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Biomedical subjects

K Yamaguchi

Publications and source records attributed to K Yamaguchi.

At least 649 records · Page 36Linked to original sources
PubMed1997

Inhibition of gene expression from the human c-erbB gene promoter by a retroviral vector expressing anti-gene RNA.

Anti-gene is a potent inhibitor of transcriptional promoter activity and subsequent gene expression. This property has been exploited to suppress the expression of a variety of oncogenes for regulating tumor proliferation or viral activities. In this paper, we describe a novel retroviral vector designed to express human c-erbB anti-gene RNA and to reduce the promoter activity in the cells. Mouse fibroblast NIH3T3 cells were stably transfected with an expression construct containing a truncated human c-erbB gene promoter fused to the firefly luciferase reporter gene. Infection into these cells of the c-erbB anti-gene retroviral vector targeted to the 26 bp pyrimidine-rich element in the human c-erbB gene promoter resulted in a dose-dependent decrease in the luciferase activity of the cells. Retroviral vector expressing anti-gene RNA may be useful as an alternative program of gene regulation in the cells.

3T3 Cells↗
PubMed1997

Persistent clonal proliferation of human T-lymphotropic virus type I-infected cells in vivo.

Clonal proliferation of human T-lymphotropic virus type I (HTLV-I)-infected cells has been detected by Southern blot analysis and inverse PCR in patients with adult T-cell leukemia, patients with HTLV-I-associated diseases, and even in asymptomatic carriers. Combining inverse PCR with long PCR, we amplified the genomic DNA regions flanking the integration sites of the HTLV-I provirus to detect clones of infected cells. Inverse long PCR revealed that increased virus load was associated with an increase of both the number of cells in each clone and the number of clones. Clonal proliferations were found in both CD4- and CD8-positive cells in a carrier and a patient with HTLV-I-associated neuropathy/tropical spastic paraparesis. These HTLV-I-infected clones persisted over several years in the same carriers, and, moreover, most of the persistent clones were CD4 positive in a HTLV-I carrier. These findings indicate that HTLV-I infection plays an important role in the clonal expansion of lymphocytes and the prolonged survival of CD4-positive cells in vivo. Surviving T-lymphocytes may be susceptible to genetic changes, leading to the onset of leukemia.

Adult↗
PubMed1997

Displacement of gold marker in immunoelectron microscopy of human respiratory cilia.

Preembedding immunogold electron microscopy was performed to evaluate the position of outer arm dynein heavy chains in normal human respiratory cilia. Anti-dynein antibody (AD2), which is specific for sea urchin sperm flagellar dynein heavy chains, was used as primary antibody. Direct cross-sections of cilia were selected, and the distance between the center of a cilium and the center of a colloidal gold particle attached to the cilium (X) was measured. The distance between the center of a cilium and the farthest edge of an outer dynein arm of the cilium was measured by ordinary electron microscopy (Yo) and by immunoelectron microscopy (Yi). X was significantly longer than Yo and Yi. If it is assumed that the structure of respiratory cilia is dense and that antibodies are located at the outer side of the actual position of the heavy chains, then the average distance difference of approximately 90-120 A may represent the length of two conjugated antibodies. This length should be kept in mind when performing immunoelectron microscopy. The data suggest that AD2 recognizes the outer arm dynein heavy chains of normal human respiratory cilia.

Adult↗
PubMed1997

Diagnosis of bladder cancer by analysis of the allelic loss of the p53 gene in urine samples using blunt-end single-strand conformation polymorphism.

The novel approach of blunt-end single-strand conformation polymorphism (SSCP) has been applied in the analysis of urine samples from bladder-cancer patients for detecting loss of heterozygosity (LOH) of 3 polymorphic markers in the p53 gene. Of the 28 urine samples examined by SSCP analysis of blunt-ended DNA fragments using a fluorescence-based automated sequencer, 16 were informative in more than 1 of the 3 polymorphic markers at the p53 locus and 8 (50.0%) showed allelic loss of the p53 gene. In analysis of resected tumor tissues, LOH of the p53 gene was detected in 8 of 8 informative samples (100%) with T1 and higher stages and/or Grade 2 and Grade 3 tumors, while it was detected in 6 (75.0%) urine samples obtained from these 8 patients. This new diagnostic modality enables sensitive detection of tumor cells in urine samples and would be applicable for diagnostic bladder cancer with invasive character.

Alleles↗
PubMed1997

Stromal and thylakoid-bound ascorbate peroxidases are produced by alternative splicing in pumpkin.

A cDNA encoding stromal ascorbate peroxidase (sAPX) was isolated using poly(A)+ RNA of pumpkin cotyledons by RT-PCR. The cDNA encodes a polypeptide with 372 amino acids and shares complete sequence identity with pumpkin thylakoid-bound ascorbate peroxidase (tAPX), except for the deletion of a putative membrane spanning region located in the carboxy domain of tAPX. Southern blot hybridization and analysis of intron structure indicated that mRNAs for sAPX and tAPX, whose suborganellar localizations in chloroplasts are different, are produced by alternative splicing. Immunoblot analysis showed that the accumulation of sAPX and tAPX was differently regulated during germination and subsequent greening of pumpkin cotyledons.

Alternative Splicing↗
PubMed1997

Functional expression and characterization of frog photoreceptor-specific calcium-binding proteins.

S-modulin (sensitivity-modulating protein) is a photoreceptor-specific calcium-binding protein which plays an important role in the light adaptation process by controlling rhodopsin phosphorylation in rods. S-modulin and its cone homologue, s26, were expressed at high level (more than 30% of total protein) in Escherichia coli and then purified. They both inhibited rhodopsin phosphorylation in a calcium dependent manner. Myristoylated recombinants of S-modulin and s26 showed calcium-dependent changes in tryptophan emission spectra with half-maxima at about 0.7 microM free calcium concentration. However, the spectral changes are distinctive from each other, suggesting that there is some difference in the structural change between S-modulin and s26.

Adenosine Triphosphate↗
PubMed1997

The multiple inhibitory mechanisms of GEM 91, a gag antisense phosphorothioate oligonucleotide, for human immunodeficiency virus type 1.

GEM 91 (gene expression modulator) is a 25-mer oligonucleotide phosphorothioate complementary to the gag initiation site of HIV-1. GEM 91 has been studied in various in vitro cell culture models to examine inhibitory effects on different stages of HIV-1 replication. Experiments were focused on the binding of virions to the cell surface, inhibition of virus entry, reverse transcription (HIV DNA production), inhibition of steady state viral mRNA levels, inhibition of virus production from chronically infected cells, and inhibition of HIV genome packaging within virions. Experiments were also performed in vitro in an attempt to generate strains of HIV with reduced sensitivity to GEM 91. We observed sequence-dependent inhibition of virus entry/reverse transcription and a reduction in steady state viral RNA levels. We also observed sequence-independent inhibition of virion binding to cells and inhibition of virus production by chronically infected cells. Using in vitro methods that were successful in generating HIV strains with reduced sensitivity to AZT, we were unable to generate strains with reduced sensitivity to GEM 91.

Anti-HIV Agents↗
PubMed1997

Crystallization and preliminary X-ray studies of azurin-I and azurin-II from denitrifying bacterium Alcaligenes xylosoxidans GIFU 1051.

Two distinct azurins named azurin-I and azurin-II were isolated from Alcaligenes xylosoxidans GIFU 1051 (AzG-I and AzG-II). They have been purified and crystallized. The crystals of AzG-I belong to the monoclinic crystal system, space group C2, unit-cell parameters a = 130.6, b = 54.4, c = 74.7 A, beta = 96.1 degrees. The crystals of AzG-II belong to the tetragonal crystal system, space group P4(1)22 or P4(3)22, unit-cell parameters a = b = 52.6, c = 100.7 A. Both crystals diffract up to 2.0 A resolution and are suitable for X-ray crystallographic studies.

Journal Article↗
PubMed1997

TAK1 mediates the ceramide signaling to stress-activated protein kinase/c-Jun N-terminal kinase.

Ceramide has been proposed as a second messenger molecule implicated in a variety of biological processes. It has recently been reported that ceramide activates stress-activated protein kinase (SAPK, also known as c-Jun NH2-terminal kinase JNK), a subfamily member of mitogen-activated protein kinase superfamily molecules and that the ceramide/SAPK/JNK signaling pathway is required for stress-induced apoptosis. However, the molecular mechanism by which ceramide induces SAPK/JNK activation is unknown. Here we show that TAK1, a member of the mitogen-activated protein kinase kinase kinase family, is activated by treatment of cells with agents and stresses that induce an increase in ceramide. Ceramide itself stimulated the kinase activity of TAK1. Expression of a constitutively active form of TAK1 resulted in activation of SAPK/JNK and SEK1/MKK4, a direct activator of SAPK/JNK. Furthermore, expression of a kinase-negative form of TAK1 interfered with the activation of SAPK/JNK induced by ceramide. These results indicate that TAK1 may function as a mediator of ceramide signaling to SAPK/JNK activation.

Animals↗
PubMed1997

Activation of the aryl hydrocarbon receptor/transcription factor and bone marrow stromal cell-dependent preB cell apoptosis.

In the absence of known endogenous ligands, investigators have exploited ubiquitous environmental pollutants, including polycyclic aromatic hydrocarbons, to gain insight into the physiologic functions of the aryl hydrocarbon (dioxin) receptor/transcription factor (AhR). AhR ligands induce cell transformation and steroid-like immunosuppression, suggesting a role for the AhR in regulation of cell growth and/or function. However, mechanisms through which the AhR influences cells in general and lymphocytes in particular remain unresolved. A murine model of B cell development was created to: 1) examine a role for the AhR in immunosuppression; 2) define mechanisms of AhR ligand immunosuppression; 3) characterize AhR expression in preB cells, in bone marrow stromal cells that support preB cells, or in primary bone marrow B cells; and 4) determine if AhR ligands suppress lymphopoiesis by acting directly on preB cells or indirectly via the microenvironment, as represented by bone marrow stromal cells. Results indicate that: 1) low doses (> or = 10(-8) M) of the prototypic AhR ligand, 7,12-dimethylbenz[a]anthracene (DMBA), induce preB cell apoptosis in 12 to 24 h; 2) alpha-naphthoflavone, an AhR and cytochrome P-450 inhibitor, blocks DMBA-induced apoptosis; 3) AhR mRNA and functional AhR protein are expressed at high levels in bone marrow stromal cells (little or no AhR is present in preB cell lines), and 4) preB cells maintained in rIL-7 do not undergo DMBA-induced apoptosis unless cultured with stromal cells. Results underscore the regulatory role played by bone marrow stromal cells in lymphopoiesis and support the hypothesis that the AhR effects immunosuppression by inducing stromal cells to deliver a death signal to lymphocytes.

9,10-Dimethyl-1,2-benzanthracene↗
PubMed1997

Absence of human T-lymphotropic virus type I in Japanese patients with cutaneous T-cell lymphoma.

Cutaneous T-cell lymphoma (CTCL) is a disease entity characterized by a primary sporadic T-cell proliferation in the skin. Human T-lymphotropic virus type 1 (HTLV-1) is a retrovirus that causes adult T-cell leukemia/lymphoma. Recently, several authors have detected the HTLV-1 genome in genomic DNA from patients with CTCL and proposed a causal relation of HTLV-1 to CTCL. However, it remains controversial because these studies contain some problems in materials used to detect HTLV-1. We investigated both fresh and cultured T lymphocytes (128 specimens) derived from 50 Japanese patients with CTCL, where HTLV-1 is endemic, by using polymerase chain reaction with four sets of primers including gag, pol, env, and pX regions of HTLV-1 to elucidate the relationship between HTLV-1 and CTCL in Japan. However, none of the 128 DNA specimens revealed positive for HTLV-1 in contrast to the previous studies. We conclude that CTCL, which does not include HTLV-1, is present although the pathogenesis of CTCL may be different by areas or races.

Adult↗
PubMed1997

Interaction of HPC-1/syntaxin 1A with the cytoskeletal protein, tubulin.

It is believed that HPC-1/syntaxin 1A regulates the intracellular membrane transport. We found a possible tubulin binding motif like sequence in residues 89 approximately 106 of HPC-1/syntaxin 1A. To determine whether or not HPC-1/syntaxin 1A binds to tubulin, we performed in vitro binding studies. We showed that both the rat brain and recombinant HPC-1/syntaxin 1A bound to tubulin in vitro. Competition experiments with synthetic peptides revealed that HPC-1/syntaxin 1A bound to tubulin at residues 89 approximately 106 which were supposed to constitute the tubulin binding consensus sequence.

Amino Acid Sequence↗
PubMed1997

Retinoic acids differentially regulate NOR-1 and its closely related orphan nuclear receptor genes in breast cancer cell line MCF-7.

NOR-1, NGFI-B, and Nurr1 are closely related orphan nuclear receptors implicated in diverse biological processes including cell growth and differentiation. We examined the effect of retinoic acids on the expression of these putative transcription factor genes in the breast cancer cell line MCF-7 by a quantitative reverse transcription and polymerase chain reaction. Both all-trans and 9-cis retinoic acids markedly induced NOR-1 mRNA and slightly increased Nurr1 mRNA. In contrast, NGFI-B mRNA was decreased. In the presence of cycloheximide, all-trans retinoic acid superinduced NOR-1 mRNA, whereas all-trans and 9-cis retinoic acids strongly suppressed the NGFI-B mRNA accumulation. The differential effects of retinoic acids on the expression of these genes are in contrast with the effects of forskolin and 12-O-tetradecanoylphorbol-13-acetate, both of which induced mRNAs of all three genes. These findings suggest that NOR-1, NGFI-B, and Nurr1 play distinct roles in the retinoic acid signaling in MCF-7 cells.

Alitretinoin↗
PubMed1997

Overexpressed syntaxin 1A/HPC-1 inhibits insulin secretion via a regulated pathway, but does not influence glucose metabolism and intracellular Ca2+ in insulinoma cell line beta TC3 cells.

We have previously established a stable beta TC3 cell line that overexpresses syntaxin 1A, designated beta TC-hpc1 cells, in which glucose-stimulated insulin release was decreased. Using beta TC-hpc1 cells, we aimed to determine whether syntaxin 1A functions in the regulatory or constitutive pathway of insulin release. We therefore examined the secretion of phorbol-12-myristate-13-acetate (TPA)-stimulated newly synthesized proinsulin/insulin and total immunoreactive insulin. beta TC3 and beta TC-hpc1 cells were simultaneously pulse-labeled with 3H-leucine for 30 min in 11 mM glucose and chased for 1 h in one of a number of different concentrations of TPA in 11 mM glucose. Total immunoreactive insulin release (IRI) by both cell types during the chase period was markedly increased by the addition of TPA in a dose-dependent manner; however, the IRI from beta TC-hpc1 cells was lower than that from beta TC3 cells. The secretion of newly synthesized proinsulin/insulin from both cell types, which in beta TC3 cells is thought to occur via a constitutive pathway, was in the same range under any condition. Thus, the evidence indicates that syntaxin 1A preferentially functions in the regulated insulin release pathway in beta TC3 cells. In order to clarify the effect of overexpressed syntaxin 1A on glucose metabolism and intracellular Ca2+ we analyzed the glucose transport system, glucose phosphorylation activity, and cytosolic concentration of free Ca2+ ([Ca2+]i). 2-Deoxy-glucose uptake and the content of GLUT1 protein in the plasma membrane fractions of beta TC-hpc1 cells were not different from those of beta TC3 cells. Radiometric assays of glucose phosphorylation activity showed that there were no differences in hexokinase activity and glucokinase activity between beta TC3 and beta TC-hpc1 cells. [Ca2+]i measured by using fura 2 demonstrated that there was no difference in [Ca2+]i between beta TC3 and beta TC-hpc 1 cells under glucose-stimulated conditions. The present experiments indicate that syntaxin 1A plays a central role in a late step of the regulatory insulin release pathway without a change in glucose metabolism and [Ca2+]i in beta TC3 cells.

Animals↗