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T Tamura

Publications and source records attributed to T Tamura.

At least 19 recordsLinked to original sources
PubMed1992

Ornithine decarboxylase is degraded by the 26S proteasome without ubiquitination.

Ornithine decarboxylase (ODC), a key enzyme in polyamine biosynthesis, is the most rapidly turned over mammalian enzyme. We have shown that its degradation is accelerated by ODC antizyme, an inhibitory protein induced by polyamines. This is a new type of enzyme regulation and may be a model for selective protein degradation. Here we report the identification of the protease responsible for ODC degradation. Using a cell-free degradation system, we demonstrate that immunodepletion of proteasomes from cell extracts causes almost complete loss of ATP- and antizyme-dependent degradation of ODC. In addition, purified 26S proteasome complex, but not the 20S proteasome, catalyses ODC degradation in the absence of ubiquitin. These results strongly suggest that the 26S proteasome, widely viewed as specific for ubiquitin-conjugated proteins, is the main enzyme responsible for ODC degradation. The 26S proteasome may therefore have a second role in ubiquitin-independent proteolysis.

Adenosine Triphosphate↗
PubMed1992

Regulation of gene expression of proteasomes (multi-protease complexes) during growth and differentiation of human hematopoietic cells.

We have reported that proteasomes are expressed at abnormally high levels in various hematopoietic tumor cells (Kumatori, A., Tanaka, K., Inamura, N., Sone, S., Ogura, T., Matsumoto, T., Tachikawa, T., Shin, S., and Ichihara, A. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 7071-7075). In the present study, we examined changes in the expressions of proteasomes during growth of peripheral T-lymphocytes from healthy adults and differentiation of human leukemic cell lines. Up-regulation of mRNAs encoding multiple proteasome subunits was observed during proliferation of resting T-cells induced by mitogens such as phytohemagglutinin and interleukin-2. In contrast, in vitro terminal differentiation into monocytic, granulocytic, and erythroid cells of various immature leukemic cell lines, such as HL-60 promyelocytic leukemia cells and K562 erythroleukemia cells, by various inducing agents caused rapid and marked down-regulation of proteasomes expression, independently of the cell type, direction of differentiation, or type of signal. The syntheses of proteasome subunits of 21-31 kDa and their associated components of 35-110 kDa, measured by [35S]methionine incorporation, were much higher in mitogen-activated T-cells and unstimulated HL-60 cells, which grow rapidly, than in resting and differentiated cells, indicating apparent correlations of the mRNA levels of proteasomes with their translational activities. However, immunochemically, no detectable difference in the cellular contents of proteasomes was found in these cells in induced and uninduced states for proliferation and differentiation, suggesting accelerated turnover of proteasomes in rapidly proliferating cells. Inhibition of proteasome expression by an antisense oligodeoxynucleotide for the largest proteasome subunit, C2, caused partial arrest of cell cycle progression of T-lymphocytes, suggesting that up-regulation of proteasomes is indispensable for proliferation of the cells. We also observed that the nuclear fraction of proteasomes increased in proliferating T-cells and that proteasomes moved rapidly between the nucleus and cytoplasm during differentiation of HL-60 cells.

Base Sequence↗
PubMed1992

Structure of a heparan sulphate oligosaccharide that binds to basic fibroblast growth factor.

Binding of basic fibroblast growth factor (bFGF) to the extracellular matrix of cultured bovine aorta smooth muscle cells is likely to be mediated via heparan sulphate, since not only exogenous addition of heparan sulphate to the culture medium but also pretreatment of the cells with heparitinase (but not chondroitinase ABC) resulted in loss of binding. Comparison of the affinity of bFGF to various glycosaminoglycan-conjugated gels showed a direct and specific binding of bFGF to heparan sulphate. Heparan sulphate also bound to a bFGF affinity gel. However, the proportion of heparan sulphate bound varied depending on the source of the HS (more than 90% and 45% with pig aorta heparan sulphate and mouse EHS tumour heparan sulphate respectively). The bound heparan sulphate had the ability to protect bFGF from proteolytic digestion, but the unbound heparan sulphate did not. The results suggest the presence in the bound heparan sulphate of a specific structure involved in binding. Limited digestion with heparitinase I of porcine aorta heparan sulphate yielded 13% oligosaccharides bound to the gel, of which the smallest were octasaccharides. Analysis of a hexadecasaccharide fraction which was obtained at the highest yield among the bound oligosaccharides was performed by h.p.l.c. of the deamination products obtained with nitrous acid and the unsaturated disaccharide products formed by heparitinase digestion. Comparison of the disaccharide unit compositions exhibited a marked difference in IdoA(2SO4)GlcNSO3 and IdoA(2SO4)GlcNSO3(6SO4) units between the bound and unbound hexadecasaccharides. The amounts measured were 3 mol and 1 mol per mol of the former and 0.4 mol and 0.6 mol per mol of the latter. It is likely that the binding of bFGF to heparan sulphate may require the domain structure of the heparan sulphate to be composed of clustering IdoA(2SO4)-GlcNSO3 units.

Animals↗
PubMed1992

New antiinflammatory agents. 2. 5-Phenyl-3H-imidazo[4,5-c][1,8]naphthyridin-4(5H)-ones: a new class of nonsteroidal antiinflammatory agents with potent activity like glucocorticoids.

We previously described new antiinflammatory agents, 4-hydroxy-2-oxo-1-phenyl-1H-1,8-naphthyridine-3-carboxamides 1. Further modification of the compounds bearing 1-phenyl-1,8-naphthyridin-2-one as a mother skeleton led to 5-phenylimidazo[4,5-c][1,8]naphthyridin-4(5H)-one derivatives 2 and 3. Regioselective synthesis of these compounds bearing a substituent at the 1- or 3-position was conducted according to the method shown in Schemes I and II. In this series of compounds, antiinflammatory activities were greatly influenced by the position and nature of substituents on imidazole. 3-Alkyl or 3-benzyl substitution result in the potent activity, but 1-substitution did not. Minor modification of the benzyl group reduced or eliminated the activity. Detailed examination of structure-activity relationships led to 3-benzyl-5-phenyl-3H-imidazo[4,5-c][1,8]naphthyridin-4(5H)-one (22), which exhibited potent oral antiinflammatory activities in carrageenan-, zymosan-, and reversed passive Arthus reaction-induced rat paw edemas (ED40 = 5.3, 0.37 mg/kg, ED50 = 0.47 mg/kg, respectively). This broad activity of 22 was like that of glucocorticoids. Compound 22 did not affect activities of CO and 5-LO enzymes and receptor binding of various ligands. As one of the mechanisms of action, induction of release of glucocorticoids was postulated. These results suggest that 22 represents a novel class of antiinflammatory agents.

Animals↗
PubMed1992

Demonstration that a human 26S proteolytic complex consists of a proteasome and multiple associated protein components and hydrolyzes ATP and ubiquitin-ligated proteins by closely linked mechanisms.

It is known that two types of high-molecular-mass protease complexes are present in the cytosol of mammalian cells; a 20S latent multicatalytic proteinase named the proteasome, and a large proteolytic complex with an apparent sedimentation coefficient of 26S that catalyzes ATP-dependent breakdown of proteins conjugated with ubiquitin. In this work, we first demonstrated that a low concentration of SDS was required for activation of the latent proteasome, whereas the 26S complex degraded substrates for proteasomes in the absence of SDS. Moreover, the 26S complex was greatly stabilized in the presence of 2 mM ATP and 20% glycerol. Based on these characteristics, we next devised a novel procedure for purification of the 26S proteolytic complexes from human kidney. In this procedure, the proteolytic complexes were precipitated from cytoplasmic extracts by ultracentrifugation for 5 h at 105000 x g, and the large 26S complexes were clearly separated from the 20S proteasomes by molecular-sieve chromatography on a Biogel A-1.5 m column. The 26S enzyme was then purified to apparent homogeneity by successive chromatographies on hydroxyapatite and Q Sepharose, then by glycerol density-gradient centrifugation. Electrophoretic and immunochemical analyses showed that the purified human 26S complex consisted of multiple subunits of proteasomes with molecular masses of 21-31 kDa and 13-15 protein components ranging in molecular mass over 35-110 kDa, which were directly associated with the proteasome. The purified 26S proteolytic complex degraded 125I-labeled lysozyme-ubiquitin conjugates in an ATP-dependent manner. The 26S enzyme also showed high ATPase activity, which was copurified with the complex. Vanadate and hemin strongly inhibited not only ATP cleavage, but also ATP-dependent breakdown of ubiquitinligated proteins, suggesting that the 26S complex hydrolyzes ATP and ubiquitinated proteins by closely linked mechanisms. These findings indicate that the 26S complex consists of a proteasome with proteolytic function and multiple other components including an ATPase that regulates energy-dependent, ubiquitin-mediated protein degradation.

Adenosine Triphosphatases↗
PubMed1992

High serum IL-6 level reflects susceptible status of the host to endotoxin and IL-1/tumor necrosis factor.

Patients with high level of serum endotoxin did not necessarily develop into lethal shock, whereas some patients died of septic shock even when their serum endotoxin levels were low. These results indicate that limiting factor which determines the host to be endotoxin shock principally depends on the host susceptibility to endotoxin instead of serum endotoxin level. To understand this susceptible status of the host to endotoxin, we used Propionibacterium acnes primed mouse endotoxin shock model. We found that P. acnes-primed mice responded to low dose of LPS by enhanced production of IL-1 and TNF. And such mice were highly susceptible to the lethal shock inducing effect of IL-1 and/or TNF, which also induced high level of serum IL-6 in these mice. Therefore, measurement of serum IL-6 level provides us with the information of the preceding exposure of the host to either LPS or IL-1 and/or TNF and the highly susceptible status of the host to these stimuli. Based on these results obtained from animal model, we investigated the relationship between serum IL-6 levels and serum endotoxin levels in the patients with malignant hematologic disorders. We found that these patients fell into two groups; an endotoxin susceptible group, equivalent to P. acnes-primed mice, showing high level of serum IL-6 with low level of serum endotoxin, and a nonendotoxin susceptible group, equivalent to P. acnes-nonprimed mice, showing low or undetectable level of serum IL-6 with high level of serum endotoxin. We propose that the measurement of serum IL-6 level in the patients positive for endotoxin is a useful tool in evaluating diagnosis and prognosis of endotoxin shock.

Adolescent↗
PubMed1992

Lack of bone resorption in osteosclerotic (oc/oc) mice is due to a defect in osteoclast progenitors rather than the local microenvironment provided by osteoblastic cells.

In a co-culture system of mouse spleen cells and osteoblastic cells, we have demonstrated that a suitable microenvironment must be provided by osteoblastic cells in order for osteoclast-like multinucleated cell (MNC) formation. Using this co-culture system, we examined the pathogenetic mechanism underlying the lack of bone resorption in osteosclerotic oc/oc mice. Numerous tartrate-resistant acid phosphatase (TRAP, an osteoclast marker enzyme)-positive MNCs were formed in response to 1 alpha,25-dihydroxyvitamin D3 [1 alpha,25(OH)2D3] both in co-cultures of oc/oc spleen cells and normal osteoblastic cells and in those of normal spleen cells and oc/oc osteoblastic cells. TRAP-positive MNCs derived from normal spleen cells tended to spread out on culture dishes, whereas those from oc/oc spleen cells remained as small, compact MNCs. When TRAP-positive MNCs enriched from co-cultures of normal spleen cells and oc/oc osteoblastic cells were cultured on dentine slices, they formed numerous resorption pits with ruffled borders and clear zones. In contrast, none of the TRAP-positive MNCs derived from oc/oc spleen cells formed either ruffled borders or resorption pits. These results indicate that the lack of bone resorption in oc/oc mice is due to a defect in osteoclast progenitors rather than the local microenvironment provided by osteoblastic cells.

Acid Phosphatase↗
PubMed1992

T cell activation through TCR/-CD3 complex. IL-2 production of T cell clones stimulated with anti-CD3 without cross-linkage.

To elucidate the Th cell activation mechanism through the TCR/CD3 complex, we examined the reactivity of T cell clones to soluble monovalent and divalent anti-CD3 without accessory cells or costimulatory factor. All T cell clones tested produced IL-2 in response to monovalent anti-CD3, although reactivity to divalent anti-CD3 was variable depending upon clones. IL-2 production of T cell clones induced by monovalent anti-CD3 was suppressed by cross-linking of the antibody with anti-hamster IgG. IL-2 mRNA expression and the increment of intracellular Ca2+ concentration were consistent with the IL-2 production. When T cell clones were stimulated with monovalent anti-CD3, they increased in size, although divalent anti-CD3 stimulation did not affect their size irrespective of their IL-2 production. These results indicate that monovalent anti-CD3 is more efficient than divalent anti-CD3 in induction of IL-2 production and that the cross-linkage of the TCR/CD3 complex is not necessarily required for the T cell clone activation.

Animals↗
PubMed1992

cDNA cloning of rat proteasome subunit RC1, a homologue of RING10 located in the human MHC class II region.

The nucleotide sequence of a cDNA that encodes a new subunit, named RCl, of rat proteasomes (multicatalytic proteinase complexes) has been determined. The polypeptide predicted from the open reading frame consisted of 208 amino acid residues with a calculated molecular mass of 23, 130, which is consistent with the size obtained by electrophoretic analysis of purified RCl. The partial amino acid sequences of several fragments of RCl, obtained by protein chemical analyses, were found to be in excellent accordance with those deduced from the cDNA sequence. Surprisingly, the overall structure of RCl was found to be almost identical to that of recently isolated RING10, whose gene is located in the class II region of the human MHC gene cluster. This finding suggests that RCl is a homologue of human RING10, supporting the proposal that proteasomes are involved in the antigen processing pathway.

Amino Acid Sequence↗
PubMed1992

Early cellular swelling during cerebral ischemia in vivo is mediated by excitatory amino acids released from nerve terminals.

This study demonstrates ischemic cellular swelling in vivo detected as changes in the concentration of 14C-sucrose pre-perfused into the extracellular space (ECS) as an ECS marker. Microdialysis was utilized as a means of perfusion and measurement of the extracellular concentration of 14C-sucrose ([14C-sucrose]e). Concomitant with an abrupt increase in [K+]e at 1-3 min following the ischemia induction, [14C-sucrose]e was also rapidly elevated. Since sucrose is not taken up by either cells or capillaries, the absolute amount of 14C-sucrose in the ECS must be unchanged. The increase therefore appears to represent a relative decrease in water volume in the ECS resulting from a movement of water into the cells, i.e. cellular swelling. Ca(2+)-free perfusate containing Co2+, which has been shown to block excitatory amino acid release during cerebral ischemia, significantly delayed the increase in [14C-sucrose]e and [K+]e. Kynurenic acid, a broad-spectrum antagonist of excitatory amino acids, administered in situ through the dialysis probe also significantly delayed the increase in [14C-sucrose]e and [K+]e. These findings indicate that the early cellular swelling occurring during cerebral ischemia is a result of massive ionic fluxes mediated by excitatory amino acids which are released by a Ca(2+)-dependent exocytotic process from the nerve terminals.

Amino Acids↗
PubMed1992

A novel synthesis and potent antiinflammatory activity of 4-hydroxy-2(1H)-oxo-1-phenyl-1,8-naphthyridine-3-carboxamides.

New antiinflammatory agents 4-hydroxy-2(1H)-oxo-1-phenyl-1,8-naphthyridine-3- carboxamides 7 were designed and synthesized via a valuable intermediate, 1-phenyl-2H-pyrido[2,3-d][1,3]oxazine-2,4(1H)-dione (9). The nature of substituents on the amide nitrogen had a pronounced effect on antiinflamatory activity. Studies of structure-activity relationships led to compounds 33 and 34 bearing a pyridine ring on the amide nitrogen. Compounds 33 and 34 were active against carrageenin-, zymosan-, and arachidonic acid-induced rat paw edemas and also potently inhibited the reversed passive Arthus reaction in rats. Thus, they possess a broader spectrum of antiinflammatory activity than the classical nonsteroidal antiinflammatory drugs (NSAIDs) such as indomethacin and piroxicam.

Animals↗
PubMed1992

Demonstration of a testis-specific trans-acting factor Tet-1 in vitro that binds to the promoter of the mouse protamine 1 gene.

We have established testis-specific in vitro transcription of the mouse protamine 1 (MP1) gene using rat testis nuclear extracts. Addition of testis nuclear extracts to brain extracts enhanced transcription from the MP1 upstream sequence-carrying adenovirus major late promoter. Moreover, the MP1 upstream region from positions -92 to -41 alone exhibited transcriptional activation in a tissue-specific manner. DNase I footprinting demonstrated the presence of a DNA-binding factor around position -60 (Tet-1) in testis nuclear extracts, but not in other tissues. Gel shift analysis also revealed the presence of testis-specific Tet-1. Since mutational analysis in transcriptional and binding assays demonstrates that the Tet-1 site is responsible for transcriptional activation, we suggest that Tet-1 is a novel tissue-specific trans-acting factor. The Tet-1-recognizing sequence was delineated to the 11-mer TGACTTCATAA at position -64. Although the first 8-mer in the Tet-1 11-mer shares homology with the cyclic AMP-responsive element, Tet-1 is demonstrated to be distinct from known cAMP-responsive element-binding factors.

Adenoviridae↗
PubMed1992

Tyrosine protein kinase is involved in anti-IgM-mediated signaling in BAL17 B lymphoma cells.

BAL17 B lymphoma cells, representing mature B lymphocytes, were used to analyze the role of tyrosine kinase in B cell activation. Anti-IgM-induced tyrosine phosphorylation was inhibited by preincubation of cells with tyrosine kinase inhibitor herbimycin A. Enzymatic activity of lyn protein was also inhibited by this drug, accompanied by down-regulation of p53lyn and p56lyn. However, a protein kinase C-mediated event was intact in the herbimycin A-pretreated cells, suggesting that the inhibitor acts selectively on tyrosine kinase. Anti-IgM failed to stimulate herbimycin A-pretreated cells to induce increases in inositol phospholipid metabolism or increased [Ca2+]i, whereas aluminum fluoride-induced metabolism was not altered. Moreover, membrane IgM density as revealed by flow cytometry was not changed by herbimycin A. These results indicate that tyrosine kinase(s) participates in the coupling of an Ag receptor cross-linkage to phospholipase C activation through a phosphorylation event in B lymphoma cells.

Animals↗
PubMed1992

PRS3 encoding an essential subunit of yeast proteasomes homologous to mammalian proteasome subunit C5.

We found by computer analysis that a putative yeast proteasome subunit gene named PRS3 that encodes a protein very similar to subunit C5 of rat and human proteasomes is located immediately 3' to the ERD2 gene of Saccharomyces cerevisiae. The similarity of the primary structures of the two suggests that this subunit may have a common function in proteasomes of all eukaryotes. The protein, deduced from the open reading frame of PRS3, consists of 242 amino acid residues with a calculated molecular weight of 27,077. Chromosomal disruption of the PRS3 gene created a recessive lethal mutation. Physical mapping by hybridization to intact S. cerevisiae chromosomal DNA showed that the PRS3 gene is located on chromosome II, unlike two other subunit genes, PRS1 and PRS2, which are located on chromosomes XV and VII, respectively. These findings indicate that the PRS3 protein is a subunit of yeast proteasomes that is essential for cell viability.

Amino Acid Sequence↗
PubMed1992

Determinants of response to the DNA topoisomerase II inhibitors doxorubicin and etoposide in human lung cancer cell lines.

BACKGROUND: Small-cell lung cancer (SCLC) is more sensitive to anticancer agents than non-small-cell lung cancer (NSCLC), but few studies have analyzed the mechanisms of natural drug resistance responsible for this difference. PURPOSE: To elucidate these mechanisms, we determined drug sensitivity and evaluated the biochemical parameters affecting response to the DNA topoisomerase II inhibitors doxorubicin and etoposide in both types of cancer cell lines, in particular the activity and content of DNA topoisomerase II, as well as etoposide uptake and cell doubling time. METHODS: Drug sensitivity and cellular uptake of etoposide were determined by clonogenic assay and accumulation of radiolabeled drug, respectively. The topoisomerase II activity was assayed by decatenation of kinetoplast DNA to minicircle DNA using nuclear protein, and the content was determined by immunoblot analysis of nuclear extracts. We also compared the topoisomerase II content in parent cell lines with that in lines with cisplatin resistance acquired in vitro. RESULTS: Sensitivities to doxorubicin and etoposide were higher in SCLC cell lines than in NSCLC lines, and the difference was statistically significant. Etoposide uptake in SCLC cells was higher than in NSCLC cells; the difference was statistically significant, but this difference may not be sufficient to account for the variation in sensitivities of the cell lines. Topoisomerase II activities of nuclear protein from SCLC cell lines were reproducibly twofold higher than those for NSCLC cell lines. The topoisomerase II content in nuclear protein appeared to be higher in SCLC cell lines than in NSCLC cell lines and corresponded to the sensitivities to doxorubicin and etoposide. In the cisplatin-resistant NSCLC cell lines PC-7/CDDP and PC-14/CDDP, the topoisomerase II content was increased compared with that in the parent lines, but the topoisomerase II content in other cisplatin-sensitive parent lines was similar to that in resistant sublines. CONCLUSIONS: These findings suggest that the topoisomerase II activity and content may be major factors in determining sensitivity to topoisomerase II inhibitors.

Carcinoma, Non-Small-Cell Lung↗
PubMed1992

Clinical significance of serum colloid osmotic pressure in relation to pulmonary edema and coronary instability in patients with unstable angina.

Several investigators demonstrated that severe reduction of colloid osmotic pressure (COP) predicts a fatal outcome in patients with cardiopulmonary failure. To evaluate the clinical significance of COP in relation to pulmonary edema, we studied 117 patients with unstable angina admitted in the hospital within 24 h after the chest pain. The mean COP of all 117 patients was 24.8 +/- 3.7 mmHg. COP was significantly lower in patients with pulmonary edema, according to the chest x-ray findings, compared with the patients without it. Among 26 patients with emergency coronary arteriography, a fairly good correlation was observed between coronary jeopardy score and COP (r = -0.57, p less than 0.005). Furthermore, COP in patients who survived 26 months after the hospital discharge was significantly higher than that of the nonsurvivors. Thus, measurement of COP is advised for monitoring patients with unstable angina.

Angina, Unstable↗
PubMed1992

Preparation and characterization of a mouse osteoclast-like multinucleated cell population.

We have reported that numerous tartrate-resistant acid phosphatase-positive osteoclast-like multinucleated cells (TRAP+ MNCs) are formed when mouse osteoblastic cells and spleen cells are cocultured in the presence of 1 alpha,25-dihydroxyvitamin D3 [1 alpha,25-(OH)2D3] (Endocrinology 123:2600, 1988). In this study, we prepared a TRAP+ MNC population using a modified coculture system and examined its osteoclastic properties. TRAP+ MNCs were formed in cocultures of mouse osteoblastic cells and marrow cells on 10 cm collagen gel-coated dishes. The TRAP+ MNC population was prepared by treating the dishes with 0.2% bacterial collagenase followed by density gradient centrifugation. The yield of TRAP+ MNCs was 20,000-40,000 cells per dish, much higher than that of osteoclasts (OCLs) isolated from neonatal rat bones (approximately 1000 cells per head). The purity of TRAP+ MNCs was 5.6 +/- 0.6% in cell number and about 30% in the number of nuclei. The recovery of TRAP+ MNCs after density gradient centrifugation was 30-40%. Acid production by MNCs was demonstrated by vital staining with acridine orange. Numerous resorption pits were formed when the MNC population was cultured for 48 h on bone slices. Autoradiography using [125I]salmon calcitonin (CT) showed abundant CT binding in most TRAP+ MNCs. Saturation analysis of [125I]salmon CT indicated a dissociation constant Kd for TRAP+ MNCs of 8.9 +/- 0.7 x 10(-10) M and 16.5 +/- 1.5 x 10(6) binding sites per cell. These results were similar to the Kd value (3.5 x 10(-10) M) and the number of binding sites (3.3 x 10(6) per cell) in isolated rat OCLs. Displacement curves for [125I]salmon CT with unlabeled salmon and human CT were similar in MNC and OCL preparations. Salmon and human CT increased cAMP production (maximal response: slmon CT at 10(-10) M, human CT at 10(-8) M; ED50s: salmon CT, 2.2 x 10(-11) M, human CT, 1.3 x 10(-9) M) in the MNC preparation. These results indicate that a large number of mouse TRAP+ MNCs possessing OCL characteristics can be easily prepared from in vitro cultures. This procedure will facilitate examination of mammalian OCL functions.

Acid Phosphatase↗