Potential association between TLR4 and chitinase 3-like 1 (CHI3L1/YKL-40) signaling on colonic epithelial cells in inflammatory bowel disease and colitis-associated cancer.

Inflammatory bowel disease (IBD) is a group of inflammatory disorders in the small and large intestines. Several studies have proved that persistent and disregulated host/microbial interactions are required for the development of IBD. It is well known that chronic IBD is strongly associated with an increased risk of developing colorectal cancer by 0.5-1% annually, 8-10 years after the initial diagnosis. To detect the tiny dysplasia or early stage of cancer in chronic IBD patients, a tremendous amount of effort is currently directed for improving colonoscopic technology and noninvasive serological marker development. However, there is only a limited amount of data available to understand the exact mechanism of how long term chronic colitis is connected to the development of colorectal tumors. Recently, our group has identified significantly increased expression of chitinase 3-like 1 (CHI3L1) molecule in non-dysplastic mucosa from patients with IBD and remote dysplasia/cancer, compared to patients with IBD without dysplasia or healthy controls. CHI3L1 seems to contribute to the proliferation, migration, and neoplastic progression of colonic epithelial cells (CECs) under inflammatory conditions. Furthermore, the TLR4-mediated intracellular signaling cascade is likely to interact with CHI3L1 signaling in CECs. In this review article, we have concisely summarized the cellular and molecular mechanisms underlining the development of IBD and colitis-associated cancer, with particular focus on the TLR4- and CHI3L1-signaling pathways in CECs.

[Membranotropic effect of some triterpene glycosides possessing immunostimulating properties].

The peculiarities of the interaction between cell membrane lipids and triterpene glycosides from holothurians Apostichopus japonicus S. and Cucumaria japonica (holotoxin A1 and cucumarioside A2-2, respectively) were studied in comparison with plant saponins from Quillaja saponaria, known as hemolytic, adjuvant, and structure-forming components of immunostimulating complexes. Similar to Quillaja saponins, the sea glycosides, holotoxin A1 and cucumarioside A2-2 were shown to possess a high hemolytic activity (2.6 and 3 microg/ml, respectively) and sterol-depending membranotropic effect mediated by the formation of nonbilayer sterol-lipid-glycoside complexes. At the same time, cucumarioside A2-2 bound exogenic cholesterol only in the presence of membrane lipids, such as phosphatidylcholine or monogalactosyldiacylglycerol, in contrast to Quillaja saponins and holotoxin A1, which bound cholesterol in the molar ratios 1:2 and 1:8, respectively. Moreover, in all cases, tree-component complexes containing cholesterol, lipid, and glycoside exhibited a lower hemolytic activity compared with two-component sterol-glycoside complexes. It was concluded that the hydrophobic medium of cell membranes performs a potentiative role in the effective interaction between triterpene glycosides and "sterol receptors". A method for decreasing the toxicity of membranotropic holothurian glycosides possessing the immunomodulating properties was suggested.

Cloning and expression of human cDNA encoding human homologue of pituitary tumor transforming gene.

Recently, a potent transforming gene which was exclusively expressed in rat pituitary tumor but not in normal pituitary had been isolated and named as pituitary tumor transforming gene (PTTG). A cDNA clone encoding human homologue of rat PTTG was isolated from human fetal liver cDNA library. It contained an open reading frame of 603 base pairs predicting a protein composed of 201 amino acids with a calculated molecular weight of 26 kDa. The deduced protein showed about 85% homology (78% identity, 7% favored substitution) with the rat PTTG. Northern blot analysis showed that the cDNA hybridized to 1.0 kb mRNA species which was expressed in fetal liver and several cancer cell lines. These results suggest that the presence of the human homologue of rat PTTG gene may not be restricted to pituitary tumor.

Increment of efficiency in the identification of noble genes by colony hybridization assay. (Screening of low redundant clones from human fetal cDNA library).

For the rapid identification of noble genes in a specific tissue by computer analysis from the cDNA sequences determined by single-pass cDNA sequencing, clone redundancy was one of the major obstacles. To facilitate the efficiency in identification of noble genes, it was necessary to reduce the number of clones to be sequenced by eliminating the redundant clones for a rapid analysis. In order to increase the probability of isolating noble sequences from the cDNA clones of human fetal liver tissue origin, colony hybridization assay was adopted and redundant clones were efficiently removed. Four cDNA clones highly redundant in the human fetal liver cDNA libraries including alpha-globin, gamma-globin, serum albumin and H19 RNA sequences were selected as the probes. Two hundreds and sixty two cDNA clones were randomly selected and tested with the probes for hybridization properties. The identity of each cDNA clone giving positive or negative signals in the hybridization assay was determined by DNA homology search with the nucleic acid databases. Among the 76 clones giving positive signals, 57 clones (75%) were found to be identical to the probe sequences and could be eliminated by colony hybridization assay before neucleotide sequencing.

Cloning and expression of human cDNA encoding Na+, K(+)-ATPase gamma-subunit.

A cDNA clone encoding the Na+, K(+)-Atpase gamma-subunit polypeptide was cloned from a human fetal liver cDNA library. The deduced amino acid sequence of the protein comprised 58 amino acids with a calculated molecular weight of 6400 Da, and showed about 86% homology when compared with those of the bovine, rat and mouse Na+, K(+)-ATPase gamma-subunits published elsewhere. Northern blot analysis showed that the cDNA hybridized to a 0.7 kb mRNA which was expressed in human kidney, pancreas and fetal liver.

Cloning and characterization of cDNA for human adenylate kinase 2A.

We have isolated and characterized a cDNA clone encoding human adenylate kinase 2A (AK2A) from cDNA libraries of fetal liver origin. The complete nucleotide sequence of the cloned 889 nucleotide cDNA fragment indicated that the deduced gene product of human AK2A is composed of 239 amino acids with a molecular weight of 26 kD. Comparison of these nucleotide and amino acid sequences with the corresponding sequences of bovine AK2A and rat AK2 revealed a high degree of conservation. It showed 91% and 98% homologies in DNA and amino acid sequences, respectively, with bovine AK2A throughout the full open reading frame. RNA blot analysis revealed that three species of mRNA were present with approximate sizes of 3.4, 2.1, and 1.0 kb. This gene was expressed in E. coli cells and the recombinant protein was enzymatically active.