As the lecture of Predisent of the 50th Spring Meeting of the Japanese Electrophoresis Society, covered the own group's research field during the past about 20 years periods. Basic aspect and clinical significance of pepsinogens were reviewed. Contents of the lecture were the cellular localization of pepsinogen granules in the one cell, the activation mechanism of it, the entire human pepsinogen A and C genes, and the nucleotide sequence of all the exon and the 5'-and 3'-flanking regions. Topics of isozymes of human pepsinogen A (I), the schema of pepsinogen-producing mechanisms from mRNA, and electrophoretic pattern of pepsinogen A (I) from human gastric mucosa using agargel, were included. On the other hand, as for the clinical significance of pepsinogens, the first case report of pepsinogen producing gastric carcinoma with high pepsinogen II levels in both serum and ascites, and pepsinogen I and II immunohistochemical staining in gastric carcinoma tissues using the monoclonal antibodies, were presented. Finally, the clinical application of the serum pepsinogen levels in the research of Helicobacter pylori infection, gastric cancer development in the course of carcinogenesis from atrophic gastritis to gastric cancer in the population level, gastric cancer screening using serum pepsinogen test kits, which were commercially available now in Japan, and the significance of low serum pepsinogen levels to detect stomach cancer associated with extentive chronic gastritis, which was a precancerous condition of gastric cancer, were discussed.

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Helicobacter pylori (Hp) has been classified as a stomach carcinogen on the basis of epidemiological findings. For detailed analysis of the role of Hp in stomach carcinogenesis, it is essential to establish a small animal model. Hirayama et al. first demonstrated that Mongolian gerbils (MGs) can be infected with Hp. We have established experimental models of stomach carcinogenesis in MGs using the chemical carcinogens, N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and N-methyl-N-nitrosourea (MNU). In the present study, male MGs were infected with Hp before or after MNU administration. Glandular stomach adenocarcinomas not only of well-differentiated but also poorly differentiated and signet-ring cell types were induced. The incidence of adenocarcinomas was significantly higher in animals treated with before or after MNU administration than in the controls. Hp infection also enhances glandular stomach carcinogenesis in MGs treated with MNNG. To further assess this latter possibility, male MGs were treated with MNU followed by inoculation with Hp, or infected with Hp followed by MNU administration, then Hp was eradicated in half of the infected animals. The incidence of tumor development in infected animals was significantly higher than in Hp eradicated animals. The incidence was significantly suppressed by the eradication procedure. Our studies have shown that Hp enhances glandular stomach carcinogenesis of MGs treated with chemical carcinogens. Moreover, Hp eradication may be useful as a prevention approach.

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Stomach epithelia proliferate and differentiate continuously throughout life, playing various organ-specific roles. The regulatory mechanisms of proliferation and differentiation of the epithelia is under the control of a genetic program called epithelial-mesenchymal interaction. Some of the factors that mediate the interaction have recently been identified: there is a great deal of evidence that the components of extracellular matrix control the morphology and function of epithelial cells in various organs. Moreover, some soluble mesenchymal factors turns out to be mitogen, motogen and morphogen for various epithelial and endothelial cells. The results of our recent studies, both in vivo and in vitro, also demonstrate that the proliferation and differentiation of gastrointestinal epithelial cells is regulated region-specifically not only by soluble factors but also by insoluble components of the ECM. With various other factors such as systemic hormones, gastrointestinal polypeptides, neuronal factors and acidic environment of the gastric lumen playing as active modulators, the epithelial-mesenchymal interaction is deeply involved in the development and maintenance of morphology and functions of the stomach.

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We review on the involvement of intracellular signaling molecules in the protection against myocardial ischemia-reperfusion injury. Ischemia or repeated brief ischemia-reperfusion (ischemic preconditioning, IP) induces translocation of PKC isoforms to the membrane or nucleus. PKC-ε isoform is involved in the IP's protective effect in the perfused heart and in vivo infarction model. In the in vivo model, IP upregulates VEGF mRNA and subsequent angiogenesis. Additionally, NO generated during reperfusion activates PKC-α, δ and ε isoforms, thereby alleviating contractile dysfunction by reperfusion. On the other hand, ischemia induces PKC-ζ translocation through PI3 kinase activation. Ischemia also induces MAP kinase, c-Jun-N-terminal kinase (JNK) to the nucleus, and reperfusion activates MAP kinase and JNK, thereby inducing c-fos and c-jun. The pathway of PI3 kinase, PKC-ζ, and MAP kinase protects myocardial cells against ischemia-reperfusion injury through apoptosis inhibition. Ischemia also induces translocation of small heat shock proteins HSP27 and MKBP to the nucleus and myofibrils, which would protect myofibrils and nucleus against oxidative stress induced by reperfusion.

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Adhesion of colon cancer cells (colo 201) and neutrophils to endothelial cells which had been briefly treated with hypoxanthine/xanthine oxidase, hydrogen peroxide, or peroxynitrite was analyzed in the absence of de novo protein synthesis using actinomycin D or cycloheximide. Hypoxanthine/xanthine oxidase treatments accelerated the adhesion of both colo 201 cells and neutrophils to endothelial cells, and these effects were blocked by SOD/catalase or EDTA. These data provided preliminary evidence for the fact that hydroxyl radicals directly affect the cell surface of endothelial cells and accelerate cell adhesion.

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There is increasing evidence that aldehydes generated endogenously during the degradation process of biological molecules are involved in many of the pathophysiologies associated with cardiovasular diseases such as atherosclerosis and the long-term complications of diabetes. Major sources of reactive aldehydes in vivo are lipid peroxidation, glycation, and amino acid oxidation. Although the types of aldehydes are varied, the important aldehydes that can exert biological effects relevant to the pathobiology of oxidant injury are represented by 2-alkenals, 4-hydroxy-2-alkenals, and ketoaldehydes. These aldehydes exhibit facile reactivity with proteins, generating stable products at the end of a series of reactions. On the other hand, a number of reactive aldehydes have been implicated as inducers in generating intracellular oxidative stress and activation of stress signaling pathways, that integrate with other signaling pathways to control cellular responses to the extracellular stimuli.

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The mechanism of metal ion-catalyzed oxidative modification of apolipoprotein E (apoE) in human very low density lipoprotein (VLDL) and its inhibition by ascorbic acid and glycosaminoglycans (GAGs) were investigated in vitro. The apoE in VLDL formed high molecular aggregates and lost its heparin-binding activity along with the lipid peroxidation catalyzed by Cu²⁺. This suggests that Cu²⁺-catalyzed oxidative modification of apoE causes impairment of lipid uptake by cells and leads deposit of the oxidized lipids in tissues. The oxidation of VLDL and modification of apoE were strongly inhibited by ascorbic acid and GAGs (heparan sulfate, heparin, and chondroitin sulfate A) in dose dependent manner. These results may indicate that endogenous ascorbic acid and GAGs preserve the biological functions of apoE from oxidative stress.

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Creatine N-phosphotransferase (CK; EC 2.7.3.2)-linked immunoglobulin (Ig) G and IgM detected in serum of a 74 year-old woman with acute renal infarction was reported. CK-BB released into the circulation from the renal tissue by renal infarction rose in the patient serum temporarily together with rises of aspartate aminotransferase and lactate dehydrogenase. The IgG and IgM had affinity for CK-B subunit but not for CK-M subunit. At the first stage, IgG as well as IgM was present, however IgM became dominant after one week and IgG became dominant after one year. IgG subclass changed from IgG 1 dominant to IgG 3 dominant after 6 months. IgG was linked to CK-B subunit at the Fab region. CK activity present in renal tissue was 16.4±7.6IU/g wet weight and CK-BB was dominant. By the immunoenzymatic method, CK-BB was detected along the vein-circulation-system, that is the endothelial cells of the arteriole, lumens of the arteriole, the glomerular capillary and of the interlobular vein but not in the cells or lumen of the renal tubules. From these results it was suggested that CK-BB released into the circulation by renal infarction played a role of trigger to produce CK-B subunit-linked IgM. The IgM changed to IgG in addition to the CK-BB-linked IgG that had already been existed in the circulating blood. In the ischemic heart diseases, it was suggested by the immunoenzymatic method that endothelial cells of the arteriole, smooth muscle of the media and parts of the sclerotic artery were sources for CK-BB consisting of CK-BB-IgG complexes.

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Fibrinogen Matsumoto II is a hereditary dysfibrinogenemia with heterozygous missense mutation, γ308 Asn->Lys. The propositus's fibrinogen analyzed by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) using the Laemmli's buffer system under reducing conditions and Western blotting revealed two distinct bands corresponding to the γ-chain; one with an apparently normal molecular weight (MW) (47, 500) and the other with a lower MW (45, 500). To better define the characteristics on SDS-PAGE of γ308 Lys mutant γ-chain, we synthesized three fibrinogens with single amino acid substitution at this residue: γ308 Lys, γ308 Ile, γ308 Ala. We found the MW of the γ-chains derived from recombinant fibrinogens, γ308 Asn, γ308 Ala, γ 308Ile, and γ308 Lys, were 48.5, 48.5, 47.0, and 46.0kDa, respectively. These results demonstrated the lower MW of mutant γ-chain derived from plasma fibrinogen of Matsumoto II and Baltimore III (γ308 Asn->Ile) propositus due to the single amino acid substitution. The increased mobility of the mutant γ-chain could be attributed to the binding properties of SDS to the peptide. It is well-known that the increased or decreased SDS binding induces complicated changes in the electrophoretic mobility, however, we speculate the mechanism of the increased mobility of the protein is followings. Since SDS preferentially binds either basic or hydrophobic amino acids, the mutant peptide increased basicity or hydrophobicity bind much more SDS. Then the increased SDS binding may lead to complicated change of conformation and/or net charge of the peptide. Finally these phenomena may result in faster mobility on SDS-PAGE and lead decreased MW of the peptide.

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T-cell-rich B-cell lymphoma is defined as a variant type of non-Hodgkin's lymphoma, which is characterized by a few neoplastic B-cells associated with predominant reactive T-cells. We describe here a patient with a lymphoma which was revealed as T-cell-rich B-cell lymphoma using molecular analysis. The patient, who had initially diagnosed as having Hodgkin's disease in 1988 at one hospital, was referred to our hospital at second relapse in 1992. A second lymph node biopsy did not include characteristic RS cells, although a few atypical histiocytes surrounded by atypical lymphocytes were observed. She died at third relapse in 1993 with the diagnosis of diffuse large cell lymphoma. Southern blot analysis of DNA derived from the second biopsy revealed faint rearranged bands detected by IgH and BCL-2 probes, while DNA derived from liver tumors at postmortem contained discrete rearranged bands which were identical in size to those of second relapse. To detect small number of tumor cells, we next developed PCR for amplifying the complementarity determining region (CDR) of IgH. The DNA at second biopsy showed a rearranged band corresponding to the CDR I, while DNA at postmortem showed rearranged bands corresponding to the CDR II and CDR III. Sequencing analysis of the PCR product from CDR II region revealed somatic mutations as compared with the germline sequence registered in the database. This indicates that diffuse large cell lymphoma was derived from germinal center B cells, and discriminates it from classical Hodgkin's disease. These results demonstrated that the tumor at second relapse was T-cell-rich B-cell lymphoma, and the clone was the same as that found in postmortem. Southern blot hybridization and PCR described here are valuable tools to diagnose confused lymphoid tumor, and will be of useful for prospective purpose.

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We devised a simple and sensitive method for the determination of oligoclonal IgG bands (OCB) in the cerebrospinal fluid (CSF) by combining agarose gel electrophoresis with immunoblotting. In this method, it is not neccessary to concentrate the CSF. This method is effective for the separation of proteins present at low concentrations, such as is CSF samples. Its specificity is excellent. We were able to clearly detect OCBs in CSF samples, even when the IgG concentration was low, and also when there was strong masking by a polyclonal IgG smear. Our method is concluded to be useful for identification of OCBs in CSF samples.

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A Japanese family with familial dysalbuminemic hyperthyroxinemia (FDH) was presented and recent research progress concerning FDH was reviewed. Two mutations in the albumin gene have been reported as a cause of FDH; Arg 218 His in Caucasian and Chinese cases, and Arg 218 Pro in a Japanese family. In the latter, higher thyroxine levels are observed than those for the former, possibly due to a conformational change favorable to the binding with thyroxine. A recent study suggested that FDH might result in altered warfarin metabolism.

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We examinied the differentiation of lipoprotein in human serum using polyacrylamide gradient gel (PAGG) electrophoresis to analyze the lipid components and to quantify the small, dense LDL. Electrophoretic patterns were classified into three types, which included two types reported by Krauss et al. and one new type containing equal same amounts of regular and small size LDL. We devised a direct staining method to stain triglyceride (TG) and cholesterol (Cho) in lipoprotein separated by the polyacrylamide gel electrophoresis and components of lipoprotein was quantitaivety analyzed with the densitogram. At the same time, the presence of apoproteins LP (a) and B in the lipoprotein was confirmed. As a result of the staining, we observed more amounts of Cho containing in the fraction of regular size LDL than small dense LDL, and more amounts of TG containing in the fraction of small dense LDL than regular size LDL. Apo B was detected in both fractions of regular size LDL and small dense LDL, but no LP (a) was detected.

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A case of multiple myeloma with various kinds of paraproteins is described in this report. IgG 3-λ type monoclonal protein, γ3-heavy chain and Bence Jones protein-λ were detected in the serum of a 60-year-old male patient. Also, γ3-heavy chain and Bence Jones protein-λ were detected in the urine. The final diagnosis of this case seems to be multiple myeloma with whole molecule and its fragments of monoclonal immunoglobulin G. More interestingly, these paraproteins partly had a characteristics to react with the agar gel, not reaction with the agarose gel.

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