ABSTRACT
A set of 70 peptides with affinity for the class I MHC HLA-A*0201 molecule was subjected to quantitative analyses of structure-affinity relationship based on the SCORE function with good results (r(2)=0.6982, q(2)=0.6188, RMS=0.280). Then the 'leave-one-out' cross-validation (LOO-CV) and an outer test set including 18 outer samples were used to validate the QSAR model, and the results show that the QSAR model has good predictability for outside samples. Statistical analysis showed that the hydrophobic and hydrogen bond interaction played a significant role in peptide-MHC molecule binding. The study also provided useful information for structure modification of CTL epitope, and laid theoretical base for molecular design of therapeutic vaccine.
Subject(s)
Algorithms , Epitopes, T-Lymphocyte/immunology , HLA-A Antigens/immunology , T-Lymphocytes, Cytotoxic/immunology , Computational Biology , Drug Design , HLA-A Antigens/chemistry , HLA-A2 Antigen , Hydrogen Bonding , Hydrophobic and Hydrophilic Interactions , Models, Molecular , Peptides/chemistry , Peptides/immunology , Protein Binding , Protein Conformation , Quantitative Structure-Activity Relationship , Reproducibility of Results , Vaccines/chemistry , Vaccines/immunologyABSTRACT
BACKGROUND: The dermal papilla is a major component of hair, which signals the follicular epithelial cells to prolong the hair growth process. To date, little is known about the significance of the specific protein(s) express in the dermal papilla cells (DPC) with regard to their aggregative behaviour. OBJECTIVES: To identify proteins involved in aggregative behaviour of DPC, we comparatively analyzed the proteome of cells with and without aggregative behaviour. METHODS: A series of methods were used, including two-dimensional gel electrophoresis (2-DE), PDQuest software analysis of 2-DE gels, peptide mass fingerprinting based on matrix-assisted laser desorption/ionisation-time of flight-mass spectrometry (MALDI-TOF-MS), and NCBInr database searching, to separate and identify differentially expressed proteins. Western blotting and reverse transcriptase polymerase chain reaction (RT-PCR) were used to validate the differentially expressed proteins. RESULTS: Image analysis revealed that averages of 618+/-22 and 568+/-47 protein spots were detected in passages 3 and 10 DPC, respectively. Twenty-four differential protein spots were measured with MALDI-TOF-MS. A total of 17 spots yielded good spectra, and 15 spots matched with known proteins after database searching. Western blotting confirmed that heat shocking protein 70 was up-regulated in passage 3 DPC. Over-expression of mitochondrial ribosomal protein S7 was confirmed by RT-PCR, indicating that they are involved in aggregation of DPC through some signaling pathway. CONCLUSIONS: The clues provided by the comparative proteome strategy utilized here will shed light on molecular mechanisms of DPC in aggregative behaviour.
Subject(s)
Dermis/cytology , Dermis/physiology , HSP70 Heat-Shock Proteins/metabolism , Hair/cytology , Hair/physiology , Proteomics/methods , Adult , Amino Acid Sequence , Blotting, Western , Cell Aggregation/physiology , Electrophoresis, Gel, Two-Dimensional/methods , Female , HSP70 Heat-Shock Proteins/analysis , Hair/growth & development , Humans , Male , Microfilament Proteins/analysis , Microfilament Proteins/metabolism , Molecular Sequence Data , Muscle Proteins/analysis , Muscle Proteins/metabolism , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization/methods , Up-RegulationABSTRACT
<p><b>OBJECTIVE</b>To explore effects of nordy on biological behaviors of human malignant glioblastoma cell line U87MG in vitro and transplanted tumor in vivo, and to identify the differential proteome upon Nordy induced differentiation.</p><p><b>METHODS</b>Glioblastoma U87MG cells were induced to differentiate by synthetic lipoxygenase inhibitor, Nordy. The drug was also given via peritoneal injection to nude mice (27 mg/kg body weight) bearing orthotopic transplanted tumors of U87MG cells in the brain. The tumor volumes and GFAP expression were measured. Total proteins of U87MG cells after Nordy treatment were analysed by two-dimensional gel electrophoresis. PDQuest 7.1 computer software was used to compare protein profiles of the treated cells with that of untreated control. Differentially expressed proteins were then selected and characterized by matrix assisted laser desorption ionization-time of flight-mass spectrometry. The functional aspects of these proteins were analyzed by bioinformatics.</p><p><b>RESULTS</b>Nordy suppressed both the proliferation of U87MG cells in vitro and the tumor growth of orthotopic transplanted tumors in vivo (P < 0.01). The differentially expressed proteins induced by Nordy included proliferation-associated gene A, alternative splicing factor ASF-3, eukaryotic translation initiation factor 5A, coffilin 1 (non-muscle), beta galactoside binding lectin, glyceraldehyde-3-phosphate dehydrogenase, enolase 1 and an unknown protein.</p><p><b>CONCLUSIONS</b>Nordy promotes the differentiation of glioblastoma cells, by which it may serve as a therapeutic agent. Various proteins identified during Nordy-induced differentiation are involved in the cell proliferation, metabolism, differentiation, apoptosis and gene transcription.</p>
Subject(s)
Animals , Female , Humans , Male , Mice , Antineoplastic Agents , Pharmacology , Brain Neoplasms , Metabolism , Pathology , Cell Differentiation , Cell Line, Tumor , Cell Proliferation , Gene Expression Regulation, Neoplastic , Glial Fibrillary Acidic Protein , Metabolism , Glioblastoma , Metabolism , Pathology , Lipoxygenase Inhibitors , Pharmacology , Masoprocol , Pharmacology , Mice, Inbred BALB C , Mice, Nude , Neoplasm Transplantation , Protein Array Analysis , Proteome , Genetics , Metabolism , Proteomics , Methods , Random Allocation , Tumor BurdenABSTRACT
<p><b>OBJECTIVE</b>To investigate the expression of B7-H1 on peripheral blood mononuclear cells from patients with chronic HBV infection.</p><p><b>METHODS</b>Immunofluorescent staining and flow cytometry assay were used to measure the expression of B7-H1 on peripheral blood CD3high T cells, CD19high B cells and CD14high monocytes from chronic HBV infected patients.</p><p><b>RESULTS</b>No significant difference was observed in B7-H1 expression on T cells and B cells between chronic HBV infected patients (CHB) and health controls (HC). B7-H1-expressing CD14high cells were significantly increased in chronic HBV-infected patients (19.17-/+11.64)% as compared with healthy controls [(7.30-/+5.49)%, P<0.01]. A significant positive correlation was found between B7-H1 expression on CD14high monocytes and serum ALT levels.</p><p><b>CONCLUSION</b>There is no significant difference in B7-H1 expression on T cells and B cells between CHB patients and healthy subjects. B7-H1, which is up-regulated on monocytes from chronic HBV-infected patients, in positively correlated to serum ALT levels, and may play a role in the persistence of HBV infection.</p>
Subject(s)
Adult , Aged , Female , Humans , Male , Middle Aged , Young Adult , Antigens, CD , Metabolism , B-Lymphocytes , Allergy and Immunology , B7-H1 Antigen , Case-Control Studies , Hepatitis B, Chronic , Allergy and Immunology , Metabolism , Leukocytes, Mononuclear , Metabolism , Monocytes , Allergy and Immunology , T-Lymphocytes , Allergy and ImmunologyABSTRACT
<p><b>OBJECTIVE</b>To explore the in vitro immune response to taxol resistance associated antigen 3 (TRAG-3)-derived cytotoxic T lymphocyte (CTL) epitope-pulsed dendritic cell (DC).</p><p><b>METHODS</b>The HLA-A2.1 restricted CTL epitope of TRAG-3 was previously identified to be a nonameric peptide sequence from 58 to 66 amino acid residues (TRAG-3(58-66)). The peptide was synthesized according to Fmos procedure, purified and molecular weight determined. Peripheral blood mononuclear cells (PBMC) of normal HLA-A2.1(+) individuals were used to isolate DC and DCs' phenotype identified by flow cytometry. Induction of CTL was achieved by in vitro stimulation of HLA-A2.1(+) PBMC with peptide-pulsed DC. CTL activity against HLA-A2.1(+)/TRAG-3(+) melanoma cell line LB373-MEL was assessed by (51)Cr release assay and IFN-gamma released by ELISA.</p><p><b>RESULTS</b>The synthetic nonameric peptide was above 90% pure and the determined values of molecular weight were conformed to their theoretical values. The phenotype of the isolate DCs was characteristic for mature ones. PBMC stimulated in vitro by TRAG-3-derived epitope-pulsed DCs for three times could specifically kill the target cells and secreted high concentration of IFN-gamma.</p><p><b>CONCLUSION</b>TRAG-3-derived epitope-pulsed DC can elicit specific anti-tumor immune response in vitro. Clinical trials using it as tumor vaccine may be feasible as specific immunotherapy for patients with TRAG-3 expressing cancers.</p>
