Biomedical subjects

Naoki Yamamoto

Publications and source records attributed to Naoki Yamamoto.

At least 127 records · Page 7Linked to original sources

PubMed2004

Role of follicular dendritic cells in the early HIV-1 infection: in vitro model without specific antibody.

About 90% of HIV-1 RNA in the lymph nodes is reported to localize in follicular dendritic cellsnetwork (FDC-NW) as early as several days after infection and as much as that in the late stage. But the mechanism remains to be fully understood. To elucidate the role of follicular dendritic cells (FDC) in the early stage of HIV-1 infection, FDC-like cell strains (FDCLC) were established and they were characterized in the co-culture system with T cells for their effect on HIV-1 trapping and replication in p24 immunoassay, immunohistochemistry as well as confocal and electronmicroscopy. Established FDCLC were positive for CNA-42, S-100alpha and intercellular desmosome-like junctions. L-SIGN and DC-SIGN were also detected in FDCLC. Alu-HIV-1 PCR analysis showed no HIV-1 integration in FDCLC. FDCLC trapped HIV-1 and transferred them to uninfected MOLT-4 T cells (MOLT-4) efficiently in the absence of specific antibody. FDCLC also accelerated HIV-1 replication in HIV-1-pre-exposed MOLT-4. These unique FDCLC effects were explained, at least partly, by the fact that FDCLC up-regulated CD4 expression in MOLT-4 and helped T cells escape from apoptosis in the co-culture. These data suggest that FDC/FDCLC engage not only in trapping but also in active expansion of HIV-1 in the absence of specific antibody.

Cells, Cultured↗

PubMed2004

Prompt tumor formation and maintenance of constitutive NF-kappaB activity of multiple myeloma cells in NOD/SCID/gammacnull mice.

Clinically and biologically relevant animal models are indispensable to evaluate both the pathophysiology and strategies for diagnosis and treatment of multiple myeloma (MM). We examined the tumorigenicity of MM cell lines KMM-1 and U-266 in an in vivo cell proliferation model using NOD/SCID/gammacnull (NOG) mice. Two cell lines were inoculated either subcutaneously (s.c.) in the post-auricular region or intravenously (i.v.) in the tail of NOG mice. The KMM-1 cell line produced a progressively growing large tumor with infiltration of the cells expressing human lambda-chain in various organs of all NOG mice, while the U-266 cell line failed to do so. Tumor cells grown in NOG mice maintained the original histomorphology, as well as expression patterns of tumor markers human lambda Ig light chain and VEGF. Tumor progression in mice also correlated with elevation of serum human soluble IL-6R and gp130. Tumor cells sustained a strong NF-kappaB activity in vivo and induced NF-kappaB components were indistinguishable from those in cells cultured in vitro. The rapid and efficient engraftment of the MM cell line in NOG mice suggests that this is a very useful animal model which could provide a novel system in which to clarify the mechanism of growth of cancer cells, as well as to develop new therapeutic regimens against MM.

Animals↗

PubMed2004

Automatic quantification of morphological traits via three-dimensional measurement of Arabidopsis.

Many mutants have been isolated from the model plant Arabidopsis thaliana, and recent important genetic resources, such as T-DNA knockout lines, facilitate the speed of identifying new mutants. However, present phenotypic analysis of mutant screens depends mainly on qualitative descriptions after visual observation of morphological traits. We propose a novel method of phenotypic analysis based on precise three-dimensional (3D) measurement by a laser range finder (LRF) and automatic data processing. We measured the 3D surfaces of young plants of two Arabidopsis ecotypes and successfully defined two new traits, the direction of the blade surface and epinasty of the blade, quantitatively. The proposed method enables us to obtain quantitative and precise descriptions of plant morphologies compared to conventional 2D measurement. The method will open a way to find new traits from mutant pools or natural ecotypes based on 3D data.

Algorithms↗

PubMed2004

Environment- and mutation-dependent aggregation behavior of Alzheimer amyloid beta-protein.

The deposition of amyloid beta-protein in the brain is a fundamental process in the development of Alzheimerís disease; however, the mechanism underlying aggregation of amyloid beta-protein remains to be determined. Here, we report that a membrane-mimicking environment, generated in the presence of detergents or a ganglioside, is sufficient per se for amyloid fibril formation from soluble amyloid beta-protein. Furthermore, hereditary variants of amyloid beta-protein, which are caused by amyloid precursor protein gene mutations, including the Dutch (E693Q), Flemish (A692G) and Arctic (E693G) types, show mutually different aggregation behavior in these environments. Notably, the Arctic-type amyloid beta-protein, in contrast to the wild-type and other variant forms, shows a markedly rapid and higher level of amyloid fibril formation in the presence of sodium dodecyl sulfate or GM1 ganglioside. These results suggest that there are favorable local environments for fibrillogenesis of amyloid beta-protein.

Alzheimer Disease↗

PubMed2004

Effects of psychotomimetic and antipsychotic agents on neocortical and striatal concentrations of various amino acids in the rat.

A subcutaneous injection of small and moderate doses (1.6, 3.2, 4.0 and 4.8 mg/kg) of the schizophrenomimetic methamphetamine caused a dose-related increase in the tissue content (the net content) of L-Arg and L-Asn in the neocortex and striatum at 60 min, but not at 360 min, after injection. The methamphetamine-induced (4.8 mg/kg) increases in levels of these amino acids were significantly attenuated by pretreatment with an antipsychotic drug, haloperidol (1 mg/kg) or clozapine (10 mg/kg). In the neocortex, a clozapine-reversible increase in the level of L-Thr was also observed 60 min after methamphetamine administration. Striatal concentrations of L-Glu, L-Ser, LThr, Gly and L-Ala were augmented by the same regimen in a haloperidol- and clozapine-sensitive fashion. A moderate dose of another schizophrenomimetic phencyclidine (7.5 mg/kg) given subcutaneously induced robust abnormal behavior, a diminution in the neocortical and striatal levels of L-Asp and an increase in the striatal L-Ala content without significant effects on the other amino acids studied. These results suggest that neocortical and striatal L-Arg, L-Asn, L-Thr, Gly, L-Ala or L-Ser may be implicated in the psychotomimetic effects of methamphetamine and might display mutual interaction with cerebral dopaminergic transmission. The differential effects of methamphetamine and phencyclidine on the net neocortical and striatal concentrations of various amino acids might, at least in part, underlie the distinct features of psychoses induced by these two drugs.

Amino Acids↗

PubMed2004

A consecutive priming-boosting vaccination of mice with simian immunodeficiency virus (SIV) gag/pol DNA and recombinant vaccinia virus strain DIs elicits effective anti-SIV immunity.

To evaluate immunity induced by a novel DNA prime-boost regimen, we constructed a DNA plasmid encoding the gag and pol genes from simian immunodeficiency virus (SIV) (SIVgag/pol DNA), in addition to a replication-deficient vaccinia virus strain DIs recombinant expressing SIV gag and pol genes (rDIsSIVgag/pol). In mice, priming with SIVgag/pol DNA, followed by rDIsSIVgag/pol induced an SIV-specific lymphoproliferative response that was mediated by a CD4+-T-lymphocyte subset. Immunization with either vaccine alone was insufficient to induce high levels of proliferation or Th1 responses in the animals. The prime-boost regimen also induced SIV Gag-specific cellular responses based on gamma interferon secretion, as well as cytotoxic-T-lymphocyte responses. Thus, the regimen of DNA priming and recombinant DIs boosting induced Th1-type cell-mediated immunity, which was associated with resistance to viral challenge with wild-type vaccinia virus expressing SIVgag/pol, suggesting that this new regimen may hold promise as a safe and effective vaccine against human immunodeficiency virus type 1.

AIDS Vaccines↗

PubMed2004

Epstein-Barr virus nuclear antigen leader protein induces expression of thymus- and activation-regulated chemokine in B cells.

Epstein-Barr virus (EBV) nuclear antigen leader protein (EBNA-LP) plays a critical role in transformation of primary B lymphocytes to continuously proliferating lymphoblastoid cell lines (LCLs). To identify cellular genes in B cells whose expression is regulated by EBNA-LP, we performed microarray expression profiling on an EBV-negative human B-cell line, BJAB cells, that were transduced by a retroviral vector expressing the EBV EBNA-LP (BJAB-LP cells) and on BJAB cells that were transduced with a control vector (BJAB-vec cells). Microarray analysis led to the identification of a cellular gene encoding the CC chemokine TARC as a novel target gene that was induced by EBNA-LP. The levels of TARC mRNA expression and TARC secretion were significantly up-regulated in BJAB-LP compared with BJAB-vec cells. Induction of TARC was also observed when a subline of BJAB cells was converted by a recombinant EBV. Among the EBV-infected B-cell lines with the latency III phenotype that were tested, the LCLs especially secreted significantly high levels of TARC. The level of TARC secretion appeared to correlate with the level of full-length EBNA-LP expression. These results indicate that EBV infection induces TARC expression in B cells and that EBNA-LP is one of the viral gene products responsible for the induction.

Animals↗

PubMed2004

Apoptosis induced by the histone deacetylase inhibitor FR901228 in human T-cell leukemia virus type 1-infected T-cell lines and primary adult T-cell leukemia cells.

Inhibition of histone deacetylase (HDAC) activity induces growth arrest, differentiation, and, in certain cell types, apoptosis. FR901228, FK228, or depsipeptide, is an HDAC inhibitor effective in T-cell lymphomas. Adult T-cell leukemia (ATL) is caused by human T-cell leukemia virus type 1 (HTLV-1) and remains incurable. We examined whether FR901228 is effective for treatment of ATL by assessing its ability to induce apoptosis of HTLV-1-infected T-cell lines and primary leukemic cells from ATL patients. FR901228 induced apoptosis of Tax-expressing and -unexpressing HTLV-1-infected T-cell lines and selective apoptosis of primary ATL cells, especially those of patients with acute ATL. FR901228 also efficiently reduced the DNA binding of NF-kappaB and AP-1 in HTLV-1-infected T-cell lines and primary ATL cells and down-regulated the expression of Bcl-x(L) and cyclin D2, regulated by NF-kappaB. Although the viral protein Tax is an activator of NF-kappaB and AP-1, FR901228-induced apoptosis was not associated with reduced expression of Tax. In vivo use of FR901228 partly inhibited the growth of tumors of HTLV-1-infected T cells transplanted subcutaneously in SCID mice. Our results indicated that FR901228 could induce apoptosis of these cells and suppress the expression of NF-kappaB and AP-1 and suggest that FR901228 could be therapeutically effective in ATL.

Adult↗

PubMed2004

In vitro and in vivo transport and delivery of phosphorothioate oligonucleotides with cationic liposomes.

A recent strategy in gene therapy has been using antiviral genes that are delivered to uninfected cells, either as RNA or DNA, to provide intracellular protection from human immunodeficiency virus type-1 (HIV-1) infection. Antisense oligonucleotides that are complementary to specific target genes suppress gene expression. A variety of techniques are available to enhance the cellular uptake and pharmacological effectiveness of antisense oligonucleotides, both in vitro and in vivo. We investigated the intracellular and tissue uptake of an oligonucleotide/cationic lipid complex, using a fluorescently labeled oligonucleotide. The antisense oligonucleotide was designed against the HIV-1 gag gene sequence. A T-cell line (MT-4) and PHA-stimulated peripheral blood mononuclear cells (PBMCs) were both infected with HIV-1(NL432) at an MOI of 0.01. One h later, both cultures were washed and treated with medium containing 1 microM antisense oligonucleotide. After a 3-day interval, the HIV-1 antigen expression was monitored by an indirect immunofluorescence assay. At 3 days post infection, we confirmed that p24 antigen production was inhibited by the antisense oligonucleotide/cationic lipid complex at a 1/10 ratio in the PBMCs, using enzyme-linked immunosorbent assay (ELISA). We also confirmed the intracellular existence of the complex by fluorescent microscopy. We investigated different means of transporting the antisense oligonucleotide/cationic lipid complex to mouse tissues by intravenous, intraperitoneal and subcutaneous injections. We observed that the anti-HIV-1 activity of the antisense oligonucleotide/cationic lipid complex was the result of enhanced cellular uptake, both in vitro and in vivo. Therefore, the antisense oligonucleotide/cationic lipid complex is an excellent system for the transport and delivery of genes to target cells, as it is effective both in vitro and in vivo.

Animals↗

PubMed2004

The normalization of guinea pig leukocyte fractions and lymphocyte subsets in blood and lymphoid tissues using a flow cytometric procedure.

Many hematological and immunological parameters remain unclear in the study of the guinea pig. In this study, we established the mean values of blood counts, the percentage of leukocyte fractions and lymphocyte subsets in blood and various lymphoid tissues of the guinea pig with a flow cytometric procedure using MIL4/SSC. The mean counts of WBC and RBC in the blood were lower, and MCV and MCH were higher than those of other rodents, resembling those of humans. Furthermore, the mean percentages of blood lymphocytes were smaller and that of granulocyte was larger than those of other rodents, resembling those of humans. We further established a flow cytometric procedure for lymphocyte subsets and clarified the mean percentages of T- and B-cells, CD4(+)-, CD8(+)- and MHC Class II(+)- T-cells, and CD4(-)CD8 (-) T-cells. The latter were morphologically larger in cell size and cytoplasm than CD4(+)- plus CD8(+) T-cells, and this subset had a significantly higher percentage in newborn animals. Furthermore, the appearance of the MHC Class II(+) T-cell subset was suggested to be a marker of hyper-activation of T-cells in BCG-immunized animals. Thus, both the novel flow cytometric procedure for leukocyte fractions and lymphocyte subsets, and the established normal values will be useful tools in studying guinea pigs as models of various diseases and biological phenomena.

Animals↗

PubMed2004

Differentiation of human and mouse embryonic stem cells along a hepatocyte lineage.

Embryonic stem (ES) cells may differentiate along a hepatocyte lineage; however, currently there are no reports of culture conditions yielding high levels of hepatocyte-specific gene expression in these cells. We investigated culture conditions for differentiating ES cells into hepatocyte-like cells in vitro. Various combinations of culture media, growth and differentiation factors, and substratum precoatings were evaluated, and it was determined that a combination of Iscove's modified Dulbecco's medium with 20% fetal bovine serum, human insulin, dexamethasone. and collagen type I precoating was optimal for directing mouse ES cells along a hepatocyte lineage. Treatment of mouse ES cell with the optimal condition led to prealbumin gene expression 20% as high, and albumin synthesis 7% as high, as in mouse liver. The optimal culture condition also induced albumin gene expression in differentiated human ES cells 1% as high as in normal human hepatocytes as shown by Western blot analysis, and cells were positive for human albumin by immunocytochemistry. In addition, our optimal condition led to high levels of albumin gene expression in primary mouse hepatocytes after 35 days of culture, levels 10-fold higher than with other hepatocyte differentiation media. In conclusion, our optimal condition directed both mouse and human ES cells along a hepatocyte lineage. This represents the initial step in establishing cell lines that can be employed in cell-based therapeutics in humans and for toxicology and pharmacology studies.

Albumins↗

PubMed2003

Functional analysis and intracellular localization of rice cryptochromes.

Blue-light-receptor cryptochrome (CRY), which mediates cotyledon expansion, increased accumulation of anthocyanin, and inhibition of hypocotyl elongation, was first identified in Arabidopsis. Two Arabidopsis cryptochromes (AtCRY1 and AtCRY2) have been reported to be localized to the nucleus. However, there is no information on the cryptochromes in monocotyledons. In this study, we isolated two cryptochrome cDNAs, OsCRY1 and OsCRY2, from rice (Oryza sativa) plants. The deduced amino acid sequences of OsCRY1 and OsCRY2 have a photolyase-like domain in their N termini and are homologous to AtCRY1. To investigate the function of OsCRY1, we overexpressed a green fluorescence protein-OsCRY1 fusion gene in Arabidopsis and assessed the phenotypes of the resulting transgenic plants. When the seedlings were germinated in the dark, no discernible effect was observed. However, light-germinated seedlings showed pronounced inhibition of hypocotyl elongation and increased accumulation of anthocyanin. These phenotypes were induced in a blue-light-dependent manner, indicating that OsCRY1 functions as a blue-light-receptor cryptochrome. We also examined the intracellular localization of green fluorescence protein-OsCRY1 in the transgenic plants. It was localized to both the nucleus and the cytoplasm. We identified two nuclear localization domains in the primary structure of OsCRY1. We discuss the relationship between the function and intracellular localization of rice cryptochromes by using additional data obtained with OsCRY2.

Amino Acid Sequence↗

PubMed2003

Synthesis of potent CXCR4 inhibitors possessing low cytotoxicity and improved biostability based on T140 derivatives.

A peptidic CXCR4 antagonist T140 efficiently blocks the entry of T cell line-tropic strains of HIV-1 (X4-HIV-1) into target cells. In this study, a series of T140 derivatives, replacing the basic amino acid residues with Glu (D-Glu) and/or L-citrulline (Cit), were synthesized in order to reduce non-specific binding and cytotoxicity. Among them, TE14011 ([Cit6, D-Glu8]-T140 with the C-terminal amide) exhibited strong anti-HIV activity and low cytotoxicity. TE14011 was found to be stable in mouse serum, but unstable in rat liver homogenate due to the deletion of the N-terminal Arg1-Arg2-L-3-(2-naphthyl)alanine (Nal)3 residues from the parent peptide. N-Terminal acetylation of TE14011 led to the development of a novel lead compound, Ac-TE 14011, which possesses a high selectivity index as well as increased stability in serum and liver homogenate.

Amino Acid Sequence↗

PubMed2003

Enhancement of the T140-based pharmacophores leads to the development of more potent and bio-stable CXCR4 antagonists.

A CXCR4 antagonistic peptide, T140, and its bio-stable analogs, such as Ac-TE14011, were previously developed. These peptides inhibit the entry of T cell line-tropic strains of HIV-1 (X4-HIV-1) into T cells. Herein, a series of TE14011 analogs having modifications in the N-terminal region were synthesized to develop effective compounds with increased biostability. Among these analogs, 4F-benzoyl-TE14011 (TF14013) showed the strongest anti-HIV activity derived from CXCR4-antagonism, suggesting that a 4-fluorobenzoyl moiety at the N-terminus of T140 analogs constitutes a novel T140-based pharmacophore for CXCR4 antagonists. Structure-activity relationship (SAR) studies on TE14011 analogs with N(alpha)-acylation by several benzoic acid derivatives have disclosed a significant relationship between the anti-HIV activity and the Hammett constant (sigma) of substituted benzoic acids. TF14013 was found to be stable in mouse serum, but not completely stable in rat liver homogenate due to deletion of the C-terminal Arg14-NH2 from the parent peptide. This biodegradation was completely suppressed by N-alkyl-amidation at the C-terminus. Taken together, the enhancement of the T140-based pharmacophores led to development of a novel CXCR4 antagonist, 4F-benzoyl-TE14011-Me (TF14013-Me), which has very high anti-HIV activity and increased biostability.

Circular Dichroism↗

PubMed2003

Soluble IL-6 receptor governs IL-6 activity in experimental arthritis: blockade of arthritis severity by soluble glycoprotein 130.

Studies in IL-6-deficient (IL-6(-/-)) mice highlight that IL-6 contributes to arthritis progression. However, the molecular mechanism controlling its activity in vivo remains unclear. Using an experimental arthritis model in IL-6(-/-) mice, we have established a critical role for the soluble IL-6R in joint inflammation. Although intra-articular administration of IL-6 itself was insufficient to reconstitute arthritis within these mice, a soluble IL-6R-IL-6 fusion protein (HYPER-IL-6) restored disease activity. Histopathological assessment of joint sections demonstrated that HYPER-IL-6 increased arthritis severity and controlled intrasynovial mononuclear leukocyte recruitment through the CC-chemokine CCL2. Activation of synovial fibroblasts by soluble IL-6R and IL-6 emphasized that these cells may represent the source of CCL2 in vivo. Specific blockade of soluble IL-6R signaling in wild-type mice using soluble gp130 ameliorated disease. Consequently, soluble IL-6R-mediated signaling represents a promising therapeutic target for the treatment of rheumatoid arthritis.

Animals↗

PubMed2003

Two carboxyl-terminal activation regions of Epstein-Barr virus latent membrane protein 1 activate NF-kappaB through distinct signaling pathways in fibroblast cell lines.

Latent membrane protein 1 (LMP1), an Epstein-Barr virus transforming protein, is able to activate NF-kappaB through its carboxyl-terminal activation region 1 (CTAR1) and 2 (CTAR2), but the exact role of each domain is not fully understood. Here we show that LMP1 activates NF-kappaB in different NF-kappaB essential modulator (NEMO)-defective cell lines, but not in cells lacking both IkappaB kinase 1 (IKK1) and 2 (IKK2). Mutational studies reveal that CTAR1, but not CTAR2, mediates NEMO-independent NF-kappaB activation and that this process largely depends on IKK1. Retroviral expression of LMP1 mutants in cells lacking either functional NF-kappaB inducing kinase (NIK), NEMO, IKK1, or IKK2 further illustrates distinct signals from the two activation regions of LMP1 for persistent NF-kappaB activation. One originates in CTAR2, operates through the canonical NEMO-dependent pathway, and induces NFKB2 p100 production; the second signal originates in CTAR1, utilizes NIK and IKK1, and induces the processing of p100. Our results thus help clarify how two functional domains of LMP1 persistently activate NF-kappaB through distinct signaling pathways.

Amino Acid Sequence↗

PubMed2003

T140 analogs as CXCR4 antagonists identified as anti-metastatic agents in the treatment of breast cancer.

A chemokine receptor, CXCR4, and its endogenous ligand, stromal cell-derived factor-1 (SDF-1), have been recognized to be involved in the metastasis of several types of cancers. T140 analogs are peptidic CXCR4 antagonists composed of 14 amino acid residues that were previously developed as anti-HIV agents having inhibitory activity against HIV-entry through its co-receptor, CXCR4. Herein, we report that these compounds effectively inhibited SDF-1-induced migration of human breast cancer cells (MDA-MB-231), human leukemia T cells (Sup-T1) and human umbilical vein endothelial cells at concentrations of 10-100 nM in vitro. Furthermore, slow release administration by subcutaneous injection using an Alzet osmotic pump of a potent and bio-stable T140 analog, 4F-benzoyl-TN14003, gave a partial, but statistically significant (P</=0.05 (t-test)) reduction in pulmonary metastasis of MDA-MB-231 in SCID mice, even though no attempt was made to inhibit other important targets such as CCR7. These results suggest that T140 analogs have potential use for cancer therapy, and that small molecular CXCR4 antagonists could potentially replace neutralizing antibodies as anti-metastatic agents for breast cancer.

Animals↗

PubMed2003

Hyperosmolar mannitol simulates expression of aquaporins 4 and 9 through a p38 mitogen-activated protein kinase-dependent pathway in rat astrocytes.

The membrane pore proteins, aquaporins (AQPs), facilitate the osmotically driven passage of water and, in some instances, small solutes. Under hyperosmotic conditions, the expression of some AQPs changes, and some studies have shown that the expression of AQP1 and AQP5 is regulated by MAPKs. However, the mechanisms regulating the expression of AQP4 and AQP9 induced by hyperosmotic stress are poorly understood. In this study, we observed that hyperosmotic stress induced by mannitol increased the expression of AQP4 and AQP9 in cultured rat astrocytes, and intraperitoneal infusion of mannitol increased AQP4 and AQP9 in the rat brain cortex. In addition, a p38 MAPK inhibitor, but not ERK and JNK inhibitors, suppressed their expression in cultured astrocytes. AQPs play important roles in maintaining brain homeostasis. The expression of AQP4 and AQP9 in astrocytes changes after brain ischemia or traumatic injury, and some studies have shown that p38 MAPK in astrocytes is activated under similar conditions. Since mannitol is commonly used to reduce brain edema, understanding the regulation of AQPs and p38 MAPK in astrocytes under hyperosmotic conditions induced with mannitol may lead to a control of water movements and a new treatment for brain edema.

Animals↗