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Volume 45, Issue 1
Displaying 1-13 of 13 articles from this issue
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Shin-ichi Fujimaki, Tadao Funato2001 Volume 45 Issue 1 Pages 1-3
Published: March 15, 2001
Released on J-STAGE: March 31, 2009
JOURNAL FREE ACCESSIn many applications, an understanding of differentially expressed genes in different tissues or owing to an applied stimulus is important. However, the wide use of polymerase chain reaction (PCR)-based techniques for the identification of differentially expressed mRNAs (RNA fingerprinting by arbitrarily primed PCR) and differential display (DD) has shown that using radioisotopes is still a problem. Although a technique has been developed that avoids some of the disadvantages, the use of radioisotopes for band detection still limits various applications. It has been improved this technique for analyzing differentially expressed mRNA by resolving the amplified products using fluorescent-labeling as a non-radioisotope. Our modified method allows the improvement of identification on differentially expressed bands with a high accuracy. This technique with a non-radioisotope representation may be possible to perform PCR-DD analysis with many applications.View full abstractDownload PDF (2118K) -
Masato Maekawa, Kokichi Sugano2001 Volume 45 Issue 1 Pages 5-8
Published: March 15, 2001
Released on J-STAGE: March 31, 2009
JOURNAL FREE ACCESSSingle-strand DNA conformation polymorphism (SSCP) analysis has been used to detect alterations in relatively short DNA fragments such as genetic polymorphisms, mutations, deletions, and so on. Here we introduce other applications of SSCP analysis, using its capacity of separating alleles. A sensitive method, designated as blunt-end SSCP analysis was developed for detecting loss of heterozygosity (LOH) in cancer tissues and urine samples. The method is fluorescence-based SSCP analysis, using blunt-end DNA fragments and applied for detecting an LOH of the p53 gene. The combination of reverse transcribed-PCR and fluorescence-based SSCP analysis is proposed for the quantitative determination of ratio of mRNA molecules with homologous sequences. The procedure is applicable to a determination of expression levels of genes such as lactate dehydrogenase subunits and cyclooxygenases 1 and 2. The combination of bisulfite treatment and PCR-SSCP analysis is proposed for quantitative methylation assay. This analytic procedure can be applied to the rapid identification of methylation status in multiple samples, quantification of methylated vs. unmethylated sequences and detection of methylation heterogeneity in the amplified DNA fragments. PCR-SSCP analysis is advantageous in simple procedure with relatively high sensitivity and is applicable to any other fields.View full abstractDownload PDF (2492K) -
Yoichi Matsubara2001 Volume 45 Issue 1 Pages 9-10
Published: March 15, 2001
Released on J-STAGE: March 31, 2009
JOURNAL FREE ACCESSMutation detection in various genetic disorders has been facilitated by recent advances in nucleotide sequencing technology. An introduction of capillary electrophoresis enabled sequencing analysis to be performed in clinical service laboratory. On the other hand, previously identified known mutations can be readily detected by TaqMan-allele specific amplification, which has been developed in our laboratory.View full abstractDownload PDF (334K) -
Eiichi Sakai, Tsutomu Tanaka, Mitsuko Mori, Kan-ichi Nakagawara2001 Volume 45 Issue 1 Pages 11-16
Published: March 15, 2001
Released on J-STAGE: March 31, 2009
JOURNAL FREE ACCESSIf I classify roughly for the quantification method of mRNA, there are the following 5 kinds. 1. Northern and dot hybridization, 2. RNase protection assay, 3. RT-PCR (the use of internal control), 4. competitive RT-PCR (the use of competitor), 5. real time monitoring PCR. In these methods, 3-5 employ PCR. Though 3, 4 are a method to quantify at an exponential increase term, it is different point that 5 is quantification method by means of PCR cycle number to exceed a detection limit of PCR product, just before entering an exponential increase term. Recently, a quantification method by the real time monitoring PCR basks in attention. Not only this method isn't necessary to confirm a cycle number of an exponential increase term in advance but have the wide quantification range in comparison to the method to quantify at an exponential increase term, there are many merits. I introduce LightCyclerTM system (Roche Diagnostics) with this draft as an equipment to be able to do a realtime monitoring.View full abstractDownload PDF (6221K) -
Michiei Oto2001 Volume 45 Issue 1 Pages 17-22
Published: March 15, 2001
Released on J-STAGE: March 31, 2009
JOURNAL FREE ACCESSPCR-based analysis of genetic alterations has been applied to the diagnosis of various diseases, however, for this technique to become widely accepted as a tool for clinical DNA diagnosis, the development of automated analytical system is essential. Capillary electrophoresis (CE) is a promising alternative to gel electrophoresis for high-speed and reproducible separation of DNA fragments, and its use is feasible for automated DNA analysis. Therefore, CE is applied for DNA sequencing and the detection of genetic changes including microsatellite instability and mutations. Recently, capillary array electrophoresis for the simultaneous analyses of many samples is applied for the automated high-throughput sequencing. Heteroduplex analysis has been reported to be a promising technique for detecting mutated sequences because of its higher sensitivity than that of SSCP analysis. In this analysis, heteroduplex DNA containing mutated sequences is separated by denaturing gradient gel electrophoresis (DGGE). However, in DGGE, PCR products joined to a GC clamp are necessary and the gel preparation is also complicated. Therefore, it is difficult to automate. Denaturing high-performance liquid chromatography (DHPLC) has been reported to be a novel automated high-throughput technique for separating heteroduplex and homoduplex DNA fragments amplified with primers without a GC clamp under controlled temperature conditions. Hetroduplex analysis involving DHPLC enable more accurate, more rapid and easier heterozygous mutation detection than SSCP analysis. Hence, it is suggested that CE is preferable for DNA sizing and sequencing analyses, and DHPLC is effective for screening of genetic mutations and polymorphisms.View full abstractDownload PDF (934K) -
2001 Volume 45 Issue 1 Pages 23-44
Published: March 15, 2001
Released on J-STAGE: March 31, 2009
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2001 Volume 45 Issue 1 Pages 45-64
Published: March 15, 2001
Released on J-STAGE: March 31, 2009
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Yan-Ping Feng, Akira Kondo, Masato Maekawa, Takashi Kanno2001 Volume 45 Issue 1 Pages 65-68
Published: March 15, 2001
Released on J-STAGE: March 31, 2009
JOURNAL FREE ACCESSWe investigated whether cholesteryl ester transfer protein (CETP) deficiency is a risk factor for atherosclerosis or other disease. Ninety-seven individuals with increased levels of high density lipoprotein-cholesterol (HDL-C) (≥85mg/dl) were selected. CETP deficiency was assessed in all 97 subjects by polymerase chain reaction (PCR) and either single stranded conformation polymorphism (SSCP) or restriction fragment length polymorphism (RFLP). Of the 97 subjects, 7 (7%) were heterozygous for the intron 14 splicing defect, which decreases CETP activity, and 9 (9%) were heterozygous for D442G missense mutation, which has no effect on CETP activity. One patient was found to be a compound heterozygote for the intron 14 and D442G mutations. These mutations were found in only 3 atherosclerotic patients; 2 carried the D442G mutation, and 1 had the intron 14 mutation. Although CETP activity was not associated with atherosclerosis, we discovered a novel relation between CETP gene mutations and thyroid gland disorders. Specifically, 5 of 7 subjects with CETP gene intron 14 mutation had thyroid disease. Regarding the D442G mutation, there was no difference between subjects with and without thyroid disease. It is possible that CETP activity affects thyroid function.View full abstractDownload PDF (735K) -
Hiroshi Ihara, Natsuko Toya, Takashi Kakinoki, Asuka Tani, Yutaka Aoki ...2001 Volume 45 Issue 1 Pages 69-74
Published: March 15, 2001
Released on J-STAGE: March 31, 2009
JOURNAL FREE ACCESSWe performed capillary zone electrophoresis (CZE) on serum specimens from 129 healthy Japanese volunteers, ages 7 to 90 years. Our goal was to estimate the reference ranges for the five major serum protein fractions: albumin, α1-globulin, α2-globulin, β-globulin, γ-globulin and for the five minor fractions: in which prealbumin, hemopexin, α1-acid glycoprotein, α1-antitrypsin or transferrin predominated. Results by CZE for the above ten fractions correlated well (p<0.001) with the assays by CAE or immunonephelometry for the same protein fraction. Owing to the Gaussian distribution of the data, we can state the reference ranges as the mean ±2SD. For α1-acid glycoprotein, α1-antitrypsin and transferrin fractions, reference values were estimated only in terms of percent composition. Our reference values (concentrations in parenthesis) for CZE were albumin, 52.7 to 67.1% (3.9-5.2g/dl); α1-globulin, 3.6 to 6.8% (0.27-0.52g/dl); α2-globulin, 4.8 to 9.0% (0.36-0.69g/dl); β-globulin, 8.2 to 13.5% (0.59-1.06g/dl); and γ-globulin, 10.7 to 22.5% (0.73-1.81g/dl); prealbumin, 0.1 to 0.9% (10.7-66.4mg/dl); hemopexin fraction, 1.1 to 2.7% (76-212mg/dl); α1-acid glycoprotein fraction, 1.3 to 3.4%; α1-antitrypsin fraction, 2.0 to 3.8%, and transferrin fraction, 3.7 to 6.1%.View full abstractDownload PDF (991K) -
Gary B. Smejkal2001 Volume 45 Issue 1 Pages 75-81
Published: March 15, 2001
Released on J-STAGE: March 31, 2009
JOURNAL FREE ACCESSSeveral dyes were evaluated for their potential use as indices of ionic boundaries and pH discontinuities during polyacrylamide gel electrophoresis (PAGE). Dye selection was based largely upon color transition over the pH intervals present in the commonly used Tris-HCl-glycine buffer systems. In the absence of sodium dodecylsulfate (SDS) nitrazine yellow, cresol purple, cresol red, and phenol red had relative mobilities (relative to the chloride boundary; RC1) of 1.00 at 20% polyacrylamide concentration (% T) compared to bromophenol blue (BPB) for which RC1=0.77. The homologue of BPB, 3, 4, 5, 6-tetrabromophenolsulfonephthalein (TBPS), exhibited a useful color transition over the pH 6.6-8.2 range but was an equally poor indicator of the chloride boundary at 20% T in the absence of SDS (RC1=0.73). The RC1 of other dyes was increased in the presence of SDS, particularly the cationic dye neutral red which complexes with micellar but not monomeric SDS. In the presence of SDS, all dyes except neutral red exhibited RC1 of 1.00 at 15% T, whereas only neutral red and nitrazine yellow had RC1 values of 1.00 at lower gel concentrations. Partial characterization of SDS, in terms of RC1 and zone broadening, was performed in a system devoid of extraneous SDS in the gel or electrolytes.View full abstractDownload PDF (4788K) -
Kinue Ohi, Yuko Sakurai, Tsutomu Nobori2001 Volume 45 Issue 1 Pages 83-88
Published: March 15, 2001
Released on J-STAGE: March 31, 2009
JOURNAL FREE ACCESSLDL fraction was qualitatively analyzed by simultaneous determination of serum cholesterol and triglyceride (TG) by agarose gel electrophoresis. Modified low density lipoprotein (LDL) showing faster electrophoretic mobility was easily distinguished from native LDL. Modification frequency is determined by a relative change of the mobility shift to the anode. Modification frequency greater 8 is defined as positive modified LDL. The modified LDL was found in 46.9% of 131 patients with higher level of TG and 4.1% of 147 patients with normal TG level. 73.4% of 53 patients with modified LDL had the level of TG≥300mg/dl. These data suggested that modified LDL was mostly consisted of small dense LDL with a high level of TG. However, the level of TG was decreased faster than that of modified LDL.View full abstractDownload PDF (1003K) -
Youji Shimazaki, Masayoshi Muro, Takashi Manabe2001 Volume 45 Issue 1 Pages 89-92
Published: March 15, 2001
Released on J-STAGE: March 31, 2009
JOURNAL FREE ACCESSAfter the albumin content of human plasma proteins was decreased by the addition of polyethylene glycol, the plasma proteins were separated in non-denaturing isoelectric focusing (IEF) gel. The separated proteins in the IEF gel were digested with one of three enzymes; trypsin, Achromobacter protease I (API) or Staphylococcus aureus V8 protease (V8). Proteins in the gel were boiled in the presence of mercaptoethanol/SDS, and constituent polypeptides of proteins were separated by SDS-PAGE. The electrophoretic pattern of polypeptide spots were compared to that of non-denaturing IEF/SDS-PAGE. The α chain of the third component of complement (C3α) was effectively digested with trypsin, but albumin and IgG were not effectively fragmented with trypsin. On the other hand, albumin was digested with API and V8 protease, but C3α was not effectively fragmented with these enzymes. These results indicate that trypsin is the most effective enzyme for the fragmentation of C3 in the IEF gel.View full abstractDownload PDF (5395K) -
Kyoko Akano, Tatsuo Tozawa2001 Volume 45 Issue 1 Pages 93-95
Published: March 15, 2001
Released on J-STAGE: March 31, 2009
JOURNAL FREE ACCESSLactate dehydrogenase (LDH) anomaly was observed in cold storage serum of 94 years old female patient. There were an extra band and tailing between LDH 3 and LDH 4 isoenzyme. The immunoprecipitation technique revealed that LDH of this patient was linked to IgG and IgA. However, in the fresh serum, the isoenzyme pattern was normal, and immunoglobulins linked to LDH weren't detected. This anomalous pattern was reduced when a cold storage serum was left for one night at room temperature. Our results indicated that this anomaly was reversible. It had already been reported that LDH anomaly sometimes appeared with cold storage of serum in which there were inhibitory factors to LDH. The present case was characterized by reversible change of LDH anomaly and no reduction of LDH activity.View full abstractDownload PDF (2313K)
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