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ABSTRACT: Because loss of chromosome 9 is known to be the most common finding in human bladder tumors, we studied the mutation of the tumor suppressor gene TSC1 (chromosome 9q34) in bladder tumors. Since another tumor suppressor gene, TSC2 (chromosome 16p13.3), is reported to interact with TSC1 in the pathway that modulates tumor suppression, we assessed loss of heterozygosity (LOH) at 16p13.3. Furthermore, we also examined the expression of p27 because the TSC1 product is reported to influence the level of p27.
Microsatellite markers were used to evaluate LOH at 9q34 or 16p13.3. Mutations of TSC1 were screened by single strand conformation polymorphism analysis and verified by direct sequencing. The expression of p27 was examined by reverse transcriptase-polymerase chain reaction and immunohistochemical examination.
We identified LOH at 9q34 in 12 of 37 bladder tumors (32.4%) but no LOH at 16p13.3 was observed. Furthermore, on single strand conformational polymorphism analysis we identified tumor specific mutations of TSC1 in 4 cases, of which all had LOH at 9q34, demonstrating the 2-hit mutations of TSC1. The expression of p27 was suppressed in all 4 cases with the 2-hit mutations of TSC1. Unexpectedly p27 suppression was detected at the transcription level, although its mechanism is unknown.
Our data suggest that the TSC1 mutation possibly has a causative role in the initiation or progression of some bladder tumors and this process is possibly related to the functional loss of p27.
The Journal of Urology 09/2003; 170(2 Pt 1):601-4. · 3.75 Impact Factor
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ABSTRACT: Alterations in the rat tuberous sclerosis gene (Tsc2) cause renal cell carcinomas (RCCs) with complete penetrance. In this study, it was shown that the minimal core promoters of the rat Tsc2 and endonuclease III 1 (Nth1) genes, lying in a 5'-to-5' arrangement, were localized in a 0.11-kb region containing two Ets binding sites (EBSs). This region worked as a bidirectional promoter in a single reporter plasmid. Mutational inactivation of each of the two EBSs significantly reduced promoter activity. Moreover, gel shift assays revealed the presence of specific EBSs-protein complexes. These results demonstrate that some members of the Ets family positively regulate the promoter activities of the Tsc2/Nth1 genes by binding to the EBSs. We identified Elf-1 as a binding factor for EBSs through super-shift assays, and detected approximately 35 kDa bands with an EBSs-containing DNA probe by Southwestern blot analysis. Forced expression of Elf-1 in cells, however, bidirectionally suppressed the activities of the Tsc2/Nth1 promoters. Elf-1 may be a negative regulator of Tsc2/Nth1 gene expression and may compete against positive regulators for binding to the EBSs. Our observations suggest that mechanisms that inactivate Tsc2 gene expression, such as promoter suppression, may exist.
Molecular Carcinogenesis 08/2003; 37(3):122-9. · 3.16 Impact Factor
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ABSTRACT: Fas (Apo-1/CD95) ligand, which is a type II membrane protein, is a major inducer of apoptosis. Osthole is a coumarin derivative present in medicinal plants. The effect of osthole on hepatitis induced by anti-Fas antibody in mice was studied. Pretreatment of mice with osthole (10, 50, and 100 mg/kg, i.p.) prevented the elevation of plasma alanine aminotransferase (ALT) caused by anti-Fas antibody (175 microg/kg, i.v.). Administration of osthole to mice even at a dose of 10 mg/kg significantly inhibited of anti-Fas antibody-induced elevation of plasma ALT. Capase-3 is a cysteine protease, and treatment of mice with anti-Fas antibody caused an elevation of caspase-3 activity at 3.5 and 6 hr. Pretreatment of mice with osthole (100 mg/kg, i.p.) inhibited the elevation of caspase-3 activity caused by anti-Fas antibody. However, the addition of osthole (up to 10(-4)M) to a liver cytosol fraction isolated from mice treated with anti-Fas antibody did not inhibit caspase-3 activity in vitro. Thus, treatment of mice with osthole inhibited caspase-3 activity by an effect upstream of caspase-3 activation. The livers of mice treated with anti-Fas antibody contained apoptotic and dead cells; osthole attenuated the development of this apoptosis and cell death. The present results show that osthole prevented anti-Fas antibody-induced hepatitis by inhibiting the Fas-mediated apoptotic pathway.
Biochemical Pharmacology 03/2003; 65(4):677-81. · 4.70 Impact Factor
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ABSTRACT: Cancer is a heritable disorder of somatic cells. Environment and heredity are both important in the carcinogenic process. The Eker rat model of hereditary renal carcinoma (RC) is an example of a Mendelian dominantly inherited predisposition to a specific cancer in an experimental animal. Forty years after the discovery of the Eker rat in Oslo, we and Knudson's group independently identified a germline retrotransposon insertion in the rat homologue of the human tuberous sclerosis (TSC2) gene. To our knowledge, this was the first isolation of a Mendelian dominantly predisposing cancer gene in a naturally occurring animal model. Recently, we discovered a new hereditary renal carcinoma in the rat. This rat was named the "Nihon" rat and its predisposing (Nihon) gene could be a novel renal tumor suppressor gene. This article will review the utility of these unique models for the study of problems in carcinogenesis; e.g., species-specific differences in tumorigenesis, cell stage and tissue/cell-type specific tumorigenesis, multistep carcinogenesis, modifier gene(s) in renal carcinogenesis, cancer prevention and the development of therapeutic treatments which can be translated to human patients, as well as how environmental factors interact with cancer susceptibility gene(s).
Cancer Science 03/2003; 94(2):142-7. · 3.33 Impact Factor
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ABSTRACT: Although it is generally accepted that cyclooxygenase (Cox)-2 is overexpressed in carcinomas, few studies have examined Cox-2 expression in renal carcinoma (RC). The Eker rat RC is an example of a Mendelian dominantly inherited carcinoma, and in the present study, expression of Cox-2 in the Eker rat RC was examined. Reverse transcription polymerase chain reaction indicated constitutive expression of Cox-2 mRNA in the normal control kidney and non-tumor part of Eker rat kidney. Unexpectedly, expression of Cox-2 mRNA was down-regulated in four Eker RCs from two Eker rats, and in the cell line Lk9ds. Immunohistochemical analysis failed to reveal Cox-2 protein staining in Eker RCs. As a control to Cox-2 expression, Cox-1 expression was examined. Interestingly, in contrast to the down-regulated Cox-2 expression, Cox-1 mRNA expression was induced in these four Eker RCs and cell lines. Cox-2 and Cox-1 expression were further examined in six additional Eker RCs. In total, Cox-2 mRNA expression was down-regulated in eight out of ten Eker RCs and cell lines, while Cox-1 mRNA expression was up-regulated in nine out of ten Eker RCs and cell lines.
Cancer Science 02/2003; 94(1):22-5. · 3.33 Impact Factor
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ABSTRACT: Hereditary renal carcinomas in the Eker rat are caused by germline retrotransposon insertion in the tuberous sclerosis-2 (Tsc2) gene. We established previously a transgenic Eker rat model into which was introduced a wild-type Tsc2 gene. The embryonic lethality of mutant homozygotes and renal carcinogenesis of heterozygotes were completely suppressed by this transgene (Tg). The function of the Tsc2 product (tuberin) is not fully understood, although several findings have been obtained mainly in vitro. Therefore, to elucidate the functional domains of Tsc2 in vivo, we generated transgenic Eker rats carrying deletion mutants of the Tsc2 gene. A Tg coding for the C-terminal region (amino acids 1425-1755) suppressed renal carcinogenesis in the Eker rat and interestingly the degree of this suppression correlated with the level of expression of the Tg. Notably, the product of this Tg lacks the ability to bind to the Tsc1 product (hamartin). Surprisingly, while a Tg lacking the C-terminus of tuberin (amino acids 1-1755) completely suppressed renal carcinogenesis, it partially rescued homozygous mutants from embryonic lethality. In conclusion, we have determined the minimal region of tuberin necessary for tumor suppression but the suppressive effect was quantitative. Tuberin could function as a tumor suppressor without binding to hamartin. The requirement of the functional domain(s) of tuberin might differ for prevention of embryonic lethality and for suppression of renal carcinogenesis.
Human Molecular Genetics 12/2002; 11(24):2997-3006. · 7.64 Impact Factor
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ABSTRACT: Human DNA-binding protein (dbpA) is a member of a Y-box binding protein family containing a cold shock domain. The increased expression of Y box binding proteins in somatic cells is associated with cell proliferation and transformation. Recently, we isolated a splicing variant of dbpA as a candidate for the cellular recombinogenic protein that leads to genomic instability and inflammation-mediated hepatocarcinogenesis. The expression of dbpA is enhanced in proliferating cells, but the manner in which it regulates transcription is largely unknown. In this study, we analyzed the transcriptional regulatory region of dbpA, and searched for the mutation in this region by a direct sequence method. In 3 of 55 human hepatocellular carcinoma (HCC) cases, we identified one nucleotide replacement (T right curved arrow G transversion) in nucleotide position -6 of the promoter region. Among 3 cases showing this transversion, one HCC case was due to a somatic mutation and the other two were due to single nucleotide polymorphism (SNP). By luciferase assay, we showed that the transcriptional activity of the promoter region with the transversion was significantly higher than that of the wild-type. Using the Southwestern blotting, we also confirmed the existence of a cellular proteins (about 25 and 50 kDa) that specifically bind to the sequence with this transversion. Our results suggested the biological significance of the transversion of dbpA's promoter region as one of the factors accelerating hepatocarcinogenesis.
International Journal of Oncology 11/2002; 21(4):847-50. · 2.40 Impact Factor
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ABSTRACT: Hereditary renal carcinomas (RCs) develop in Tsc2 gene mutant (Eker) rats around the age of 1 year. We previously reported that Tsc2 mutations were detected in chemically (N-ethyl-N-hydroxyethylnitrosamine (EHEN) and diethylnitrosamine)-induced non-Eker rat RCs, suggesting an involvement of Tsc2 alteration in rat RC development. In this study, we evaluated the effect of extra copies of the Tsc2 gene on renal and hepatocarcinogenesis that was induced by EHEN in vivo. The incidence of RCs in non-transgenic rats (2/17) is slightly higher than in transgenic rats (0/32), although it is statistically not significant. These results suggest the presence of other target RC gene(s) in chemically (EHEN)-induced renal carcinogenesis. We observed no difference in the numbers and areas of the hepatic glutathione S-transferase placental type positive foci.
Cancer Letters 11/2002; 184(2):157-63. · 4.24 Impact Factor
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ABSTRACT: Target of Rapamycin (TOR) mediates a signalling pathway that couples amino acid availability to S6 kinase (S6K) activation, translational initiation and cell growth. Here, we show that tuberous sclerosis 1 (Tsc1) and Tsc2, tumour suppressors that are responsible for the tuberous sclerosis syndrome, antagonize this amino acid-TOR signalling pathway. We show that Tsc1 and Tsc2 can physically associate with TOR and function upstream of TOR genetically. In Drosophila melanogaster and mammalian cells, loss of Tsc1 and Tsc2 results in a TOR-dependent increase of S6K activity. Furthermore, although S6K is normally inactivated in animal cells in response to amino acid starvation, loss of Tsc1-Tsc2 renders cells resistant to amino acid starvation. We propose that the Tsc1-Tsc2 complex antagonizes the TOR-mediated response to amino acid availability. Our studies identify Tsc1 and Tsc2 as regulators of the amino acid-TOR pathway and provide a new paradigm for how proteins involved in nutrient sensing function as tumour suppressors.
Nature Cell Biology 10/2002; 4(9):699-704. · 19.49 Impact Factor
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ABSTRACT: Human hepatocellular carcinomas (HCCs) are preceded by chronic hepatitis and cirrhosis. Despite a clear viral etiology (hepatitis B virus [HBV] and hepatitis C virus [HCV] of human hepatocarcinogenesis, the mechanism is complex and the distinct molecular pathway or molecules that explain this phenomenon are not yet known. Viral hepatitis, "inflammation-mediated" hepatocarcinogenesis, greatly influences the incidence of somatic genetic events in hepatocytes, by increasing the number of target cells or the proliferation of once-hit hepatocytes, eventually leading to HCC. We propose that hepatitis virus can cause HCC by a combination of two mechanisms; (i) cell killing and stimulation of mitosis, leading to an accumulation of events necessary for transformation; and (ii) an increase in chromosomal instability mediated by induced recombinogeneic protein(s) during chronic hepatitis. These conditions may be designated as the "hypercarcinogenic state". Our goal is to change the "hypercarcinogenic state" to the "normo- or hypocarcinogenic" state and to prevent HCC development.
Journal of Gastroenterology 02/2002; 37(11):883-7. · 4.16 Impact Factor
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ABSTRACT: In spite of great efforts in the research relating to human hepatocarcinogenesis, its mechanism on the molecular level is yet to be determined. Chronic viral hepatitis may increase the chances of genetic events in hepatocytes of the host, by increasing the number of target cells or through proliferation of initiated hepatocytes, toward the eventual development of hepatocellular carcinoma. These conditions are referred to comprehensively as the 'hypercarcinogenic state'. Our goal, then, should be directed to the reversion of the 'hypercarcinogenic state' to the 'normo- or hypocarcinogenic state' so as to hopefully prevent or at least postpone the development of hepatocellular carcinoma.
Oncology 02/2002; 62 Suppl 1:38-42. · 2.27 Impact Factor
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ABSTRACT: Pulmonary lymphangioleiomyomatosis (LAM) is a destructive lung disease characterized by a diffuse hamartomatous proliferation of smooth muscle cells (LAM cells) in the lungs. Pulmonary LAM can occur as an isolated form (sporadic LAM) or in association with tuberous sclerosis complex (TSC) (TSC-LAM), a genetic disorder with autosomal dominant inheritance with various expressivity resulting from mutations of either the TSC1 or TSC2 gene. We examined mutations of both TSC genes in 6 Japanese patients with TSC-LAM and 22 patients with sporadic LAM and identified six unique and novel mutations. TSC2 germline mutations were detected in 2 (33.3%) of 6 patients with TSC-LAM and TSC1 germline mutation in 1 (4.5%) of 22 sporadic LAM patients. In accordance with the tumor-suppressor model, loss of heterozygosity (LOH) was detected in LAM cells from 3 of 4 patients with TSC-LAM and from 4 of 8 patients with sporadic LAM. Furthermore, an identical LOH or two identical somatic mutations were demonstrated in LAM cells microdissected from several tissues, suggesting LAM cells can spread from one lesion to another. Our results from Japanese patients with LAM confirmed the current concept of pathogenesis of LAM: TSC-LAM has a germline mutation but sporadic LAM does not; sporadic LAM is a TSC2 disease with two somatic mutations; and a variety of TSC mutations causes LAM. However, our study indicates that a fraction of sporadic LAM can be a TSC1 disease; therefore, both TSC genes should be examined, even for patients with sporadic LAM.
Journal of Human Genetics 02/2002; 47(1):20-8. · 2.57 Impact Factor
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ABSTRACT: Antigen presenting cells, especially the antigen presenting dendritic cells (DC) in the tissue, regulate the magnitude of antigen-specific immune response. A role of impaired and narrowly focused specific immune response has been implicated in the pathogenesis of chronic hepatitis due to hepatitis B virus and hepatitis C virus. In order to clarify this role, we studied liver DC from interferon gamma (IFN-gamma) transgenic mouse (TgM), an animal model of chronic hepatitis. These mice had high serum levels of alanine transaminase and histological evidence of chronic hepatitis. Transgene negative offspring (littermate control) with normal serum transaminase levels and without any evidence of hepatitis were used as controls. The stimulatory capacity of the liver DC from IFN-gamma TgM in allogenic mixed leukocyte reaction was significantly lower than that of the liver DC from control mouse. The endocytosis capacity was significantly lower in liver DC from IFN-gamma TgM than in that from the control mouse. Most importantly, liver DC from IFN-gamma TgM were unable to induce antigen-specific proliferation. The impaired function of liver DC from these mice may be attributable to increased production or induction of suppressor cytokines such as interleukin-10 and nitric oxide. Defective capacity of liver DC from mouse with chronic hepatitis (IFN-gamma TgM) may be related to impaired magnitude of specific immune response in the liver.
International Journal of Molecular Medicine 02/2002; 9(1):71-6. · 1.98 Impact Factor
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ABSTRACT: Twenty-seven Japanese patients with the tuberous sclerosis complex (TSC), consisting of 23 sporadic and 4 familial cases, were tested for mutations in the TSC1 and TSC2 genes, using single-strand conformational polymorphism analysis and direct sequencing. Four possible pathogenic mutations were found in the TSC1 gene, including three frame shifts and a nonsense mutation in a familial case. All mutations were expected to result in a truncated hamartin gene product. The TSC2 gene analysis identified six possible pathogenic mutations only in the sporadic cases, including two frame shifts, one in-frame deletion, and three missense mutations. Two of the TSC2 mutations were expected to result in a truncated tuberin gene product. These results of the Japanese TSC patients were compatible with the reports from Europe and the United States, i.e., (1) TSC1 mutations are rarer in sporadic cases than in familial cases, (2) substantial numbers of sporadic cases arise from mutations in the TSC2 gene, and (3) mutations of the TSC1 gene may cause premature truncation of hamartin. Am. J. Med. Genet. 90:123–126, 2000 © 2000 Wiley-Liss, Inc.
American Journal of Medical Genetics 03/2000; 90(2):123 - 126.
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ABSTRACT: We and others have demonstrated that a mutation in Tsc2 is the rate-limiting step for renal carcinogenesis in the Eker rat model. Although inactivation of Tsc2 results in development of renal tumors, it is not sufficient for metastatic renal cell carcinomas (RCs) in the Eker rat. To investigate the additional genetic event(s) necessary for cancer metastasis, we have established highly metastatic S-Lk9d-SLM cell lines from a non-metastatic RC cell line (Lk9dL) by co-implantation with a foreign body (gelatin sponge). Since these cell lines were remarkably different in metastatic performance (all and none, respectively) despite having the same genetic background, they should be useful experimental tools to investigate metastasis-promoting events in renal carcinogenesis.
Cancer Science 10/1998; 89(11):1104 - 1108. · 3.33 Impact Factor
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ABSTRACT: We searched for the rat homologue of the human tuberous sclerosis (TSC2) gene mutations in loss of heterozygosity (LOH)-negative Eker rat renal carcinomas (RCs) by polymerase chain reactionsingle-strand conformational polymorphism (PCR-SSCP) analysis using 45 primer sets covering all 41 coding exons and one leader exon including splicing donor/acceptor sites. We have identified intragenic somatic mutations in 7 of 21 spontaneous RCs, including one cell line (33%), and in 3 of 9 (33%) N-ethyl-N-nitrosourea (ENU)-induced LOH-negative RCs. Interestingly, five mutations in the spontaneous RCs were either deletion or duplication (5/7=71%). In contrast, all three in ENU-induced RCs were base substitutions (3/3 = 100%), as expected. Thus, a qualitative difference in the second hit might exist between spontaneous and ENU-induced mutations (e.g., deletion or duplication versus point mutation). By a direct cloning approach utilizing the restriction length difference caused by germline insertional mutation or reverse transcriptase-PCR analysis in two applicable cases, we could clearly show the presence of intragenic somatic mutations in the second copy (wild-type) of the Tsc2 gene. This is the first demonstration at the DNA sequence level of the validity of Knudson's two-hits hypothesis in the Tsc2 gene.
Cancer Science 02/1997; 88(3):254 - 261. · 3.33 Impact Factor
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ABSTRACT: Recently, von Hippel-Lindau (VHL) gene mutations were detected in non-inherited, sporadic human renal cell carcinomas (RCs) at a high frequency. In order to determine whether or not the VHL gene is also a critical gene in rat RCs, we cloned and sequenced the rat homologue of human VHL gene and searched for mutations of the VHL gene in rat RCs. Mutations in the VHL gene were not detected in spontaneous RCs of the Eker rat model or in ferric nitrilotriacetate-induced rat RCs using the polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) method. These data indicate that mutation of the VHL tumor suppressor gene is not an event in rat renal carcinogenesis, at least in our present systems.
Cancer Science 09/1995; 86(10):905 - 909. · 3.33 Impact Factor
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ABSTRACT: We have recently identified on rat chromosome 10q a germline mutation in the tuberous sclerosis gene(Tsc2), the gene predisposing to renal carcinoma(a) in the Eker rat. The homozygous mutant condition is lethal at around the 13th day of fetal life. In heterozygotes, RCs invariably develop in the first year of life. Histologically, RCs develop through multiple stages from early preneoplastic lesions(i.e., phenotypically altered tubules) to adenomas. The wild-type allele mutation has been found even in the earliest preneoplastic lesions, fitting Knudson's two-hit hypothesis and supporting the hypothesis that Tsc2 is a tumor suppressor gene. In this study, homozygous deletion of the Ink4 homologue on rat chromosome 5q was observed in 14 of 24(58%) RC-derived cell lines. This may represent involvement of a second tumor suppressor gene, contributing to tumor progression. Considering previous results of studies of homozygous deletion of the ifn gene in five of 24 cases(21%) and the Ifnβ gene in one of 24 cases(4%), the order of the genes may be Ink4–Ifna— Ifnβ Microsatellite instability was not observed in 26 Eker rat tumors.© 1995 Wiley-Liss, Inc
Molecular Carcinogenesis 08/1995; 14(1):23 - 27. · 3.16 Impact Factor
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ABSTRACT: The Yoshida sarcoma (YS) is characterized by growth as “free cells” in ascites. Long-survival Yoshida sarcoma (LY) variants, which develop after transplantation of YS into immunologically conditioned Donryu rats, in contrast, form “islands” in ascites. A representational difference analysis (RDA) approach was adopted to isolate genes differentially expressed between YS and LY variants to elucidate the molecular mechanism of their development. Fifteen clones presenting differences in expression were characterized. Nine genes (those encoding for the high-affinity IgE receptor chain, pJG116 repetitive sequence, non neuronal enolase, proteasome subunit RC1, cytotoxic T lymphocyte-associated gene transcript CTLA-1, interleukin-2 receptor chain, and three unknown sequences) expressed mRNA in YS, but showed lower or no expression of mRNA in LYs. The mRNAs of the other six genes (those encoding for cytokeratin 8, cytokeratinlS (Endo B), TIMP2 and three unknown sequences) were not found in YS, but were present in LYs. Interestingly, CTLA-1 is a non-epithelial (hematopoietic) cell-specific gene in terms of transcription, while cytokeratin 8 and cytokeratin 18 are both epithelium-specific genes. Immunohistochemically, YS expressed T-cell specific antigens CD2 and CDS, and T cell receptor β and chain genes were rearranged in YS, but not in LYs. Moreover, using restriction fragment length polymorphism probes, we found that LYs exhibited different cell lineage from YS. Thus, our present findings, unexpectedly, raise fundamental questions concerning the cellular origins of YS and LY variants rather than pointing to any specific mechanism to explain the LY phenomenon.
Cancer Science 10/1994; 85(11):1099 - 1104. · 3.33 Impact Factor
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Intervirology 08/1970; 42(2-3):205-210. · 2.34 Impact Factor
