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T Hara

Publications and source records attributed to T Hara.

At least 577 records · Page 32Linked to original sources
PubMed1995

Polyol pathway, 2,3-diphosphoglycerate in erythrocytes and diabetic neuropathy in rats.

The relationship between the 2,3-diphosphoglycerate concentration in red blood cells as a biological indicator of tissue hypoxia and diabetic neuropathy, and the effect of a potent aldose reductase inhibitor, (2S,4S)-6-fluoro-2'5'-dioxospiro [chroman-4,4'-imidazolidine]-2-carboxamide (SNK-860), on both were investigated in streptozotocin-induced diabetic rats. Diabetic rats demonstrated significantly delayed motor nerve conduction velocity and reduced sciatic nerve blood flow. Altered biochemical features in the sciatic nerves, including a marked accumulation of sorbitol and fructose, myo-inositol depletion and decreased Na+/K(+)-ATPase activity were also detected in diabetic rats. These defects were accompanied by a decrease in the red blood cell 2,3-diphosphoglycerate concentration. Treatment with SNK-860 partially or completely ameliorated these abnormalities. These observations suggest that a decrease in the red blood cell 2,3-diphosphoglycerate concentration is one of the factors contributing to tissue hypoxia, which results in diabetic neuropathy, and that this decrease is mediated through an aldose reductase inhibitor-sensitive pathway.

2,3-Diphosphoglycerate↗
PubMed1995

Molecular cloning and functional characterization of a novel member of the C-C chemokine family.

Chemokines play an important role in immune and inflammatory responses by inducing migration and adhesion of leukocytes. We have isolated a novel chemokine cDNA, designated CCF18, from a cDNA library of an IL-3-dependent murine pro-B cell line, Ba/F3. The cDNA encodes a protein structurally related to the C-C chemokine members. Among this family, C10 shows the highest homology to CCF18, and MIP-1 alpha also has a significant homology but to a lesser extent. CCF18 produced from COS cells induced chemotaxis and Ca2+ flux in CD4+ T cell clones. Moreover, prior administration of MIP-1 alpha desensitized the cells to CCF18. The CCF18 gene (Scya10) was mapped to a middle region of murine chromosome 11, where other genes for several C-C chemokine members are localized. These results clearly indicate that CCF18 is a new member of the C-C chemokine family. Since a high level of CCF18 mRNA is constitutively expressed in macrophage and myeloid cell lines, CCF18 may play a role in inflammatory processes.

Amino Acid Sequence↗
PubMed1995

Detection of high density lipoprotein binding proteins with a new method using apolipoprotein AI-DNP as a ligand.

To detect high density lipoprotein binding proteins, a new ligand blotting method that uses apolipoprotein AI-dinitrophenol as a ligand was established. Apolipoprotein AI-dinitrophenol was prepared by incubating the mixture of apolipoprotein AI and 2,4-dinitrobenzene sulfonic acid sodium salt at pH 8.5. Four to six dinitrophenol groups were found to be conjugated with an apolipoprotein AI molecule without any obvious impairment of its binding activity to the binding proteins. By this detection procedure, we detected two apolipoprotein AI binding proteins in plasma membrane fractions from bovine liver and adrenal cortex with molecular masses of 120 kDa and 95 kDa. Since this new ligand blotting method is simple and time-saving, it is expected to become a useful tool in the studies on high density lipoprotein binding proteins.

Adrenal Cortex↗
PubMed1995

Species differences in anticoagulant and anti-Xa activity of DX-9065a, a highly selective factor Xa inhibitor.

Activation of the blood coagulation cascade results in the formation of factor Xa (FXa) in either its intrinsic or extrinsic pathways, which in turn converts prothrombin to thrombin. We recently described the synthesis and characterization of DX-9065a, a highly selective FXa inhibitor, as an orally active anticoagulant agent (1). Although DX-9065a potently inhibited human FXa (2), a much larger dosage was required to inhibit thrombus formation in a rat thrombosis model (3): The plasma concentration of DX-9065a after administration of a dose which reduced thrombus formation by 50% in rats was in the microM range, or 40 times higher than the Ki value for FXa. From this, several assumptions arise, namely that an unknown mechanism other than FXa inhibition contributes to the antithrombotic effect of DX-9065a, or that the efficacy of DX-9065a differs among species. However, as the anticoagulant effect of DX-9065a was closely consistent with anti-Xa activity in plasma and as DX-9065a inhibited only FXa activity (2), the former assumption could be disregarded. To clarify the latter, we examined the inhibitory effect of DX-9065a on FXa from several animal species and its anticoagulant effect ex vivo in these species. Further, we also compared the anticoagulant effect of DX-9065a in plasma from each species with that of NAPAP, a benzamidine-derived direct thrombin inhibitor (4).

Animals↗
PubMed1995

Receptor-mediated transfer of pSV2CAT DNA to a human hepatoblastoma cell line HepG2 using asialofetuin-labeled cationic liposomes.

Asialofetuin-labeled liposomes (AF-lps) were developed as a vector for gene transfer to hepatocytes. Plasmid pSV2CAT DNA which encodes bacterial chloramphenicol acetyltransferase (CAT) was associated with (meaning, in this report, the sum of 'to be adsorbed on the surface of' and 'to be encapsulated into the internal phase of') AF-lps (AF-lps-pSV2CAT) prepared by a tandem combination of the detergent removal and freeze-thaw methods. Ninety-six percent of input pSV2CAT was associated with AF-lps containing N-(alpha-trimethylammonioacetyl)-didodecyl-D-glutamate chloride, and approx. two-thirds of the associated DNA was encapsulated into the internal phase. The uptake of AF-lps by the cultured human hepatoblastoma cell line HepG2, having asialoglycoprotein receptors (AGPR) on their plasma membrane, was decreased by the addition of free AF and cytochalasin B. AF-lps bound to HepG2 cells through specific interaction with AGPR, and were internalized into the cells by the receptor-mediated endocytotic pathway. HepG2 cells transfected by AF-lps-pSV2CAT showed a significantly higher CAT activity than those transfected by pSV2CAT associated with non-labeled control lps (N-lps-pSV2CAT) or a mixture of pSV2CAT and empty AF-lps. Pretreatment with EDTA-encapsulated AF-lps increased the transfection efficiency of AF-lps-pSV2CAT. The CAT activity in A431 and Swiss/3T3 cells transfected with AF-lps-pSV2CAT was low and almost the same as those transfected with N-lps-pSV2CAT. Since DNA encapsulated in lps is likely to be protected against digestion by nucleases in the blood circulation, AF-lps could be used as a gene transfer vector targeting the hepatocytes in vivo.

Asialoglycoprotein Receptor↗
PubMed1995

A novel cytokine-inducible gene CIS encodes an SH2-containing protein that binds to tyrosine-phosphorylated interleukin 3 and erythropoietin receptors.

Cytokines manifest their function through alteration of gene expression. However, target genes for signals from cytokine receptors are largely unknown. We therefore searched for immediate-early cytokine-responsive genes and isolated a novel gene, CIS (cytokine inducible SH2-containing protein) which is induced in hematopoietic cells by a subset of cytokines including interleukin 2 (IL2), IL3, granulocyte-macrophage colony-stimulating factor (GM-CSF) and erythropoietin (EPO), but not by stem cell factor, granulocyte colony-stimulating factor and IL6. The CIS message encodes a polypeptide of 257 amino acids that contains an SH2 domain of 96 amino acids in the middle. To clarify the function of CIS in cytokine signal transduction, we expressed CIS in IL3-dependent hematopoietic cell lines under the control of a steroid-inducible promoter. The CIS product stably associated with the tyrosine-phosphorylated beta chain of the IL3 receptor as well as the tyrosine-phosphorylated EPO receptor. Forced expression of CIS by steroid reduced the growth rate of these transformants, suggesting a negative role of CIS in signal transduction. CIS induction requires the membrane-proximal region of the cytoplasmic domain of the EPO receptor as well as that of the common beta chain of the IL3, IL5 and GM-CSF receptor, whereas CIS binds to the receptor that is tyrosine phosphorylated by cytokine stimulation. Thus CIS appears to be a unique regulatory molecule for cytokine signal transduction.

Amino Acid Sequence↗
PubMed1995

Radiation-induced G1 arrest is selectively mediated by the p53-WAF1/Cip1 pathway in human thyroid cells.

We investigated the sensitivity and sequential events that take place in thyroid epithelial cells after irradiation. Cell survival ratios at a dose of 2 Gy were 18 +/- 2.5%, 58 +/- 1.0%, 59 +/- 1.5%, and 98 +/- 1.8% in primary thyroid cells, papillary thyroid carcinoma cells, follicular thyroid carcinoma cells, and anaplastic thyroid carcinoma cells, respectively. Thyroid carcinoma cell lines carrying mutations in the p53 gene were resistant to ionizing radiation. Although irradiated cells were accumulated at G1 in primary thyroid cells even after low-dose irradiation (0.2 Gy), this phenomenon was not observed in the thyroid carcinoma cell lines. Wild-type p53 expression in primary thyroid cell was increased following irradiation, but mutated p53 in the thyroid carcinoma cell lines was unchanged. To clarify the signal transduction in the G1 arrest following irradiation, levels of expression of the p53 putative downstream effectors GADD45 and WAF1/Cip1 were examined. Despite the consistent level of GADD45 mRNA, the level of WAF1/Cip1 transcripts was increased in a radiation dose-dependent manner in primary thyroid cells. This increase in the WAF1/Cip1 mRNA level was observed 30 min after irradiation and continued for at least 48 h. A mobility shift assay performed using the sequence of the putative p53 DNA binding site on the WAF1/Cip1 and GADD45 genes as a probe showed that nuclear protein extracted from primary thyroid cells, anti-p53 antibody, and probe oligonucleotide-bound complex was clearly shifted. An increase in binding activity of the p53/antibody/DNA complex was observed following irradiation. In contrast, the nuclear extract from thyroid carcinoma cells could not bind the specific DNA site, suggesting that mutant p53 has lost its binding ability. Actinomycin D inhibited WAF1/Cip1 and GADD45 mRNA levels and cycloheximide stimulated up-regulation of both basal mRNA levels, but an additional increase of the mRNA expression following irradiation was observed only in the WAF1/Cip1 gene. These data suggest that p53 in postradiation acts at a transcriptional level on WAF1/Cip1 gene expression and that de novo protein synthesis is not required for this effect. These results suggest that the p53-WAF1/Cip1 pathway may play a central role in induction of G1 arrest following irradiation in human thyroid epithelial cells.

Base Sequence↗
PubMed1995

Interleukin-3 (IL-3) poor-responsive inbred mouse strains carry the identical deletion of a branch point in the IL-3 receptor alpha subunit gene.

Interleukin-3 (IL-3) stimulates colony formation of multiple lineages of hematopoietic cells. Bone marrow cells of A/J mice are nonresponsive to IL-3, and this observation has recently been correlated with aberrant mRNA splicing and impaired expression of the IL-3 receptor alpha subunit (IL-3R alpha), a binding component of the high-affinity receptors. We examined the IL-3R alpha gene in 27 inbred mouse strains and found the identical mutation, a 5-bp deletion at the branch point of intron 7, in 10 of these mouse strains. Bone marrow cells isolated from these 10 mouse strains did not express IL-3R alpha on the cell surface and did not form colonies in response to IL-3. Because the defective IL-3R alpha gene was found in several distantly related mouse strains, it appears to be a recessive allele rather than a sporadic mutation. In contrast, only 1 of 21 wild-derived mouse strains carried the 5-bp deletion in the IL-3R alpha gene. This study suggests that IL-3 function is not required for normal hematopoiesis in mice, but the retention of the IL-3 and IL-3R system may be of some selective advantage in wild populations.

Alleles↗
PubMed1995

Molecular cloning, sequencing, and expression of the genes encoding adenosylcobalamin-dependent diol dehydrase of Klebsiella oxytoca.

The pdd genes encoding adenosylcobalamin-dependent diol dehydrase of Klebsiella oxytoca were cloned by using a synthetic oligodeoxyribonucleotide as a hybridization probe followed by measuring the enzyme activity of each clone. Five clones of Escherichia coli exhibited diol dehydrase activity. At least one of them was shown to express diol dehydrase genes under control of their own promoter. Sequence analysis of the DNA fragments found in common in the inserts of these five clones and the flanking regions revealed four open reading frames separated by 10-18 base pairs. The sequential three open reading frames from the second to the fourth (pddA, pddB, and pddC genes) encoded polypeptides of 554, 224, and 173 amino acid residues with predicted molecular weights of 60,348 (alpha), 24,113 (beta), and 19,173 (gamma), respectively. Overexpression of these three genes in E. coli produced more than 50-fold higher level of functional apodiol dehydrase than that in K. oxytoca. The recombinant enzyme was indistinguishable from the wild-type one of K. oxytoca by the criteria of polyacrylamide gel electrophoretic and immunochemical properties. It was thus concluded that these three gene products are the subunits of functional diol dehydrase. Comparisons of the deduced amino acid sequences of the three subunits with other proteins failed to reveal any apparent homology.

Amino Acid Sequence↗
PubMed1995

Purification and characterization of a newly identified isoform of cytochrome P450 responsible for 3-hydroxylation of 2,5,2',5'-tetrachlorobiphenyl in hamster liver.

In the hamster liver, 2,5,2',5'-tetrachlorobiphenyl (TCB) is metabolized to 3-hydroxy- and 4-hydroxy-2,5,2',5'-TCB to a similar extent, and formation of the former metabolite is stimulated by phenobarbital pretreatment of the animals, while that of the latter metabolite is stimulated by 3-methylcholanthrene pretreatment. In the present study, we identified a new isoform (designated P450HPB-1) of cytochrome P450 which proved to be phenobarbital-inducible and responsible for 3-hydroxylation of this TCB isomer. This isoform was purified from liver microsomes of phenobarbital-treated hamsters and characterized. P450HPB-1 has a molecular mass of 50 kDa, determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and the absorption maxima of the oxidized form at 417 nm and of the reduced CO-complex form at 450 nm. The sequence of 28 amino acids of P450HPB-1 at the amino-terminal has a 68% similarity with those of rat P450 2B1 and mouse P450 2b10, 57% similarity with that of guinea pig P450GP-1, and 54% similarity with that of guinea pig P450GP-1, and 54% similarity with those of rabbit P450 2B4 and dog P450 2B11. P450HPB-1 in the reconstituted system catalyzed the 3- but not 4-hydroxylation of 2,5,2',5'-TCB, at a rate of 19.0 pmol/min/nmol P450. The isoform also has high catalytic activity for 17-oxidation of testosterone but low activity for the N-demethylation of benzphetamine and 16 alpha- and 16 beta-hydroxylations of testosterone. In microsomal metabolism of 2,5,2',5'-TCB, rabbit antiserum against P450HPB-1 almost completely inhibited 3- but not 4-hydroxylation. Immunoblot analysis of hamster liver microsomes revealed that P450HPB-1 was constitutive and phenobarbital-inducible but was decreased by pretreatment with 3-methylcholanthrene or 3,4,5,3',4'-pentachlorobiphenyl. These results suggest that P450HPB-1 belongs in the P450 2B subfamily and apparently plays a major role in the 3-hydroxylation of 2,5,2',5'-TCB, in hamster liver.

Amino Acid Sequence↗
PubMed1995

Impaired interleukin-3 (IL-3) response of the A/J mouse is caused by a branch point deletion in the IL-3 receptor alpha subunit gene.

Interleukin-3 (IL-3) alone does not support hematopoietic colony formation of bone marrow cells from the A/J mouse. To elucidate the molecular lesion in A/J mice, we examined expression of the alpha and beta subunits of the IL-3 receptor (IL-3R). While IL-3R beta was normally expressed, IL-3R alpha was not detectable on the surface of A/J-derived cells by antibody staining. Genetic linkage analysis using recombinant inbred mouse strains between A/J and IL-3-responsive C57BL/6 indicated that the IL-3R alpha gene locus was responsible for the impaired IL-3 response in A/J mice. Molecular cloning and characterization of A/J-derived IL-3R alpha cDNA revealed that it lacked the sequence corresponding to exon 8, which encodes 10 amino acid residues in the extracellular domain. The aberrant splicing was due to a 5 base pair deletion at the branch point in intron 7 and was reproduced in heterologous cells by transfecting with an IL-3R alpha minigene carrying the deleterious intron. The A/J-specific abnormal form of IL-3R alpha was localized inside the cells, but not on the cell surface, providing the molecular basis for the impaired IL-3 response in the A/J mouse.

Amino Acid Sequence↗
PubMed1995

Multi-colony stimulating activity of interleukin 5 (IL-5) on hematopoietic progenitors from transgenic mice that express IL-5 receptor alpha subunit constitutively.

The interleukin 3 (IL-3), IL-5, and granulocyte/macrophage colony-stimulating factor receptors consist of a cytokine-specific alpha subunit and the common beta subunit. Whereas IL-3 stimulates various lineages of hematopoietic cells, including multipotential progenitors, IL-5 acts mainly as an eosinophil lineage-specific factor. To investigate whether the lineage specificity of IL-5 is due to restricted expression of the IL-5 receptor alpha subunit (IL-5R alpha), we generated transgenic mice that express the mouse IL-5R alpha constitutively by phosphoglycerate kinase promoter. The transgenic mouse expressed IL-5R alpha ubiquitously, and the bone marrow cells formed various types of colonies, including multi-lineage colonies, in response to IL-5. IL-5 also supported formation of both multi-lineage and blast cell colonies from dormant progenitors of the 5-fluorouracil-treated transgenic mice. The cells composing the blast cell colony gave rise to many colonies including multi-lineage colonies when they were replated in secondary culture containing either Il-5 or IL-3. There was no significant difference in replating efficiency or in types of secondary colonies between IL-5- and IL-3-stimulated cultures. Conversely, the cells from the IL-3-induced blast cell colonies of the transgenic mice proliferated in response to either IL-3 or IL-5. Thus, the development of the progenitors can be equally supported by either IL-5 or IL-3, suggesting that intracellular signals from the IL-3R can be replaced by those from IL-5. These results strongly suggest that the lineage specificity of IL-5 is mainly due to the restricted expression of IL-5R alpha.

Animals↗
PubMed1995

The murine interleukin-3 receptor alpha subunit gene: chromosomal localization, genomic structure, and promoter function.

The interleukin-3 receptor (IL-3R) is composed of alpha and beta subunits, members of the class I cytokine receptor family. Here we describe isolation and characterization of the chromosomal gene for the mouse IL-3R alpha subunit (mIL-3R alpha). Whereas the human IL-3R alpha gene is tightly linked with the granulocyte-macrophage colony-stimulating factor receptor alpha subunit (GM-CSFR alpha) gene in the pseudoautosomal region of the X and Y chromosomes, the mIL-3R alpha gene (II3ra) is located in the proximal region of mouse chromosome 14, separated from the mouse GM-CSFR alpha gene, which is on chromosome 19. The mIL-3R alpha gene spans about 10 kb and is divided into 12 exons. All the exon-intron boundaries possess the splicing junction consensus sequences (5'GT-AG3'), and the whole genomic structure is similar to those of the previously reported class I cytokine receptor genes. There are two major transcription initiation sites that are located at 215 and 188 nucleotides upstream of the initiator codon. The promoter region is GC-rich and contains potential binding sites for GATA, Ets, c-myb,, Sp1, Ap-2, and G-C boxes, but not a typical TATA or CAAT sequence. A fusion gene containing 0.8 kb of the 5' noncoding sequence linked to the firefly luciferase gene directed the transcription in mouse mast cells but not in fibroblasts or T cells, suggesting that this promoter functions in a cell type-specific manner. Further sequential deletion of the 5' region suggests two potential regulatory regions for transcription of the mIL-3R alpha gene.

3T3 Cells↗
PubMed1995

Efficacy of equator rings in an experimental rabbit study.

OBJECTIVE: To determine the efficacy of the equator ring in maintaining the circular contour of the capsule bag equator and transparency of the posterior capsule after crystalline lens extraction in 12 rabbit eyes. METHODS: After phacoemulsification through a 5-mm window made by continuous circular capsulorrhexis in the central anterior capsule, a flexible silicone ring (outer diameter, 11.0 mm; width, 1.5 mm; and thickness, 1.5 mm) was implanted in the capsule in seven rabbit eyes. A 13.0-mm Sinskey-style posterior chamber intraocular lens was then implanted inside the ring. Three other eyes received only equator rings, and two others received only intraocular lenses. Eyes were followed up for 3.5 +/- 1.4 months (mean +/- SD) before enucleation and evaluation by stereomicroscopy; they then underwent light microscopic examination. RESULTS: The two eyes that received only the intraocular lens had equatorial distortion and severe posterior capsule opacification. In nine (90%) of the 10 eyes that were implanted with the rings, the circular contour of the equator was preserved, and seven (70%) of these 10 eyes had transparent posterior capsules. CONCLUSION: Implantation of the equator ring effectively maintained the circular contour of the capsule bag equator and inhibited cells at the bag equator from proliferating toward the center of the posterior capsule in the rabbit.

Animals↗
PubMed1995

Cloning and expression of a porcine UDP-GalNAc: polypeptide N-acetylgalactosaminyl transferase.

By employing a bovine UDP-N-acetylgalactosamine: polypeptide N-acetylgalactosaminyl transferase (O-GalNAc transferase) cDNA as a probe, we isolated four overlapping cDNAs from a porcine lung cDNA library. Both the nucleotide sequence of the porcine cDNA and the predicted primary structure of the protein (559 amino acids) proved to be very similar to those of the bovine enzyme (95% and 99% identity, respectively). Transient expression of the clone in COS-7 cells, followed by enzymatic activity assays, demonstrated that this cDNA sequence encodes a porcine O-GalNAc transferase. The intracellular O-GalNAc transferase activity was increased approximately 100-fold by transfecting cells with the porcine cDNA.

Amino Acid Sequence↗
PubMed1995

Doxorubicin-heparin complex: reduction of cardiotoxicity of doxorubicin.

We have compared the antitumor activity and cardiotoxicity of free doxorubicin (Dox) and doxorubicin-heparin complex in vivo and in vitro. Dox and Dox-heparin complex equally inhibited the DNA synthesis of leukemic cells and showed a similar anticancer activity against tumor-bearing mice. Acute toxicity of Dox at the dose of 20 mg/kg or 30 mg/kg was significantly more profound than that of the Dox-heparin complex, which was demonstrated by survival rate (P < 0.01). Chronic toxicities of Dox and the Dox-heparin complex were compared by giving the respective reagent (2 mg/kg) weekly for 20 weeks. The weight gains of the mice given Dox-heparin complex were greater than those of the mice given Dox alone (P < 0.01). The pathological damage to the cardiac tissue in mice treated with Dox-heparin complex was significantly less severe than that of mice treated with Dox. Thus, the present study indicates that complexing with heparin diminished the acute and chronic toxicity of Dox without reducing its antitumor activity in mice, and suggests a possible clinical application of Dox-heparin complex in humans.

Animals↗