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Volume 43, Issue 2
Displaying 1-13 of 13 articles from this issue
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Haruo Saito, Mutsuhiro Takekawa1999 Volume 43 Issue 2 Pages 49-55
Published: June 15, 1999
Released on J-STAGE: March 31, 2009
JOURNAL FREE ACCESSThe MAP kinase signaling cascade (MAPKKK-MAPKK-MAPK) is well conserved in all eukaryotic cells. The yeast Hog cascade (Ssk2/Ssk22-Pbs2-Hog1) is structurally and functionally homologous to the mammalian stress-responsive p38 and JNK pathways that regulate apoptotic cell death. In order to identify positive or negative regulators for the p38 and JNK pathways, we devised several cloning strategies using the yeast HOG pathway mutants. First, we screened for human proteins whose expression in yeast complement the osmosensitivity of ssk2/ssk22Δ mutations. Thus, we found a human homolog of yeast Ssk2/22 MAPKKKs, termed MTK1, which mediates the stress-induced activation of the p38 and JNK pathways in mammalian cells. Second, we identified three distinct GADD45-related proteins that bound to an N-terminal domain of MTK1 using a yeast two-hybrid method. The GADD45-related genes are induced by environmental stresses. Moreover, these proteins activated MTK1 kinase activity both in vivo and in vitro, resulting in induction of p38/JNK activation and apoptosis which can be partially suppressed by coexpression of a dominant inhibitory MTK1 mutant protein. These results indicate that the GADD45-related proteins mediate activation of the p38 and JNK pathways, via MTK1, in response to environmental stresses. Third, we screened for human cDNA clones that down-regulate the mutational hyperactivation of the yeast HOG pathway. The human PP2Cα was found to negatively regulate the HOG pathway. Expression of PP2Cα in mammalian cells inhibited activation of the p38 and JNK cascades but not the ERK pathway. These findings indicate that PP2Cα plays a role in negative regulation of mammalian stress responses.View full abstractDownload PDF (8010K) -
Misao Ohki1999 Volume 43 Issue 2 Pages 57-61
Published: June 15, 1999
Released on J-STAGE: March 31, 2009
JOURNAL FREE ACCESSWe have isolated the NotI sites that exist on the long arms of human chromosomes 21 and 11 as NotI linking clones and constructed complete NotI restriction maps spanning the entire regions. These maps, which provide the most reliable ordering and distance estimation in the regions from pericentromeric loci to the termini, have led to the identification of four fusion genes including AML1-MTG8 in the breakpoints of chromosome translocations associated with myeloid leukemia.View full abstractDownload PDF (2647K) -
Functions of matrilysin, trypsin and laminin-5Kaoru Miyazaki, Satoshi Miyata, Naohiko Koshikawa, Hiroto Mizushima, H ...1999 Volume 43 Issue 2 Pages 63-67
Published: June 15, 1999
Released on J-STAGE: March 31, 2009
JOURNAL FREE ACCESSDuring complex process of tumor metastasis, tumor cells interact with various extracellular matrix proteins. These proteins regulate tumor invasion and metastasis positively or negatively. Tumor cells utilize various types of matrix-degrading proteinases to invade through basement membranes and connective tissues. These matrix proteinases are classified into two major groups: the matrix metalloproteinases (MMPs) such as gelatinases A and B, interstitial collagenase, stromelysin, matrilysin and MT-MMPs, and the matrix serine proteinases such as plasminogen activators, plasmin and trypsin. On the other hand, we have found laminin-5 (ladsin) as a tumor-derived cell scattering factor with potent cell adhesion and cell motility activities. This protein is assumed to contribute to tumor cell migration. This article describes our recent results on functions of matrilysin, trypsin and laminin-5 in the malignant growth of tumor cells.View full abstractDownload PDF (6059K) -
Ikunosuke Sakurabayashi1999 Volume 43 Issue 2 Pages 69-73
Published: June 15, 1999
Released on J-STAGE: March 31, 2009
JOURNAL FREE ACCESSSerum protein fractionation using capillary zone electrophoresis and the application is described. There is 20-30 major components in human serum protein, even though the several hundred proteins are exist. However, in serum protein fractionation by cellulose acetate membrane electrophoresis applied to the clinically, it is only five fractions which intermingle many proteins ingredients with one fraction. Clinically, combining change of the 5 fractions has been made to be the assistance of the diagnosis. In the meantime, capillary zone electrophoresis for excellent separation can be consequentially divided the serum proteins into 10 fractions. However, it becomes a result of being inconvenient on the contrary. The clinical significance equal to conventional electrophoresis of protein would be found when divided protein fractions are specially integrated for 5 fractions. The purpose was achieved by the CZE 2000 instrument (Beckman-Coulter Co., USA) and was obtained by the preparation of the software which arranges the serum protein fractions are originally divided into 10 peaks for 5 fractionation. Therefore, it is possible that can fundamentally follow clinical significance by cellulose acetate membrane electrophoresis and moreover, the data of 10 fractionation is also taken out. And, for the identification of regarded M-protein in cases of multiple myeloma and MGUS (monoclonal gammopathy undetermined significance), it is conveniently possible by subtraction method using the capillary electrophoresis. There is dissociation of α1 fraction value between cellulose acetate membrane electrophoresis and capillary electrophoresis. From our detailed experimental results, it is proved that is due to destaining of α1-acid glycoprotein.View full abstractDownload PDF (2514K) -
Koji Otsuka, Shigeru Terabe1999 Volume 43 Issue 2 Pages 75-78
Published: June 15, 1999
Released on J-STAGE: March 31, 2009
JOURNAL FREE ACCESSA brief review of interfaces for on-line coupling of capillary electrophoresis (CE) and mass spectrometry (MS) is presented. To enable the CE-MS system, electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI) were introduced as MS interfaces for connecting with CE instruments, while the partial-filling method was developed to suppress the introduction of non-volatile components or additives in CE buffers as well as ionic species such as ionic micelles into the MS interface. Strategy for the coupling of micellar electrokinetic chromatography (MEKC) and MS is also discussed. Examples of CE-MS are briefly shown.View full abstractDownload PDF (627K) -
Toyofumi Nakanishi1999 Volume 43 Issue 2 Pages 79-84
Published: June 15, 1999
Released on J-STAGE: March 31, 2009
JOURNAL FREE ACCESSProgress in medicine and molecular research has been increased by the need for simple and reliable methods of detection and characterization of aberrant proteins caused either by mutation or by post-translational modification. We applied electrospray ionization mass spectrometry (ESIMS) to detect and characterize abnormal structure proteins, and to determine the ratio of mutant and wild type proteins. Direct examination of hemolysate might well lead to rapid ascertainment of variant hemoglobins (Hbs) provided that the mass difference between the normal and the abnormal chain is larger than the resolution power of standard instruments. Various procedures are required in the presentation of most plasma and cell proteins other than Hb prior to MS. Two simple methods were deviced to prepare proteins. One was immunoprecipitation with antisera against target proteins, followed by reversed-phase HPLC/ESIMS, and the other was 2-dimensional LC (a strong anion exchange region and a reversed-phase resin) connected to ESIMS. With both methods, the ions of intact normal and variant proteins were clearly observed in samples from patients with neurodegenerative diseases such as familial amyloidotic polyneuropathy (FAP) and amyotrophic lateral screlosis (FALS). Using these procedures, we detected more than 40 cases of transthyretins (TTRs) mutants including to 10 different types. Two of these, [101 Gly->Ser] and [38 Asp->Ala], were new. We also successfully detected 4 different mutants of Cu/Zn-binding superoxide dismutase inerythrocytes, and a variant in spinal cord from patients with FALS by immunoprecipitation mehods. Two of these, [4Ala->Ser] and [111Cys->Tyr], were new. Finally we also measured glycated β-globin N-ternimus hexapeptide (HbA1c) by Poroszyme V8 protease digestion and nanoESIMS technique.View full abstractDownload PDF (831K) -
Yoshinobu Baba1999 Volume 43 Issue 2 Pages 85-89
Published: June 15, 1999
Released on J-STAGE: March 31, 2009
JOURNAL FREE ACCESSThe application of micromachining techniques to prepare microfabricated system for capillary electrophoresis is reviewed. Microstructures and capillaries, integrated detector systems, and integrated PCR micro-chamber are concepts that can be combined in a variety of ways to produce unique, miniaturized analytical systems for biomolecules. Microfabrication of 48 microchannels produces high throughput DNA analysis system. Integration of PCR and capillary electrophoresis was realized on a chip and allowed to amplification of DNA and fast analysis of the amplified fragments. Coupling of microfabricated capillary electrophoretic channels with mass spectrometer provides high throughput screening system for protein analysis, drug analysis, and DNA analysis.View full abstractDownload PDF (3060K) -
Johji Inazawa1999 Volume 43 Issue 2 Pages 91-95
Published: June 15, 1999
Released on J-STAGE: March 31, 2009
JOURNAL FREE ACCESSComparative genomic hybridization (CGH) is a powerful technique to identify novel amplification sites through all human chromosomes. We employed CGH to explore genomic imbalances in 19 malignant fibrous histiocytomas (MFHs). Together with losses and gains in various chromosome regions, distinct high-level amplifications were found in six loci (4q12-21, 8p21-pter, 8q24.1-qter, 9p12-13, 12p11.2-pter, and 15q11.2-15), suggesting that those regions may contain unknown genes responsible for carcinogenesis. We focused on the 8p amplicon, and a novel gene designated MASL1 (MFH-amplified sequences with leucine-rich repeats 1) was isolated from within this narrowly defined region. Expression of the MASL1 gene was enchanced significantly in MFH tumors bearing the 8p amplicon. The primary structure of its deduced product revealed an ATP/GTP-binding site, three leucine zipper domains, and a leucine-rich tandem repeat, all of which are important structural or functional elements for interactions among proteins related to the cell cycle. These features suggest that overexpression of MASL1 might well be oncogenic with respect to MFH.View full abstractDownload PDF (4500K) -
Fuyuki Ishikawa1999 Volume 43 Issue 2 Pages 97-102
Published: June 15, 1999
Released on J-STAGE: March 31, 2009
JOURNAL FREE ACCESSThe chromosome ends, called telomeres, are essential for stable transmission of chromosomes. Recent progress in molecular understanding of telomere structures and functions have enabled us to reason how telomere insufficiencies lead to chromosome instability. Examples that can be explained by this hypothesis include cellular aging and cancer progression.View full abstractDownload PDF (956K) -
Tetsuya Moriuchi, Keiji Furuuchi, Hidenori Yashima, Tetsuya Aoyama, Hi ...1999 Volume 43 Issue 2 Pages 103-106
Published: June 15, 1999
Released on J-STAGE: March 31, 2009
JOURNAL FREE ACCESSRecent research on genetic alterations found in human cancers has shown that inactivation of a tumor suppressor gene is a major step in the development of cancers. Two strategies have so far been widely used to study tumor suppressor genes in clinical materials: screening with antibodies and DNA structure-based screening. Recently, a different approach based on a biological assay for tumor suppressor gene function has been devised by using yeast as a living test tube. This method takes advantage of the fact that gapped plasmid is repaired by homologous recombination between the linear plasmid and DNA fragment in yeast. The yeast assay in which yeast change color according to the status of tumor suppressor gene raises the very real possibility of simple and large-scale screening for the genetic mutations in clinical samples. We describe here two conceptually different yeast assays: p53 yeast functional assay and APC premature termination assay.View full abstractDownload PDF (3635K) -
Fumio Itoh, Hiroyuki Yamamoto, Yuji Hinoda, Kohzoh Imai1999 Volume 43 Issue 2 Pages 107-109
Published: June 15, 1999
Released on J-STAGE: March 31, 2009
JOURNAL FREE ACCESSWidespread microsatellite instability (MSI) due to the defective DNA mismatch repair underlies the pathogenesis of the majority of hereditary non-polyposis colorectal cancer and a subset of various sporadic malignant tumors. Using 5 microsatellite markers and the criteria of MSI proposed by the National Cancer Institute (NCI) workshop, we analyzed 205 gastric adenocarcinomas for MSI. Based on the number of markers showing instability per tumor, the tumors were divided into three groups; those with two or more of the five markers displaying instability (high MSI, MSI-H), those with one of five markers displaying instability (low MSI, MSI-L), and those with no instability (microsatellite stable, MSS). Among 205 tumors, 30 (15%) were MSI-H. 15 (7%) were MSI-L, and 160 (78%) were MSS. All of the 30 MSI-H tumors demonstrated instability at BAT26, a sensitive marker for the widespread MSI, while none of the 15 MSI-L tumors did. MSI-H tumors were significantly associated with distal location and well or moderate differentiation, but MSI-H tumors were indistinguishable from MSS tumors. Bax frameshift mutations were detected in 60% of the 30 MSI-H tumors, while not in any of the 15 MSI-L tumors. These results suggest that microsatellite analysis using the criteria proposed by the NCI workshop may appropriate for gastric cancers because it unveils real differences in genotype and phenotype.View full abstractDownload PDF (437K) -
Toshio Okazaki, Takayuki Nakagawa, Tomoyuki Kamakura, Yuko Yanagisawa, ...1999 Volume 43 Issue 2 Pages 111-115
Published: June 15, 1999
Released on J-STAGE: March 31, 2009
JOURNAL FREE ACCESSHemoglobin (Hb) in the blood exhibited rapidly release of heme under free forms, and the resulting heme was transferred to albumin from Hb. Heme-liberating from Hb was not observed when the Hb was combined with haptoglobin (Hp) molecule. They were clarified using zymographical staining of heme. Moreover, it is proved that albumin incubated with fatty acid can induce the releasing of heme from Hb molecules.View full abstractDownload PDF (4553K) -
Katsumi Kawano, Nozomu Hibi, Katsuo Kubono, Mitsunori Yagame, Hideto S ...1999 Volume 43 Issue 2 Pages 117-121
Published: June 15, 1999
Released on J-STAGE: March 31, 2009
JOURNAL FREE ACCESSAdvanced glycation end products (AGEs) produced at the end stage of Maillard reaction are suggested to be one of the substances causing diabetic complications. We have developed EIA for serum AGE and reported the relationship between AGE levels and diabetic complications. In this study, we investigated AGE with respect to its mode of existence in peritoneal dialysate, as the substitute for serum, from patients with renal failure. By using gel filtration, immunoaffinity chromatography and western blotting, it was found that major part of AGE was present in relatively high molecular weight fractions, and bound to serum proteins. Of these proteins IgG, albumin and β2-microglobulin were identified.View full abstractDownload PDF (3510K)
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