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Biomedical subjects

K Shimotohno

Publications and source records attributed to K Shimotohno.

At least 163 records · Page 9Linked to original sources
PubMed1992

An antigenic structure of the trans-activator protein encoded by human T-cell leukemia virus type-I (HTLV-I), as defined by a panel of monoclonal antibodies.

In order to study an antigenic structure of the trans-activator protein encoded by human T-cell leukemia virus type-I (HTLV-I), tax1 antigen, we generated and characterized a panel of rat anti-tax1 monoclonal antibodies (MAbs) designated WATM-1, WATM-2, WATM-3, and WATM-4. These MAbs were derived from WKA rats immunized with HTLV-I-transformed (HTLV-I+) syngeneic T cells. Immunoblot assays showed that: (1) All the MAbs reacted with the tax1 antigen in HTLV-I+ cell lines and a recombinant tax1 antigen, PX141 (containing entire tax1 polypeptide); (2) WATM-3 and WATM-4, but not WATM-1 or WATM-2, reacted with a truncated tax1 antigen, XD59 (tax1 amino acids 180-338); (3) None of them reacted with another truncated tax1 antigen, XD128 (tax1 amino acids 1-47 and 286-353); and (4) each of the four MAbs had different reactivity with tax1-related antigens in the range 38-41 kDa expressed in simian cell lines infected with various HTLV-I-related simian retroviruses (STLV-I). None of the MAbs reacted with HTLV-II tax antigen. Human sera containing anti-tax1 antibodies interfered specifically with the antigen-specific binding of all the MAbs. These results suggest that the present rat MAbs are directed against various epitopes on the tax1 antigen. An antigenic structure of the tax1 antigen deduced from reactivity of a panel of anti-tax1 MAbs including the present rat MAbs is discussed.

Animals↗
PubMed1992

Serodiagnostic assay of hepatitis C virus infection using viral proteins expressed in Escherichia coli.

Infection with hepatitis C virus (HCV) was analyzed by an enzyme-linked immunosorbent assay based on recombinant viral proteins encoded by regions of the putative viral core, NS3, NS4 and NS5, which were expressed in E. coli. Results showed that 106 of 124 cases (85.5%) of non-A, non-B chronic hepatitis and 43 of 45 cases (95.5%) of hepatocellular carcinoma, negative for HBV marker, were positive for antibodies against at least one of these viral proteins. One of 87 healthy individuals with normal alanine aminotransferase activity was positive for antibody against only the viral core, but was negative for HCV RNA. The serum of one patient with chronic hepatitis was positive for one of these proteins, but negative for HCV RNA. These findings in combination with results on detection of HCV RNA in the sera of patients with non-A, non-B chronic hepatitis indicated that 105 of 124 cases (84.6%) were positive for HCV infection. Sera that were negative for HCV antibodies against all these proteins were also negative for HCV RNA assayed by reverse transcription followed by the polymerase chain reaction. Screening of HCV infection by detecting viral antibodies in circulating blood using all these viral proteins is useful for reducing the number of ambiguous results in screening for viral infection. Thus, this assay system may be useful diagnostic purposes.

Carcinoma, Hepatocellular↗
PubMed1991

Distribution of plural HCV types in Japan.

A detection system was developed to distinguish the four different HCV genomes [HCV-J, HCV-US, HCV-K2 and group II HCV (HCV-GII)], involving reverse transcription followed by a nested polymerase chain reaction using specific primers for each HCV type. The putative non-structural (NS) 5 regions of HCV-J, HCV-US and HCV-K2 and the putative NS3 region of HCV-GII were amplified. Of 95 specimens from patients with acute hepatitis, chronic hepatitis, liver cirrhosis or hepatocellular carcinoma, 67 specimens were positive for HCV-J, 2 for HCV-US, 23 for HCV-K2 and 11 for HCV-GII. About half the specimens that were positive for HCV-K2 or HCV-GII were coinfected with HCV-J and all those that were positive for HCV-GII were also positive for HCV-K2. Nucleotide sequence analysis of several amplified cDNA products revealed that HCV-K2 and HCV-GII could each be classified into two groups, and the pattern of classification of HCV-K2 was identical with that of HCV-GII. Therefore, our results strongly suggest that HCV-K2 is the same as HCV-GII.

Base Sequence↗
PubMed1991

Identification of transcriptional suppressor proteins that bind to the negative regulatory element of the human immunodeficiency virus type 1.

Two different proteins which independently bound to neighboring sequences within the negative regulatory element (NRE) of human immunodeficiency virus type 1 (HIV-1) were detected in the nuclear extract of a virus-infected human T cell line. One of the factors bound to a novel dyad symmetrical sequence. This sequence is well conserved in various HIV-1 isolates and partial homology was found with the promoter region of the human retinoblastoma gene. Similar DNA binding activity was detected in a variety of virus-uninfected human T cell lines and HeLa cells by means of a gel mobility shift assay. The other factor bound to a putative AP-1 recognition sequence predicted for the HIV-1 NRE. However, this factor did not bind to a typical AP-1 site. The insertion of multiple copies of the binding site for the former or latter factor into a heterologous promoter reduced the promoter activity to one-tenth or one-third, respectively. Thus, each factor may function as a novel negative regulator of transcription.

Base Sequence↗
PubMed1991

Infection of rats with HTLV-1: a small-animal model for HTLV-1 carriers.

A human T-cell line producing human T-cell leukemia virus type I (HTLV-I), MT-2, was injected intravenously into female F344 rats aged 5 weeks to make HTLV-I carrier rats. Antibody against HTLV-I was detected at the 5th week after MT-2 injection, and its titer reached a high plateau which continued from the 15th to the 27th week. The antibodies were against p19, p24, p28 and p53 of HTLV-I antigens from MT-2 cells. The gag, pX and LTR nucleotide sequences of HTLV-I provirus were demonstrated by using polymerase chain reaction (PCR) in the peripheral-blood mononuclear cells of 3 rats at the 44th week and 2 at the 66th to 68th week out of 8 F344 rats injected with MT-2 cells. Quantification of the HTLV-I proviral sequence revealed that 30 to 60 molecules were present in 10(5) peripheral-blood mononuclear cells, indicating that the rats were chronically infected with HTLV-I. HTLV-I-infected rats could serve as a small-animal model for studying the pathophysiological state of HTLV-I carriers and also that of HTLV-I infection on various HTLV-I-related diseases, including adult T-cell leukemia and HTLV-I-associated myelopathy.

Animals↗
PubMed1991

Murine retroviral vectors expressing the tax1 gene of human T-cell leukemia virus type 1.

Murine retroviral vectors which express the tax1 gene of human T-cell leukemia virus type 1 (HTLV-1) have been constructed. A significant increase in virus titer was achieved by inserting a portion of the gag region of Moloney murine leukemia virus. Using these vectors, tax1 was stably introduced into primary human T-cells derived from peripheral blood lymphocytes. Expression of the functional tax1 in infected cells was confirmed by trans-activation of HTLV-1 long terminal repeat-directed transcription. These vectors provide a useful means of investigating the function of tax1 in natural target cells for HTLV-1.

Animals↗
PubMed1991

A new method of gene transfer into hematopoietic progenitors using liquid culture with interleukin-3 and interleukin-6.

The technique of gene transfer into hematopoietic cells is expected to offer a new form of therapeutics. As a result of studies on a gene-delivery system using granulocyte-macrophage colony-forming units (CFU-GM), a type of hematopoietic progenitor, we have established a technique for efficient gene transfer into CFU-GM. DGL, a retrovirus vector containing the SV40 promoter and the neomycin resistance gene, was constructed and found to transfer genes effectively into murine CFU-GM, which subsequently expressed the neomycin resistance gene. After gene transfer of murine non-adherent bone marrow cells precultured in liquid culture with recombinant murine IL-3 (rmIL-3) and recombinant human IL-6 (rhIL-6) for 6 days, gene transferred CFU-GM in bone marrow cells were able to proliferate 5-10-fold and the ratio of gene transferred CFU-GM to total CFU-GM reached 70-100% from less than 1% in the liquid culture with rmIL-3, rhIL-6 and neomycin for 6 days. Using this protocol, we have been able to obtain large amounts of highly concentrated gene-transferred CFU-GM for fundamental research on CFU-GM gene-delivery systems.

Animals↗
PubMed1991

Gene mapping of the putative structural region of the hepatitis C virus genome by in vitro processing analysis.

Processing of the putative structural proteins of hepatitis C virus was examined by using an in vitro expression system. An RNA transcript for cell-free translation was prepared from a cDNA construct that encompasses the region encoding the 980 amino-terminal residues of the viral polyprotein precursor. Processing of the in vitro translation product proceeded cotranslationally in the presence of microsomal membranes and generated four major membrane-associated products. Two of these four major products, named gp35 and gp70, were shown to be transported into microsomes and heavily glycosylated, suggesting that the processing events are partly mediated by the signal peptidase of the endoplasmic reticulum. The other two products, p19 and p21, were probably associated with the outer surface of the microsomal membrane. Analysis of processed proteins translated from a series of truncated forms of the cDNA construct as well as determination of amino-terminal amino acid sequences of gp35 and gp70 indicated that these four products are arranged from the amino-terminal end of the polyprotein precursor in the order: NH2-p22-gp35-gp70-p19. Both gp35 and gp70 could be candidates of initially processed forms of envelope proteins of the hepatitis C virus.

Acetylglucosaminidase↗
PubMed1991

Production of a recombinant human T-cell leukemia virus type-I trans-activator (tax1) antigen and its utilization for generation of monoclonal antibodies against various epitopes on the tax1 antigen.

A 42-kDa recombinant protein, PX141, consisting of the trans-activator protein encoded by human T-cell leukemia virus (HTLV-1) (tax1 antigen) and the amino-terminal fusion peptide of 12 amino acid residues of the alpha-peptide encoded by the plasmid pUC19 was produced. In order to investigate the immunogenicity of the tax1 antigen, mice were immunized with the purified PX141 and 4 anti-tax1 monoclonal antibodies (MAbs) designated TAXY-1, TAXY-6, TAXY-7 and TAXY-8 were generated, and their reactivity was characterized along with another anti-tax1 MAb, Lt-4. Immunoblot assays showed that all the MAbs reacted with the PX141, the native tax1 antigen expressed in various HTLV-1-infected cell lines and the gp68 of MT-2 cells expressing the tax1 amino acids 94-353. Immunoblot assays using recombinant, truncated tax1 antigens, XD59 (expressing amino acids 180-338) and XD128 (expressing amino acids 1-47 and 286-353) showed that: (1) TAXY-1 and Lt-4 did not react with either antigen; (2) TAXY-6 and TAXY-8 reacted with only XD128: and (3) TAXY-7 reacted with both. In addition, TAXY-1, but not the other MAbs, reacted with a putative tax antigen of an STLV-I-infected cell line, designated RfM26-I. Competitive binding assays showed that TAXY-6 and TAXY-8 did not compete against each other. Sera from HTLV-I-infected humans interfered with the binding of all of these anti-tax1 MAbs. These results indicate that the tax1 antigen and the PX141 express at least 5 distinct epitopes recognized by human and mouse antibodies.

Amino Acid Sequence↗
PubMed1991

Molecular structure of the Japanese hepatitis C viral genome.

The amino acid sequence of the polyprotein deduced from the nucleotide sequence of the Japanese hepatitis C virus genome (N. Kato et al. (1990) Proc. Natl. Acad. Sci. USA 87, 9524-9528) indicated that this virus is a member of a new class of positive-stranded RNA viruses. Several domains of this polyprotein also showed weak homology with those of flaviviruses and pestiviruses including the chymotrypsin-like serine proteinase, NTPase and RNA-dependent RNA polymerase.

Amino Acid Sequence↗
PubMed1991

Hypervariable regions in the putative glycoprotein of hepatitis C virus.

A comparison of the sequences of the putative glycoprotein region in three independent cDNA clones of hepatitis C virus and of sequences of four other clones revealed extensive genetic variation clustered and interspersed with highly conserved amino acid sequences. We obtained evidence for two hypervariable regions in the putative envelope glycoprotein, one region was assumed to be a potential antigenic site, as deduced from the hydrophilicity and analyses of secondary structures. These observations suggest the existence of a large pool of antigenic variants of hepatitis C virus, in Japan.

Amino Acid Sequence↗
PubMed1991

Sequence variations in LTR and env regions of HTLV-I do not discriminate between the virus from patients with HTLV-I-associated myelopathy and adult T-cell leukemia.

For detailed comparison of human T-cell leukemia virus type I (HTLV-I) in adult T-cell leukemia (ATL) and HTLV-I-associated myelopathy (HAM), the nucleotide sequences of parts of the long terminal repeat (LTR) and env regions of the HTLV-I proviruses from 12 patients with HAM, 8 patients with ATL and one with both diseases were analyzed. About 340 bp of the LTR U3 region, about 450 bp of the 5' region and about 280 bp of the 3' region of env were sequenced directly in DNAs amplified by the polymerase chain reaction (PCR) with 2 or 3 sets of primers for each region. Nucleotide insertions, deletions or point mutations were observed at 50 positions in these regions of about 1,000 nucleotides length. None of these changes was specific to either HAM or ATL, and some changes were observed in proviruses from both cases of HAM and ATL. Moreover, the sequences of proviruses isolated from pairs of cell lines established from cerebrospinal fluid (CSF) and peripheral blood mononuclear cells (PBMCs) of the 4 patients with HAM also had different sequences. These results indicate that the proviruses from HAM and ATL are indistinguishable in these sequenced regions, suggesting that these 2 diseases are caused by infection with genetically indistinguishable HTLV-I. Therefore, the reason why these two distinct diseases, HAM and ATL, develop in HTLV-I carriers may be based on a host factor(s) or some other factor(s) rather than variation in the virus itself.

Base Sequence↗
PubMed1991

Massive sero-epidemiological survey of hepatitis C virus: clustering of carriers on the southwest coast of Tsushima, Japan.

We conducted a mass screening survey for anti-hepatitis C virus antibody (anti-HCV-Ab) among 1,009 inhabitants, 341 males and 668 females, older than 40 years, in south Tsushima in 1989. The overall positive rate for anti-HCV-Ab was 2.3% (2.9% in males and 1.9% in females). The positive rate for anti-HCV-Ab among people with histories of blood transfusions was 14.8% (8/54), which was higher than than (1.6%, 15/955) in people without blood transfusions (P less 0.001). The positive rate (15.1%, 8/53) in people with an elevated level of glutamic oxalacetic transaminase (GOT, greater than or equal to 41 U/liter) or glutamic pyruvic transaminase (GPT, greater than or equal to 36 U/liter) was significantly higher than that (1.6%, 15/956) for people with normal values of GOT and GPT (P less than 0.001). The most interesting finding was that the anti-HCV-Ab positives without histories of blood transfusions were clustered in a few villages located in the southwestern coastal areas of Tsushima, where the positive rate of 3.7% (13/355) was significantly higher (P less than 0.001) than that of 0.3% (2/600) in other villages.

Adult↗
PubMed1991

Detection of hepatitis C virus infection by enzyme-linked immunosorbent assay system using core protein expressed in Escherichia coli.

Enzyme-linked immunosorbent assay of the core protein of hepatitis C virus (HCV) expressed in E. coli led to detection of the antibody against this virus in patients with chronic hepatitis. Some of the negative results obtained using a different viral protein became positive with this E. coli-expressed viral protein, and were also positive for the viral RNA. Thus, use of the core protein of HCV facilitates accurate detection of HCV infection.

Base Sequence↗
PubMed1991

Isolation of cDNAs for DNA-binding proteins which specifically bind to a tax-responsive enhancer element in the long terminal repeat of human T-cell leukemia virus type I.

One of the gene products of human T-cell leukemia virus type I (HTLV-I), p40tax, activates its own viral transcription in trans through tax-responsive enhancers in viral long terminal repeats. Five species of cDNA clones for proteins that bind to the tax-responsive enhancer element in HTLV-I were isolated from the Jurkat cell library. The beta-galactosidase fusion protein prepared from the lysogen of a clone specifically recognized the cyclic AMP-responsive element in HTLV-I enhancer. The nucleotide sequence of a full-length cDNA clone (TAXREB67) had a coding capacity of 351 amino acids, which contained a basic motif followed by a leucine zipper structure near the carboxy terminus. Its mRNA was detected in human cell lines, including HTLV-I-infected or noninfected hematopoietic cell lines. The mRNA level in Jurkat cells was decreased temporarily by increasing cyclic AMP concentration but increased by increasing Ca2+ concentration. Polyclonal antibodies against the fusion protein specifically recognized a 52-kDa protein in Jurkat cells. Analyses of the function of this protein and its interactions with other cellular factors will be useful to help understand the regulatory mechanism through tax-responsive enhancers in HTLV-I.

Amino Acid Sequence↗