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45 巻, 1 号
選択された号の論文の13件中1～13を表示しています
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藤巻 慎一, 舩渡 忠男2001 年 45 巻 1 号 p. 1-3
発行日: 2001/03/15
公開日: 2009/03/31
ジャーナル フリーIn 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.抄録全体を表示PDF形式でダウンロード (2118K) -
前川 真人, 菅野 康吉2001 年 45 巻 1 号 p. 5-8
発行日: 2001/03/15
公開日: 2009/03/31
ジャーナル フリーSingle-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.抄録全体を表示PDF形式でダウンロード (2492K) -
松原 洋一2001 年 45 巻 1 号 p. 9-10
発行日: 2001/03/15
公開日: 2009/03/31
ジャーナル フリーMutation 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.抄録全体を表示PDF形式でダウンロード (334K) -
酒井 栄一, 田中 勉, 森 光子, 中川原 寛一2001 年 45 巻 1 号 p. 11-16
発行日: 2001/03/15
公開日: 2009/03/31
ジャーナル フリーIf 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.抄録全体を表示PDF形式でダウンロード (6221K) -
大藤 道衛2001 年 45 巻 1 号 p. 17-22
発行日: 2001/03/15
公開日: 2009/03/31
ジャーナル フリーPCR-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.抄録全体を表示PDF形式でダウンロード (934K) -
電気泳動の将来は如何にあるべきか?2001 年 45 巻 1 号 p. 23-44
発行日: 2001/03/15
公開日: 2009/03/31
ジャーナル フリーPDF形式でダウンロード (5908K) -
2001 年 45 巻 1 号 p. 45-64
発行日: 2001/03/15
公開日: 2009/03/31
ジャーナル フリーPDF形式でダウンロード (10596K) -
Yan-Ping Feng, 近藤 明, 前川 真人, 菅野 剛史2001 年 45 巻 1 号 p. 65-68
発行日: 2001/03/15
公開日: 2009/03/31
ジャーナル フリーWe 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.抄録全体を表示PDF形式でダウンロード (735K) -
渭原 博, 戸谷 夏子, 柿木 孝志, 谷 あすか, 青木 豊, 橋詰 直孝, 稲田 義信, 難波 俊二, 浦山 功, 吉田 光孝2001 年 45 巻 1 号 p. 69-74
発行日: 2001/03/15
公開日: 2009/03/31
ジャーナル フリーWe 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%.抄録全体を表示PDF形式でダウンロード (991K) -
Gary B. Smejkal2001 年 45 巻 1 号 p. 75-81
発行日: 2001/03/15
公開日: 2009/03/31
ジャーナル フリーSeveral 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.抄録全体を表示PDF形式でダウンロード (4788K) -
大井 絹枝, 櫻井 裕子, 登 勉2001 年 45 巻 1 号 p. 83-88
発行日: 2001/03/15
公開日: 2009/03/31
ジャーナル フリーアガロースゲル電気泳動法によるコレステロール分画・トリグリセライド分画同時解析法により, 変性LDLの検討を行った. 変性LDLは, 陽極側への相対的移動度を数値化して変性度数とし, 8以上を陽性とした. 変性LDLはTG高値で高率に出現し, LDL分画においてTGの増加を認めた. 変性LDL出現の主な原因はTG高値によるTG rich な小粒子LDLと考えられ, TGが高値から低下した例ではTGの低下よりも変性LDLの消失のほうが遅れる傾向にあった.抄録全体を表示PDF形式でダウンロード (1003K) -
島崎 洋次, 室 征芳, 真鍋 敬2001 年 45 巻 1 号 p. 89-92
発行日: 2001/03/15
公開日: 2009/03/31
ジャーナル フリーポリエチレングリコール分画法によりアルブミン量を減らしたヒト血漿タンパク質を未変性条件の等電点電気泳動法 (IEF) により分離した. この等電点ゲル内で分離されているタンパク質を, トリプシン, Achromobacterprotease I (API) または, Staphylococcus aureus V8 protease (V8) で酵素処理した後, SDS-メルカプトエタノール存在下で, 変性還元させ, タンパク質を構成するポリペプチドをSDS-PAGEで分離した. 酵素未処理やそれぞれの酵素処理をしたもので, 得られたポリペプチドスポットのIEF-SDS-PAGEパターンを比較した. 補体第3成分のα鎖 (C3α) がトリプシンにより効率よく断片化されているのに対し, アルブミンやIgGなどはこの酵素では断片化されにくいことがわかった. 一方, APIやV8では, アルブミンが効率よく断片化されているのに対し, C3αが断片化されにくいことがわかった. これらの結果から, 未変性条件のIEF内で, トリプシンはC3を効率よく断片化する酵素であることがわかった.抄録全体を表示PDF形式でダウンロード (5395K) -
赤野 香子, 戸沢 辰雄2001 年 45 巻 1 号 p. 93-95
発行日: 2001/03/15
公開日: 2009/03/31
ジャーナル フリーLactate 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.抄録全体を表示PDF形式でダウンロード (2313K)
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