The widely-distributed domains are thought to affect structure and function of proteins．However, it is still a challenge to investigate potential mechanisms of these effects combing the structural information (e.g domain number, domain length and domain coverage)．The proteins were classified into single- or multi- domain proteins, then human protein structural interaction network was constructed with classification information by integrating the protein-protein interaction and the domain- domain interaction data．Furthermore, comparing with the protein-protein interaction network, specific structure characteristics of human protein structural interaction network were studied. And with respect to single-/multi- domain proteins, function enrichment analysis was carried out for their functions. With domain-domain interaction considered, human protein structural interaction network could provide more detailed information distinct from networks based on protein-protein interaction datasets only, and might reveal the underlying complexity of protein-protein interaction network from the perspective of protein classification. Human protein structural interaction network could exploit domain information to provide additional and crucial protein interaction details necessary for understanding what human structural interactions imply.

Abstract:Extensive studies have been performed on function of PD-1 in immunological regulation, while as so far, studies on the exact regulation mechanism of PD-1 expression have not been reported. PD-1 expression in EL4 and Sp2/0-Ag cell lines was detected with PE-Anti Mouse-PD-1 antibody through FACS. Genomic DNA from C57BL/6J mouse was used to produce different lengths of PD-1 promoter fragments. The PCR products were cloned into the luciferase reporter vector pGL3-Basic and co-transfected with pRL-SV40 into EL4 and Sp2/0-Ag cell lines to investigate the PD-1 promoter activity. FACS results showed that EL4 cells express PD-1 protein constituently, while Sp2/0-Ag cells express high level of PD-1 after stimulation with phorbol 12-myristate 13-acetate (PMA) plus ionomycin (IO). Luciferase assays revealed that the activity of different lengths of PD-1 promoter region was very alterable. This staggering negative-positive-negative arrangement indicates the complex regulation of PD-1 expression and provides important clues for elucidating the mechanisms of the PD-1 gene transcriptional regulation.

Abstract:Osteopontin (OPN) is able to enhance migration of tumor cells through activation of metastasis-related genes involving many signal pathways. Calpain small subunit 1 (Capn4) plays important roles in tumor metastasis. The mechanism of migration of hepatoma cells mediated by OPN was investigated by reporter gene assay, RT-PCR, Western blot analysis and wound healing assay. It was found that OPN was able to upregulate the expression of Capn4 at the levels of promoter activity, mRNA and protein in human hepatoma HepG2 cells. The treatment with PDTC (an inhibitor of NF-κB) could abolish the upregulation of Capn4 mediated by OPN. Moreover, wound healing assay showed that OPN was able to promote the migration ability of hepatoma cells through upregulating Capn4. Thus, It was concluded that OPN is able to upregulate Capn4 through NF-κB in promotion of migration of hepatoma cells.

Abstract:The immnue-inflammatory response mediated by LPS is firmly related to the development of atherosclerosis. ABCA1 has been identified to play a key role in the cellular cholesterol efflux which is regarded to anti-atherosclerosis. To investigate the changes of cholesterol efflux, ATP-binding cassette transporter A1 (ABCA1) mRNA and protein expression in THP-1 macrophage derived foam cells treated with LPS, and to discover the role of TLR4/NF-κB pathway and LXRs in this process. The foam cells were exposed to different concentration of LPS for 24 h or exposed to LPS for different times, and more, the cells were treated with LPS along or together with N-p-Tosyl-L-phenylalanine chloromethyl ketone (TPCK) for 24 h. The mRNA levels of ABCA1, TLR4 and LXRα were measured by reverse transcriptase-polymerase chain reaction，and the protein levels of ABCA1, LXRα and intranuclear NF-κB p65 were measured by Western blotting. Cellular lipid accumulation was determined by high performance liquid chromatography analysis. Cholesterol efflux was determined by FJ-2107P type liquid scintillator. The results show that the expression of ABCA1 were decreased in a dose- and time-dependent manner after treated with LPS, while TLR4 expression and the intranuclear NF-κB p65 protein level were increased by LPS, and these changes can be reversed partly by pretreatment with TPCK. LPS and TPCK can not effect LXRα expression. The results also show that cellular lipid accumulation was increased, while the cellular cholesterol efflux was decreased in THP-1 macrophage derived foam cells after exposured to LPS for 24 h. It was concluded that LPS can down-regulate the expression of ABCA1, promote the accumulation of lipid and decrease cellular cholesterol efflux in THP-1 macrophage derived foam cells, which may be related to the TLR4/NF-κB dependent and LXRα-independent pathway.

Abstract:Absorption and fluorescence spectra of the chromoprotein changed during the ApcD of core subunit from Anabaena sp. PCC 7120 bound PCB with reconstitution in E. coli, the λ_max of absorption and fluorescence spectra were 605 nm and 633 nm before purification, while the λ_max of absorption and fluorescence spectra were 650 nm and 665 nm after purification. To study the phenomenon above, eight mutants were constructed. It is indicated with reconstitution in E. coli that: the mutant ApcD (Y88I) had one more absorbance and fluorescence peaks after purification , the λ_max of absorption and fluorescence spectra were 668 nm and 690 nm. The spectra of ApcD (W59Q), ApcD (Y73A), ApcD (W87E) had no change during the purification. The absorption spectra of ApcD (M126S), ApcD (Y116S), ApcD (M160T) had no change during the purification, but the fluorescence spectra had red shifts by 5 nm, 7 nm and 10 nm after purification, respectively. The λ_max of absorption and fluorescence spectra of ApcD (M115I) had changed from 605 nm and 633 nm to 638 nm and 655 nm. Under acidic urea conditions, those chromoproteins had maximal absorption at 662 nm, indicating that they had PCB chromophore. Under the study of CD spectra of PCB-ApcD, PCB-ApcD (Y116S) and PCB-ApcD (M160T), the two mutants influenced the conformation of chromophore which bound with apoprotein, but not affected the secondary structure of the reconstitution proteins.

Abstract:In order to reveal the mechanisms of cell osmo-adaptation to salt stress, a newly identified moderately halophilic Bacillus sp. I121 was used to study the salt-stress responding proteins. Differential proteomic analysis of the purified plasma membrane fractions were performed by two dimensional Blue-Native/SDS-PAGE. Eight proteins corresponding to NaCl were identified by MALDI-TOF/TOF. Up-regulated proteins under salt stress include an ABC transporter permease, glycerol-3-phosphate permease, pyrimidine nucleotide transporter, formate dehydrogenase, and down-regulated proteins include succinate dehydrogenase iron-sulfur subunit, flavoprotein subunit, cytochrome b-556 subunit, and a hypothetical membrane protein similar to charperone DnaJ. Some of these changes were further verified by enzyme activity assay. Most of these proteins are highly hydrophobic trans-membrane protein, and responsible for material transport across membrane and energy metabolism. These results indicated that in halophilic Bacillus sp. I121, higher salt stress activated material transport across plasma membrane, and compatible solutes, such as proline and ectoine, could be synthesized through suppressed TCA cycle. The research further demonstrated that besides the protein complexes in mitochondria and chloroplast, BN/SDS-PAGE is also a powerful tool for the protein complexes in plasma membrane.

Abstract:Upon the rennin-angiotensin system, angiotensin Ⅰ-converting enzyme (ACE) inhibitors play critical roles in alleviating and suppressing hypertension. The study was performed to isolate and purify an angiotensinⅠ-converting enzyme inhibitory peptide from papain digests of Spirulina platensis by ultra-filtration, gel filtration chromatography and reverse-phase high-performance liquid chromatography. The purified peptide was identified by matrix assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and amino acid sequencing. Furthermore, the inhibition pattern of the peptide was also investigated and the stability was evaluated under simulated gastrointestinal condition. The results demonstrated that the digests with molecular mass ranging from 0 to 3 000 ku had the most potent ACE inhibitory activity with an IC₅₀ value of (1.03 ± 0.04) g/L. An ACE inhibitory peptide with an IC₅₀ value of (0.009 4 ± 0.000 2) g/L which was equivalent to (27.36 ± 0.14) μmol/L was obtained from that fraction of digests, and was identified as Val-Glu-Pro. The Lineweaver-Burk plot and the Dixon plot indicated the ACE inhibitory peptide was a non-competitive inhibitor with a K_i value of (23.59 ± 0.54) μmol/L. In vitro stability assay showed that the peptide could keep its inhibitory activity well after incubation with gastrointestinal proteases including pepsin, chymotrypsin, and trypsin, suggesting the ACE inhibitory peptide from Spirulina platensis be of great prospects as an ingredient of functional foods or pharmaceuticals in prevention and treatment of hypertension.