Isao Kawabata

Activation of PPARγ inhibits cell growth and induces apoptosis in human gastric cancer cells

We investigated the expression of peroxisome proliferator‐activated receptor γ (PPARγ) and the role of PPARγ in cell growth in human gastric cancer cells. Reverse transcription‐polymerase chain reaction, Northern blot and Western blot analyses showed that a human gastric cancer cell line, MKN45, expressed PPARγ mRNA and protein. Luciferase assay in MKN45 cells showed that troglitazone, a selective ligand for PPARγ, transactivated the transcription of a peroxisome proliferator response element‐driven promoter. Troglitazone or pioglitazone, selective ligands for PPARγ, inhibited the growth of MKN45 cells in a dose‐dependent manner. Co‐incubation of MKN45 cells with troglitazone induced DNA ladder formation. These results suggest that human gastric cancer cells express PPARγ and that activation of PPARγ inhibits cell growth and induces apoptosis in gastric cancer cells.

Eos: a novel member of the Ikaros gene family expressed predominantly in the developing nervous system

We identified a novel member of the Ikaros gene family, which has critical roles in the development of lymphoid lineages. This gene, which we named Eos, was expressed predominantly in the developing central and peripheral nervous system. Eos protein could interact with itself and Ikaros protein through its C‐terminal portion in the yeast two hybrid assay. These findings suggested that Eos may have important roles in neural development similarly to the Ikaros family in the development of hemolymphoid tissue.

Clinical aspects of Clara cell 10-kDa protein/ uteroglobin (secretoglobin 1A1).

Clara cell 10-kDa protein (CC10)/ uteroglobin (UG) is a nonglycoprotein with a molecular mass of 16 kilodaltons, which is produced by mucosal epithelial cells in the lung (Clara cells), uterus and prostate. Like other low molecular weigh proteins it is catabolized in renal proximal tubules. Structurally it is a homodimer of subunits of 70 amino acids covalently bound in an antiparallel manner. It belongs to secretogobin (SCGB) family and is assigned as subgroup 1A1. The function of the protein so far elucidated is immunoregulatory and anti-inflammatory in innate immunity. The knockout mouse of UG gene resulted in aggravation of inflammation by allergic and hyperoxic stimuli. It also showed very similar pathological features with human IgA nephropathy. The value is changed in the lung fluid and serum of various inflammatory and allergic lung diseases. Several kinds of single nucleotide polymorphisms (SNPs) in human CC10/UG gene were recently discovered; Adenine allele accumulation in G38A SNP has possible association with asthma and IgA nephropathy, being paralleled with disease severity of IgA nephropathy. Its expression is enhanced by some transcriptional factors induced by cytokines such as interferon-gamma. For cancer cells, the protein functions as an antagonist of neoplastic phenotype. CC10/UG forms one of intra- and intercellular regulators involved in inflammation and malignant transformation in the respiratory and urogenital fields.

Impaired interleukin-2 production in T-cells from a patient with Wiskott-Aldrich syndrome: basis of clinical effect of interleukin-2 replacement therapy

Interleukin-2 production may be one of the underlying causes of Wiskott-Aldrich syndrome immunodeficiency and recombinant interleukin-2 administration (or an infusion of T-cells expanded by CD3 stimulation and rIL-2) is able, to some extent, to restore defective T-cell function.

Evaluation of adsorption of urine cystatin C to the polymer materials on the microplate by an antigen capture enzyme-linked immunosorbent assay.

BACKGROUND Cystatin C is a low molecular weight protein of 13 kDa with an isoelectric point of 9.3. Its adsorption on the urine sampling containers may cause the underestimation of cystatin C levels. We newly developed an antigen capture enzyme-linked immunosorbent assay (ELISA) of sandwich method for measurement of adsorbed level. METHODS We used a polystyrene microplates with 3 different polymers. These include high hydrophobic, low hydrophobic, and hydrophilic materials. Using the same microplate, the absorbed protein was measured by an antigen Capture ELISA, and calibration was conducted by an ordinary ELISA. RESULTS In normal urine the concentrations of absorbed cystatin C levels to the 3 materials at day 1 were 0.50, 0.32-0.84 microg/l (median, interquartile range), 0.28, 0.21-0.37 microg/l, and <0.08, <0.08-0.09 microg/l in high hydrophobic, low hydrophobic, and high hydrophilic material, respectively. The absorption rate was 6%, 3%, and 1%, respectively. The adsorption is dependent on urine pH. It changes reciprocally with urine protein concentration. In pathologic urine, the absolute absorption level was <0.08 microg/l on the median, and the adsorption ratio (absorption level/urine level) was much less than 0.5% of that in normal urine. CONCLUSION In the clinical setting, the absorption of cystatin C to sample containers is negligible since the rate of adsorption is low both in normal and pathologic urine. The material with high hydrophilic surface processing may be used for other proteins when interaction of the proteins with surface material affects the value to clinical decision.

Urinary protein 1/Clara cell 16 concentrations and lung functions in male subjects with pneumoconiosis

Background: Protein 1 (P1)/Clara cell 16 kDa protein (CC16, previously named CC10), a potentially immunosuppressive protein secreted by non-ciliated cells of the tracheobronchial epithelium, has been found to be a new useful lung-specific biomarker in several pathological lung conditions. Particularly, urinary P1 (uP1) may reflect the altered lung functions in pneumoconiosis. Methods: We investigated the relationship between uP1 values and lung functions in 31 non-smoking pneumoconiotic males (mean age 73 years) with a history of dust exposure work in shipbuilding. The protein was measured using an originally prepared enzyme-linked immunosorbent assay system. The forced expiratory volume in 1 s % (FEV1.0%) and % vital capacity (%VC) were tested with a spirometer. Results: The mean values of uP1 were 4.62 ± 4.82 (mean ± standard deviation) ng/mol creatinine. A univariable correlation test showed a significant positive correlation between uP1 and %VC (r = 0.356, P = 0.049). Also, a multiple regression analysis, when adjusted for age, disease duration, FEV1.0% and %VC, showed a significant correlation of uP1 with %VC (β = 0.467, P = 0.030). Conclusion: The results suggest that a decreased uP1, corroborated by a decreased %VC, may be the result of damage to secretory cells. Measurement of uP1 may become a possible index of fibrotic changes in pneumoconiosis.

Signal transduction mechanisms of Ia induction in B cells by interleukin 4 and immunoglobulin receptors

Molecular mechanisms of signal transduction through receptors for interleukin 4 (IL-4) are still largely unknown. To elucidate the second messenger(s) of IL-4 action in mature B cells, we performed blocking experiments with inhibitors of various aspects of cellular responses, using Ia-inducing activity of IL-4 as a readout system. In the event, only agents that are shown to inhibit calcium ion (Ca2+) release from intracellular stores, such as 3,4,5-trimethoxybenzoic acid 8-(diethylamino)octyl ester (TMB-8) and ryanodine, could block the IL-4 action in a dose-dependent fashion. These results suggest that the process leading to the final expression of IL-4 action may be mediated, at some point, by the release of Ca2+ from intracellular stores. In the parallel experiments with antiimmunoglobulin (Ig) antibody, we found that amiloride, an inhibitor of Na+/H+ pump, blocks the Ia induction by anti-IgM antibody. Thus the Na+/H+ exchange system activated by anti-Ig antibody may be present in mature B cells.

Expression of uteroglobin in normal and carcinogenic endometrium and influence of hormone replacement therapy.

Uteroglobin, first reported in 1968 as a steroid secreted in rabbit uterine fluid during early pregnancy, is a progesterone‐regulated and progesterone‐binding protein. There is evidence that indicates that uteroglobin is inversely correlated to neoplastic growth but its role to endometrial carcinogenesis is not known. Therefore we analyzed the expression of uteroglobin in 13 normal endometrium, 19 hyperplasia and 21 endometrial carcinoma samples and the relation to estrogen receptor‐α (ER‐α) and progesterone receptor (PR) by immunohistochemistry and Western blotting. We also analyzed the expression of uteroglobin in 15 menopausal women who received hormone replacement therapy (HRT). The expression of uteroglobin was higher during the secretory phase than in the proliferative phase; however, it was detected in endometrial hyperplasia as weakly as in the proliferative phase and decreased according to the loss of differentiation in endometrial carcinoma. The results were basically in accord with those for PR; however, the expression of uteroglobin was weak, though PR was most detected in endometrial hyperplasia. In menopausal endometrium, the group treated with estrogen plus progesterone exhibited higher expression of uteroglobin than the group treated only with estrogen. The evidence suggests that uteroglobin expression is regulated by progesterone in the normal endometrium but that the regulation by PR is lost in endometrial hyperplasia and carcinoma according to acquirement of tumorigenesis and that estrogen plus progesterone therapy reduces the risk for endometrial carcinoma by restoring uteroglobin.

Structural features of the Lyb-2 molecule

The Lyb-2 system of the mouse is a B cell-specific cell surface molecule (Sato and Boyse 1976) that has been reported to be a monomer of relative mass (M r) of 45 000 (Tung et al. 1977). The genetic locus is on a segment of chromosome 4 (Sato et al. 1977, Taylor and Shen 1977) which contains genes coding for other lymphocyte cell surface antigen systems such as Ly-19 (Tada et al. 1981) and Ly-32 (Tada et al. 1987). Recent serological and biochemical analyses have revealed that the Lyb-2 system is composed of five antigenic specificities defined by four alleles (Tung et al. 1986). Functional studies have already shown that Lyb-2 is involved in an early phase of B-cell activation (Yakura et al. 1981, 1982, Subbarao and Mosier 1983, 1984), possibly by regulating a process mediated by B-cell stimulatory factor 1 (BSF-1) or interleukin 4 (IL-4) (Yakura et al. 1986, 1988). To clarify the relationship between Lyb-2 and the BSF-1/IL-4 receptor and to obtain a structural basis of the function of Lyb-2, we decided to reexamine the molecular nature of Lyb-2 at the protein level. Spleen cells from C57BL/6 (B6) (Lyb-2.2) or B6-Lyb-2 ~ congenic mice were treated with monoclonal Thy-l .2 antibody and complement, and were used as a source of B cells. Breeding pairs of B6-Lyb-2 ~ congenic mice were generously supplied to us by Dr. E. A. Boyse (Sloan-Kettering Institute, New York, New York). Splenic B cells were surface-labeled with 125I by lactoperoxidase method as described (Jones 1980). Labeled cells were solubilized with extraction buffer containing 0.5 % Nonidet P-40 and were precipitated with monoclonal Lyb-2.1 antibody or normal mouse serum (NMS) coupled to protein A-Sepharose beads (Pharmacia Fine Chemicals, Piscataway, New Jersey). Immunoprecipitates were then resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) through 8 % acrylamide under reducing conditions. Figure 1 illustrates that