Photoaffinity labeling is widely applied to demonstrate targets of small molecule ligands. In this paper, biotin photoaffinity labeled molecule with propargyl group 1 has been designed and synthesized, followed it's labeling of N2-acetyl-2'-O-propargyl guanosine 9 by "click chemistry". This technology presents delight development potential in labeling of second messenger cyclic nucleotide, antisense oligonucleotide or siRNA.
Biotin ; chemistry ; Click Chemistry ; Guanosine ; chemical synthesis ; chemistry ; Ligands ; Photoaffinity Labels

Objective To observe the effect of liver disease special-purpose enteral nutrition preparation on protein metabolism and liver function in children with liver injury.Methods Sixty cases of severe ill with liver injury in hospital,with mean age of (7.8?6.3) years old.All patients were randomly divided into experimental group (n=30) and control group(n=30).The experimental group was treated by adding the liver disease special-purpose enteral nutrition preparation homogenized diet and control group was treated by adding entire protein entire nutrition type enteral nutrition preparation.All patients in both 2 groups were nasally fed with intestinal nutrition,which contained 418-628 kJ/(kg?d).One day before nutritional support and 14 days after nutritional support,the liver function,total serum protein,albumin,hemoglobin were recorded.SPSS 11.5 software was used to analyze the data.Results The baseline indicators were similar before nutritional supports.Fourteen days after nutritional support,alanine aminotransferase(ALT) and aspartate aminotransferase(AST) were all significantly lower in experimental group than in control group(Pa

OBJECTIVETo investigate the distribution of ER isoforms in endometriosis and eutopic endometrium.

METHODSTissue samples of patients with ovarian endometriosis, treated in People's Liberation Army General Hospital from January 2004 to December 2006, were retrieved. A total of 60 cases of ovarian endometriotic cysts with their corresponding eutopic endometrium (30 cases of proliferation phase and 30 of secretary phase eutopic endometrium) and 30 cases of normal endometrium (15 proliferative and 15 secretary phase endometrial samples respectively) were included. Expressions of ERalpha and ERbeta were analyzed using immunohistochemistry and the expression ratio was statistically analyzed by using SPSS 12.0 software.

RESULTSExpressions of both ERalpha and ERbeta in epithelial cells were positively correlated with that of the stromal cells. The expression of ERalpha in eutopic endometrium (73.3% in epithelium and 76.7% in stroma) was significantly higher than that in ovarian endometriotic cysts (43.3% in epithelium and 46.7% in stroma), or normal control (56.7% in epithelium and 50.0% in stroma, respectively, each P < 0.05. However, the expression of ERbeta (90.0% in epithelium and 76.7% in stroma) was higher in ovarian endometriotic cysts than that in the eutopic endometrium (68.0% in epithelium and 63.3% in stroma respectively, P < 0.05), and ERbeta expression in eutopic endometrium was higher than that in the normal control endometrium (36.7% in epithelium and 26.7% in stroma, respectively, P < 0.05). The expressions of both ERalpha and ERbeta changed periodically in eutopic and normal endometrium, whereas ERalpha and ERbeta level were less variable in the ectopic endometrium. The expression of ERbeta was statistically higher than that of ERalpha (P < 0.05) in ectopic endometrium, whereas no significant difference was seen between the two isoforms in the eutopic or normal endometrium.

CONCLUSIONSBoth ERalpha and ERbeta have higher expression levels in eutopic endometrium of patients with ovarian endometriotic cysts. ERbeta is predominantly expressed in endometriotic cysts, where the expression of ERalpha is limited. The different distribution of ERalpha and ERbeta may play an important role in the development of ovarian endometriosis.

Adult ; Choristoma ; pathology ; Endometriosis ; metabolism ; pathology ; Endometrium ; metabolism ; pathology ; Epithelium ; Estrogen Receptor alpha ; analysis ; Estrogen Receptor beta ; analysis ; Female ; Humans ; Immunohistochemistry ; Middle Aged ; Protein Isoforms ; analysis ; Receptors, Estrogen ; analysis ; Stromal Cells ; metabolism

OBJECTIVETo prepare HDAg with biological activities as a candidate of diagnostic reagent.

METHODSTo synthesize HDV gene fragment after codon optimization. To construct a thio-fused recombinant plasmid based on M48 expression vector. To express in E. coli induced by IPTG. To purify the protein by affinity chromatography followed by characterization in ELISA:

RESULTSPlasmid construction was verified by enzyme digestion. SDS-PAGE indicated the molecular weight of the protein was the same as we expectation. ELISA proved its affinity with HDV antibodies.

CONCLUSIONHDAg was obtained successfully and it will pave the road to the research of HDV diagnostic reagent.

Enzyme-Linked Immunosorbent Assay ; Hepatitis D ; diagnosis ; Hepatitis delta Antigens ; genetics ; immunology ; isolation & purification ; Humans ; Plasmids ; Recombinant Proteins ; biosynthesis ; immunology ; isolation & purification

Objective To explore the value of seeking tumor cells through cerebrospinal fluid in the metastasis and prognosis of retinoblastoma.Methods Two hundred and fifty-six cases clinic diagnosed retinoblastoma were collected,154 cases were boys and 102 cases were girls.There were 85 children with bilateral disease and 171 children with unilateral disease.Lumbar puncture was examined before treatment,compare the result and prognosis.Children who were found tumor cells in their cerebrospinal fluid were treated with 6 to 9 cycles of systemic chemotherapy with CTV(carboplatin,teniposide and vincristine)protocol and 8 to 10 intrathecal injections with cytrarabine,methotrexate and dexametha-sone.Follow-up were between 8 to 23 months,the mean time was 14.6 months.Results There were 8 cases found tumor cells in their cerebrospinal fluid in all 256 children,one of them died of wide spread intracranial metastasis;the other patient was given high dose chemotherapy with autologous perip-heral stem cell transplantation(APBSCT) because of intracranial metastasis,follow-up to now the patient′s condition was stable.The rest 6 children were enucleated in follow-up period,there were 4 cases(67%) with the histopathology of the eye indicates spread of tumour cells beyond the lamina cribrosa,and 2 cases with invading the cut end of the optic nerve.Six children were no metabasis evidences at other tissues.Conclusions Intracranial metastasis is the common metastasis style in retinoblastoma,so lumbar puncture to seeking tumor cells was important to evaluate intracranial metastasis,early treatment and prognosis.

OBJECTIVETo study the expression of tau-related protein in spinal cord of Chinese patients with Alzheimer's disease.

METHODSGallays-Braak stain and immunohistochemical study for tau protein (AT8) were carried out in the spinal cord tissue (T2, T8, T10, L2 and S2 segments) of 3 Chinese patients with Alzheimer's disease. Seven age-matched cases without evidence of dementia or neurologic disease were used as controls.

RESULTSNeurofibrillary tangles were identified in the neurons of anterior horn in 2 Alzheimer's disease cases but none was observed in the controls. Tau-positive axons and astroglia were detected in all Alzheimer's disease cases. Tau immunoreactivity in spinal cord of the patients correlated with that in brain tissue.

CONCLUSIONThe expression of tau-related protein is demonstrated in the spinal cord of Alzheimer's disease patients suggesting that axonal transport defect may play a role in the pathogenesis of Alzheimer's disease.

Aged ; Alzheimer Disease ; metabolism ; pathology ; Axonal Transport ; Axons ; metabolism ; pathology ; Humans ; Male ; Neurofibrillary Tangles ; metabolism ; pathology ; Phosphorylation ; Spinal Cord ; metabolism ; pathology ; tau Proteins ; metabolism

BACKGROUNDGlobally, 180 million people suffer from diabetes mellitus. Islet transplantation is believed to be an almost ideal therapy for insulin-dependent patients. How to maintain the viability and the function of isolated human islets is a challenge in clinical practice. Sertoli cells are considered 'nurse cells' in the seminiferous tubules and have been used in cell graft protocols for neurodegenerative diseases and diabetes in many studies. Many researchers have used immature murine testes as the primarily source of Sertoli cells in islet transplantation because they are easily purified. Mature human Sertoli cells have been seldom investigated. In the present study, we developed a method for the isolation and culture of Sertoli cells derived from adult human testes, and investigated their effects on the function of allogeneic islets when they were cultured together in vitro.

METHODSAdult Sertoli cells were prepared successfully by two-step enzyme digestion with trypsin, collagenase and hyaluronidase. They were identified by morphological characteristics and their activity was determined by MTT colorimetry over a 28-day culture time in vitro. A glucose-stimulated insulin secretion test was performed to detect the effects of Sertoli cells on allogeneic islets' function when they were co-cultured for 21 days in vitro.

RESULTSIn cultured cells, mature human Sertoli cells accounted for more than 90% of total cells. The activity of Sertoli cells reached 95% and they remained highly cytoactive for a long time in vitro (P > 0.05). Compared with the islets cultured alone, the co-cultured islets with allogeneic Sertoli cells maintained higher sensitivity to glucose stimulation for the duration of the experiment (P < 0.01).

CONCLUSIONSA method of isolation and culture of Sertoli cells from adult testes has been established. Sertoli cells could enhance allogeneic islets' function when they were co-cultured in vitro. They could be a helper cell in islet transplantation.

Adult ; Cell Separation ; methods ; Cell Survival ; Cells, Cultured ; Coculture Techniques ; Humans ; Islets of Langerhans ; physiology ; Islets of Langerhans Transplantation ; Male ; Sertoli Cells ; cytology ; physiology

OBJECTIVETo study the effect of gene optimization on the expression and purification of HDV small antigen produced by genetic engineering.

METHODSBased on the colon preference of E. coli, the HDV small antigen original gene from GenBank was optimized. Both the original gene and the optimized gene expressed in prokaryotic cells, SDS-PAGE was made to analyze the protein expression yield and to decide which protein expression style was more proportion than the other. Furthermore, two antigens were purified by chromatography in order to compare the purity by SDS-PAGE and Image Lab software.

RESULTSSDS-PAGE indicated that the molecular weight of target proteins from two groups were the same as we expected. Gene optimization resulted in the higher yield and it could make the product more soluble. After chromatography, the purity of target protein from optimized gene was up to 96.3%, obviously purer than that from original gene.

CONCLUSIONGene optimization could increase the protein expression yield and solubility of genetic engineering HDV small antigen. In addition, the product from the optimized gene group was easier to be purified for diagnosis usage.

Electrophoresis, Polyacrylamide Gel ; Genetic Engineering ; methods ; Hepatitis D ; diagnosis ; Hepatitis delta Antigens ; genetics ; isolation & purification ; Recombinant Proteins ; biosynthesis ; isolation & purification

OBJECTIVETo study the immunologic function of dendritic cells (DCs) cultured in two kinds of hepatoma cell line's supernatant and the enhancing effects of carboxymethylpachymaran (CMP) on DCs.

METHODSDCs were harvested after stimulation by granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin (IL)-4 from umbilical cord blood using density-gradient centrifugation method. Cultured supernatant of two hepatoma cell lines (HepG2 and HepG2.2.15) were collected for condition medium (CM) according to a volume ratio of supernatant to incomplete RPMI-1640 medium, which was 3:1. CMP was dissolved in incomplete RPMI-1640 medium. Experimental groups were divided according to the culture medium, either CM or with CMP in it. DCs subsets CD83, CD86, CD1a, and d-related human leukocyte antigens (HLA-DR) were analyzed by flow cytometry. The proliferation ability of allogeneic T cells in mixed lymphocyte reaction (MLR) stimulated by DCs was examined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) analysis. IL-12p70, interferon-γ (IFN-γ), and nuclear factor κB (NF-κB) were detected by enzyme-linked immunosorbent assay analysis.

RESULTSThe proliferation of lymphocytes and secreting level of IL-12 and expression of phenotype of DCs cultured in two kinds of CM were lower than those of normal group (P <0.01). Compared with the normal group, groups treated with CMP showed a higher expression level of DCs subsets, lymphocyte reproductive activity, as well as IL-12 and IFN-γ secretion levels. Groups treated with CMP also demonstrated higher levels of DCs phenotype expression and IL-12 and IFN-γ secretion in supernatant of MLR and higher lymphocyte reproductive activity compared with CM group (P <0.05). Compared with the normal group, the expression level of NF-κB in DCs nuclear was higher in CMP groups but lower in two CM groups (P <0.05). After CMP was added, the NF-κB expression levels of two CM groups were increased compared with levels before CMP was added (P <0.05). However, there was no significant difference between the two CM groups (P >0.05).

CONCLUSIONSTwo kinds of hepatoma cell line's supernatant can inhibit the immunologic function of DCs. This suppressive effect may be related to the inhibition of NF-κB/Rel pathway. CMP may up-regulate the DCs function by activating the NF-κB/Rel pathway.

Carcinoma, Hepatocellular ; pathology ; ultrastructure ; Cell Line, Tumor ; Cell Shape ; Dendritic Cells ; drug effects ; immunology ; Glucans ; pharmacology ; Humans ; Immunophenotyping ; Interferon-gamma ; metabolism ; Interleukin-12 ; metabolism ; Liver Neoplasms ; pathology ; ultrastructure ; Lymphocyte Culture Test, Mixed ; Signal Transduction ; drug effects ; Subcellular Fractions ; drug effects ; Transcription Factor RelA ; metabolism