[Comparative proteomic analysis of the promastigotes and amastigotes of Leishmania donovani].

OBJECTIVE: To explore the protein profile and identify developmentally regulated proteins of the promastigotes and axenic amastigotes with comparative proteomics technique. METHODS: The total proteins of promastigotes and axenic amastigotes of Leishmania donovani SC6 strain were separated by two-dimensional electrophoresis (2-DE) in a broad pH range (3-10), and the gel was stained with Coomassie blue. The images were analyzed by PDQuest 1.0 software, and the major developmentally regulated proteins were identified by electrospray mass spectrometry. RESULTS: Approximately 700 protein spots were revealed in equivalent proteins of the promastigotes and axenic amastigotes separated by 2-DE, among which more than 90% protein spots showed equivalent quantity and distribution, with 6 proteins up-regulated and 3 proteins down-regulated in axenic amastigotes compared with promastigotes. Five of the 6 up-regulated proteins were with known function, respectively ascribed as Rieske iron-sulfur protein precursor, alpha-tubulin, peroxidoxin 1, dihydrolipoamide acetyltransferase precursor, and mannose-1-phosphate guanylyltransferase. Two of the 3 down-regulated proteins were identified as heat shock protein 70 and beta-tubulin. The functions of the developmentally regulated proteins were related to the carbohydrate/energy metabolism, stress response, or formation of cell membrane/cytoskeleton. CONCLUSION: The findings demonstrate the differences in protein expression profiles between promastigotes and amastigotes.

Comparative proteome analysis of breast cancer and adjacent normal breast tissues in human.

Two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and matrix-assisted laser desorption/ionization tandem time-of-flight mass spectrometry (MALDI-TOF/TOF-MS), incorporated with online database searching, were performed to investigate differential proteins of breast cancer and adjacent normal breast tissues. Considering that serum albumin is abundantly presented in normal control samples, 15 differential spots detected in 11 out of 12 (91.7%) breast cancer samples were identified by online SIENA-2DPAGE database searching and MALDI-TOF/TOF-MS analysis. The results indicate that pathological changes of breast cancer are concerned with augmentation of substance metabolism, promotion of proteolytic activity, decline of activity of some inhibitors of enzymes, and so on. Some important proteins involved in the pathological process of breast cancer with changed expression may be useful biomarkers, such as alpha-1-antitrypsin, EF-1-beta, cathepsin D, TCTP, SMT3A, RPS12, and PSMA1, among which SMT3A, RPS12, and PSMA1 were first reported for breast cancer in this study.

[Analysis of differential expression genes related to different metastasis potential of adenoid cystic carcinoma using restriction fragments differential display PCR].

OBJECTIVE: To construct differential expression profiles of adenoid cystic carcinoma cell lines for screening candidate genes related to metastasis and to verify some candidate genes in adenoid cystic carcinoma. METHODS: Restriction fragments differential display PCR (RFDD-PCR) was used to set up gene expression profiles of adenoid cystic carcinoma cell lines-ACC-M and ACC-2, with high and low metastasis potential respectively. Candidate genes were screened through bioinformatics analysis. Then, a gene family of these candidate genes was checked using semi-quantitative reverse transcription-PCR(RT-PCR). RESULTS: Two gene expression profiles including 5420 gene fragments were constructed, 12 genes of a family called matrix metalloproteinase genes (MMPs) were observed obvious differentially expressed between two cell lines. Results of semi-quantitative RT-PCR also identified this different expression of MMP2,MMP7,MMP9,MMP14,MMP15 and MMP24. CONCLUSION: The construction of gene expression profiles of ACC-M and ACC-2 cell lines makes the foundation for seeking the target genes of adenoid cystic carcinoma. MMP2,MMP7,MMP9 and MMP15 may be relevant with carcinogenesis, development and metastasis of adenoid cystic carcinoma, and different metastasis potential may result from different subtype of MMPs gene family.

[The expression of matrix metalloproteinases and tissue inhibitor of metalloproteinases in transplanted tumor of salivary adenoid cystic carcinoma cell lines].

PURPOSE: To study the differential expression pattern of MMPs and TIMPs in ACC-M and ACC-2 tumor model. METHODS: High and low metastatic tumor models were set up by transplantion of ACC-M and ACC-2 to nude mouse respectively. Then 3 mice in each group were selected randomly to detect the mRNA level of MMP-2, MMP-7, MMP-9, TIMP-1 and TIMP-2 using semiquantitative RT-PCR,GAPDH was used as internal control. Meanwhile, the expression of MMP-2, MMP-7, MMP-9, TIMP-1 and TIMP-2 in each transplanted tumor were determined by immunohistochemical assay, and IOD value of immunohistochemical reaction was measured. All data were analyzed by SPSS11.5 software package for Student's t test. RESULTS: The results of RT-PCR and immunohistochemistry showed an up-regulation of MMP-2, MMP-7, MMP-9 and TIMP-1 in ACC-M tumor model compared with ACC-2 model (P < 0.05), while the expression of TIMP-2 showed no significant difference between the two models (P > 0.05). CONCLUSION: The differential expression pattern of MMP-2, MMP-7, MMP-9, and TIMP-1 in 2 tumor models may result in different metastasis potential; The 2 tumor models provide good study model for investigation of the metastatic mechanism of salivary adenoid cystic carcinoma.