Intestinal trefoil factor induces decay-accelerating factor expression and enhances the protective activities against complement activation in intestinal epithelial cells

Mucosal damage induces a massive influx of serum complement components into the lumen. The epithelium produces a number of factors that can potentially ameliorate injury including intestinal trefoil factor (ITF), a small protease-resistant peptide produced and secreted onto the mucosal surface by goblet cells, and decay-accelerating factor (DAF), a protein produced by columnar epithelium which protects the host tissue from autologous complement injury. However, coordination of these intrinsic defensive products has not been delineated. DAF protein and mRNA expression were evaluated by immunoblotting and Northern blotting, respectively. NF-kappaB-DNA binding activity and DAF promoter activity were assessed by an electrophoretic gel mobility shift assay and a reporter gene luciferase assay, respectively. ITF induced a dose- and time-dependent increase in DAF protein and mRNA expression in human (HT-29 and T84) and rat (IEC-6) intestinal epithelial cells. In differentiated T84 cells grown on cell culture inserts, basolateral stimulation with ITF strongly enhanced DAF expression, but apical stimulation had no effects. The C3 deposition induced by complement activation was significantly blocked by the treatment with ITF. In HT-29 cells, ITF increased the stability of DAF mRNA. ITF also enhanced the promoter activity of the DAF gene via NF-kappaB motif and induced activation of NF-kappaB-DNA binding activity. ITF promotes protection of epithelial cells from complement activation via up-regulation of DAF expression, contributing to a robust mucosal defense.     

Rapid purification and characterization of DNA topoisomerase I from cultured mouse mammary carcinoma FM3A cells

We have previously shown that a DNA topoisomerase I from mouse mammary carcinoma cells is inhibited by heparin. Taking advantage of this enzyme-heparin interaction, we developed a rapid and efficient method of purification of this enzyme to near homogeneity by extraction of chromatin with 0.15 M phosphate buffer followed by two-step column chromatography on heparin-Sepharose and phenyl-Sepharose. Electrophoresis on sodium dodecyl sulfate-polyacrylamide gels revealed that the final preparation is composed of two polypeptides with apparent Mr approximately 98,000 (p98) and 102,000 (p102), p98 comprising 70% and p102 30%. Extraction and renaturation of the polypeptides from the gel shows that both p98 and p102 seem to possess topoisomerase activity. Partial proteolytic digestion of p98 and p102 with Staphylococcus aureus V8 and chymotrypsin yielded a series of identical peptides, indicating that the two polypeptides are structurally related. The enzyme sedimented through sucrose density gradient with s20,w of 4.0 S, and thus is monomeric in solution.     

Growth Characteristics in Cultured Cucumber Tissues Infected with Hop Stunt Viroid

Leaf disks from hop stunt viroid (HSVd)-infected cucumber plants and healthy controls were cultured in vitro under different auxin/cytokinin regimes. Amounts of tissue growth and root formation were consistently reduced as a result of HSVd infection. Histological observations indicated that anomalies in the distribution of peripheral meristems and tracheary elements associated with cambial cells might be involved in the hypoplastic syndrome incited by HSVd.     

Absence of a gamma-melanocyte-stimulating hormone sequence in proopiomelanocortin mRNA of chum salmon Oncorhynchus keta

1. Complete nucleotide sequence of one of the salmon proopiomelanocortin mRNAs (POMC mRNAs) was determined. 2. The region corresponding to gamma-melanocyte-stimulating hormone (gamma-MSH) was lacking in salmon POMC mRNA, although overall organization of the multi-hormone structure was exactly the same as that of mammalian POMC mRNAs. 3. The possible evolutional history of POMC mRNA in mammalian species may be revealed from the finding of this characteristic that salmon POMC mRNA lacks the region corresponding to gamma-MSH.     

Acyl-homoserine lactones suppresses IEC-6 cell proliferation and increase permeability of isolated rat colon

We investigated to determine whether a variety of acyl-homoserine lactones (AHLs) influences epithelial cell proliferation and mucosal permeability. 3-Oxo-C₁₂-homoserine lactone (HSL) and 3-oxo-C₁₄-HSL significantly suppressed IEC-6 cell proliferation. A significant increase in mucosal permeability was observed in isolated rat colon tissue exposed to C₁₂-HSL, 3-oxo-C₁₂-HSL, and 3-oxo-C₁₄-HSL. These data indicate that AHLs suppress epithelial proliferation and disrupt barrier function in intestinal mucosa.     

Inhibitory action of leptin on early follicular growth differs in immature and adult female mice.

In order to investigate the action of leptin on early follicular growth, preantral follicles, 95-115 microm in diameter were mechanically isolated from the ovaries of BDF1 hybrid immature (11-day-old) and adult (8-wk-old) mice, and cultured for 4 days in vitro. Follicular growth was assessed by daily changes in follicular diameter and by the amount of estradiol and immunoreactive (IR)-inhibin released into the culture medium at Day 4. Preantral follicles from immature mice showed a significant development in follicular growth as a result of stimulation by GH (1 mIU/ml), insulin-like growth factor (IGF)-I (100 ng/ml) + FSH (100 mIU/ml), and GH (1 mIU/ml) + FSH (100 mIU/ml). Although leptin at concentrations of 1-1000 ng/ml did not have any significant effect on follicular growth stimulated by IGF-I or GH, it significantly inhibited follicular growth in a dose-related manner when follicles were stimulated by IGF-I + FSH and GH + FSH, respectively, suggesting that leptin attenuated the additive effect of FSH. On the other hand, preantral follicles from adult mice were cultured in the presence of FSH, and FSH-dependent follicular growth was inhibited by leptin in a dose-related manner. Because FSH stimulates cAMP production, we investigated the involvement of cAMP in the inhibitory mechanisms of leptin. Preantral follicles from immature and adult mice were cultured in the presence of either 8-Br-cAMP or forskolin. Both 8-Br-cAMP and forskolin significantly increased follicular diameter and hormone secretion in both immature and adult mice. However, 8-Br-cAMP and forskolin-stimulated follicle growth and hormone secretion were significantly inhibited in immature mice by coadministration of leptin, whereas growth of preantral follicles from adult mice was not inhibited by addition of leptin to cultures. These results indicate that leptin causes an inhibitory effect on the early follicular development of both immature and adult mice, but the inhibitory mechanisms of leptin are different.     

Application of 2-aminopyridine fluorescence labeling in the analysis of in vivo and in vitro metabolism of dextran sulfate sodium by size-exclusion high-performance liquid chromatography

The present study describes a size-exclusion high-performance liquid chromatographic method for the separation and quantification of sulfated polysaccharides, such as dextran sulfate sodium (DSS). Pyridylamination of DSS was achieved without difficulty using 2-aminopyridine as a fluorometric label. In addition, 0.1–0.2 M phosphate buffer (pH 3.0) was found to be the mobile phase which produced the best separation. In vitro enzymatic degradation of the pyridylamino-DSS (PA-DSS₅₀₀₀, M_r 5000) using α-amylase and the in vivo metabolism in the rat feces after oral administration of PA-DSS₅₀₀₀ were then evaluated. Two small peaks of approximately M_r 380 and 600 appeared after co-incubation with α-amylase, indicating PA-DSS₅₀₀₀ may be considerably depolymerized. In vivo, however, PA-DSS₅₀₀₀ excreted in the feces was mainly of PA-DSS₅₀₀₀ polymer. No peaks of less than M_r 5000 were not clearly detectable in the feces because of background fluorescence attributable to gut lumen contents. This method of fluorometric analysis allows fairly selective detection of sulfated polysaccharides in biological materials.