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Biomedical subjects

Naoki Yamamoto

Publications and source records attributed to Naoki Yamamoto.

At least 73 records · Page 4Linked to original sources
PubMed2005

The development of vaccines against SARS corona virus in mice and SCID-PBL/hu mice.

We have investigated to develop novel vaccines against SARS CoV using cDNA constructs encoding the structural antigen; spike protein (S), membrane protein (M), envelope protein (E), or nucleocapsid (N) protein, derived from SARS CoV. Mice vaccinated with SARS-N or -M DNA using pcDNA 3.1(+) plasmid vector showed T cell immune responses (CTL induction and proliferation) against N or M protein, respectively. CTL responses were also detected to SARS DNA-transfected type II alveolar epithelial cells (T7 cell clone), which are thought to be initial target cells for SARS virus infection in human. To determine whether these DNA vaccines could induce T cell immune responses in humans as well as in mice, SCID-PBL/hu mice was immunized with these DNA vaccines. As expected, virus-specific CTL responses and T cell proliferation were induced from human T cells. SARS-N and SARS-M DNA vaccines and SCID-PBL/hu mouse model will be important in the development of protective vaccines.

Animals↗
PubMed2005

A novel NF-kappaB inhibitor DHMEQ selectively targets constitutive NF-kappaB activity and induces apoptosis of multiple myeloma cells in vitro and in vivo.

Multiple myeloma (MM) is a fatal lymphoid malignancy that is incurable with conventional modalities of chemotherapy. Strong and constitutive activation of nuclear factor kappa B (NF-kappaB) is a common characteristic of MM cells. In our study we successfully target NF-kappaB with a novel NF-kappaB inhibitor dehydroxymethylepoxyquinomycin (DHMEQ). DHMEQ completely abrogates constitutive NF-kappaB activity and induces apoptosis of MM cells, whereas control peripheral blood mononuclear cells (PBMC) are resistant to NF-kappaB inhibition and apoptosis by DHMEQ treatment. DHMEQ inhibition of NF-kappaB triggers activation of caspases 8 and 9, as well as G0/G1 cell cycle arrest accompanied by downregulation of antiapoptotic genes Bcl-XL and c-FLIP and cell cycle progression gene cyclins D1 and D2. DHMEQ-mediated inhibition of vascular endothelial growth factor (VEGF) production in MM cells raises the possibility that DHMEQ abrogates the autocrine VEGF loop and enhances its antitumor effects by inhibiting neovascularization in the bone marrow. Using an in vivo NOD/SCID/gammac(null) (NOG) mice model, we show that DHMEQ has a potent inhibitory effect on the growth of MM cells. Compared to other compounds having the potential to inhibit NF-kappaB, DHMEQ is a unique compound that blocks the translocation of NF-kappaB p65 into the nucleus and selectively targets NF-kappaB activated in tumor cells. Therefore, our study presents a new molecular target therapy in MM.

Animals↗
PubMed2005

A20 is a negative regulator of IFN regulatory factor 3 signaling.

IFN regulatory factor 3 (IRF-3) is a critical transcription factor that regulates an establishment of innate immune status following detection of viral pathogens. Recent studies have revealed that two IkappaB kinase (IKK)-like kinases, NF-kappaB-activating kinase/Traf family member-associated NF-kappaB activator-binding kinase 1 and IKK-i/IKKepsilon, are responsible for activation of IRF-3, but the regulatory mechanism of the IRF-3 signaling pathway has not been fully understood. In this study, we report that IRF-3 activation is suppressed by A20, which was initially identified as an inhibitor of apoptosis and inducibly expressed by dsRNA. A20 physically interacts with NF-kappaB-activating kinase/Traf family member-associated NF-kappaB activator-binding kinase 1 and IKK-i/IKKepsilon, and inhibits dimerization of IRF-3 following engagement of TLR3 by dsRNA or Newcastle disease virus infection, leading to suppression of the IFN stimulation response element- and IFN-beta promoter-dependent transcription. Importantly, knocking down of A20 expression by RNA interference results in enhanced IRF-3-dependent transcription triggered by the stimulation of TLR3 or virus infection. Our study thus demonstrates that A20 is a candidate negative regulator of the signaling cascade to IRF-3 activation in the innate antiviral response.

Cell Line↗
PubMed2005

Stereoselective synthesis of [L-Arg-L/D-3-(2-naphthyl)alanine]-type (E)-alkene dipeptide isosteres and its application to the synthesis and biological evaluation of pseudopeptide analogues of the CXCR4 antagonist FC131.

L,L-Type and L,D-type (E)-alkene dipeptide isosteres (EADIs) that have unnatural side chains at the alpha-position were synthesized by the combination of stereoselective aziridinyl ring-opening reactions and organozinc-copper-mediated anti-S(N)2' reactions toward a single substrate of gamma,delta-cis-gamma,delta-epimino (E)-alpha,beta-enoate. The utility of this methodology was demonstrated by the stereoselective synthesis of a set of diastereomeric EADIs of L-Arg-L/D-3-(2-naphthyl)alanine (Nal) that is contained in a small CXCR4 antagonist FC131 [cyclo(-D-Tyr-Arg-Arg-Nal-Gly-)]. Furthermore, a (Nal-Gly)-type EADI was synthesized by samarium diiodide (SmI(2))-induced reduction of a gamma-acetoxy-alpha,beta-enoate. Several FC131 analogues, in which these EADIs were inserted for reduction of their peptide character, were synthesized with analogues containing reduced amide-type dipeptide isosteres to investigate the importance of these amide bonds for anti-HIV and CXCR4-antagonistic activity.

Alanine↗
PubMed2005

Mouse serum factor(s) down-modulate the CD4 and CXCR4 molecules on human T cells conferring resistance to HIV infection in NOG mice.

Human cells have developed innate immunity, exploiting several means to block virus infection, and viruses have evolved diverse strategies to resist these. We show here that the human immunodeficiency virus 1 (HIV-1) could neither progressively infect engrafted human leukemic T cells nor repress their growth in NOG mice. However, ED-40515(-) cells infected with HIV-1 before inoculation were found to significantly delay the onset of tumor growth and increased the survival period of NOG mice. ED-40515(-) tumor cells showed resistance to HIV-1 which was apparently correlated with the down-regulation of CD4 and CXCR4 molecules in NOG mice. Serum from three different mouse strains, including NOG, retained a suppressive effect on the CD4 molecule of ED-40515(-) cells in vitro. ED-40515(-) cells obtained from mice re-expressed CD4 and CXCR4 molecules upon in vitro culture and were again successfully infected with HIV-1. These findings indicate that HIV-1 may initially successfully delay or regress tumor growth in NOG mice, but eventually fails to do so because of the evolution of HIV-resistant cells due to a rapid down-modulation of CD4 and CXCR4. Our data also demonstrated that some unknown soluble factor(s) present in mouse serum was responsible for conferring resistance to HIV infection to human T cells.

Animals↗
PubMed2005

Suppressed expression of GTP cyclohydrolase I mRNA and accelerated expression of inducible nitric oxide synthase mRNA in endomyocardial biopsy specimens from patients with dilated cardiomyopathy.

BACKGROUND: Tetrahydrobiopterin (BH4) is an essential cofactor of nitric oxide synthase, and GTP cyclohydrolase I (GCHI) is a rate-limiting enzyme in the biosynthesis of BH4. The expression of inducible nitric oxide synthase (iNOS) was earlier demonstrated in the ventricles of patients with dilated cardiomyopathy (DCM) although that of GCHI was not clarified. The present study was designed to determine the GCHI mRNA expression as well as to confirm iNOS mRNA expression in endomyocardial biopsy specimens from patients with DCM. METHODS: Clinical details were assessed in 19 patients with DCM and in 9 control subjects. The real-time reverse transcription polymerase chain reaction (PCR) was performed on total RNA extracted from endomyocardial biopsy specimens. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA was quantified for use as an internal control. RESULTS: iNOS/GAPDH for the DCM samples was 4.8-fold greater than that for the control ones (P<0.01), whereas the GCHI/GAPDH for the DCM samples was reduced to 31.1% of the control (P<0.05). CONCLUSIONS: The increased expression of iNOS mRNA was confirmed in endomyocardial biopsy specimens from patients with DCM. The GCHI mRNA level was suppressed in these specimens.

Base Sequence↗
PubMed2005

The middle to 3' end of the HIV-1 vif gene sequence is important for vif biological activity and could be used for antisense oligonucleotide targets.

The human immunodeficiency virus type-1 (HIV-1)-encoded vif protein is essential for viral replication, virion production, and pathogenicity. HIV-1 Vif interacts with the endogenous human APOBEC3G protein (an mRNA editor) in target cells to prevent its encapsidation into virions. Some studies have established targets within the HIV-1 vif gene that are important for its biologic function; however, it is important to determine effective therapeutic targets in vif because of its critical role in HIV-1 infectivity and pathogenicity. The present study demonstrates that virions generated in transfected HeLa-CD4+ cells, especially from HIV-1 vif frame-shift mutant (3' delta vif; 5561-5849), were affected in splicing and had low infectivity in MT-4 cells. In addition, HIV-1 vif antisense RNA fragments constructed within the same region, notably the region spanning nucleic acid positions 5561-5705 (M-3'-AS), which corresponds to amino acid residues 96-144, significantly inhibited HIV-1 replication in MT-4 and reduced the HIV-1 vif mRNA transcripts and reporter gene (EGFP) expression. The generated virions showed low secondary infection in H9 cells. These data therefore suggest that the middle to the 3' end of vif is important for its biological activity in the target cells.

3' Flanking Region↗
PubMed2005

Identification of HIV-1 epitopes that induce the synthesis of a R5 HIV-1 suppression factor by human CD4+ T cells isolated from HIV-1 immunized hu-PBL SCID mice.

We have previously reported that immunization of the severe combined immunodeficiency (SCID) mice reconstituted with human peripheral blood mononuclear cells (PBMC) (hu-PBL-SCID mice) with inactivated human immunodeficiency virus type-1 (HIV-1)-pulsed-autologous dendritic cells (HIV-DC) elicits HIV-1-reactive CD4(+) T cells that produce an as yet to be defined novel soluble factor in vitro with anti-viral properties against CCR5 tropic (R5) HIV-1 infection. These findings led us to perform studies designed to identify the lineage of the cell that synthesizes such a factor in vivo and define the epitopes of HIV-1 protein that have specificity for the induction of such anti-viral factor. Results of our studies show that this property is a function of CD4(+) but not CD8(+) T cells. Human CD4(+) T cells were thus recovered from the HIV-DC-immunized hu-PBL-SCID mice and were re-stimulated in vitro by co-culture for 2 days with autologous adherent PBMC as antigen presenting cells, APC previously pulsed with inactivated HIV in IL-2-containing medium to expand HIV-1-reactive CD4(+) T cells. Aliquots of these re-stimulated CD4(+) T cells were then co-cultured with similar APC's that were previously pulsed with 10 microg/ml of a panel of HIV peptides for an additional 2 days, and their culture supernatants were examined for the production of both the R5 HIV-1 suppression factor and IFN-gamma. The data presented herein show that the HIV-1 primed CD4(+) T cells produced the R5 suppression factor in response to a wide variety of HIV-1 gag, env, pol, nef or vif peptides, depending on the donor of the CD4(+) T cells. Simultaneous production of human interferon (IFN)-gamma was observed in some cases. These results indicate that human CD4(+) T cells in PBMC of HIV-1 naive donors have a wide variety of HIV-1 epitope-specific CD4(+) T cell precursors that are capable of producing the R5 HIV-1 suppression factor upon DC-based vaccination with whole inactivated HIV-1.

Animals↗
PubMed2005

Lentiviral-mediated delivery of combined HIV-1 decoy TAR and Vif siRNA as a single RNA molecule that cleaves to inhibit HIV-1 in transduced cells.

RNA interference (RNAi) silences gene expression via short interfering 21-23 mer double-stranded RNA (siRNA) segments that guide cognate mRNA degradation in a sequence-specific manner. On the other hand, HIV-1 decoy TAR RNA are known to competitively interact with the HIV-1 Tat protein, to downregulate the enhanced gene expression from the long terminal repeat (LTR) promoters. Here we report that a novel expression construct, encoding both HIV-1 decoy TAR and Vif siRNA, as a single RNA substrate, was expressed under the control of the human U6 promoter, and later the TAR and siRNA were cleaved into their respective separate RNA by the endogenous RNase III-like enzyme. Each of the cleaved HIV-1 anti-genes then synergistically contributed toward enhancing the inhibition efficacy (>80%) of HIV-1 replication in transduced Jurkat cells. These results suggest that targeting HIV-1 mRNA with simultaneously expressed intracellular decoy TAR and Vif-siRNA could lead to an effective gene therapy strategy for the control and management of HIV-AIDS.

Acquired Immunodeficiency Syndrome↗
PubMed2005

Phylogenetic heterogeneity of new HTLV type 1 isolates from southern India in subgroup A.

Seven isolates of human T cell leukemia virus type 1 (HTLV-1) were taken in southern India and phylogenetically analyzed to gain new insights into the origin and dissemination of HTLV-1 in the subcontinent. The new Indian HTLV-1s were found to be members of subgroup A (Transcontinental subgroup) of the Cosmopolitan group. They formed three different clusters (South African/Caribbean, Middle Eastern, and East Asian clusters). These results demonstrate that Indian HTLV-1s are genetically heterogeneous and include the most divergent strain of subgroup A. On the basis of these results, we speculate that subgroup A HTLV- 1s may have been present for thousands of years in India.

Adult↗
PubMed2005

Predominance of three NF-kappaB binding sites in the long terminal repeat region of HIV Type 1 subtype C isolates from Zambia.

Human immunodeficiency virus type-1 (HIV-1) is a leading cause of mortality and morbidity in the world, with almost 46 million people infected globally. HIV-1 subtype C accounts for 55% of these infections. In Zambia, the majority of HIV-1 infections are subtype C. However, to its north most countries have non-subtype C as the most predominant HIV-1 subtype while to its south most of them are predominantly subtype C. The aim of this study was to determine the subtype distribution and to analyze the long terminal repeat (LTR) region of HIV-1 isolates from the northern part of Zambia. We amplified as well as directly sequenced the LTR, gag, and env regions of 78 HIV-1 peripheral blood samples from adult Zambians. Our results show 95% (74/78) of our isolates were HIV-1 subtype C. Furthermore, of the subtype C samples analyzed across the LTR, 61% (25/41) carried 3 NF-kappaB signature binding site sequences.

Base Sequence↗
PubMed2005

Molecular epidemiology of the heterosexual HIV-1 transmission in Kunming, Yunnan Province of China suggests origin from the local IDU epidemic.

Molecular epidemiological investigation was conducted among injecting drug users (IDUs) (n = 11) and heterosexuals (n = 15) in Kunming, Yunnan Province of China. HIV-1 genotypes were determined based on the nucleotide sequences of 2.6-kb gag-RT region. The distribution of genotypes among IDUs was as follows: CRF07_BC (5/11) and CRF08_BC (5/11); subtype B' (1/11). Similarly, a majority of Kunming heterosexuals (14/15) were infected with CRF07_BC (4/15), CRF08_BC (6 /15), or subtype B' (4/15), known to predominate among IDUs in China. This contrasts with trends in the coastal regions of China and surrounding southeastern Asian countries, where CRF01_AE predominates among heterosexuals. The heterosexual HIV-1 epidemic in Kunming thus appears to derive from the local IDU epidemic. Of note, subtype B' was the most prevalent strain among heterosexuals before 1997, while CRF07_BC and CRF08_BC became predominant in 2002, indicating a transition of HIV-1 genotype distribution between the early and the more recent samples from Kunming heterosexuals.

China↗
PubMed2005

Production and characterization of a monoclonal antibody specific to Nef-associated factor 1 (Naf1)/A20-binding inhibitor of NF-kappaB activation (ABIN-1).

Cellular protein Naf1 (Nef-associated factor 1) or ABIN-1 (A20-binding inhibitor of NF-kappaB activation) is an important cellular protein, expressed in various human tissues and T-cell lines. Naf1 protein has two isoforms (Naf1alpha and Naf1beta) with different C-termini, produced by alternative splicing. Naf1alpha and Naf1beta have approximately 2800 and 2600 nucleotides, with an open reading frame of 1941 and 1781 nucleotides, encoding the 72-kDa Naf1alpha and 68-kDa Naf1beta proteins, respectively. In the present study, we generated a monoclonal antibody (MAb) against human Naf1, which recognizes full-length, endogenous Naf1 of both isotypes. For this purpose, recombinant 6xHis and myc-tagged N-terminal Naf1(38135), Naf1(N) protein was produced by using the baculovirus expression system. Recombinant Naf1(N) protein was used to immunize Balb/c mice, and a hybridoma cell line producing stable and highly specific MAb with strong affinity to Naf1 was established. We further characterized this antibody by immunofluorescent assay and Western blot analysis to confirm effectiveness in detecting recombinant and endogenous Naf1. By Western blot analysis of recombinant Naf1-N fusion proteins with overlapping N-terminal sequences, the epitope targeted by anti-Naf1 MAb was determined as the 81-88-amino acid region of human Naf1.

Animals↗
PubMed2005

Solution RNA structures of the HIV-1 dimerization initiation site in the kissing-loop and extended-duplex dimers.

Dimer formation of HIV-1 genomic RNA through its dimerization initiation site (DIS) is crucial to maintaining infectivity. Two types of dimers, the initially generated kissing-loop dimer and the subsequent product of the extended-duplex dimer, are formed in the stem-bulge-stem region with a loop including a self-complementary sequence. NMR chemical shift analysis of a 39mer RNA corresponding to DIS, DIS39, in the kissing-loop and extended-duplex dimers revealed that the three dimensional structures of the stem-bulge-stem region are extremely similar between the two types of dimers. Therefore, we designed two shorter RNA molecules, loop25 and bulge34, corresponding to the loop-stem region and the stem-bulge-stem region of DIS39, respectively. Based upon the chemical shift analysis, the conformation of the loop region of loop25 is identical to that of DIS39 for each of the two types of dimers. The conformation of bulge34 was also found to be the same as that of the corresponding region of DIS39. Thus, we determined the solution structures of loop25 in each of the two types of dimers as well as that of bulge34. Finally, the solution structures of DIS39 in the kissing-loop and extended-duplex dimers were determined by combining the parts of the structures. The solution structures of the two types of dimers were similar to each other in general with a difference found only in the A16 residue. The elucidation of the structures of DIS39 is important to understanding the molecular mechanism of the conformational dynamics of viral RNA molecules.

Base Sequence↗
PubMed2005

Replication-defective HIV isolated from seronegative individuals at high risk for HIV infection.

We have identified a mutant human immunodeficiency virus type 1 (HIV-1) CRF01_AE that contains a single nucleotide mutation in gag gene from 4 HIV-1 seronegative drug users in Thailand. We found A to G mutation at the nucleotide position 75 of gag p17 gene (A75G) not changing the amino acid sequence. The mutant HIV-1 molecular clones were examined for their replication capability. Although the mutation dramatically reduced the level of virion production, it did not affect the amounts of viral protein synthesis within the transfected cells. In addition, this mutation did not affect the levels of Gag polyproteins. Furthermore, electron microscopic examinations have revealed a dramatic reduction of the virion production and perturbation of viral morphogenesis at the cytoplasmic membrane. These results indicate that the A75G mutation is attributable to the long-term sero-negativity of individuals at high risk of HIV-1 infection and suggest a novel mechanism that regulates HIV production.

Gene Products, gag↗
PubMed2005

Development of a monoclonal antibody against Epstein-Barr virus nuclear antigen leader protein (EBNA-LP) that can detect EBNA-LP expressed in P3HR1 cells.

A mouse monoclonal antibody, LP4D3, was raised against purified Epstein-Barr virus nuclear antigen leader protein (EBNA-LP) fused to glutathione-S-transferase. The antibody detected endogenous and exogenous EBNA-LP in immunoblotting, immunofluorescence and immunoprecipitation assays, and the epitope of the antibody was mapped in the W2 domain of EBNA-LP. While another monoclonal antibody to EBNA-LP, JF186, which is widely used for analyses of the viral protein, did not react with truncated forms of EBNA-LP expressed in P3HR1 cells, as reported earlier, the LP4D3 antibody did. The LP4D3 antibody will be a useful tool for further studies of EBNA-LP, especially investigations into the phenotypes of mutant EBNA-LP expressed in P3HR1 cells.

Antibodies, Monoclonal↗
PubMed2005

Hodgkin's lymphoma cells are efficiently engrafted and tumor marker CD30 is expressed with constitutive nuclear factor-kappaB activity in unconditioned NOD/SCID/gammac(null) mice.

As there are very few reproducible animal models without conditioning available for the study of human B-cell-type Hodgkin's lymphoma (HL), we investigated the ability of HL cells to induce tumors using novel NOD/SCID/gammac(null) (NOG) mice. Four human Epstein-Barr virus-negative cell lines (KM-H2 and L428 originated from B cells, L540 and HDLM2 originated from T cells) were inoculated either subcutaneously in the postauricular region or intravenously in the tail of unmanipulated NOG mice. All cell lines successfully engrafted and produced tumors with infiltration of cells in various organs of all mice. Tumor cells had classical histomorphology as well as expression patterns of the tumor marker CD30, which is a cell surface antigen expressed on HL. Tumor progression in mice inoculated with B-cell-type, but not T-cell-type, HL cells correlated with an elevation in serum human interleukin-6 levels. Tumor cells from the mice also retained strong nuclear factor (NF)-kappaB DNA binding activity, and the induced NF-kappaB components were indistinguishable from those cultured in vitro. The reproducible growth behavior and preservation of characteristic features of both B-cell-type and T-cell-type HL in the mice suggest that this new xenotransplant model can provide a unique opportunity to understand and investigate the mechanism of pathogenesis and malignant cell growth, and to develop novel anticancer therapies.

Animals↗