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Motokazu Mukaide,
Yasuhito Tanaka,
Hirokazu Kakuda,
Kei Fujiwara,
Fuat Kurbanov,
Eturo Orito,
Kentaro Yoshioka,
Kiyotaka Fujise,
Shoji Harada,
Takazumi Kozaki,
Kazuo Takemura, Kazumasa Hikiji,
Masashi Mizokami
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ABSTRACT: To develop a new sensitive and inexpensive hepatitis C virus (HCV) combination test (HCV Guideline test) that enables the determination of HCV genotypes 1, 2 and 3, and simultaneous determination of HCV viral load using commercial Amplicor GT HCV MONITOR test v2.0 (microwell version).
The HCV Guideline test used the PCR product generated in commercial Amplicor GT HCV Monitor test v2.0 for viral load measurement using microwell plate version of Amplicor HCV Monitor and also captured on separate plates containing capture probes and competitive oligonucleotide probes specific for HCV genotypes 1, 2 and 3, The HCV genotype was subsequently determined using the biotin-labeled PCR product and five biotin-labeled HCV-specific probes.
The sensitivity of the HCV Guideline test was 0.5 KIU/mL. Specificity of the HCV Guideline test was confirmed by direct sequencing of HCV core region and molecular evolutionary analyses based on a panel of 31 samples. The comparison of the HCV Guideline test and an in-house HCV core genotyping assay using 252 samples from chronic hepatitis C patients indicated concordant results for 97.2% of samples (59.5% genotype 1, 33.7% genotype 2, 6.0% genotype 3, and 0.8% mixed genotypes). Similarly, the HCV Guideline test showed concordance with a serological test, and the serological test failed to assign any serotype in 12.7% of the samples, indicating a better sensitivity of the HCV Guideline test.
Clinically, both viral load and genotypes (1, 2 and 3) have been found to be major predictors of antiviral therapy outcome regarding chronic hepatitis C based on guidelines and they are, in normal circumstances, performed as separate stand-alone assays. The HCV Guideline test is a useful method for screening large cohorts in a routine clinical setting for determining the treatment regimen and for predicting the outcome of antiviral therapy of chronic hepatitis C.
World Journal of Gastroenterology 02/2005; 11(4):469-75. · 2.47 Impact Factor
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ABSTRACT: Telomerase is a cellular RNA-dependent DNA polymerase that serves to maintain the tandem arrays of telomeric TTAGGG repeats at eukaryotic chromosome ends. One of the human telomerase components is hTERT, which has three alternative spliced sites that introduce eight isoforms of hTERT mRNA. The expression of these isoforms in gastrointestinal cell lines is unknown. We developed a PCR-based assay for detecting these splicing variants. In gastric and hepatocellular carcinoma cell lines, the gamma deletion variant and its combination variants, alpha- and gamma-, beta- and gamma-, and alpha-, beta- and gamma-deletion variants were frequently detected, while they were not detected in colorectal carcinoma cell lines. Our results provide important information of use for more detailed studies on the regulation of telomerase activity.
Oncology Reports 02/2004; 11(1):127-31. · 1.84 Impact Factor
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ABSTRACT: The human progesterone receptor (PR) is a ligand-activated nuclear transcription factor that mediates progesterone action in target tissues. We found a novel alternatively spliced variant (ASV) of the PR mRNA in breast cancer tissues. The deleted transcript was characterized by an out-of-frame deletion of 52 bp in exon 6 (PR delta6/2 ASV). The PR delta6/2 ASV mRNA results in a partial defect in the region of the ligand-binding domain of the hormone receptor, where conserved residues are missing from the core of the protein. To clarify the clinical significance of the PR delta6/2 ASV, we investigated the expression of this ASV in noncancerous and cancerous tissues from patients with breast cancer using RT-PCR. The novel PR delta6/2 mRNA was detected in 24 of 39 (61.5%) cancerous tissues and in 3 of 39 (7.7%) noncancerous tissues from patients with breast cancer. PR delta6/2 ASV mRNA was expressed more frequently in breast cancer tissues than in noncancerous tissues (p < 0.0001), which suggests a possible relationship between the expression of PR delta6/2 and breast cancer. Our observations may provide a novel strategy for the genetic diagnosis of breast cancer.
International Journal of Cancer 06/2003; 105(2):182-5. · 5.44 Impact Factor
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ABSTRACT: We identified two novel deletion variants of the thymidylate synthase transcript in gastric cell lines. Sequence analyses indicate that none of these variants results in introduction of a premature stop-codon or a frame shifts. In 39 gastric cancer samples, both the full-length and one-deletion variant messages were detected in cancerous as well as non-cancerous tissues. However, another isoform was found in only seven of 39 cancerous tissues. Our results provide important information to assist more detailed studies on the regulation of thymidylate synthase activity.
Cancer Letters 05/2003; 193(2):127-31. · 4.24 Impact Factor
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Hisashi Hisatomi,
Kumi Nagao,
Mutsuji Kawakita,
Tadashi Matsuda,
Hiroyuki Hirata,
Shigeki Yamamoto,
Takaaki Nakamoto,
Hiroshi Harasawa,
Noboru Kaneko, Kazumasa Hikiji,
Yutaka Tsukada
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ABSTRACT: RT-nested PCR has been introduced as a highly specific and sensitive assay method to detect the prostate-specific membrane antigen (PSM) mRNA in peripheral blood. However, appreciable percentages of false-positive cases have been reported. Additionally, primer sets reported previously could not discriminate between PSM and PSM', an alternatively spliced variant, mRNA. These isoforms can be produced from a single gene. Switches in alternative splicing patterns are often controlled with strict cell-type or developmental-stage specificity. Therefore, it is most important to discriminate between PSM mRNA and PSM' mRNA. Using our highly specific primer sets, PSM mRNA was detected in 3 of 24 peripheral blood samples of normal male volunteers (12.5%) and was not detected in peripheral blood of 11 normal female volunteers. PSM' mRNA was detected in 5 of 24 peripheral blood samples of normal male volunteers (20.8%) and in 4 of 11 of normal female volunteers (36.4%). PSM' mRNA induced false-positive results, it is important for genetic diagnosis of prostate cancer to discriminate between PSM and PSM' using our primer sets with high specificity. The advances in the uniquely designed primer sets may allow researchers to detect a real PSM mRNA without PSM' mRNA.
International Journal of Molecular Medicine 12/2002; 10(5):619-22. · 1.98 Impact Factor
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ABSTRACT: Many researchers have investigated the expressions of candidates for a suitable reverse transcription nested polymerase chain reaction (RT-PCR) marker. But typically biomarkers often have false-positive results. We assessed whether epidermal growth factor receptor (EGFR), carcinoembryonic antigen (CEA) and prostate-specific membrane antigen (PSM) could be detected in 28 different types of normal human sources. Using RT-nested PCR assay, EGFR mRNA was also detected in various types of normal tissue, including pancreas, prostate and uterus. CEA was detected in various types of normal tissue, including prostate, uterus, bladder and spleen. PSM mRNA was also detected in various types of normal tissue, including kidney, liver, skeletal muscle, spleen, bladder and ovary. We report here that the expression of these biomarkers in normal cells might have induced false-positives, and that further enhancement of sensitivity might compromise specificity. Conversely, these biomarkers can be utilized for attempts to define micrometastases in various types of tumors whose cells express these tissue-specific genes.
International Journal of Molecular Medicine 10/2002; 10(3):307-10. · 1.98 Impact Factor
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Yutaka Kondo,
Masashi Mizokami,
Tatsunori Nakano,
Takanobu Kato,
Ryuzo Ueda,
Motokazu Mukaide, Kazumasa Hikiji,
Takafumi Ishida,
Dorjibal Dorjsuren,
Bumbein Dashnyam,
Tsendsuren Oyunsuren
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ABSTRACT: We studied the prevalence of GB virus C/hepatitis G virus (GBV-C/HGV) infection among 112 patients with liver disease and 121 blood donors in Ulaanbaatar, Mongolia. Reverse transcription and polymerase chain reaction were employed to detect GBV-C/HGV RNA using the specific primers derived from the 5′-untranslated region (5′-UTR) of the GBV-C/HGV genome. Nucleotide sequences of all positive samples for GBV-C/HGV RNA were determined. The sequences were analyzed by a molecular evolutionary method. Twenty-five (10.7%) of 233 people were positive for GBV-C/HGV RNA. Eight (6.6%), 11 (9.1%), and 30 (24.8%) blood donors were positive for GBV-C/HGV RNA, HBsAg, and anti-HCV, respectively, although 17 (15.2%), 65 (58.0%), and 64 (54.5%) patients with liver disease were positive for each viral marker. The prevalences of GBV-C/HGV RNA, HBV, and HCV in the patients were significantly higher than those in blood donors (P < 0.05). There was no significant difference in the prevalence of anti-HCV among people with and without GBV-C/HGV RNA, while the prevalence of HBsAg among people with GBV-C/HGV RNA was significantly higher than among those without GBV-C/HGV RNA (P < 0.05). The molecular evolutionary tree showed that GBV-C/HGV was a heterogeneous virus and all strains could be divided into 2 types. One is the same phylogenetic type as HGV, and the other is a new type that is different from GBV-C and HGV. J. Med. Virol. 52:143–148, 1997. © 1997 Wiley-Liss, Inc.
Journal of Medical Virology 12/1998; 52(2):143 - 148. · 2.82 Impact Factor
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Yutaka Kondo,
Masashi Mizokami,
Tatsunori Nakano,
Takanobu Kato,
Ken-ichi Ohba,
Etsuro Orito,
Ryuzo Ueda,
Motokazu Mukaide, Kazumasa Hikiji,
Tsendsuren Oyunsuren,
W.Graham Cooksley
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ABSTRACT: GB virus C/hepatitis G virus is a newly described virus. Classification of GB virus C/hepatitis G virus into genotypes has not been established. We analyzed nucleotide sequences within the 5′ untranslated region of GB virus C/hepatitis G virus isolates and segregated these isolates into genotypes. Twenty serum samples with GB virus C/hepatitis G virus RNA from Australia, Cameroon, the Congo, Japan, Mongolia, and Bangladesh were studied. Reverse transcription and polymerase chain reaction were used to obtain GB virus C/hepatitis G virus RNA. After nucleotide sequences from the 5′ untranslated region were determined, 68 nucleotide sequences, including 48 previously reported sequences, were analyzed by molecular evolutionary methods. The phylogenetic tree of the 5′ untranslated region showed that all strains could be divided into three major genotypes, GB type (type 1), HG type (type 2), and Asian type (type 3). Bootstrap analysis indicated that the strains could be divided into three major genotypes but could not be further subdivided. Moreover, frequency histograms of pairwise distances between nucleotide sequences demonstrated only one peak. These result indicated that GB virus C/hepatitis G virus can be classified into three major genotypes, GB type (type 1), HG type (type 2), and Asian type (type 3), and should not be divided into minor subtypes.
Virus Research.
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Hiroyuki Hirata,
Hisashi Hisatomi,
Mutsushi Kawakita,
Kumi Nagao,
Shigeki Yamamoto, Kazumasa Hikiji,
Takaaki Nakamoto,
Hiroshi Harasawa,
Noboru Kaneko,
Tadashi Matsuda,
Masahiro Yamamoto,
Taichi Kanamaru
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ABSTRACT: The presence of circulatory metastasis is one of the most significant factors for poor-prognosis in patients with several types of cancer. To establish a sensitive reverse transcription PCR assay to detect micrometastasis in blood containing several cancer types, we first investigated Uroplakin II (UP II), a novel molecular marker for human transitional cell carcinoma of the bladder, in 25 types of normal organs. In our study, UP II mRNA was detected in 10 types of organs, including bladder, kidney, lung and pancreas, but was not detected in normal lymph nodes or leukocytes. The data indicated evidence of UP II expression in various types of normal tissues by RT-nested PCR analysis. UP II mRNA was detected in 2 of 11 (18.2%) peripheral blood samples from lung cancer patients with no metastasis, and in 5 of 12 (41.7%) peripheral blood samples of lung cancer patients with metastasis. UP II was also detected in 6 of 16 (37.5%) peripheral blood samples of patients with pancreatic cancer. The data are particularly important in that the molecular detection of micrometastasis in the blood by means of UP II mRNA identification is feasible for UP II-positive neoplasms, including lung and pancreatic cancers.
Oncology Reports 10(4):963-6. · 1.84 Impact Factor
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ABSTRACT: We identified a novel alternatively spliced isoform of PR mRNA in breast cancer tissues. The deleted transcript was characterized by an out-of-frame deletion of 437 bp, corresponding to the complete loss of exons 4 and 6 (PR delta4+6 ASV). PR delta4+6 ASV will result in a partial defect in the region of the ligand-binding domain of hormone receptors, suggesting that the conserved residues are missing from the core of the protein. In the limited number of samples studied, a novel PR delta4+6 mRNA was detected in 1 of 45 (2.2%) non-cancerous tissues of patients with breast cancer, in 5 of 45 (11.1%) cancerous tissues of patients with breast cancer. Loss of both exons 4 and 6 will be induced by incomplete splicing and/or repair mechanism. Further studies are necessary to establish the biological significance of this alternative splicing. The expressions of ASVs that induced the mimic PR transcripts need to be considered when designing strategies for regulation analysis of the PR gene.
Oncology Reports 10(2):305-8. · 1.84 Impact Factor
