OBJECTIVE: To express and purify NR4A1-DNA binding domain (DBD) protein of nuclear receptors. METHODS: The fusion protein PET28a-NR4A1-DBD was constructed and purified with the nickel affinity chromatography, cation-exchange chromatography and gel filtration chromatography. RESULTS: The protein PET28a-NR4A1-DBD was mostly soluable at 24 °C. A total of 2-3 mg/L pure NR4A1 proteins were yielded in bacterial culture and the purity for final fractions of NR4A1-DBD protein were great than 95% by SDS-PAGE analysis. CONCLUSION Nickel affinity chromatography is effective to purify protein. The protein purity can be further improved by the following methods including cation-exchange chromatography and gel filtration chromatography.
Electrophoresis, Polyacrylamide Gel ; Nuclear Receptor Subfamily 4, Group A, Member 1 ; chemistry ; Recombinant Fusion Proteins ; chemistry

Objective To investigate the biological significance of differentially expressed proteins from human primary lung adenocarcinoma with lymph node metastasis adenocarcinoma (LNM AdC) and without metastasis (non-LNM AdC) according to clinical diagnosis of lymph node metastasis and distant metastasis,with bioinformatics approach.Methods Cytoscape software was used to analyze a functional enrichment analysis and a protein-protein interaction network from differentially expressed proteins from LNM AdC and non-LNM AdC.Results The top biological processes were related to glucose catabolic process,hexose catabolic process,monosaccharide catabolic process,alcohol catabolic process,and cellular carbohydrate catabolic process.The top molecular functions were related to phospholipase inhibitor activity,lipase inhibitor activity,calcium-dependent phospholipid binding,phosphlipase A2 inhibitor activity,and lipid binding.A protein-protein interaction network of differentially expressed proteins was generated with literature data.Conclusions This bioinformatics analysis demonstrated that glucose catabolic process,alcohol catabolic process,calcium-dependent phospholipid binding,phosphlipase A2 inhibitor activity,ACTB,ANXA1,ANXA2,ANXA3,VCP,NPM1,KRT1,and SUMO4 are significantly associated with a lung adenocarcinoma.These network data provide new insights into the metastasis mechanisms of human lung adenocarcinoma.

Objective In order to investigate the effect of SH2B1 on leptin signal transduction JAK2/IRS2 and its biological function.Methods Vitro kinase assay and Western blot were used to analyse tyrosine phosphorylatin of key molecule JAK2 and insulin receptor substrate-2 (IRS2). ELISA was used to measure the plasma leptin levels in mice. The postnatal growth of mice was monitored over 27 weeks. Results SH2B1 dramatically enhanced the leptin-stimulated tyrosine phosphorylation of JAK2 and IRS2 in HEK293 cells stably expressing LRb (HEK239~(LRb)). Leptin-stimulated activation of hypothalamic JAK2 and phosphorylation of hyphothalamic IRS2 were significantly impaired in SH2B1~(-/-) mice. The deletion of SH2B1 led to leptin resistance,and fasting and randomly fed plasma leptin levels were respectively 3.2 times and 5.1 times higher in SH2B1~(-/-) males than wild-type littermates at 15 weeks of age. SH2B1~(-/-) males gained body weight rapidly and exceeded wild-type littermates from 5~(th) week. SH2B1(-/-) (at 21 weeks) was approximately twice heavier than wild-type littermates.Conclusion SH2B1 is an endogenous enhancer of leptin sensitivity and required for maintaining normal bodyweight in mice via leptin JAK2/IRS2 pathway.

The molecular techniques were used to analyse tyrosine phosphorylation of JAK2 and STAT3 in leptin receptor overpression cell lines and SH2-Bβ knockout (SH2-Bβ-/-) mice. The serum level of leptin in SH2-Bβ mice was measured by ELISA. The results showed that SH2-Bβ dramatically enhanced the leptin-stimulated tyrosine phosphorylation of JAK2 and STAT3 in vitro. Leptin-stimulated activation of JAK2 and phosphorylation of STAT3 were significantly impaired in hypothalamus of SH2-Bβ-/- mice. The fasting and postprandial serum levels of leptin and body weight were markedly increased in SH2-Bβ-/- mice. Therefore, SH2-Bβ is an endogenous enhancer of leptin sensitivity and regulates body weight via leptin/ JAK2/STAT3 pathway.

Objective To search for lymph node metastasis-associated proteins in human lung squamous carcinoma (hLSC).Methods Laser capture microdissection (LCM) was used to purify the target cells from lung primary tumor and matched lymph node metastatic tumor in hLSC. Two-dimensional gel electrophoresis (2-DE) was performed to separate the total proteins of microdissected tumor cells from lung primary tumor and matched lymph node metastatic tumor. PDQuest software was applied to analyze 2-DE images. Differential protein spots between the two types of tissues were identified by matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF-MS). The expression of Rho-GDIα, one of the differential proteins, in the microdissected lung primary tumor cells (LPTC) and matched lymph node metastatic tumor cells (LNMTC) was detected by Western blot. Results In the present study, 2-DE patterns of microdissected LPTC and LNMTC were established, and 22 differential proteins in the above two tissues were identified, of which 14 were down-regulated in LNMTC and 8 were up-regulated in LNMTC.Conclusion The 22 differential proteins may play some roles in the process of lymph node metastasis in hLSC, and the data provide new clues for metastasis-associated biomarker screen and mechanism of hLSC.

Objective To identify proteins eliciting a humoral immune response in patients with nasopharyngeal carcinoma (NPC) by a serological proteome analysis, and provide candidate biomarkers for diagnosis and treatment of NPC.Methods Two-dimensional (2-DE) electropboresis was used to separate total cellular proteins from 19 NPC tissues.Separated proteins were transferred onto PVDF membranes and sera from 19 NPC patients and 19 healthy subjects were individually screened by western blotting for antibodies that react against separated proteins.Each tissue samples was subjected to three 2-DE gels and coomassie staining was performed in one of them.The protein spots which selectively reacted with the patient sera were excised from the preparative gels and subjected to further analysis of MOLDI-TOF MS and ESI-QTOF MS/MS.The proteins were identified based on peptide mass fingerprints and peptide sequence tags followed by searching database.Results In this study, 13 NPC associated antigens (HSP 70, HAS,HSP 60, CK 15, LAP 3, α-enolase, EBP 1, CK 19, ribosomal protein P 0, pyrovate dehydrogenase E1,guanine nucleotide-binding protein, prohibitin, Rho-GDI 2) that elicited an antibody response in most of NTC patients were identified.The positivities of these proteins were more than 21% all in NPC patients, but were lower or even absent in normal subjects.Conclusion These 13 NPC associated antigens and their autoantibodies may be useful for NPC diagnosis and treatment.

OBJECTIVE: To establish the protein expression map of nasopharyngeal carcinoma (NPC), and provide a basis for proteomic study of NPC. METHODS: Laser capture microdissection (LCM) was used to isolate cancer cells from NPC tissues. Two-dimensional gel electrophoresis(2-DE) was used to separate the total proteins of LCM purified NPC cells. Matrix-assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS) was performed to identify the proteins, and bioinformatics was used to construct the 2-DE database of NPC proteome. RESULTS: A 2-DE reference map of NPC was established. On the 2-DE map, a total of (1 312+/-30) protein spots were detected, and 427 protein spots representing 241 non-redundant proteins were identified. The 2-DE database of NPC proteome was constructed. These data could be accessed at our website (http://www.xyproteomics.org). CONCLUSION A protein expression profile of LCM purified NPC tissues has been established for the first time, which provides useful information and source for proteomic study of NPC.
Computational Biology ; Gene Expression Profiling ; Humans ; Lasers ; Microdissection ; methods ; Nasopharyngeal Neoplasms ; genetics ; metabolism ; Neoplasm Proteins ; genetics ; isolation & purification ; metabolism ; Proteome ; metabolism ; Proteomics ; methods ; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization

OBJECTIVE: To detect the methylation and expression of glioma pathogenesis-related protein 1(GLIPR1) gene in the acute myeloid leukemia (AML) cell lines and bone marrow cells from AML patients, and to determine the relationship between promoter methylation and expression of GLIPR1. METHODS: Five leukemia cell lines, 54 bone marrows from the newly diagnosed AML patients, 48 bone marrows from the acute lymphoblastic leukemia (ALL )patients, 40 bone marrows from the chronic myeloid leukemia (CML) patients,35 bone marrows from control patients, and 8 bone marrows from the complete remission AML patients were collected. RT-PCR and methylation-PCR (MSP) were used to detect the mRNA expression and promoter methylation of GLIPR1, respectively, and the relationship between them was analyzed. RESULTS: The level of GLIPR1 mRNA in the AML cell lines was lower than that in the chronic myeloid leukemia (CML) and ALL cell lines, whereas the methylation level of GLIPR1 in the former was higher than that in the latter. The level of GLIPR1 mRNA in the AML cell lines was significantly increased, but had no obvious changes in the CML and ALL cell lines after 5-aza-2dC treatment. The mRNA level of GLIPR1 in the AML bone marrows (0.38+/-0.20)was obviously lower than that in the ALL bone marrows (0.76+/-0.18), CML bone marrows (0.80+/-0.14), and control bone marrows(0.85+/-0.12). The level of GLIPR1 mRNA in the bone marrows with complete remission AML was obviously higher than that in the AML bone marrows before the treatment (0.78+/-0.13 vs. 0.36+/-0.20); but there was no obvious difference between the ALL bone marrows and the control bone marrows, and the CML bone marrows and the control bone marrows (both P>0.05). The positive rate of GLIPR1 gene methylation in the AML bone marrows (81.5%) was obviously higher than that in the ALL bone marrows(37.5%), CML bone marrows (27.5%) and the control bone marrows(14.3%). The positive rate of GLIPR1 gene in the bone marrows with complete remission AML was obviously lower than that in the bone marrows before the treatment (12.5% vs. 75.0%), but there was no obvious difference between the ALL bone marrows and between the control bone marrows,and the CML bone marrows and the control bone marrows (both P>0.05). There was a negative correlation between the mRNA level and methylation status of GLIPR1 in the AML bone marrows. CONCLUSION GLIPR1 expression is downregulated or even lost by promoter methylation in AML, and the expression and methylation level of GLIPR1 gene may have some significance in evaluating the curative effect of AML patients.
Adolescent ; Adult ; Aged ; Aged, 80 and over ; DNA Methylation ; Female ; HL-60 Cells ; Humans ; K562 Cells ; Leukemia, Myeloid, Acute ; genetics ; metabolism ; Male ; Middle Aged ; Neoplasm Proteins ; genetics ; metabolism ; Nerve Tissue Proteins ; genetics ; metabolism ; Promoter Regions, Genetic ; RNA, Messenger ; genetics ; metabolism ; Young Adult

Objective To investigate the methylation status of 14-3-3σ promoter in nasopharyngeal carcinoma cell lines and the influence of de-methylation treatment on 14-3-3σ expression. Methods Methylation status of 14-3-3σ gene promoter and 14-3-3σ mRNA expression were detected by methylation specific PCR (MSP) and RT-PCR in nasopharyngeal carcinoma cell lines CNE1, CNE2,5-8F,6-10B and immortalized nonneoplastic human nasopharyngeal epithelial cell line, NP69. Four nasopharyngeal carcinoma cell lines were treated with 5-asa-2' -deoxycytidine(5-aza-2dC) in different concentration for 72 h, then 14-3-3σ promoter meth-ylation status and m RNA expression were assessed, and western-blot was performed to detect the expression of 14-3-3σ protein. Results 14-3-3σ promoter methylation was detected by MSP in all of the four nasopharyn-geal carcinoma cell lines untreated by 5-aza-2dC whereas not in the treated ones or the immortalized human na-sopharyngeal epithelial cell line, NP69. Accordingly, 14-3-3σ mRNA expression was significantly discounted in untreated nasopharyngeal carcinoma cell lines as compared with NP69. 5-aza-2dC treatment dose-depend-ently reversed 14-3-3σ promoter methylation status and consequently upregulated the expression of 14-3-3σmRNA and protein in 4 nasopharyngeal carcinoma cell lines. High-differentiated CNE1 was more sensitive to 5-aza-2dC than lowly-differentiated CNE2, 5-8F and 6-10B. Conclusion Promoter methylation directly leads to decreased 14-3-3σ gene expression in nasopharyngeal carcinoma cell lines, and 14-3-3σ promoter de-methylation perhaps indicates a new target for nasopharyngeal carcinoma treatment.

Objectives To establish the two-dimensional electrophoresis(2-DE)profile of cell. Secreted proteins.Difierential expression profiling of fibroblast cell secreted proteins between nasopharyngeal carcinoma and normal nasopharyngeal tissue was analyzed.Methods Five tissue specimens each from patients with nasopharyngeal carcinoma and nasal polyp were collected individually.Fibroblast eells from above-mentioned tissue were cultured in serum-free medium,and cell-secreted proteins from the cultured medium were harvested by uhrafihration concentration and desalination.Samples were analyzed by 2-DE,and the differentially expressed proteins were analyzed and identified by matrix-assisted laser desorption/ionization time of flight mass spectrometry.Galectin-1 wa8 analyzed by EUSA test.Results 2-DE diagram of fibroblast cell-secreted proteins Was constructed.1 8 protein spots displayed quantitative changes in expression,and 11 protein spots among them were identified by mass speetrometrv.3 proteins including cystatin C,complement subcomponent C1S precursor,heterogeneous nuclear ribonueleoprotein A1 were down-regulated in the cultured medium of nasopharyngeal carcinoma associated fibroblast cells(CAFs). Nevertheless,the rest cell-secreted proteins including galectin-1,14-3-3 protein sigma,eathepsin L and etc,were up-regulated.Meanwhile,the expression of galectin-1 in the cultured medium was also analyzed and Its results were compared between CAFs and the normal fibroblast cells by ELISA.There Was statistical significance difference between them,and galectin-1 was up-regulated in the cIlltured medium of CAFs.Conclusions The changes of fibroblast cell-secreted proteins during nasopharyngeal carcinogenesi8 are analyzed by 2-DE analysis.The variation of pattern of secreted proteins is involved in signal transduction,protein synthesis,degradation and other pathways.CAFs may regulate tumor microenvironment by the abeve-mentioned pathways,and influence nasopharyngeal carcinogenesis,progress,invasion and metastasis.This study provided experimental basis for the eell secreted proteomics studv in future.