The Society of Electrophoresis was founded in 1950 by a group of the researchers in Japan who were engaged with the Tiselius Electrophoresis, and the name of the Society was changed to The Japanese Electrophoresis Society in 1993. The main activities of the Society include biannual scientific meetings (the General Meeting and the Spring Meeting), publications of the Physico-Chemical Biology (renamed to the Japanese Journal of Electrophoresis since 1995) and the monographs on Electrophoretic Methods, proposal of the standard electrophoretic methods (Tiselius, filter paper and cellulose acetate electrophoresis, and the standard films for densitometry), annual training courses of electrophoresis for beginners since 1963, and presentation of two types of academic awards (the Kodama Prize and the Hirai International Prize).

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During fifty years' history of the Japanese Electrophoresis Society, many types of handmade electrophoresis experiments by many members of the Society contributed to the progress of electrophoretic methods. Starting from the handmade Tiselius' electrophoresis apparatus by Hirai and Shimao, recollections of the members active in the handmade electrophoreses in the first fifteen years of the Society are described. Then, two examples of handmade electrophoresis by the author, namely, a buffer gradient gel electrophoresis and a comparative study of computer simulations of glycine and tricine discontinuous sodium dodecyl sulfate-polyacrylamide gel electrophoresis made in the last twenty years are shown.

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An immunoassay system was integrated into a glass microchip. Polystyrene beads were introduced into a microchannel, and then human secretory immunoglobulin A (s-IgA) adsorbed on the bead surface was reacted with anti-s-IgA antibody-colloidal gold conjugate, and detected using a thermal lens microscope. The integration reduced the time necessary for the antigen-antibody reaction by 1/90, thus shortening the overall analysis time from 24h to about 45min. The scale merits of liquid microspace on the molecular behavior remarkably contributed to reduced assay time. Moreover, troublesome operations required for conventional immunosorbent assays could be replaced by simple operations. This integrated immunosorbent assay system will be useful to be put into practical use including clinical diagnosis and biochemical researches.

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One of the key issues in Human Genome project was to develop a high throughput DNA sequencer that should have one order of magnitude higher throughput in base reading than the conventional DNA sequencer. Various technologies have been developed along this direction including DNA chips and capillary array DNA sequencers. By the use of replaceable gel as well as a highly sensitive fluorescence detector, a very high throughput DNA sequencer has been achieved and commercialized with capillary array gel electrophoresis. Now it is expected that the human genome sequencing is completed in a few years. The function analysis of genes and their application to medical and pharmaceutical fields are the next research target where an ultra-high speed and throughput analyzer, from the view point of analyzable sample numbers, is required. The required analyses are gene expression profiling, comparative genome analysis including SNPs (single nucleotide polymorphisms) analysis. A capillary array gel electrophoresis with short capillaries, micro-fabricated multi-channel electro-devices, or DNA chips will take the major role in the post genome era. Besides, the development of sample preparation devises for various applications are getting important.

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Human genome project has been moving on to the post genomic sequencing era including SNP (single nucleotide polymorphism) analysis, genomic function analysis, and proteome analysis. Towards post genomic sequencing era, laser confocal fluorescence detection system has been developed for the imaging of single DNA molecule migrating in the microchannel on a microchip. The separating process for a mixture of DNA fragments is visualised and the conditions for ultra fast separation of DNA fragments are optimised. The nanochip technology coupled with AFM (atomic force microscopy) has been developed for the manipulation of single DNA molecule. It will be applicable to the SNP analysis of single genomic DNA molecule.

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A capillary column liquid chromatography-tandem mass spectrometry (LC-MS/MS) system for the analysis of proteome (the protein complement expressed by a genome, a cell or a tissue type) is described. The system has a power to determine accurate molecular mass, amino acid sequence and post-translational modification of micro-quantity of protein. The system further integrated with surface plasmon resonance based biosensor (BIA) to obtain sequence information of the affinity-bound proteins on the BIA sensorchip. Because BIA provides quantitative molecular interaction information and because LC-MS/MS provides structural information with femtomole amount of samples, the integrated system of BIA and LC-MS described here should allow wide application to molecular studies of stable and unstable interaction.

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The electrospray ionization mass spectrometry (ESIMS) is a powerful tool to detect and characterize abnormal proteins and to determine the ratio of mutant and wild proteins and peptides. Using this technique, the ions of intact normal and variant proteins were clearly observed in samples from patients with hemoglobinopathies and neurodegenerative diseases such as familial amyloidotic polyneuropathy (FAP) and amyotrophic lateral screlosis (FALS). We detected 23 kinds (43 cases) of abnormal hemoglobins (Hbs), 10 kinds (38 cases) of transthyretins (TTRs) mutants and 4 kinds of Cu/Zn superoxide dismutases (SOD-1s) for past 6 years. We also measured glycated β-globin N-terminus hexapeptide (HbA_1c) by Poroszyme V8 protease digestion/LC-ESIMS and proposed a calculation “weights sum method” for the quantification of true HbA_1c values.

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Since the first introduction of cancer gene therapy using TNF (tumor necrosis factor)-α gene transduced tumor infiltrated lymphocytes (TIL) in 1991 by Rosenberg et al., there have already been officially approved more than 230 clinical protocols in US. These protocols include immunogene therapy, prodrug (suicide) gene therapy, tumor suppressor gene therapy, and chemoprotection gene therapy. Among these protocols, immunogene therapy has been most extensively studied recently to enhance traditional cancer immunotherapy. We have prepared cancer immunogene therapy using GM-CSF gene against renal cell cancer for the last 8 years and have administered GM-CSF gene transduced renal cancer cells to 3 patients suffered from stage IV renal cell cancer. In this review, current status of cancer gene therapy is summarized. Also, background and current status of GM-CSF immunogene therapy is reported.

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The α-fetoprotein (AFP) gene is reexpressed in hepatoma cells. We have previously shown that the retrovirus vector (LNAF0.3TK) carrying the herpes simplex virus thymidine kinase (HSVTK) gene regulated by the 0.3-kb human AFP promoter provides ganciclovir (GCV)-mediated cytotoxicity in AFP-producing hepatoma cells in parallel with the ability of AFP production. Since the recent reports have revealed that a G to A substitution in the human AFP promoter region is relevant to the hereditary persistence of human AFP, the same substitution was generated in LNAF0.3TK to construct LNAFM0.3TK. LNAFM0.3TK infection into intermediate and low AFP-producing human hepatoma cells, PLC/PRF/5 and huH1/cl. 2, respectively, resulted in more pronounced growth inhibition by GCV treatment than LNAF0.3TK infection. In addition, to improve the efficacy of retrovirus-mediated gene therapy for the intermediate or low AFP-producing hepatoma cells, human AFP domain B enhancer region was linked to 5'-end of the 0.3-kb AFP promoter region. The infections with sense and reverse-oriented retrovirus vectors, LNAFE0.3TK and LN (AFE0.3TK) R, respectively, sensitized PLC/PRF/5 and huH1/cl.2 cells to GCV, in which LN (AFE0.3TK) R infection showed more pronounced tumoricidal effect. These results suggest that modifications of the AFP promoter ensure the therapeutic gene expression in gene therapy for the low AFP-producing hepatoma cells.

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It has become clear that cancers develop and progress througt the accumulation of various genetic alterations. Among various human malignancies, none is better understood at the molecular genetic level than colorectal cancer. Colorectal cancer is one of the commonest malignant tumors and has a relatively poor prognosis. Recent advances in the molecular genetics of colorectal cancer have stimulated attempt to evaluate the prognostic significance of specific genetic alterations in this tumor. Although conventional pathological staging has served as the standard measure of prognosis in colorectal cancer, certain molecular genetic markers are now considered to be useful for predicting the behavior of cancer and the clinical outcome of patients. We have described here the two different genetic route of colorectal carcinogenesis and the recent advances in prognostic factors of target genes of different carcinogenic route, such as bax on microsatellite instability (MIN) tumor, DCC on chromosomal instability (CIN) route. Also, we describe a matrix metalloproteinase matrilysin as an clinically useful prognostic markers. We hope that these molecular genetic markers with prognostic significance play an important role in disease management of patients with colorectal cancer near future.

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Hepatocyte growth factor (HGF) exhibits mitogenic, motogenic, morphogenic, and anti-apoptotic activities, through Met/HGF receptor. In a variety of tumor tissues, HGF is involved in malignant behavior of cancers as a mediator in tumor-stromal interaction, enhancing tumor invasion and metastasis. To establish a new strategy to inhibit tumor invasion and metastasis, we prepared an antagonistic molecule for HGF. The antagonist, NK4, is an internal fragment of HGF that encompasses the N-terminal hairpin and four kringle domains. Notably, NK4 inhibits tumor angiogenesis, growth and metastasis, as an angiogenesis inhibitor, as well as HGF-antagonist. In vitro, NK4 specifically inhibited tumor invasion induced by HGF. In contrast, NK4 inhibited growth and migration of microvascular endothelial cells induced by basic fibroblast growth factor (bFGF), vascular endothelial cell growth factor (VEGF), and HGF. NK4 inhibited bFGF-induced angiogenesis in rabbit cornea. We concluded that NK4 is bifunctional: it is HGF-antagonist and angiogenesis inhibitor. When the anti-tumor activity of NK4 was examined in murine metastatic tumors, Lewis lung carcinoma, administration of NK4 suppressed the growth of primary tumors subcutaneously implanted in mice, and decreased the number of metastatic nodules in the lung. NK4 suppressed neovascularization of tumor cells. Likewise, NK4 inhibited invasion, tumor angiogenesis, peritoneal dissemination, and liver metastasis of pancreatic cancer. The bifunctional properties of NK4 to act as an angiogenesis inhibitor and as a HGF antagonist raises possibility that NK4 may prove a therapeutic for cancer patients.

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A novel member of the epidermal growth factor (EGF) family, the neural- and thymus-derived activator for ErbB kinases (NTAK), has been purified and cloned. Four alternative spliced isoforms have been detected in the rat adrenal pheochromocytoma cell line, PC-12 cells. In vivo, NTAK is only expressed in the brain of rat E11.5 embryos, and in the brain and thymus of adult rats. The soluble 46kDa form binds directly to ErbB3 and B4, but not to ErbB1 or B2. NTAK, however, trans-activates ErbB1 and B2 via heterodimerization with ErbB3 or B4. NTAK stimulates the differentiation of MDA-MB-453 cells and competitively inhibits the binding of ¹²⁵I-neuregulin to these cells. In addition to these neuregulin-like properties, NTAK exhibits limited structural homology to neuregulins in the immunoglobulin (Ig)-like, EGF-like and hydrophobic domains. Thus, NTAK appears to be a new member of the EGF family displaying neuregulin properties.

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Isolated stellate cell on plastic dish indicated the proliferation and the expression of procollagen type I mRNA during 14 days culture. Thus culture system of stellate cell in vitro may reflect pathophysiology in vivo. A choline deficient L-amino acid defined (CDAA) diet led to the development of liver cirrhosis in 100% of male Wistar rats after 16 weeks and developed hepatocellular carcinoma in 90% of rats after one year. Concurrent administration of a prolyl 4-hydroxylase inhibitor, 2, 4-pyridine dicarboxylic acid bis [(2-methoxyethyl amide)] (HOE 077), to rats fed a CDAA diet reduced the increase in liver hydroxyproline content in a dose-dependent manner for doses up to 200ppm in parallel with the percent area of GSTP-positive lesions. Also 200ppm HOE 077 reduced the incidence of development of hepatocellular carcinoma from 90% to 50% one year after a CDAA diet administration. These data suggest that inhibition of fibrosis may limit the development of subsequent neoplasms in the liver.

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We have studied a relationship between high-molecular mass intestinal alkaline phosphatase (HIAP) found in blood group B and O secretors on 6.0% polyacrylamide disc gel electrophoresis in the presence of 1.0% TritonX-100 and the intestinal variant alkaline phosphatase (IAP-variant) on agarose gel electrophoretic method. The serum of 53 healthy subjects was tested by above two electrophoretic methods. The HIAP and IAP-variant were detected in the same subjects, and the levels of their activity were higher in serum of blood group B and O secretors. In addition, the HIAP and IAP-variant can be normalized to usual IAP by brief treatment with subtilisin or papain on respective methods. These results suggest that HIAP and TAP-variant are the same IAP isoform.

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We purified purple acid phosphatase (PAPase) from kidney bean (Phaseolus vulgaris L. Ohfuku) seeds, a Japanese cultivar of the kidney bean. The molecular mass of the purified native enzyme was estimated approximately 110kDa by gel filtration. Following SDS-PAGE in the presence of 2-mercaptoethanol, glycosylated polypeptides of major 61kDa and minor 59kDa were observed. The N-terminal amino acid sequences for both bands were determined to be GKSSNFVRKTNKNRDMPLDS, suggesting they were two different subunits with the same N-terminal but probably different C-terminal or degree of glycosylation. The partial nucleotide sequences covering the N-terminal region were also determined using polymerase chain reaction (PCR). Comparison with the deduced amino acid sequence from the nucleotide sequence and that of the purified enzyme revealed that the N-terminal amino acid residue corresponded to Gly 23 from the initiation colon (Met). For the deduced amino acid sequence of the PAPase gene, a SignalP (ver. 2.0) analysis program predicted that a 22 amino acid sequence at the N-terminus of a precursor protein was signal peptide. This prediction was in agreement with the fact that PAPase with a signal peptide at the N-terminus was cleaved between Gly 22 and Gly 23 by putative signal peptidase.

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Three types of the hereditary variant human amylase (AMY) of pancreatic (P) origin are known, dominant P₂, dominant P_2S, and slow P. A 67-year-old woman visited our clinic with chief complaints of epigastralgia and back pain, and she had hyperamylasemia during laboratory tests. Then, we found an extra band which migrated between P₁ and dominant P_2S by AMY isoenzyme analysis with cellulose acetate membrane electrophoresis of the patient's serum. The extra band did not react with the anti-human salivary-type AMY inhibitory monoclonal antibody, suggesting that the extra band seemed to be derived from P-type AMY, a novel P-type AMY variant. When the pedigree of this patient was investigated, the same P-type AMY variant could be detected in 7 of 14 family members, and we also found to be the novel band in both males and females of this family. Therefore, the type of inheritance of the AMY variant was considered to be autosomal dominant. The appearance rate of this novel P-type AMY variant should be investigated in future.

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