Carcinoma, Hepatocellular ; metabolism ; Glycoproteins ; biosynthesis ; genetics ; Humans ; Hyaluronan Receptors ; biosynthesis ; genetics ; Liver ; metabolism ; Liver Neoplasms ; metabolism ; Osteopontin ; biosynthesis ; genetics

Glycosylation is vital for activity, higher structure and function of protein. Glycoproteins derived from yeast contain N-glycan of high mannose type and are usually hyperglycosylated, while those from mammalian cells contain N-glycan of hybrid or complex type. We introduced the alpha-1,2-mannosidase I (MDSI) into yeast cells, which catalyzed an essential proceeding of N-glycan structures from Man8GlcNAc2 to Man5GlcNAc2. The plasmids contained MDSI genes from Homo sapiens [HMDSI(delta185)] or Arabidopsis thaliana [ATMDSI(delta48)], and three ER-signals were used to be transformed a mutant Pichia pastoris GJK01, respectively. The reporter protein HSA/GM-CSF (human serum albumin and granulocyte-macrophage colony stimulating factor fusion protein) was expressed and its N-glycans were analyzed by DSA-FACE (DNA sequencer assisted fluorophore-assisted carbohydrate electrophoresis). The plasmid contained ER-ScMnsI-ATMDSI(delta48) was expressed in Pichia pastoris, the Man5GlcNAc2 N-glycan on secreted glycoprotein HSA/GM-CSF was observed. The research reported here provided basic substrate to obtain the hybrid- and complex-type glycans in mammalian cell.
Gene Transfer Techniques ; Genetic Vectors ; genetics ; Glycoproteins ; biosynthesis ; Glycosylation ; Humans ; Mannose ; biosynthesis ; Oligosaccharides ; biosynthesis ; genetics ; Pichia ; genetics ; metabolism ; Recombinant Proteins ; biosynthesis ; genetics ; alpha-Mannosidase ; genetics

Mammalian zona pellucida 3(ZP3) plays an important role in the induction of capacitating sperm acrosome reaction. In this study, we obtained the soluble mZP3 fusion protein and identified its immunoreactivity. mZP3 cDNA was cloned into plasmid pMAL-p2x, and the recombinant plasmid was transformed into Escherichia coli BL21. To get the soluble mZP3 fusion protein, we tried to optimize the expression conditions, including additives, IPTG concentrations, temperatures and induction duration. Then, Western blotting and ELISA were used to identify the immunoreactivity of the purified protein. Based on the optimization experiments, we concluded that the best soluble expression conditions for the mZP3 fusion protein involved incubation to an A600 of 0.6, addition of glucose to a final concentration of 0.02 mol/L, addition of IPTG to a final concentration of 0.6 mmol/L and then further incubation for 4 h at 25 degrees C. Western blotting and ELISA showed that the mZP3 fusion protein retained immunoreactivity. The fusion protein can be used as solubility antigens for developing the immunocontraception vaccines of mZP3 and detecting the immune effects of the vaccine.
Animals ; Egg Proteins ; biosynthesis ; genetics ; immunology ; Escherichia coli ; genetics ; metabolism ; Female ; Membrane Glycoproteins ; biosynthesis ; genetics ; immunology ; Mice ; Receptors, Cell Surface ; biosynthesis ; genetics ; immunology ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; immunology ; Solubility ; Vaccines, Contraceptive ; immunology ; Zona Pellucida Glycoproteins

Nucleases is an important enzyme widely used in biotechnology. A codon optimized nuclease gene (SNU) from Northern Shrimps was inserted into pPICZα A vector, and expressed extracellularly in strain SMD1168H. On the basis of multi-copy recombinant strain, we further optimized the expression condition and characterized SNU. SNU was highly expressed and stable after 1% methanol induction for 72 h, yield reached 1.4×10⁵ U/mL. SDS-PAGE electrophoresis demonstrated that this is a N-linked glycoprotein of 50 kDa. It was purified by one step DEAE Sephadex chromatography to the purity of about 15 mg/L with a specific activity of 6.291×10⁶ U/mg. Functional analysis on the nuclease activity indicated that it was stimulated by bivalent iron, such as Ca²⁺, Mn²⁺, Co²⁺ and Mg²⁺, but inhibited by Zn²⁺, Cu²⁺ and high salt. Meanwhile, it was irreversibly inactivated at 70 ℃ for 10 min.
Animals ; Codon ; Electrophoresis, Polyacrylamide Gel ; Endonucleases ; biosynthesis ; Glycoproteins ; Hot Temperature ; Penaeidae ; enzymology ; Pichia ; metabolism ; Recombinant Proteins ; biosynthesis

OBJECTIVE: To explore expression of the glycoprotein in early myocardial ischemic. METHODS: The glycoprotein changes occurred at the early acute cardiac ischemic area induced experimentally by ligation of left coronary artery of 32 SD rats. 6 lectins were measured by means of immunohistochemical methods. RESULTS: Positive staining of PNA could be observed in ischemic area at 5 min after ischemia, and the positive area increased with the prolongation of ischemic period. It became the strongest for 2 h and then decreased. CONCLUSION This experiment proved that myocardial cell membrane in ischemia expressed D-galactose. This may be of some value in forensic medicine practice.
Animals ; Female ; Immunohistochemistry ; Male ; Membrane Glycoproteins/biosynthesis* ; Myocardial Ischemia/metabolism* ; Myocardium/metabolism* ; Rats ; Rats, Sprague-Dawley

OBJECTIVETo study the effects of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] on the expression of osteoprotegerin (OPG) and receptor activator of NF-kappaB ligand (RANKL) mRNA in mouse osteoblasts.

METHODSCalvariae derived from CD-1 neonatal mouse (after born 24 h). Bone samples were processed by the collagenase/trypsin digestion method. Mouse osteoblasts were cultured in vitro. After 48 hours of addition of 1,25(OH)2D3 (0, 10(-8), 10(-9), 10(-11) mol/L) to the culture medium of mouse osteoblasts, the content of the OPG protein in culture medium was estimated with enzyme linked immunosorbent assay. Total RNA was prepared from mouse osteoblasts. mRNA expression of OPG and RANKL were detected by reverse transcription-polymerase chain reaction.

RESULTSThe mRNA expression of OPG in osteoblasts added with 1,25(OH)2D3 significantly decreased compared with the controls, which was markedly dose-dependent. OPG protein production in the medium decreased after treatment with 1,25(OH)2D3. In contrast, RANKL mRNA expression levels in osteoblasts significantly increased after 48 h of culture with 1,25(OH)2D3.

CONCLUSION1,25 (OH)2D3 can stimulate RANKL mRNA expression, but decrease OPG mRNA levels in vitro in mouse osteoblasts.

Animals ; Animals, Newborn ; Calcitriol ; pharmacology ; Carrier Proteins ; biosynthesis ; genetics ; Glycoproteins ; biosynthesis ; genetics ; physiology ; Ligands ; Membrane Glycoproteins ; biosynthesis ; genetics ; Mice ; NF-kappa B ; biosynthesis ; genetics ; Osteoclasts ; metabolism ; physiology ; Osteoprotegerin ; RANK Ligand ; RNA, Messenger ; biosynthesis ; genetics ; Receptor Activator of Nuclear Factor-kappa B ; Receptors, Cytoplasmic and Nuclear ; analysis ; biosynthesis ; genetics ; physiology ; Receptors, Tumor Necrosis Factor ; biosynthesis ; genetics ; Tumor Necrosis Factor-alpha ; biosynthesis ; genetics

The fundamental event of cancer invasion and metastasis is the complicated interaction of cancer cells with host cells, in which event, a number of proteases and their inhibitors are involved. Matrix metalloproteinases are the potent proteases in degrading the basement membrane and extra cellular matrix and are inhibited by specific endogeneous inhibitors, tissue inhibitors of metalloproteinases-1(TIMP-1) and TIMP-2. The expression of mRNA for TIMP-1 and -2 was investigated by Northern blot analysis in specimens taken from 27 patients with primary gastric adenocarcinoma; 25 samples from the primary site, six from the metastatic lymph nodes and two from the peritoneal fluids. The expression for TIMP-1 and -2 was compared in primary gastric cancer tissues, metastatic lymph nodes and normal gastric mucosae. TIMP-1 mRNA was overexpressed in 24 (96%) out of 25 primary cancer tissues compared with the paired normal mucosae, while TIMP-2 was in 10 (40%). In six specimens of metastatic lymph nodes, TIMP-1 and -2 were overexpressed in 6 (100%) and 4 (67%) specimens, respectively. Of two specimens prepared from the peritoneal fluids, all specimens overexpressed TIMP-1 compared with the those of primary cancer tissues, while one (50%) specimen overexpressed TIMP-2. Immunohistochemical staining was done to investigate the localization of TIMP-1 and -2, demonstrating that the immunoreactivity for TIMP-1 and -2 was clearly detected in the cytoplasm of the stromal cells. These results suggest that both TIMP-1 and -2 are overexpressed by stromal cells in most of primary and some metastatic gastric cancer tissues and that TIMP-1 and TIMP-2, produced by stromal cells, may play an important role in inhibiting the proteolytic activity of matrix metalloproteinases originated from cancer cells, in gastric cancer.
Adenocarcinoma/*enzymology ; Blotting, Northern ; Glycoproteins/*biosynthesis/genetics ; Human ; Proteins/*biosynthesis/genetics ; RNA, Messenger/biosynthesis ; Stomach Neoplasms/*enzymology ; Support, Non-U.S. Gov't ; Tissue Inhibitor of Metalloproteinases ; Tissue Inhibitor-of Metalloproteinase-2

The fundamental event of cancer invasion and metastasis is the complicated interaction of cancer cells with host cells, in which event, a number of proteases and their inhibitors are involved. Matrix metalloproteinases are the potent proteases in degrading the basement membrane and extra cellular matrix and are inhibited by specific endogeneous inhibitors, tissue inhibitors of metalloproteinases-1(TIMP-1) and TIMP-2. The expression of mRNA for TIMP-1 and -2 was investigated by Northern blot analysis in specimens taken from 27 patients with primary gastric adenocarcinoma; 25 samples from the primary site, six from the metastatic lymph nodes and two from the peritoneal fluids. The expression for TIMP-1 and -2 was compared in primary gastric cancer tissues, metastatic lymph nodes and normal gastric mucosae. TIMP-1 mRNA was overexpressed in 24 (96%) out of 25 primary cancer tissues compared with the paired normal mucosae, while TIMP-2 was in 10 (40%). In six specimens of metastatic lymph nodes, TIMP-1 and -2 were overexpressed in 6 (100%) and 4 (67%) specimens, respectively. Of two specimens prepared from the peritoneal fluids, all specimens overexpressed TIMP-1 compared with the those of primary cancer tissues, while one (50%) specimen overexpressed TIMP-2. Immunohistochemical staining was done to investigate the localization of TIMP-1 and -2, demonstrating that the immunoreactivity for TIMP-1 and -2 was clearly detected in the cytoplasm of the stromal cells. These results suggest that both TIMP-1 and -2 are overexpressed by stromal cells in most of primary and some metastatic gastric cancer tissues and that TIMP-1 and TIMP-2, produced by stromal cells, may play an important role in inhibiting the proteolytic activity of matrix metalloproteinases originated from cancer cells, in gastric cancer.
Adenocarcinoma/*enzymology ; Blotting, Northern ; Glycoproteins/*biosynthesis/genetics ; Human ; Proteins/*biosynthesis/genetics ; RNA, Messenger/biosynthesis ; Stomach Neoplasms/*enzymology ; Support, Non-U.S. Gov't ; Tissue Inhibitor of Metalloproteinases ; Tissue Inhibitor-of Metalloproteinase-2

As one important member of B7/CD28/CTLA-4 costimulatory signal pathway, B7-2 molecule plays a critical role in regulating T-cell response. In order to further explore its effects on regulation of T cell activation, proliferation and associated signal pathways, the cDNA encoding extracellular region of human B7-2 was amplified via PCR and subcloned into some prokaryotic expression vectors to express target protein in host strains. The expressed protein was identified with Western blot and MTT. Results showed that after screening, the expression level of the protein of interest attained the yield of over 20% total bacterial protein by using pGEX-4T-2 vector and E. coli BL21 (DE3)-CodonPlus-RIL host cells. The recombinant protein could specially react with B7-2 McAb and could stimulate T-cell proliferation combined with anti-CD3 antibody. In conclusion, the recombinant protein was bioactive, therefore the study will make it possible for the research of relationship between B7-2 structure and its function.
Antigens, CD ; biosynthesis ; genetics ; pharmacology ; B7-2 Antigen ; Blotting, Western ; Cloning, Molecular ; DNA, Complementary ; analysis ; Humans ; Membrane Glycoproteins ; biosynthesis ; genetics ; pharmacology ; Recombinant Proteins ; biosynthesis ; isolation & purification ; pharmacology

BACKGROUNDTo sequence, analyze and express the nucleotide sequences of S and M segments of hantavirus strain 84Fli.

METHODSS and M segments of hantavirus 84Fli strain were amplified by RT-PCR, the PCR products were cloned into plasmid pCR2.1-TOPOr. Three clones of each segment which have been sequenced were randomly selected. The coding region of S and M segments were amplified by PCR, and cloned into expressing vector pcDNA3.0. Transient expression of nucleocapsid protein, G1 and G2 glycoproteins in COS7 cells were performed by Lipofectin transfection. The expression of NP, G1 and G2 have been conformed by using immunofluorescence, Western blot and immuno-precipitation methods.

RESULTSThe full length S and M segments cDNA have been amplified and cloned. The S and M segments of hantavirus strain 84Fli contained 1 688 and 3 616 nucleotides respectively.

CONCLUSIONSDeduced amino acid sequences of NP and glycoproteins revealed high homologue to other Hantaan viruses. NP, G1 and G2 proteins of 84Fli can be transiently expressed in COS7 cells.

Animals ; COS Cells ; metabolism ; Cloning, Molecular ; Gene Expression ; Glycoproteins ; biosynthesis ; Hantavirus ; genetics ; metabolism ; Nucleocapsid Proteins ; biosynthesis ; Sequence Analysis ; Viral Proteins ; biosynthesis