KIKUCHI, K., TAMURA, S., SHINEHA, R. and TSUIKI, S. Characterization ofProtein Phosphatases Associated with the Particulate Fraction from Rat Liver. Tohoku J. Exp. Med., 1990, 160 (4), 287-300 - Protein phosphatases associated with the particulate fraction from rat liver were studied by chromatographing the fraction on a DEAE-cellulose column and assaying the eluate with phosphorylase a and glycogen synthase D as substrates. Phosphorylase phosphatase activity emerged as two peaks, termed P-1 and P-2 in order of elution, both of which were inhibited by Mn2+ and Mg2+. P-1 and P-2 were Mr=50, 000 and 32, 000 proteins, respectively, and when treated with trypsin, P-1 converted to a form indistinguishable from P-2, to which protein phosphatase inhibitor-2 was a potent inhibitor. Thus P-2 appears to be the catalytic subunit of type-1 protein phosphatase even though it has been degrated proteolytically as evidenced by its relatively low Mr. The elution profile of glycogen synthase phosphatase activity was entirely different. The activity obtained with 5mM Mn2+ resolved into three peaks, the second-migrating M-2 being the largest. M-2 is an Mr=70, 000 protein; but an attempt to purify it has been unsuccessful giving a product of Mr=40, 000 and closely similar to the type-1 catalytic subunit in properties including inhibition by inhibitor-2. These results suggest that phosphatases P-1 and M-2 have a common catalytic subunit (type-1), which is bound to different “regulatory” subunits. M-2 distributes in glycogen particles and microsomes evenly while P-1 is almost exclusively in microsomes.-protein phosphatase; glycogen synthase; phosphorylase phosphatase; rat liver; rat liver particulate fraction
ETO, M., WATANABE, K., MORIYAMA, T. and MAKINO, I. Apolipoprotein EPhenotyping from Plasma by Isoelectric Focusing and Immunoblotting. Tohoku J. Exp. Med., 1990, 160 (4), 301-309-A method for apolipoprotein (apo) E phenotyping directly from plasma by isoelectric focusing (IEF) and immunoblotting was confirmed. Ten μl plasma were delipidated. IEF in 5% polyacrylamide flat gel with 6.4mol/l urea and 2.8% pharmalyte (PH 4-6.5) was carried out at 3, 000V for 1hr. Seventeen samples were applied per one flat gel, and IEF of two flat gels was made. Then, Western blotting on nitrocellulose membrane was done at 75V for 3hr. Immunostaining was performed using goat-anti-human apo E as first antibody and biotinylated anti-goat IgG as second antibody, and 4-chlorodel-1-naphthol as a substrate. In approximately 5% of the samples, we had difficulty in discriminating between homozygotes and heterozygotes (i.e., apo E3/3 and apo E3/2, or apo E4/4 and apo E4/3) because of equally strong sialated band, but this problem was solved by sialidase treatment of plasma before delipidation. As a result, six apo E phenotypes were clearly demonstrated. Apo E phenotyping of 34 samples could be made simultaneously in 2 days. It is concluded that the polyacrylamide gel IEF and immunoblotting method is useful for apo E phenotyping if it is made up for by sialidase treatment.
HORIE, Y., CHIBA, M., IIZUKA, M., IGARASHI, K. and MASAMUNE, O. HLAAntigens on Colorectal Adenoma and Cancer Cells. Tohoku J. Exp. Med., 1990, 160 (4), 311-322-HLA-DR and -ABC antigens on adenoma and cancer cells of the colon and rectum were investigated. Fifteen specimens of adenomas from 15 patients with sporadic colorectal adenoma, seven specimens of adenomas from seven patients with familial polyposis coli (FPC), and 10 specimens of cancers from 10 patients with colorectal cancer were obtained. Normal colonic mucosa far from the lesions, taken from 15 patients with sporadic adenoma or cancer, served as normal control mucosa. HLA antigens were identified using an immunoperoxidase staining method. Epithelia of all normal control mucosa (n=15) expressed HLA-ABC antigens, but not HLA-DR antigens. HLA-DR antigens were expressed on 47% (7/15) of sporadic adenomas, 71% (5/7) of adenomas in FPC, and 100% (10/10) of cancers. The extent of HLA-DR expression on adenoma and cancer cells became broader with more severe dysplasia in adenoma, and increased undifferentiation in cancer. It also became broader with increasing mononuclear cell infiltration in both adenomas and cancers. HLA-DR antigens on adenoma and cancer cells appeared to be related to the neoplastic transformation of the epithelia, and to the mononuclear cell infiltration. Partial disappearance of HLA-ABC antigens on adenoma and cancer cells was observed in a few specimens of both adenomas and cancers.
KIKUCHI, H., SAGAMI, I., FUJII, H., OHMACHI, T. and WATANABE, M. Complementary DNA Sequence of 3-Methylcholanthrene-Inducible P-450 from theRat Lung. Tohoku J. Exp. Med., 1990, 160 (4), 323-332-Cytochrome P-450MC was induced in pulmonary microsomes of 3-methylcholanthrene-treated rats and also at low level in that of isosafrole-treated rats. Cytochrome P-450d was not detected in the lungs of 3-methylcholanthrene- or isosafrole-treated rats by the method of Western blot analysis with a polyclonal antibody raised against cytochrome P-450c which is equally effective against P-450d, nor by the method of Northern hybridization probed with pcP450mc3 (P-450d probe). Complementary DNA of P-450MC was isolated from rat pulmonary cDNA library and the nucleotide sequence of pulmonary cDNA was compared with that of hepatic P-450c cDNA reported by Yabusaki et al. There was no gross change in nucleotide sequences of cDNA between pulmonary P-450MC and hepatic P-450c.
OHTANI, H. Ultrastructural Characterization of Enterochromaffin-Type Cellsin Human Gastric Carcinoma. Tohoku J. Exp. Med., 1990, 160 (4), 333-342-Ultrastructural characterization has been performed on eosinophilic granular cells occurring in gastric carcinomas. Of these, the present paper dealt with enterochromaffin (EC)-type cells. EC-type cells were identified by the presence of eosinophilic granules, immunoreactivity for serotonin (5HT) and argentaffinity. Conventional electron microscopy revealed that they bore membrane bound, electron-dense granules of varying size (100-800nm in shorter diameter), which were larger than those of normal EC cells. Their shapes are round, rod-shaped and concave. The granules are immunolabeled for 5HT by both pre- and post-embedding methods. The preembedding method also disclosed diffuse cytosolic immunoreactivity. Both differentiated-type and undifferentiated-type gastric carcinomas showed essentially the same results. General trends of occurrence of Paneth-like and EC-type cells in gastric carcinomas were also discussed. The present study confirmed that EC-type cells in gastric carcinoma retained basic characteristics of EC cells despite of morphological deviation from the normal cells. It is speculated that they are derived from neoplastic precursor cells with a special differentiation.
YOKOYAMA, M., KOIKE, K., KANNO, S. and OHTSUKI, K. Biochemical Characterizationof the Membrane-Associated Phosphorylating Proteins Involved in theAnti-Proliferative Effect of Human Recombinant Interferon-Alpha 2a (rIFN-α2a)in Daudi Cells. Tohoku J. Exp. Med., 1990, 160 (4), 343-359-The anti-proliferative effect of interferons (IFNs) was investigated, focusing on the physiological activities of membrane-associated proteins involved in the progress of cell proliferation. In preliminary screening of the inhibitory effect of IFNs against the cell growth of various human hematopoietic tumor cells, Daudi cell was the most sensitive to rIFN-α2a. SDS-PAGE followed by autoradiography detected that treatment of Daudi cells with rIFN-α2a significantly reduced phosphorylation of the membrane-associated Mr 98, 000 and 70, 000 polypeptides. To determine the physiological significance of these phosphorylating polypeptides in mediation of the IFN effects, they were purified from Daudi cells. It was found that the molecular weight of the purified polypeptides was approximately 330, 000 and it (designated 330-kDa protein) was consist of two distinct subunits [α-subunit (Mr 98, 000) and β-subunit (Mr 70, 000)] . The biochemical properties, such as subunit structure, subunit phosphorylation, requirements for phosphorylating activity and protease sensitivity of the 330-kDa protein were similar to those reported for Na+, K+-ATPase. Evidence provided here suggests that the anti-proliferative action of IFNs may, at least in part, be implicated in the IFN-induced physiological impairment of the membrane-associated 330-kDa protein.
KANO, Y., NAKURA, K., NAKANE, T., UEDA, Y., UEMURA, Y., YOKOYAMA, K., ISHIDA, H., OHYANAGI, H. and SAITOH, Y. Development of Monoclonal Antibodiesagainst Human Gastrointestinal Cancer. Tohoku J. Exp. Med., 1990, 160 (4), 361-373-We have evaluated approximately 10, 000 monoclonal antibodies resulting from 14 hybridization of spleen cells from mice immunized with established cancer cell lines. They were screened by binding assays using cancer cell lines or normal fibroblast cells, followed by immunohistochemical study on tissue sections. Ninety-one monoclonal antibodies were obtained by above binding assays. One of them, named KM10 was further investigated. Isotype of KM10 was IgG1 with k-light chain. It was shown that KM10 has strong reactivity with tumors of colon, stomach, papilla Vater, and pancreas, while the limited reactivity displayed with normal tissues. The electron microscopic observation revealed that the antigen recognized by KM10 is expressed on the surface of cancer cells. KM10 could mediate ADCC. These results indicate that KM10 is a good candidate for a probe of diagnosis and therapy of cancer.
ONUMA, T., TSUTSUI, M., OCHIAI, S., BOKU, A., YANADA, A., HIRAI, Y., NAKAHATA, H. and TAKEBE, K. Acid Cholesteryl Ester Hydrolase Activity ofMononuclear Leukocyte in Type 2 (Non-Insulin-Dependent) Diabetic Patients. Tohoku J. Exp. Med., 1990, 160 (4), 375-381-Acid cholesteryl ester hydrolase activity of mononuclear leukocytes was measured in 52 Type 2 (non-insulin-dependent) diabetic patients. Enzyme activity was significantly lower in the diabetic patients than in 14 age-matched control subjects (0.89±0.08 (mean±S.E.) vs. 2.20±0.17nmol/mg protein/hr, p<0.01). In diabetic patients undergoing diet treatment only, the enzyme activity was significantly lower in poorly controlled patients than in well controlled patients (0.43±0.03 vs. 1.15±0.24 nmol/mg protein/hr, p<0.01). In the diabetic patients, there was a significant negative correlation between the enzyme activity and serum total cholesterol or low density lipoprotein cholesterol level (r=-0.361, p<0.01, n=52 or r=-0.630, p<0.01, n=28). These results suggest that a low level of acid cholesteryl ester hydrolase activity in mononuclear leukocyte might play an important role in the progression of atherosclerosis in Type 2 diabetes.
KONNO, R., SHIKANO, K., HORIGUCHI, M., ENDO, A., CHIBA, H., YAEGASHI, N., SATO, S., YAJIMA, H., TASE, T. and YAJIMA, A. Detection of Human PapillomavirusDNA in Genital Condylomata in Women and Their Male Partners by Using InSitu Hybridization with Digoxygenin Labeled Probes. Tohoku J. Exp. Med., 1990, 160 (4), 383-390-Twelve couples (12 women and their male partners) presenting genital warts were investigated in order to evaluate the sexual transmission of human papillomavirus (HPV) in mutual partners and the localization of HPV DNA. Formalin-fixed, paraffin-embedded biopsy samples of 12 vulvar condylomata, and 12 penile condylomata from male partners were analyzed for the presence of HPV DNA-6, -11, and 16/18 by using in situ hybridization with digoxygenin labeled DNA probes. HPV DNA was identified in 9 women (75%) and in 9 men (75%). HPV-6 was frequently identified, being revealed in 42% of the vulvar speciemens, in 67% of the cervical specimens and 58% of the penile specimens. Seven of 9 (77%) positive couples shared the same HPV DNA, and 2 couples harbored different HPV DNA types between the partners. The signal intensity of the HPV DNA was generally strong in superficial cell layers, weak in parabasal or basal cell layers. No malignant lesions resulted from the condyloma acuminatum caused by HPV-6 or -11. There were only mild dysplasia in the both sexes.