Masayuki Kurobe

Biological and Immunological Properties of Human Hepatocyte Growth Factor from Plasma of Patients with Fulminant Hepatic Failure.

We have recently purified human hepatocyte growth factor (hHGF), a heterodimer with molecular weight of about 83,000, from plasma of patients with fulminant hepatic failure (Gohda, E. et al., J. Clin. Invest. 81, 414-419, 1988). Biological and immunological properties of hHGF were examined. Out of the well-known growth factors tested, only epidermal growth factor (EGF) and transforming growth factor-alpha (TGF-alpha) stimulated DNA synthesis of adult rat hepatocytes in primary culture. hHGF enhanced the DNA synthesis at less than one-tenth of the molar concentrations of EGF and TGF-alpha. Half-maximal stimulations by hHGF, EGF and TGF-alpha were observed at 30, 400 and 900 pM, respectively. Maximal stimulation by TGF-alpha, however, was greater than those caused by hHGF and EGF. The effect of hHGF was additive with the maximal effects of EGF and TGF-alpha. Anti-hHGF antiserum was prepared in a rabbit by injecting with purified hHGF. This antiserum recognized nonreduced hHGF, but not reduced hHGF. The antiserum for hHGF did not inhibit growth-promoting activity of EGF, that was neutralized by incubation with anti-EGF antiserum. The activity of hHGF was completely inhibited by anti-hHGF antiserum, but not by anti-EGF antiserum. hHGF did not show any cross-reactivity to anti-EGF antiserum as measured by enzyme immunoassay for EGF. Thus, biological and immunological properties of hHGF are different from those of EGF and TGF-alpha.

Anti-skeletal muscle antibodies in the sera from myasthenic patients with thymoma: identification of anti-myosin, actomyosin, actin, and α-actinin antibodies by a solid-phase radioimmunoassay and a Western blotting analysis

We recently reported a strong association between the occurrence of anti-skeletal muscle (SM) antibodies and the presence of thymoma in patients with myasthenia gravis (MG). To further examine the immunoreactivity of MG sera against human muscle antigens, we developed a solid-phase radioimmunoassay (RIA) using purified muscle antigens and a Western blotting analysis in MG sera with high titers of anti-SM antibodies. Our results showed that MG patients with thymoma (thymoma group) had markedly high titers of anti-myosin and anti-actomyosin antibodies than those without thymoma (non-thymoma group). Furthermore, a close correlation was found between titers of anti-SM, anti-myosin and anti-actomyosin antibodies. The antibody titers against actin, alpha-actinin and tropomyosin were all low and did not correlate with titers of anti-SM antibodies. But, significant levels of these three antibodies were found in the thymoma group. By a Western blotting analysis, immunoreactivity of sera from the thymoma group appeared to be predominantly directed against myosin, actin and alpha-actinin.

Human Breast Cancer Cells Synthesize and Secrete an EGF-Like Immunoreactive Factor in Culture.

A human breast cancer cell line, strain MCF-7, in culture synthesized and secreted a large amount of a polypeptide (designated as MCF-7 EGF) immunologically related to human epidermal growth factor (hEGF). The molecular weight of MCF-7 EGF estimated by gel filtration on Sephadex G-75 was similar to that of hEGF from human urine. On isoelectric focusing analysis, MCF-7 EGF gave a major peak at pH 4.6 and a minor peak at pH 5.0. In our enzyme immunoassay system, however, the dose-response curve of MCF-7 EGF did not show good parallelism with that of standard hEGF. From these results, it is suggested that MCF-7 cells synthesize and secrete a polypeptide immunologically related to hEGF into the culture medium.

Synthesis and secretion of an epidermal growth factor (EGF) by human fibroblast cells in culture

Human fibroblast (WS-1) cells in culture synthesized and secreted an epidermal growth factor which cross-reacted with human epidermal growth factor (hEGF) purified from human urine. hEGF secreted by the cells (designated as WS-1 EGF or fibroblast EGF) and hEGF isolated from urine (designated as urine EGF) were immunologically indistinguishable. The molecular weight of fibroblast EGF estimated by gel filtration was identical with that of hEGF from urine. On chromatofocusing chromatography, fibroblast EGF was eluted mainly at pH 4.26 as a sharp symmetric peak with a minor peak at pH 4.62, like urine EGF. These results suggested that EGF synthesized and secreted by human fibroblast cells is an identical molecule to that of hEGF in human urine.

Production of an hEGF-like immunoreactive factor by human gastric cancer cells depends on differentiational state of the cells

We have extended our recent observation that human gastric cancer cells (MKN-45) synthesize and secrete an hEGF-like immunoreactive factor (designated as EGF-LI) by characterization of EGF-LI produced by five human gastric cancer cell lines in culture. Two cell lines (MKN-45 and KATO-III) derived from poorly differentiated adenocarcinoma synthesized and secreted a much larger amount of EGF-LI than three cell lines (MKN-1, MKN-28, and MKN-74) derived from well-differentiated adenocarcinoma. Treatment of MKN-45 cells by retinoic acid reduced significantly synthesis and secretion of EGF-LI, suggesting that production of EGF-LI is dependent on differentiational state of gastric cancer cells.

A sensitive two-site enzyme immunoassay for human pancreatic secretory trypsin inhibitor (PSTI) using monoclonal antibodies

By using monoclonal antibody against human pancreatic secretory trypsin inhibitor (PSTI), we developed a highly sensitive, simple, and reliable two-site enzyme immunoassay system. The minimum amount of PSTI detected by this EIA is approximately 10 pg/ml when a 100 microliter aliquot of the sample is used. Good reproducibilities of within- and between-assay series and excellent recovery of exogenous PSTI from serum were observed. The correlation between the values obtained by the EIA and RIA methods was given by the linear regression equation, y = 1.09x + 4.6, for which the correlation coefficient (r) was 0.980 (n = 20). Antigenicity of the trypsin-PSTI complexes was found to be approximately 10% of that of PSTI. From these results, it seems that our recently developed EIA system for PSTI is useful in clinical testing for quantitation of PSTI in body fluids, for biochemical studies on synthesis and secretion of PSTI, and also for study of pathophysiological mechanisms involved in the development of acute pancreatitis and certain malignant neoplasms.