对比分析黑斑息肉综合征合并大肠癌组织肿瘤基因

Objective: The aim of the study was to screen the differentially expressed genes of Peutz-Jeghers syndrome (PJS) and colorectal carcinoma (CRC). Methods: This study used cDNA microarray to comparatively analyze the gene expression profiles of 4 cases of PJS combined with colorectal adenocarcinoma vs. normal mucosae. The cDNA microarray contained 8064 human genes, and then using RT-PCR to test three genes of all. Results: The experimental data showed that fourteen genes were differentially expressed, which were up-regulated in PJS. Fifty-one genes expressions were altered in CRCs, of which 32 were up-regulated, as compared to the normal mucosae. In addition, 5 genes were similarly altered in both PJS and CRCs. RT-PCR analyses confirmed the cDNA microarray data for three of those genes: LATS2, APC and MADH4. Conclusion: LCN2, USP4, GRO3, HYAL1 and APC - these differentially expressed genes likely represent biomarkers for early detection of CRC and may be potential therapeutic targets.     

Identification of genes differentially expressed in Mycobacterium tuberculosis by differential display PCR

RNA arbitrarily-primed differential display PCR (RAP-PCR) was used to identify and isolate genes differentially expressed between attenuated (H37Ra) and virulent (H37Rv, Erdman) laboratory strains of Mycobacterium tuberculosis (Mtb). Using this method, cDNA fragments showing homology to three known mycobacterial genes and six putative novel genes in mycobacterial cosmid vectors were identified. Among the putative novel Mtb genes identified, we found: (1) gene MTV041.29, containing multiple tandem repetitive sequences and encoding a putative Gly-, Ala, Asn-rich protein (PPE family); (2) gene MTV004.03, containing the AT10S repetitive gene sequence; (3) gene MTV028.09, encoding a hypothetical protein of unknown function; (4) genes MTCY78.20,21, possibly encoding two hypothetical proteins of unknown function; (5) gene MTCY01A6.09, encoding a putative novel ferrodoxin dependent glutamate synthase; and (6) gene MTCY31.20, encoding a putative cyclohexanone monooxygenase. Using gene specific primers in a second differential display PCR and by RT-PCR amplification, novel genes 1, 2, 3 and 4 were shown to be differentially up-regulated in the attenuated Mtb strain H37Ra compared to H37Rv and Erdman strain. Overall, we demonstrated that RAP-PCR, as a first step, is a quick and sensitive method for the identification and isolation of novel genes expressed in Mtb. Because of limitations inherent to the lack of specificity of arbitrary primers in the RAP-PCR method, a second differential display PCR and RT-PCR amplification with gene-specific primers was necessary in order to confirm differential expression of the identified genes.     

A- and B-type lamins are differentially expressed in normal human tissues

A selection of normal human tissues was investigated for the presence of lamins B1, B2, and A-type lamins, using a panel of antibodies specific for the individual lamin subtypes. By use of immunoprecipitation and two-dimensional immunoblotting techniques we demonstrated that these antibodies do not cross-react with other lamin subtypes and that a range of different phosphorylation isoforms is recognized by each antibody. The lamin B2 antibodies appeared to decorate the nuclear lamina in all tissues examined, except hepatocytes, in which very little lamin B2 expression was observed. In contrast to previous studies, which suggested the ubiquitous expression of lamin B1 in mammalian tissues, we show that lamin B1 is not as universally distributed throughout normal human tissues as was to be expected from previous studies. Muscle and connective tissues are negative, while in epithelial cells lamin B1 seemed to be preferentially detected in proliferating cells. These results correspond well with those obtained for lamin B1 in chicken tissues. The expression of A-type lamins is most prominent in well-differentiated epithelial cells. Relatively undifferentiated and proliferating cells in epithelia showed a clearly reduced expression of A-type lamins. Furthermore, most cells of neuroendocrine origin as well as most hematopoietic cells were negative for A-type lamin antibodies.     

A novel protease homolog differentially expressed in breast and ovarian cancer

BACKGROUND: Using differential display (DD), we discovered a new member of the serine protease family of protein-cleaving enzymes, named protease M. The gene is most closely related by sequence to the kallikreins, to prostate-specific antigen (PSA), and to trypsin. The diagnostic use of PSA in prostate cancer suggested that a related molecule might be a predictor for breast or ovarian cancer. This, in turn, led to studies designed to characterize the protein and to screen for its expression in cancer. MATERIALS AND METHODS: The isolation of protease M by DD, the cloning and sequencing of the cDNA, and the comparison of the predicted protein structure with related proteins are described, as are methods to produce recombinant proteins and polyclonal antibody preparations. Protease M expression was examined in mammary, prostate, and ovarian cancer, as well as normal, cells and tissues. Stable transfectants expressing the protease M gene were produced in mammary carcinoma cells. RESULTS: Protease M was localized by fluorescent in situ hybridization analysis to chromosome 19q13.3, in a region to which other kallikreins and PSA also map. The gene is expressed in the primary mammary carcinoma lines tested but not in the corresponding cell lines of metastatic origin. It is strongly expressed in ovarian cancer tissues and cell lines. The enzyme activity could not be established, because of difficulties in producing sufficient recombinant protein, a common problem with proteases. Transfectants were selected that overexpress the mRNA, but the protein levels remained very low. CONCLUSIONS: Protease M expression (mRNA) may be a useful marker in the detection of primary mammary carcinomas, as well as primary ovarian cancers. Other medical applications are also likely, based on sequence relatedness to trypsin and PSA.     

Identification of proteins that are abnormally regulated in differentiated cultured human keratinocytes

The inhabitants living in the neighbourhood of a deserted mercury-contaminated industrial site are subjected to an age-group differentiated mercury exposure assessment based on a scenario-linked calculation. Analytical input data for the calculation procedure are provided for from soil, air and plants in a large number. The most sensitive group are small children being mainly exposed by soil ingestion which makes up nearly 80% of the ADI, followed by inhalation of mercury contaminated indoor air. On the other hand, inhalation of indoor air has a predominant impact on youth and adults.     

Cyclic nucleotide phosphodiesterase 4 subtypes are differentially expressed by primary keratinocytes and human epidermoid cell lines

The monoclonal antibody 5B5 reacts with the beta subunit of proline-4-hydroxylase, the enzyme which catalyses the formation of 4-hydroxyl proline in collagen and other proteins with collagen-like amino acid sequences. This study aims to assess the production and tissue distribution of this enzyme in normal and diseased synovia from patients with various joint diseases, on the basis that it is a putative marker of collagen-producing cells and, therefore, in this context, of fibroblasts. Sections from five normal, 10 osteoarthritic (OA) and 26 rheumatoid arthritic (RA) synovia were labelled with a mouse monoclonal antibody to proline-4-hydroxylase. The enzyme was found to be expressed by a proportion of synovial intimal cells and by fibroblasts in the underlying connective tissue in normal, OA and RA synovia. Labelling was more pronounced in OA and RA cases. The intimal cells labelling positively showed type B synoviocyte morphology, which was confirmed by subsequent double immunolabelling with 5B5 and antibody against type IV collagen using immunocytochemistry and immunoelectron microscopy.     

Using 3'' untranslated sequences to identify differentially expressed genes in Leishmania

Because garbage collectors work in the street, they are exposed to polycyclic aromatic hydrocarbons (PAHs) in motor vehicle exhaust gas as they work. Urinary 1-hydroxypyrene (1-OH-pyrene) began to be used as a biological monitoring index for human exposure to high concentrations of PAHs. The objective of this study was to examine the applicability of urinary 1-OH-pyrene as a biological monitoring index for human low-level PAH exposure, such as the PAH exposure experienced while working in the street. The subjects were fifteen male garbage collectors. We measured individual exposure to PAHs, urinary 1-OH-pyrene concentrations and urinary cotinine concentrations. Individual air samplers were attached to the collar of the clothing of five workers to capture PAHs. Urine samples were collected before work, around noon and after finishing the day's work. In all, five PAH samples and 45 urine samples were collected. As control data, we analyzed the urinary 1-OH-pyrene and urinary cotinine levels of six smoking and four non-smoking control subjects who were not occupationally exposed to PAHs. The benzo[a]pyrene level in the air sampled for 5-6 h was 2.5-10.5 ng/m3, and the pyrene level as 10.3-70.3 ng/m3. These levels were similar to those in the vicinity of streets in Japan. A positive correlation between total PAH levels and the pyrene levels was observed. The average urinary 1-OH-pyrene level of the smokers was 0.21 +/- 0.13 mumol/mol creatinine, vs. 0.15 +/- 0.11 mumol/mol creatinine in the non-smokers. The urinary 1-OH-pyrene level obtained in this study was slightly higher than in the control group. No correlation was found between pyrene exposure and the urinary 1-OH-pyrene level of the five workers who wore the personal samplers. A significant positive correlation was observed between the urinary 1-OH-pyrene level and urinary cotinine level of the smokers. A significant positive correlation was also observed between the urinary 1-OH-pyrene and urinary cotinine levels of the control group smokers. In conclusion, urinary 1-OH-pyrene is not applicable for biological monitoring of extremely low levels of exposure to PAHs, as in the case of working in the street. Caution is required to exclude the effects of smoking when evaluating PAH exposure.     

Identification of a novel AMP-activated protein kinase beta subunit isoform that is highly expressed in skeletal muscle

The AMP-activated protein kinase (AMPK) is a member of a growing family of related kinases, including the SNF1 complex in yeast, which respond to nutritional stress. AMPK is a heterotrimeric complex of a catalytic subunit (alpha) and two regulatory subunits (beta and gamma), and proteins related to all three subunits have been identified in the SNF1 complex. We have used the two-hybrid system in order to identify proteins interacting with the catalytic subunit (alpha2). Using this approach, we have isolated a novel AMPKbeta isoform, which we designate AMPKbeta2. The N-terminal region of beta2 differs significantly from that of the previously characterized isoform (beta1), suggesting that this region could play a role in isoform-specific AMPK activity. Comparison of the C-terminal sequences of beta1 and beta2 with their related proteins in yeast identifies two highly conserved regions predicted to be involved in binding of the alpha and gamma subunits. The expression of beta1 and beta2 was examined in a number of tissues, revealing that the beta1 isoform is highly expressed in liver with low expression in skeletal muscle, whereas the opposite pattern is observed for the beta2 isoform. These results suggest that the beta isoforms have tissue-specific roles, which may involve altered responses to upstream signaling and/or downstream targeting of the AMPK complex.     

Ectopic expression of carbohydrate antigens that determine the metastatic potential of human colorectal carcinoma

Highly malignant and metastatic tumor cells are thought to arise within primary tumors and become predominant during cancer progression. We demonstrated, by the analysis of > 500 surgical specimens, that colorectal carcinomas with increased metastatic potential were characterized by an increased expression of sialyl-Le(x) antigens expressed on mucins. The biological role of sialyl-Le(x) antigens expressed on mucins produced by colon carcinoma cells has been investigated using variant cell lines selected for their expression of this antigen. KM12-HX and KM12-LX, high and low expresser variant cells, differed in their metastatic potential in nude mice after intrasplenic injection. KM12-HX cells contain higher levels of polyA+mRNA for alpha(1-3/4) fucosyltransferase than KM12-LX cells. Sialyl-Le(x) antigenic carbohydrate chains were attached to mucins as well as glycoproteins with various M(r). KM12-HX cells adhered more strongly than KM12-LX cells to human umbilical vein endothelial cells treated with tumor necrosis factor-alpha and to mouse hepatic sinusoidal endothelial cells. We have retrospectively evaluated post-operative survival of colon carcinoma patients for their sialyl-Le(x) antigen levels in the primary tumors according to the percentage of stained cells by specific antibodies. The adjusted survival rate of the patients with high levels of sialyl-Le(x) antigen in their primary tumors was much lower due to recurrence and metastasis than that of the patients with tumors containing low levels of sialyl-Le(x) antigen. The results suggested that sialyl-Le(x) antigen has a potential to be used as a predictive marker for colorectal cancer metastasis.