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Volume 44, Issue 3
Displaying 1-12 of 12 articles from this issue
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Minoru Toyota, Fumio Itoh, Kohzoh Imai2000 Volume 44 Issue 3 Pages 157-161
Published: September 15, 2000
Released on J-STAGE: March 31, 2009
JOURNAL FREE ACCESSMethylation plays an important role in gene silencing in cancer. Recent progress in analyzing methylation status of the promoter region allowed us to explore screening of multiple samples and detail methylation mapping with less laborious work. Among these, PCR based methylation assay using the DNA treated with sodium-bisulfite greatly contribute to examine methylation profiles in cancer. Using bisulfite-PCR techniques, such as MSP, COBRA and B-SSCP, it is possible to perform accurate and semiquantitative methylation mapping of the genes of interest.View full abstractDownload PDF (712K) -
Katsunori Aizawa, Yoshihide Hayashizaki2000 Volume 44 Issue 3 Pages 163-171
Published: September 15, 2000
Released on J-STAGE: March 31, 2009
JOURNAL FREE ACCESSDNA sequencing method is still limited by the resolution attainable in the electrophoretic separation. Conventionally, DNA sequencing has been achieved with polyacrylamide slab gel electrophoresis. Rapid sequencing methods are now being developed in the forms of capillary electrophoresis or microchip electrophoresis using entangled polymers as alternative media. However, the lower resolution separations of DNA was frequently found in the ultra-fast analyses. The development of new media would be the best way to attain the reliable and rapid DNA sequencing with higher resolution.View full abstractDownload PDF (1759K) -
Izuho Hatada2000 Volume 44 Issue 3 Pages 173-175
Published: September 15, 2000
Released on J-STAGE: March 31, 2009
JOURNAL FREE ACCESSMassive analysis of DNA methylation has become important because it is an effective way to detect changes of expression level of genes with low expression level. Changes of expression level can be detected as changes of DNA methylation because expression level is correlated with DNA methylation level. Massive analysis of DNA methylation can be achieved by using Restriction Landmark Genome Scanning (RLGS) method. I have been successfully used this method for searching new imprinted genes.View full abstractDownload PDF (475K) -
Jun-ichi Asakawa, Mieko Kodaira, Naofumi Ishikawa, Kunihiko Itoh, Koic ...2000 Volume 44 Issue 3 Pages 177-180
Published: September 15, 2000
Released on J-STAGE: March 31, 2009
JOURNAL FREE ACCESSWe have developed a novel two-dimensional display method of cDNA and compared the gene expression profile between human normal thyroid and papillary carcinoma. The cDNAs were synthesized with a Not I anchor primer from mRNAs prepared from surgery materials. The cDNAs were digested with three restriction enzymes, Not I, Eco RV, and Pvu II. The protruding Not I ends were filled in with 32P deoxynucleotides and the isotope-labeled cDNA fragments were separated into two dimensions. The cDNAs were in situ digested with Hinf I before the second dimension electrophoresis. Using our conditions, approximately 500 cDNA fragments were visualized as major discrete spots on a single autoradiogram without probes. We cloned two cDNA spots selected from among a number of spots which showed apparently different intensities between normal thyroid and papillary carcinoma. One spot (Nc-4) was more intense in the normal tissue than in the tumor and the other (Pc-1) was more intense in the tumor. A Northern blot analysis with clone Nc-4 showed high expression in a normal thyroid sample but low expression in a papillary carcinoma sample. A Northern blot analysis with the Pc-1 clone confirmed the over-expression of this mRNA in papillary carcinomas. This method of separating the labeled cDNA into two dimensions is much more accurate and powerful than conventional differential display methods. In addition, this method is quite rapid; one can conduct a series of experiments, gel preparation and cloning, within about 10 days. We believe that this approach, in combination with 2D genomic DNA analysis, is useful for cancer research.View full abstractDownload PDF (5471K) -
Tosifusa Toda2000 Volume 44 Issue 3 Pages 181-184
Published: September 15, 2000
Released on J-STAGE: March 31, 2009
JOURNAL FREE ACCESS“PROTEOME”is a compound term of“PROTEin”and“GenOME”, that means all translated proteins and post-translationally modified products in a celltype. For proteome profiling, proteins are separated by two-dimensional gel electrophoresis in an IPG-DALT system and identified by mass spectrometry. A huge number of dataobtained by the proteome profiling are compiled in a WWW homepage for serving the proteome database through the Internet.View full abstractDownload PDF (2804K) -
Towards large-scale analysis and identification of disease-related proteinsTakao Kawakami, Fumihiko Usui, Toshihide Nishimura2000 Volume 44 Issue 3 Pages 185-190
Published: September 15, 2000
Released on J-STAGE: March 31, 2009
JOURNAL FREE ACCESSWe describe an integrated liquid chromatography (LC) -nanoelectrospray/tandem mass spectrometry (nanoES/MS/MS) system for identification of protein in mid atto mole amount. Peptides from enzymatic or chemical digest of protein were injected into the nanoLC column by the auto-sampler linked with the system. The peptide effluents were directly interfaced with the home-made nanoelectrospray ion source on an ion-trap (IT) mass spectrometer equipped with a nanospray capillary needle. Peptides were analyzed by MS and MS/MS. MS/MS scans, containing peptide sequence information, were subjected to the tandem mass correlation algorithm SEQUESTTM and searched against protein sequence databases to identify the authentic protein. Database search with amino acid sequencing is conclusive for protein identification. The present on-line analysis system is capable of analyzing forty samples per day, and applicable to in-gel enzymatic digests of protein stained by silver or a fluorescent dye, SYPROTM Orange, on one- or two-dimensional polyacrylamide gel. These advantages satisfy the requirements for proteomics research, all-inclusive study of proteins expressed by a genome or tissue.View full abstractDownload PDF (959K) -
Hideyuki Kajiwara2000 Volume 44 Issue 3 Pages 191-194
Published: September 15, 2000
Released on J-STAGE: March 31, 2009
JOURNAL FREE ACCESSOne of Australian graduated student made a keyword“proteome”based on“PROTEin” and“genOME”in 1995. Since then, proteomics became an important research theme in the post-genome era. Australian Proteome Analysis Facility was established in 1996 and the group at Melbourne started the analysis of cancer based on the new concepts of proteomics. I introduced the organisations and the targets of the proteome research in Australia in this review.View full abstractDownload PDF (705K) -
Hisashi Hirano2000 Volume 44 Issue 3 Pages 195-199
Published: September 15, 2000
Released on J-STAGE: March 31, 2009
JOURNAL FREE ACCESSComplete genome sequences have been determined in many organisms so far and abundant information of the gene products was obtained from the sequences, but majority of them has unknown functions. Therefore, the analysis of numerous gene products, proteome analysis, is necessary to understand the functions of genes and proteins in the post-genome research era. The pioneer works on a large scale proteome analysis were performed in the United States, European countries and Australia in 1990's. Unlike these countries, Japan failed to start systematically to analyze the proteomes at the early stage. This review describes briefly the current status of proteome analysis in Japan.View full abstractDownload PDF (851K) -
Strategy to clear the discrepancy between histological findings and RT-PCR results in the detection of micrometastasisToyoji Sato, Anura Weerasinghe, Yuh Kuwano, Takushi Kaneko, Takakazu I ...2000 Volume 44 Issue 3 Pages 201-204
Published: September 15, 2000
Released on J-STAGE: March 31, 2009
JOURNAL FREE ACCESSWe have studied the expression of keratin 19 mRNA (K-19) in the axillary lymph nodes of breast cancer patients (n=100, serially from October 1997 to May 1998) in order to detect the micrometastasis by reverse transcriptase-polymerase chain reaction (RT-PCR). During the course of this study, (1) we encountered the 6 cases showing [n+, K-19(-)]. (2) To clear the discrepancy of the finding between histology and RT-PCR, some other primer pairs were used. In one case out of 6, K-19 was detected. (3) Further, the sequence of RT-PCR products showed diversity and could be roughly classified into 4 groups. Namely, group a (same sequence to the report of Genbank except for two bases), group b (almost same to group a except for one deletion and several point mutations), group c (approximate 50 bases were deleted from group a and with approximate 50 point mutations) and group d (almost same to group c except for several point mutations). Until now, we have not read the reports describing [n+, K-19(-)]. The discrepancy like these cases we experienced were partially cleared by using appropriate primer pairs.View full abstractDownload PDF (2243K) -
Kenji Ito, Yayoi Kimura, Hidenori Sassa, Hisashi Hirano2000 Volume 44 Issue 3 Pages 205-210
Published: September 15, 2000
Released on J-STAGE: March 31, 2009
JOURNAL FREE ACCESSN-Acetylation, catalyzed with N-acetyltransferase (NAT), is one of the most common co- and post-translational modifications of the α amino group of eukaryotic proteins. The eukaryotic 20S proteasome contains seven α-type and seven β-type subunits. We have found that the N-terminus of α1, α2, α3, α4, α5, α6, α7, β3 and β4 subunits of yeast proteasome is acetylated with NAT. Experiments using the yeast NAT deletion mutant suggested that N-acetylation be related to the proteolytic activity of the 20S proteasome (Kimura et al. J Biol Chem 2000; 275: 4635-9). In the present study, the rice 20S proteasome was purified by ion-exchange chromatography, gel filtration and glycerol density gradient centrifugation from the rice bran, and 14 subunits contained in the 20S proteasome were separated by two-dimensional electrophoresis and the partial amino acid sequence of them was analyzed by Edman degradation. The sequence analysis indicated that among 14 subunits, the α1, α2, α3, α4, α5, α6, α7, β3 and β4 subunits are N-terminally blocked in rice as well as yeast, and besides these subunits, the β6 subunit is blocked in rice, but not in yeast. This suggests that the β6 subunit in rice may have different functions from that in yeast.View full abstractDownload PDF (2903K) -
Takaho Tanaka2000 Volume 44 Issue 3 Pages 211-216
Published: September 15, 2000
Released on J-STAGE: March 31, 2009
JOURNAL FREE ACCESSSenescence accelerated mice (SAM-P8), which have been established as a model of early senescence, were used for analyses of glutamate decarboxylase 67kDa isoform (GAD67) and extracellular regulated kinase-2 (ERK-2) which were altered by aging. The mice was characterized by neurological abnormal signs such as loss of memory, disorientation and weak response to stimuli, which appeared from 8 to 10 weeks-old. Those neurological disorders might be associated with changes in functions of brain proteins and enzymes regulating metabolic conversion of neurotransmitters and signal transduction. SDS-polyacrylamide gel electrophoresis of proteins in brains of male SAM-P8 showed that alterations in electrophoretic patterns of proteins, which would be involved in the development of brain, according to aging at 8 and 12 weeks in comparison with control mice (SAM-R1) although the alterations were not evidenced in female SAM-P8. Examinations of enzymes of GAD67 and ERK-2 by immunoblotting showed the decrease in their immunoreactivity in male SAM-P8 8 weeks-old, but not in female SAM-P8. These results suggest that the neurological disorder in male SAM-P8 is associated with the impairment of protein biosynthesis for the normal development of brain and with the decrease in enzymes for converting glutamate to gamma-aminobutyrate and for signal transduction of the response to stimuli. Furthermore, there are sex differences in the decrease of those enzymes by aging.View full abstractDownload PDF (4708K) -
2000 Volume 44 Issue 3 Pages 226
Published: 2000
Released on J-STAGE: March 31, 2009
JOURNAL FREE ACCESSDownload PDF (69K)
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