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Biomedical subjects

H Yoshikura

Publications and source records attributed to H Yoshikura.

At least 19 recordsLinked to original sources
PubMed2006

Detection of a 5' end subgenome of hepatitis C virus terminating at nucleotide 384 in patients' plasma and liver tissues.

Quadri and Negro [Dig Liver Dis 2001; 33: 480] reported greater distribution of 5' end genomic RNA of hepatitis C virus (HCV) over its 3' end in the liver of patients with recurrent hepatitis C after liver transplantation. We not only confirmed their results by quantifying the 5' end subgenomes in various specimens by using dilution and real-time polymerase chain reaction methods, but also discovered that such subgenomes terminated at nucleotide (nt) 384 of the viral genome or in its immediate upstream. The subgenomes in the plasma uniformly, with a few exceptions, ended at this position, while those in the liver more heterogeneously at various points upstream of nt 384. Subgenome populations ending some points in the downstream of nt 384 were not detected. The amount of the 5' end subgenome, while fluctuating during the clinical course of the patients, exceeded that of the longer sized HCV genomes which included the intact genome, and, when the relative ratio of the 5' end subgenome increased, the amount of longer sized HCV RNA tended to decrease, suggesting a suppressive effect of the 5' end subgenome on viral replication.

Alanine Transaminase↗
PubMed2001

Cross-clade neutralizing antibody production against human immunodeficiency virus type 1 clade E and B' strains by recombinant Mycobacterium bovis BCG-based candidate vaccine.

The recombinant Mycobacterium bovis BCG (rBCG) vector-based vaccine secreting the V3 principal neutralizing epitope of human immunodeficiency virus type 1 (HIV-1) Japanese strain was reported to induce both humoral and cellular immune responses effectively [Proc. Natl. Acad. Sci. USA. 92 (1995) 10693]. The antigen-secreting rBCG system was applied to the V3 epitope of clade E HIV-1 in this study. The V3 sequence of 19 amino acids (aa) and 15aa fused with mycobacterial alpha-antigen was not secreted while 12aa and 11aa sequences were successfully secreted from BCG cells. Serum IgG from guinea pig which was immunized with 12aa epitope-secreting recombinant BCG neutralized the WHO reference strain as well as primary field isolates of clade E virus. The serum IgG could also neutralize Thai B (clade B') strains which possessed a conserved GPGQ motif in their V3 sequences. These data suggest that the rBCG construct secreting the 12aa epitope is implicated in the development of a prophylactic vaccine in Thailand in which both clade E and B' viruses are prevalent.

AIDS Vaccines↗
PubMed2000

Packageable antiviral therapeutics against human immunodeficiency virus type 1: virion-targeted virus inactivation by incorporation of a single-chain antibody against viral integrase into progeny virions.

To determine their activities as an antiviral agent packageable within virions and suitable for continued expression in cells, we tested a single-chain antibody (scAb) against human immunodeficiency virus type 1 (HIV-1) integrase and its three fusion proteins: fused to viral protein R (scab-Vpr), a double-cassette of the WXXF motif binding to Vpr (scAb-WXXF), and viral major capsid protein (scAb-CA), respectively. Cotransfection of human 293T cells with expression plasmid for scAb-Vpr or -WXXF along with HIV-1 clone pLAI resulted in the production of a normal amount of progeny virions with infectivity decreased by more than 10(3)-fold. Immunoblot analyses showed that scAb-Vpr or -WXXF was associated with virions, whereas scAb or scAb-CA was not, suggesting that scAb-Vpr or -WXXF was incorporated into virions. The incorporation of scAb-WXXF appeared to be Vpr dependent, because the fusion protein was associated with the wild-type but not with Vpr-truncated HIV-1 virions. Since G418-selected HeLa clones carrying expression plasmid for scAb-WXXF were obtained much more frequently than those for scAb-Vpr, scAb-WXXF was inferred to be less toxic to cells than scAb-Vpr. These results suggest that scAb-WXXF may serve as a novel class of antiviral therapeutic that inactivates progeny HIV virions from within.

Antibodies↗
PubMed1999

Application of the hybridization AT-tailing method for detection of human immunodeficiency virus RNA in cells and simian immunodeficiency virus RNA in formalin-fixed and paraffin-embedded tissues.

An in situ hybridization (ISH) AT-tailing method (HybrAT) was developed for the detection of viral genomes in infected cells and tissues. The method consists of hybridization with oligonucleotide probe which has a 3' end oligo d(A-T) tag, followed by elongation of the oligo d(A-T) by deltaTth DNA polymerase in the presence of the labeled nucleotide. The in situ HybrAT detected human immunodeficiency virus type 1 (HIV-1) in cells and simian immunodeficiency virus (SIV) in formalin-fixed and paraffin embedded sections with a sensitivity comparable to RNA ISH. The advantage of this method over other methods is discussed.

Animals↗
PubMed1999

Replication of GB virus C (hepatitis G virus) in interferon-resistant Daudi cells.

We previously reported that Daudi cells, a Burkitt's lymphoma cell line, were capable of supporting productive infection of hepatitis C virus (HCV). During continual cultivation after HCV infection, the culture became resistant to interferons (IFNs). This resistant cell line, coded as H-903, was used as host cells for replication of GB virus C (GBV-C), also known as hepatitis G virus. GBV-C RNA was detected in the culture by reverse transcription-PCR for more than 130 days after inoculation, while it was detected for 44 days but not later in the parental IFN-sensitive Daudi cells. Productive infection of GBV-C in the H-903 system was confirmed by serially inoculating supernatants from infected cultures into uninfected cells. The viral E2 antigen was detected by immunofluorescence in the cells inoculated with the fifth passage of GBV-C. The presumed capsid-coding region of the viral genome in the inoculum, in the serially passaged virus, or in the virus produced by a long-term culture was only 16 amino acids long, suggesting that the GBV-C with a short core sequence was replication competent.

Amino Acid Sequence↗
PubMed1998

Hybridization-AT-tailing (HybrAT) method for sensitive and strand-specific detection of DNA and RNA.

delta Tth DNA polymerase catalyzed polymerization of dATP and dTTP into a high-molecular-weight d(A-T) copolymer using oligo-d(A-T) as the template/primer (Hanaki et al., Biochem. Biophys. Res. Commun. 244, 210-219). Taking advantage of this reaction, we developed a highly sensitive method for strand-specific detection of DNA or RNA. The probe consisted of a 40- to 50-base-long complementary sequence on the 5' side and 10 repeats of AT on the 3' side. After hybridization using the 5' side, the 3' side AT repeat region was elongated by delta Tth DNA polymerase in the presence of the dATP, dTTP, and digoxigenin (dig)-11-dUTP. The elongation condition was 52-62 degrees C for 3 h. The method named HybrAT (hybridization-AT-tailing) was at least 100-fold more sensitive than the conventional hybridization with 5' end dig-11-dUTP labeled probe.

Adenine↗
PubMed1998

Two different reactions involved in the primer/template-independent polymerization of dATP and dTTP by Taq DNA polymerase.

Taq and Tth DNA polymerases catalyzed polymerization of dATP and dTTP into poly d(A-T) without requiring added primer/template (Hanaki et al., Biochem. Biophys. Res. Commun. 238, 113-118), while the Stoffel fragment of Taq DNA polymerase and delta Tth DNA polymerase with respective deletions of ca. 290 and 250 N-terminal amino acids did not. The primer/template-independent polymerization appeared to proceed via two reactions, the slow process of formation of 16-19 nt long oligo d(A-T) without primer/template and the rapid process of elongation of the oligo d(A-T) by self-priming. As the former step was more sensitive to N-ethylmaleimide than the elongation reaction, probably the formation of the oligonucleotide preceded the elongation. But when the substrates were depleted, Taq DNA polymerase degraded the high molecular weigh d(A-T) polymer to the oligomers which were resistant to the further digestion by the 5'-->3' exonuclease activity of Taq DNA polymerase. Probably, the elongation and the degradation reactions proceeded simultaneously, the former process being faster than the latter in the presence of enough dATP and dTTP.

DNA↗
PubMed1998

Infection of a chimpanzee with hepatitis C virus grown in cell culture.

Culture supernatant harvested from Daudi cells, a lymphoplastoid cell line, after 58 days of infection with the H77 strain of hepatitis C virus (HCV), was inoculated into a chimpanzee. HCV RNA, as detected by RT-PCR, first appeared in the serum and liver 5 and 6 weeks, respectively, after inoculation. Peripheral blood mononuclear cells (PBMC) collected on week 7 were also positive for HCV RNA. The major sequences of hypervariable region 1 (HVR1) of the viral genome recovered from the inoculated chimpanzee were the ones which were the majority in the original H77 inoculum and not those which were in the majority in the culture supernatant. Only the sequence recovered from PBMC was the same as the major one found in the cell culture.

Animals↗
PubMed1998

Threshold number of provirus copies required per cell for efficient virus production and interference in moloney murine leukemia virus-infected NIH 3T3 cells.

The gag-pol readthrough mutant of Moloney murine leukemia virus, MLV-B(CAG) (T. Odawara, H. Yoshikura, M. Oshima, T. Tanaka, D. S. Jones, F. Nemoto, Y. Kuchino, and A. Iwamoto, J. Virol. 65:6376-6379, 1991), was poorly complemented by a mutant encoding only Gag. This is because with all the genetic elements necessary for env expression present in MLV-B(CAG), insufficient Env protein was produced by the cells expressing MLV-B(CAG) for efficient virus production. Since the env mRNA expression per provirus in the MLV-B(CAG)- and wild-type-MLV-producing cells were the same and since the cells expressing the former contained eightfold fewer proviral copies, the insufficient Env expression by the former was found to be due to insufficient proviral copies in the cells. Examination of the cell clones having various proviral copies of Deltawt MLV (M. Oshima, T. Odawara, T. Matano, H. Sakahira, Y. Kuchino, A. Iwamoto, and H. Yoshikura, J. Virol. 70:2286-2295, 1996) showed that mRNA level was proportional to the number of proviral copies while interference and virus production followed a sigmoid curve with a sharp rise at the threshold number of proviral copies of around four per cell. Multicycle infection probably continues until the threshold level of proviral copies is attained in natural infection too.

3T3 Cells↗