Kei Hashimoto

EFFECT OF CAPSIANOSIDE, A DITERPENE GLYCOSIDE, ON TIGHT-JUNCTIONAL PERMEABILITY

Previous work (Hashimoto et al., (1994) Biosci. Biotech. Biochem. 58, 1345) revealed that a sweet pepper extract enhanced the tight-junctional (TJ) permeability of a human intestinal Caco-2 cell monolayer. In the present study, the substance which modulated the TJ permeability was chromatographically purified from the extract. The active substances were identified as capsianosides A-F, diterpene glycosides. Treatment of the cells with capsianoside F, the most active compound, decreased the cellular G-actin content by 40% and increased the F-actin content by 16%. The effect of capsianoside F was significantly suppressed by disturbing the cytoskeletal structure with cytochalasin D at a low dose (50 ng/ml). These results suggest that capsianosides affected the cytoskeletal function by modulating the reorganization of actin filaments, by which the TJ structure and permeability were changed. The possible involvement of a PKC inhibition in the mechanism of an increase in TJ permeability is also suggested.

Epithelial properties of human intestinal Caco-2 cells cultured in a serum-free medium

Human intestinal Caco-2 cells were cultured under serum-free conditions on an insoluble collagen and FCS matrix (Caco-2-SF), and a comparison was made between several characteristics of Caco-2 and Caco-2-SF cells. Their morphological appearance was identical. Slight differences were found in cell growth and expression of brush border enzymes between Caco-2 and Caco-2-SF cells. Similar levels of activity of Gly-Gly transport were expressed in both types of cell. Caco-2 cells cultured on permeable filters showed high transepithelial electrical resistance (TEER), indicating the high monolayer integrity. The transepithelial transport activity for glucose, alanine and Gly-Gly was detected by measuring the change in short-circuit current (ΔIsc) after adding each of these nutrients to the apical chamber. In Caco-2-SF cells, such parameters as TEER and ΔIsc were reduced drastically, suggesting that the monolayer integrity and cell polarity that are important for transepithelial transport were not attained. These parameters, however, could be restored by adding FCS or by milk whey. The result suggested that FCS and milk whey contain factors which regulate the formation of the tight junctions and, consequently, the development of cell polarity. Thus the Caco-2-SF cell-culture system will provide a useful model for studying factors which regulate the intestinal transepithelial transport functions.

A 4-base pair deletion mutation of Gsα gene in a Japanese patient with pseudohypoparathyroidism

Mutations in the guanine nucleotide binding protein α subunit (Gsα) have been found in patients with pseudohypoparathyroidism (PHP). We have screened the Gsα gene for mutations in a Japanese patient with this disorder and identified a novel 4-base pair deletion in exon 7 in codons 189–190. This deletion causes a frameshift and if synthesis of a truncated form of Gsα occurred, it would likely be biologically inactive. The patient was heterozygous for this deletion. The patient’s mother and an unaffected brother were tested for the presence of this mutation. His mother had the same mutation, and although her serum calcium and parathyroid hormone levels were within the normal range, she had subcutaneous calcifications. Thus, this mutation appears to be necessary but not sufficient to cause the complete pseudohypoparathyroidism phenotype and thus, other unknown factors, either genetic or acquired, may be necessary for the full syndrome to occur.

Comparative studies on molecular stability of immunoglobulin G from different species

1. Molecular stability of immunoglobulin G (IgG) from chicken and four mammalian species (cow, goat, pig and rabbit) was compared from immunochemical and biochemical view-points. 2. IgG from cow, goat and pig showed similar stability against denaturation caused by heat, acid and denaturant treatments, while rabbit IgG was shown to be more resistant to all of the treatments than other mammalian IgGs, suggesting that rabbit IgG has a highly stable molecular structure. 3. The heat- and acid-stability of chicken IgG measured by immunochemical methods were similar to those of cow, goat and pig IgG. Conformational stability of chicken IgG measured by fluorometric and enzymatic methods, however, was lower than the mammalian IgGs, suggesting that chicken IgG has a more flexible and unstable molecular structure.

Superoxide Dismutase Inhibition of Oxidation of Ubiquinol and Concomitant Formation of Hydrogen Peroxide

We measured ubiquinone (CoQ0) and hydrogen peroxide (H2O2) formed in the process of oxidation of ubiquinol (CoQ0H2). We found that copper-zinc superoxide dismutase and manganese superoxide dismutase inhibited both the CoQ0 formation and the H2O2 formation only in the presence of chelators such as DTPA (diethylenetriaminepentaacetic acid). The amount of H2O2 was almost equal to that of CoQ0, indicating that the H2O2 formation was coupled with the CoQ0 formation. The lack of inhibitory effects of the corresponding heat-inactivated superoxide dismutase (SOD) confirmed that the inhibition by the original SOD was due to its enzymatic activity. We propose that CoQ0H2 oxidation occurs as a chain reaction with superoxide as the chain carrier and that SOD inhibits this reaction by lowering the superoxide concentration.