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Biomedical subjects

K Ikuta

Publications and source records attributed to K Ikuta.

At least 163 records · Page 9Linked to original sources
PubMed1997

Myeloid differentiation is impaired in transgenic mice with targeted expression of a dominant negative form of retinoid X receptor beta.

To investigate the in vivo function of retinoid X receptor (RXR) on myelopoiesis, We generated transgenic (Tg) mice with targeted expression of a dominant negative form of RXR beta in myeloid cells. In these Tg mice the transgene is expected to suppress the function of hetero dimeric receptors composed of RXR and its counterparts, such as retinoic acid receptor. Out of 12 mice analysed, one Tg mouse exhibited a severe maturation arrest at the promyelocytic stage. Three other Tg mice showed a mild inhibition of myeloid differentiation, which was further augmented when mice were treated with 5-fluorouracil (5-FU). Furthermore, four Tg mice showed impaired myeloid differentiation in response to the treatment by 5-FU on granulocyte-colony stimulating factor (G-CSF), although they exhibited apparently normal myelopoiesis in the untreated state. The phenotype of Tg mice observed after G-CSF treatment correlated with the expression level of the transgene, although the correlation was not found in untreated mice. These results indicated that myeloid differentiation is perturbed in the Tg mice by the dominant negative effect of the transgenic RXR, indicating that RXR plays a role in myelopoiesis.

Animals↗
PubMed1997

Exposure of resting peripheral blood T cells to HIV-1 particles generates CD25+ killer cells in a small subset, leading to induction of apoptosis in bystander cells.

Apoptosis is a major mechanism whereby HIV-1 depletes uninfected CD4+ and CD8+ T cells. We previously showed that resting peripheral blood T cells derived from healthy donors were killed by an apoptotic mechanism after adsorption to gp120-containing, protease-defective HIV-1 (L-2) particles, more effectively than parental wild-type LAI adsorption or rgp 120-mediated CD4 cross-linking, followed by mitogenic stimulation. Here, we present evidence that the L-2 particle-based apoptosis was induced both in CD4+ and CD8+ cells by generation of effector cells which were mainly derived from a resting memory CD4+CD38- subset. This subset enhanced the CD25 expression on the surface and secreted IFN-gamma in the culture supernatant after L-2 particle exposure. Significant elevation of Fas ligand mRNA was found in the subset by L-2 particle exposure, while expression of Fas antigen on uninfected T cells was induced by exposure to IFN-gamma. These results indicate that L-2 particles can shift the CD4+CD38- subpopulation from a resting to an activated state, and this activation leads to killing of bystander CD4+ and CD8+ T cells by a Fas-mediated mechanism. In fact, purified CD4+CD38- cells exposed to L-2 particles were converted into effector cells that were able to kill autologous as well as allogenic target T cells pretreated with IFN-gamma. Further, we found that the observation of apoptosis due to L-2 particles was a more general phenomenon, that also occurred with Thai primary HIV-1 isolates. These results suggest that such specific types of HIV-1 particles may play a major role in the induction of apoptosis for both bystander CD4+ and CD8+ T cells, through inappropriate activation of CD4+CD38- cells.

Apoptosis↗
PubMed1997

Insertions, duplications and substitutions in restricted gp90 regions of equine infectious anaemia virus during febrile episodes in an experimentally infected horse.

We have studied a horse which exhibited typical clinical signs of disease when experimentally infected with a non-adapted virulent strain of equine infectious anaemia virus (EIAV), designated V70. Five viruses (F1V, F2V, F3V, F4V and F5V) were recovered during periodic febrile episodes. Cross-neutralization tests revealed that all of these variants and the parental V70 were antigenically distinct. Sequencing of their full-length env gp90 genes and gp45 5' sequences revealed novel mutations at a limited number of nucleotide positions, consisting of insertions and duplications in the gp90 principal neutralizing domain (PND) in F1V, F3V and F5V. Parts or all of small units (6, 9 and 12 nucleotides) located just before the insertion site were used for the duplications. Furthermore, amino acid substitutions in the env PND and hypervariable region were also observed in all five viruses. These mutations may contribute to the generation of serial variants. Consequently, the full-length gp90 sequences showed close relationships between V70, F2V and F4V, and between F1V, F3V and F5V. In addition to the two domains (PND and hypervariable region), a comparison of these viruses with the reported env gp90 sequences revealed four additional variable domains, although these four domains were highly conserved among the five variants.

Amino Acid Sequence↗
PubMed1997

Amplification of a full-length Borna disease virus (BDV) cDNA from total RNA of cells persistently infected with BDV.

We have developed a novel reverse transcriptase-polymerase chain reaction (RT-PCR) to amplify the full-length 8.9 kilobase (kbp) cDNA of the Borna disease virus (BDV) RNA genome from the total cellular RNA of MDCK cells persistently infected with BDV (MDCK/BDV). Antigenomic BDV cDNA was reverse transcribed using a 53-mer oligonucleotide primer, corresponding to the 5'-terminus of a putative 3'-leader sequence of the BDV RNA genome, for 2 hr at 42 C followed by 30 min at 55 C. PCR was performed in the presence of this 53-mer antigenomic primer and a 25-mer primer, corresponding to the 3'-terminus of the BDV antigenomic cDNA, by use of an rTth DNA polymerase with proof-reading activity. The amplified full-length BDV cDNA was detected in as little as 20 ng of total cellular RNA of MDCK/BDV. This RT-PCR method should be a useful technique to study the molecular quasispecies of BDV.

Animals↗
PubMed1997

Clinical investigation of the relationship between Borna disease virus (BDV) infection and schizophrenia in 67 patients in Japan.

The relationship between Borna disease virus (BDV) infection and schizophrenia in the clinical time course was investigated. By nested reverse-transcribed polymerase chain reaction (RT-PCR) and Western blotting, BDV-specific RNA and anti-BDV antibodies were examined in the EDTA-treated blood from 67 schizophrenic patients (according to DSM-III-R) in Japan. A significantly higher proportion (45%) of anti-BDV antibody and/or BDV RNA carriers were found among these 67 schizophrenic patients than in 26 controls (0%). There were no apparent associations of BDV infection with age, age at onset, period of hospitalization, accompanying somatic diseases, a past history of tuberculosis, a history of transfusion, a family history, or doses of psychotropic drugs. It is possible that, at least, BDV infection in schizophrenic patients may not be a nosocomial (hospital-acquired) infection, although the route of BDV infection in humans remains unidentified. More studies on the relationship between BDV infection and clinical psychosomatic features should be performed in order to elucidate the pathogenesis of schizophrenia.

Animals↗
PubMed1997

Photoinactivation of virus infectivity by hypocrellin A.

We investigated the photoinactivation of virus infectivity by hypocrellin A and its mechanism. The titers of vesicular stomatitis virus (VSV) and human immunodeficiency virus type 1 (HIV-1), both of which are enveloped viruses, were reduced upon illumination with hypocrellin A in a concentration-dependent manner, whereas canine parvovirus, a nonenveloped virus, was not killed. The removal of oxygen or addition of sodium azide or beta-carotene both inhibited VSV inactivation. Mannitol and superoxide dismutase had no effect on VSV inactivation. These results indicate that singlet oxygen was involved in the process of VSV inactivation. Of the three major VSV membrane proteins, peripheral membrane protein M was most damaged by the hypocrellin A phototreatment.

Animals↗
PubMed1997

Decoy approach using RNA-DNA chimera oligonucleotides to inhibit the regulatory function of human immunodeficiency virus type 1 Rev protein.

Human immunodeficiency virus type 1 (HIV-1) encodes two regulatory proteins, Tat and Rev, that bind to target RNA sequences. These are the trans-activation responsive (TAR) RNA and the Rev-responsive element (RRE), respectively. The Rev protein shifts RNA synthesis to viral transcripts by binding to the RRE within the env gene. In the present study we prepared a RNA-DNA chimera consisting of 29 or 31 nucleotides to inhibit the Rev regulatory function by means of the decoy approach. The chimera oligonucleotides (anti-Rev oligonucleotides [AROs]) contained an RNA "bubble" structure (13 oligonucleotides; the Rev-binding element in RRE) that bound Rev with a high affinity in an in vitro assay. The controls were RNA-DNA chimera oligonucleotides (negative control oligonucleotides [NCOs]) similar to ARO, but without the bubble structure, that bound with considerably less affinity to Rev. When the inhibitory effects of these decoys on HIV-1 replication were examined, we found that AROs, but no NCOs, reduced more than 90% of the HIV-1 production generated by productively infected human T-cell lines. The production of primary HIV-1 isolates in healthy donor-derived peripheral blood mononuclear cells was also similarly inhibited by AROs. In addition, the induction of viral mRNAs and antigens in latently HIV-1-infected ACH-2 cells by tumor necrosis factor alpha was specifically inhibited by AROs, but not by NCOs. No apparent cytotoxicity was caused by either decoy. Thus, the use of a Rev-binding element-based decoy, the RNA-DNA chimera oligonucleotide, may represent a safer approach to gene therapy for reducing the virus load in HIV-1-infected individuals.

Cell Line↗
PubMed1997

High prevalence of Borna disease virus infection in healthy sheep in Japan.

Previous seroepidemiological and molecular epidemiological studies of Borna disease virus (BDV) showed considerably high rates of infection in horses, cattle, cats, and humans in Hokkaido, Japan. Here, we further demonstrate high rates of specific antibodies to BDV and BDV RNA in peripheral blood mononuclear cells (PBMCs) from healthy sheep bred on the same island. The BDV prevalences by immunoblotting and/or reverse transcriptase PCR were 0% (0 of 19) in newborns (<1 month old), 51.7% (15 of 29) in lambs (1 to 6 months old), and 36.7% (11 of 30) in adults (>2 years old). Among animals positive for BDV, 60% of lambs and 45.5% of adults contained BDV RNA in PBMCs while 46.7% of lambs and 90.9% of adults contained specific antibodies to BDV. Thus, it is suggested that virus replication in the blood, as observed in lambs, is usually reduced in adulthood by raising immune responses to BDV.

Animals↗
PubMed1997

Microplate hybridization for Borna disease virus RNA in human peripheral blood mononuclear cells.

We developed a simple and sensitive microplate hybridization procedure with which to identify Borna disease virus cDNA in amplified products from human peripheral blood mononuclear cells. The mean values for the positive PCR products were significant compared with those for any of the negative products, indicating that this method can be applied to rapidly diagnose a large number of clinical specimens.

Animals↗
PubMed1997

Protease-defective, gp120-containing human immunodeficiency virus type 1 particles induce apoptosis more efficiently than does wild-type virus or recombinant gp120 protein in healthy donor-derived peripheral blood T cells.

Apoptosis and syncytium formation are two mechanisms by which human immunodeficiency virus type 1 (HIV-1) impairs uninfected CD4+ T-cell function and are mainly involved in the progression of the disease to AIDS. Previously, we showed that gp120-containing, protease-deficient HIV-1 (L-2) particles generated syncytia by particle-mediated fusion with uninfected cultured CD4+ T cells. Here, we present evidence that such L-2 particles can induce apoptosis in 40 to 50% of T cells which were enriched from HIV-1-negative healthy donor-derived peripheral blood mononuclear cells (PBMC-Ts). Activation of PBMC-Ts with phytohemagglutinin, concanavalin A, or ionomycin after incubation with L-2 particles resulted in the loss of proliferative capacity and gradual induction of apoptosis over 3 days. Wild-type strain LAI particles or recombinant gp120 were markedly less efficient (< or = 15%) at inducing such apoptosis. Western blot (immunoblot) analysis revealed that L-2 particles contained a larger amount of Env gp120 than LAI particles. Either preincubation of PBMC-Ts with a Fas antagonist or preincubation of L-2 particles with soluble CD4 blocked most of the apoptosis. This suggests that L-2-like particles can play a major role in HIV-1-induced apoptosis of uninfected bystander cells.

Apoptosis↗
PubMed1997

In vivo dynamics of equine infectious anemia viruses emerging during febrile episodes: insertions/duplications at the principal neutralizing domain.

Equine infectious anemia virus (EIAV) is a good model for studying mechanisms generating escaped retrovirus variants. We previously sequenced the entire gp90-encoding region of 22 cDNA clones obtained from five antigenically distinct isolates (F1V to F5V) recovered during febrile episodes in horse 493 experimentally infected with the Japanese virulent EIAV strain V70. The results showed that the mutations occurred in the principal neutralizing domain (PND) by insertions/duplications. In this study, we further characterized the PND of virus isolates sequentially recovered during 22 febrile episodes in seven horses newly infected with V70 or one of the V70-derived variants. Sequencing of 70 cDNA clones derived from the 22 episodes confirmed the generation of various new viral quasispecies with insertions/duplications in the PND. Although the insertion/duplication sequences in a total of 92 cDNA clones were extensively heterogeneous, we hypothesized that all the insertions/duplications occurred during reverse transcription from viral genomic RNA to minus strand DNA. The insertion/duplication regions were derived from a part of the PND sequence, which consisted of five small units. These small units, some with various substitutions and/or deletions, were also generated, especially in regions with insertions/duplications. Of particular note was that all these virus variants, except for two cDNA variants, were generated by essentially four different duplication pathways. Thus, these results extend the significance of insertions/duplications in the PND to the novel generation of EIAV in vivo during febrile episodes.

Amino Acid Sequence↗
PubMed1997

Inhibition of HIV-1 replication by targeting the Rev protein.

Human immunodeficiency virus type 1 (HIV-1) encodes two regulatory proteins, Tat and Rev. The Rev protein facilitates the transport of unspliced and singly-spliced RNA to the cytoplasm in infected host cells by binding to target RNA (Rev response element: RRE). A variety of approaches targeting Rev function, including gene therapy, have been developed that inhibit HIV-1 replication in cells cultured in vitro. This minireview summarizes the recent developments as well as our application of the Rev-binding element-based decoy approach using RNA-DNA chimera oligonucleotide modeling.

Anti-HIV Agents↗
PubMed1997

Higher prevalence of Borna disease virus infection in blood donors living near thoroughbred horse farms.

It is believed that Borna disease virus (BDV), an etiological agent of progressive polioencephalomyelitis in horses and sheep, is closely associated with psychiatric disorders in humans since the prevalence of BDV is higher in psychiatric patients than in blood donors. We investigated whether or not BDVs in humans are derived from infected domestic animals, by characterizing the BDVs in blood donors and horses derived from the same region of Hokkaido island, Japan. The seroprevalences (2.6 to 14.8%) of BDV were significantly higher in the blood donors from four regions where most horse farms are concentrated, compared with only 1% in the blood donors from Sapporo, the largest city in Hokkaido.BDV RNA was also detected in peripheral blood mononuclear cells from most of the seropositive horses and blood donors by nested reverse transcriptase-polymerase chain reaction. These findings support that BDV may be horizontally transmitted, at least in part, from infected horses to humans.

Animals↗
PubMed1997

[Demonstration on Borna disease virus in patients with chronic fatigue syndrome].

Chronic fatigue syndrome (CFS), a recently named heterogeneous disorder, is an illness of unknown etiology. The association between CFS and several viral infection has been suggested. Here, we centered on the possible link between CFS and Borna disease virus (BDV) infection. BDV is a neurotropic, nonsegmented negative-strand (NNS) RNA virus. Recent epidemiological data have suggested that BDV may be closely associated with depression and schizophrenia in humans. In Japanese patients with CFS, the prevalence of BDV infection was 34% (30/89) and 12% (7/57) by immunoblotting and PCR analysis, respectively. Furthermore, anti-BDV antibodies and BDV RNA were detected in a family cluster with CFS. These results suggested that this virus contributes to or initiates CFS, although the single etiologic role of BDV is unlikely.

Adult↗
PubMed1996

Growth dominance of a revertant virus generated during in vitro serial passage of nef frameshift mutant of HIV-1.

We prepared a series of nef mutant HIV-1 with a frameshift mutation at the Xho I site by up to 50 serial transfers into MT-4 cells. Here, we studied revertants. Immunofluorescence using an anti-Nef monoclonal antibody revealed that cells first became Nef antigen-positive at the 23rd passage. The percentage of Nef antigen-positive cells gradually increased and reached almost 100% by the 27th passage. The sequence of the provirus in the cells supported the generation of a revertant. This revertant mutated at the site immediately after the initially introduced frameshift mutation. This resulted in the substitution of only three amino acids and the insertion of two, which restored the proline-rich domain, a conserved region believed essential to viral replication, at the middle of Nef. Thus, the growth dominance of the revertant virus, compared with the original nef mutant, was directly demonstrated in vitro using serial passages consisting of mixed HIV-1 populations in a single cell.

Amino Acid Sequence↗
PubMed1996

Structure and antigenicity of the mannans of Candida famata and Candida saitoana: comparative study with the mannan of Candida guilliermondii.

The chemical structure of the mannans of antigenic factor 9-expressing yeast, Candida famata and Candida saitoana, was analyzed by acetolysis and NMR. The structural study of the oligosaccharides and mannans using one- and two-dimensional NMR indicated that the mannan of C. saitoana contains a third type of beta-1,2-linked mannose unit. On the other hand, the mannan of C. famata does not contain any beta-1,2-linked mannose units. The mannan of C. saitoana gave two groups of beta-1,2 linkage-containing oligosaccharides by acetolysis. One contains one beta-1,2-linked mannose unit and the other contains two consecutive beta-1,2-linked mannose units at the nonreducing terminal. The inhibition of the reactivity of factor 9 serum on an enzyme-linked immunosorbent assay (ELISA) with these oligosaccharides indicated that the inhibition activity of the former oligosaccharide is 1/20 of that of the latter ones. The ELISA of the mannans of Candida guilliermondii, C. saitoana, and Saccharomyces kluyveri, all of which contain the third type of beta-1,2-linked mannose unit, indicated that Man(beta)1--> 2Man(beta)1-->2Man(alpha)1-->3Man(alpha)1-->works as the antigenic factor 9 but Man(beta)1-->2Man(alpha)1-->3Man(alpha)1--> weakly behaves as both antigenic factors 6 and 9. The epitope structures of the side chain oligosaccharides agreed well with that proposed from the 2D-HOHAHA spectra of the mannans. This result demonstrates the usefulness of the H-1 - H-2-correlated cross-peak pattern, which was reported in a preceding paper (Shibata, N., Akagi, R., Hosoya, T., Kawahara, K., Suzuki, A., Ikuta, K., Kobayashi, H., Hisamichi, K., Okawa, Y., and Suzuki, S. (1996) J. Biol. Chem. 271, 9259-9266) for the determination of the epitope structure of Candida mannans without any chemical fragmentation.

Acetic Acid↗
PubMed1996

The V-J recombination of T cell receptor-gamma genes is blocked in interleukin-7 receptor-deficient mice.

IL-7R-deficient mice have severely impaired expansion of early lymphocytes and lack gamma delta T cells. To elucidate the role of IL-7R on gamma delta T cell development, we analyzed the rearrangements of TCR-alpha, beta, gamma, and delta genes in the thymus of the IL-7R-deficient mice. Southern blot analysis with a J gamma 1 probe revealed that more than 70% of J gamma 1 and J gamma 2 alleles are recombined to form distinct V gamma 1.2-J gamma 2 and V gamma 2-J gamma 1 fragments in control mice. On the contrary, no such recombination was detected in the mutant mice. The rearrangements in the TCR-alpha, beta, and delta loci were comparably observed in control and mutant mice. PCR analysis indicated that the V-J recombination of all the V gamma genes is severely hampered in the mutant mice. The mRNA of RAG-1, RAG-2, Ku-80, and terminal deoxynucleotidyl transferase (TdT) genes was equally detected between control and mutant thymi, suggesting that the expression of common recombination machinery is not affected. These data demonstrated that the V-J recombination of the TCR gamma genes is specifically blocked in the IL-7R-deficient mice and suggested the presence of highly specific regulation for TCR gamma gene rearrangement.

Animals↗