Hepatocellular carcinoma (HCC) is a highly malignant tumor, and chronic infection with hepatitis B virus is one of its major risk factors. To identify the proteins involved in HCC carcinogenesis, we used two-dimensional fluorescence DIGE to study the differentially expressed proteins in tumor and adjacent nontumor tissue samples. Samples from 12 hepatitis B virus-associated HCC patients were analyzed. A total of 61 spots were significantly up-regulated (ratio > 2, p < 0.01) in tumor samples, whereas 158 spots were down-regulated (ratio < ؊2, p < 0.01). Seventyone gene products were identified among these spots. Members of the heat shock protein 70 and 90 families were simultaneously up-regulated, whereas metabolismassociated proteins were decreased in HCC samples. The down-regulation of mitochondrial and peroxisomal proteins in these results suggested loss of special organelle functions during HCC carcinogenesis. Four metabolic enzymes involved in the methylation cycle in the liver were down-regulated in HCC tissues, indicating S-adenosylmethionine deficiency in HCC. Two gene products, glyceraldehyde-3-phosphate dehydrogenase and formimidoyltransferase-cyclodeaminase, were identified from inversely altered spots, suggesting that different isoforms or post-translational modifications of these two proteins might play different roles in HCC. For the first time, the overexpression of Hcp70/Hsp90-organizing protein and heterogeneous nuclear ribonucleoproteins C1/C2 in HCC tissues was confirmed by Western blot and then by immunohistochemistry staining in 70 HCC samples, suggesting their potential as protein tumor markers. In summary, we profiled proteome alterations in HCC tissues, and these results may provide useful insights for understanding the mechanism involved in the process of Proteomics analysis is currently considered to be a powerful tool for global evaluation of protein expression, and proteomics has been widely applied in analysis of diseases, especially in fields of cancer research. Quantitative protein expression profiling is a crucial part of proteomics, and such profiling requires methods that are able to efficiently provide accurate and reproducible differential expression values for proteins in two or more biological samples. Two-dimensional electrophoresis (2DE) 1 was a technique that was widely used for proteomics research. However, intergel variation and excessive time/labor costs have been common problems with standard 2DE. Two-dimensional (2D) DIGE might therefore be considered as one of the most significant advances in quantitative proteomics. Using the 2D DIGE approach, different samples prelabeled with mass-and charge-matched fluorescent cyanine dyes are co-separated in the same 2D gel, and an internal standard is used in every gel that has negated the problem of intergel variation (1). Moreover with the great sensitivity and dynamic range that is afforded by these dyes, 2D DIGE can give greater accuracy of quantitation than silver staining (2). It has been reported that the correlation betw...

It becomes increasingly clear that separation of pure cell populations provides a uniquely sensitive and accurate approach to protein profiling in biological systems and opens up a new area for proteomic analysis. The method we described could simultaneously isolate population of hepatocytes (HCs), hepatic stellate cells (HSCs), Kupffer cells (KCs) and liver sinusoidal endothelial cells (LSECs) by a combination of collagenase-based density gradient centrifugation and magnetic activated cell sorting with high purity and yield for the first time. More than 98% of the isolated HCs were positive for cytokeratin 18, with a viability of 91%. Approximately 97% of the isolated HSCs expressed glial fibrillary acidic protein with a viability of 95%. Nearly 98% of isolated KCs expressed F4/80 with a viability of 94%. And the purity of LSECs reached up to 91% with a viability of 94%. And yield for HCs, HSCs, LSECs and KCs were 6.3, 1.3, 2.6 and 5.0 million per mouse. This systematic isolation method enables us to study the proteome profiling of different types of liver cells with high purity and yield, which is especially useful for sample preparation of Human Liver Proteome Project.

NDRG1 (N-Myc downstream regulated) is upregulated during cell differentiation, repressed by N-myc and c-myc in embryonic cells, and suppressed in several tumor cells. A nonsense mutation in the NDRG1 gene has been reported to be causative for hereditary motor and sensory neuropathy-Lom (HMSNL), indicating that NDRG1 functions in the peripheral nervous system necessary for axonal survival. Here, we cloned three human cDNAs encoding NDRG2 (371aa), NDRG3 (375aa) and NDRG4 (339aa), which are homologous to NDRG1. These three genes, together with NDRG1, constitute the NDRG gene family. The phylogenetic analysis of the family demonstrated that human NDRG1 and NDRG3 belong to a subfamily, and NDRG2 and NDRG4 to another. At amino acid (aa) level, the four members share 53-65% identity. Each of the four proteins contains an alpha/beta hydrolase fold as in human lysosomal acid lipase. Expression of the fusion proteins NDRG2/GFP, NDRG3/GFP and NDRG4/GFP in COS-7 cells showed that all of them are cytosolic proteins. Based on UniGene cluster analysis, the genes NDRG2, NDRG3 and NDRG4 are located at chromosome 14q11.1-11.2, 20q12-11.23 and 16q21-22.1, respectively. Northern and dot blot analysis shows that all of the three genes are highly expressed in adult brain and almost not detected in the eight human cancer lines. In addition, in contrast to the relatively ubiquitous expression of NDRG1, NDRG2 is highly expressed in adult skeletal muscle and brain, NDRG3 highly expressed in brain and testis, and NDRG4 specifically expressed in brain and heart, suggesting that they might display different specific functions in distinct tissues.

Recently, a new coronavirus was isolated from the lung tissue of autopsy sample and nasal/throat swabs of the patients with Severe Acute Respiratory Syndrome (SARS) and the causative association with SARS was determined. To reveal further the characteristics of the virus and to provide insight about the molecular mechanism of SARS etiology, a proteomic strategy was utilized to identify the structural proteins of SARS coronavirus (SARS-CoV) isolated from Vero E6 cells infected with the BJ-01 strain of the virus. At first, Western blotting with the convalescent sera from SARS patients demonstrated that there were various structural proteins of SARS-CoV in the cultured supernatant of virus infected-Vero E6 cells and that nucleocaspid (N) protein had a prominent immunogenicity to the convalescent sera from the patients with SARS, while the immune response of spike (S) protein probably binding with membrane (M) glycoprotein was much weaker. Then, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was used to separate the complex protein constituents, and the strategy of continuous slicing from loading well to the bottom of the gels was utilized to search thoroughly the structural proteins of the virus. The proteins in sliced slots were trypsinized in-gel and identified by mass spectrometry. Three structural proteins named S, N and M proteins of SARS-CoV were uncovered with the sequence coverage of 38.9, 93.1 and 28.1% respectively. Glycosylation modification in S protein was also analyzed and four glycosylation sites were discovered by comparing the mass spectra before and after deglycosylation of the peptides with PNGase F digestion. Matrix-assisted laser desorption/ionization-mass spectrometry determination showed that relative molecular weight of intact N protein is 45 929 Da, which is very close to its theoretically calculated molecular weight 45 935 Da based on the amino acid sequence deduced from the genome with the first amino acid methionine at the N-terminus depleted and second, serine, acetylated, indicating that phosphorylation does not happen at all in the predicted phosphorylation sites within infected cells nor in virus particles. Intriguingly, a series of shorter isoforms of N protein was observed by SDS-PAGE and identified by mass spectrometry characterization. For further confirmation of this phenomenon and its related mechanism, recombinant N protein of SARS-CoV was cleaved in vitro by caspase-3 and -6 respectively. The results demonstrated that these shorter isoforms could be the products from cleavage of caspase-3 rather than that of caspase-6. Further, the relationship between the caspase cleavage and the viral infection to the host cell is discussed.

Hepatopoietin (HPO) is a novel polypeptide mitogen specific for hepatocytes and hepatoma cell lines, which is derived from liver and supports its regeneration. To determine whether HPO acts via a receptor-based signal transduction, recombinant human hepatopoietin was labeled by iodination and used to characterize its binding activity by specific displacement test and Scatchard analysis in primarily cultured rat hepatocytes and human hepatoma Hep-G2 cells. The binding was saturable and specific because it was replaceable by HPO but not by epidermal growth factor, transforming growth factor-␣, or insulin. Scatchard analysis indicated the presence of a single class of high affinity receptor with dissociation constant (K d ) of 2 and 0.7 pM, and a receptor density of about 10,000 sites/cell and 55,000 sites/cell in the rat hepatocytes and human hepatoma cells, respectively. The K d values were consistent with the half-maximum dose of HPO activity. Affinity cross-linking of the receptor with 125 I-HPO revealed a polypeptide of molecular mass approximately 90 kDa by SDS-polyacrylamide gel electrophoresis. Thus, the molecular mass of the HPO receptor was calculated to be about 75 kDa. These data demonstrated the existence of an HPO receptor in hepatocytes and hepatoma cells, which may account for biological effect.Previous studies implicate that a small molecule derived from liver itself specifically stimulates hepatocytes proliferation and supports liver regeneration (1-3). In 1975, LaBrecque et al. (3) first reported that in the liver of a weaning rat and the regenerating liver of a partially hepatectomized rat, there existed hepatic stimulator substance (HSS) 1 that could specifically stimulate DNA synthesis in hepatic cells. Other groups have also carried out extensive research on HSS derived from other species (2). At the same time, experiments and clinical research on human fetal liver cells demonstrated its therapeutic effect on hematopoietic diseases and severe liver diseases (2, 4). Since the 1980s, we began to isolate and purify the effective component from fetal liver. We identified hepatic stimulatory activity in the fraction with molecular size ranging from 10 to 30 kDa of human fetal liver lysate (5-7). The activity was target-specific, which was different from various well known nonspecific hepatic stimulators such as insulin, EGF, insulinlike growth factor, and TGF-␣. The characteristics of the effective component derived from human fetal liver were consistent with those of HSSs derived from other species, suggesting that the effective component could be the human-derived homologue of the animal's HSS. Then, we purified this activity and demonstrated that the biological activity of its pure form is identical to those of the crude form and consistent with those of animal-derived HSSs, but evidently different from those of serum-derived hepatocyte growth factor (8). The factor was named as hepatopoietin (HPO). Later, we proved that HPO is encoded by mRNA of fetal liver (9) and further cloned (10) its ...