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Biomedical subjects

T Yoshida

Publications and source records attributed to T Yoshida.

At least 19 recordsLinked to original sources
PubMed1992

Purification and characterization of glycerate kinase from a serine-producing methylotroph, Hyphomicrobium methylovorum GM2.

The glycerate kinase of a serine-producing methylotroph, Hyphomicrobium methylovorum GM2, was purified to complete homogeneity and characterized, the first time for an enzyme from a methylotroph. The enzyme was a monomer with a molecular mass about 41-52 kDa. The enzyme was stable against heating at 35 degrees C for 30 min at pH values over 6-10. Maximum activity was observed at pH 8.0 and around 50 degrees C. The Km values for D-glycerate and ATP were 0.13 mM and 0.13 mM, respectively. The enzyme showed high specificity for D-glycerate, and was activated by potassium and ammonium ions. The reaction product of the enzyme was identified as 2-phosphoglycerate.

Bacteria↗
PubMed1992

de novo synthesis of calmodulin binding protein in substance P-induced steroidogenesis in bovine adrenocortical cells.

In order to clarify the mechanism of substance P (SP)-induced cortisol secretion from bovine adrenocortical (BAC) cells, protein synthesis at the early stage of SP-stimulation in BAC cells was investigated. Both SP and adrenocorticotropic hormone (ACTH) increased [3H]leucine uptake into BAC cells in a dose-dependent fashion. Although the SP-induced [3H]leucine uptake precedes the cortisol secretion, ACTH was slower in inducing [3H]leucine uptake and cortisol secretion. Protein synthesis inhibitors, actinomycin D and cycloheximide, were potent in inhibiting the SP-induced cortisol secretion. SDS-PAGE analysis, revealed that a 240 kDa protein is newly synthesized in BAC cells in response to SP but not ACTH. It was also indicated that the production of this 240 kDa protein was elicited about 30 min after stimulation by SP. Moreover, A23187 and 12-O-tetradecanoyl-phorbol-13-acetate (TPA) also caused a rapid [3H]leucine uptake and production of 240 kDa protein. In contrast, dibutyryl cAMP did not induce the synthesis of this 240 kDa protein. Calmidazolium, a calmodulin inhibitor, effectively inhibited not only [3H]leucine uptake but also 240 kDa protein production due to SP. On the other hand, KT-5720, an inhibitor of protein kinase A, had no effect on [3H]leucine uptake or 240 kDa production. Using the [125I]calmodulin-membrane overlay method, it was found that the 240 kDa protein was a newly synthesized calmodulin binding protein. From the present study, it was concluded that the de novo synthesis of this 240 kDa protein may be intimately related to the cortisol secretion in SP-stimulated BAC cells associated with an activation of the Ca-calmodulin pathway.

Adrenal Cortex↗
PubMed1992

Recombinant mouse tumor necrosis factor expressed in mammalian cells: effect of glycosylation on cytotoxic activity.

A mouse tumor necrosis factor-alpha (TNF) expression vector, pTNFNeo, was constructed by inserting a 1.3 kb cDNA coding for a full structural region of mouse TNF into an expression plasmid BCMGSNeo. COS7 cells were transfected with the pTNFNeo and a G418-resistant transfectant, BK-2, which stably secreted lytic activity to L929 cells was cloned. The lytic activity in the BK-2 culture spent medium reached up to 6000 U/ml, and was completely and specifically inhibited with antiserum to mouse TNF. Gel filtration chromatography and Western blot analysis indicated that the recombinant TNF in the medium existed in associated forms composed of a mixture of 22 kDa and 17.5 kDa components. Glycopeptidase F digestion indicated that the 22 kDa species was an N-glycosylated form of the 17.5 kDa species. Specific activities of the 22 kDa and the 17.5 kDa species isolated were 6.9 x 10(5) U/mg and 8.1 x 10(6) U/mg, respectively, suggesting that carbohydrate moiety impaired the lytic activity.

Animals↗
PubMed1992

Induction by bufalin of differentiation of human leukemia cells HL60, U937, and ML1 toward macrophage/monocyte-like cells and its potent synergistic effect on the differentiation of human leukemia cells in combination with other inducers.

We have recently demonstrated that bufalin is a new potent inducer of the differentiation of human myeloid leukemia cells. The present work was carried out to examine further the effect of bufalin on the growth and characteristics of human leukemia-derived cell lines U937, ML1, and HL60. At concentrations of 5-10 nM, bufalin decreased the growth of ML1 cells preferentially at the G2 phase and U937 cells at the S and G2 phases of the cell cycle. Bufalin, under these conditions, induced the differentiation of U937, ML1, and HL60 cells to monocyte/macrophage-like cells by measuring the expression of various differentiation markers, as assessed by morphology and histochemistry, and ability to phagocytose latex particles, to reduce nitroblue tetrazolium, and to develop Fc receptors. U937 and ML1 cells started to differentiate at 4 and 6 h, respectively, after treatment with 10 nM bufalin and showed maximum differentiation 72 h later. At present, a mechanism for the bufalin-mediated induction of the differentiation of these human leukemia cells remains to be determined. The combination of bufalin with all-trans retinoic acid, 1 alpha,25-dihydroxyvitamin D3, 4'-demethylepipodophyllotoxin ethylidene-beta-D-glucoside (VP16), or human gamma-interferon synergistically induced the differentiation of HL60 and U937 cells. A similar effect on ML1 cells was observed with the combination of bufalin with VP16 or human rTNF-alpha. These results suggest that bufalin in combination with VP16, all-trans retinoic acid, 1 alpha,25-dihydroxyvitamin D3, rTNF-alpha, or gamma-interferon may be very useful in the differentiation of human leukemia.

Antineoplastic Agents↗
PubMed1992

Evidence that rat liver mitochondrial and cytosolic fumarases are synthesized from one species of mRNA by alternative translational initiation at two in-phase AUG codons.

Rat liver contains two isozymes of fumarase, mitochondrial and cytosolic enzymes. Recently, we suggested that the precursors of both isozymes might be synthesized by one species of mRNA [Suzuki, T., Sato, M., Yoshida, T. & Tuboi, S. (1989) J. Biol. Chem. 264, 2581-2586]. To examine this possibility, we have isolated and characterized rat genomic clones for fumarase. The isolated clones covered almost all of the 5' half of the fumarase gene consisting of five exons. The first exon contained the whole 5' non-coding region and the signal peptide of mitochondrial precursor. The second exon encoded 45 amino acid residues of both mature proteins, starting from the N-terminal alanine. By using the boundary region of the first intron and the second exon as an S1-nuclease-analysis probe, we obtained conclusive evidence that rat liver contains no other mRNA specific for the cytosolic isozyme of fumarase. Two transcription-initiation sites were identified by further S1-nuclease-mapping analysis and were shown to be located very close to each other, differing by only four bases in length. Therefore, these sites were considered to be functionally the same. The results obtained by hybrid-selected translation, with a DNA fragment of the 5' non-coding region as a hybridization probe for selecting mRNA, were consistent with the above findings. We found a plausible secondary structure within the 5' non-coding mRNA sequence that may impede initiation and so alter the efficiency of translation. We also discuss the mechanism regulating translational initiation.

Amino Acid Sequence↗
PubMed1992

Lateral conductance parallel to membrane surfaces: effects of anesthetics and electrolytes at pre-transition.

The effects of dilute salts and anesthetics were studied on the impedance dispersion in the dipalmitoylphosphatidylcholine (DPPC) liposomes. Below the pre-transition temperature, the apparent activation energy for conductance in DPPC-H2O without salts was equivalent to pure water, 18.2 kJ mol-1. This suggests that the mobile ions (H3O+ and OH-) interact negligibly with the lipid surface below the pre-transition temperature. At pre-transition temperature, the apparent activation energy of the conductance decreased by the increase in the DPPC concentrations. The effects of various salts (LiCl, NaCl, KCl, KBr, and KI) on the apparent activation energy of the conductance were studied. Changes in anions, but not in cations, affected the activation energy. The order of the effect was Cl- less than Br- less than I-. Cations appear to be highly immobilized by hydrogen bonding to the phosphate moiety of DPPC. The smaller the ionic radius, the more ions are fixed on the surface at the expense of the free-moving species. The apparent activation energy of the transfer of ions at the vesicle surface was estimated from the temperature-dependence of the dielectric constant, and was 61.0 kJ mol-1 in the absence of electrolytes. In the presence of electrolytes, the order of the activation energy was F- greater than Cl- greater than Br- greater than I-. When the ionic radius is smaller, these anions interact with the hydration layer at the vesicle surface and the ionic transfer may become sluggish. In the absence of electrolytes, the apparent activation energy of the dielectric constant decreased by the increase in halothane concentrations. In the presence of electrolytes, however, the addition of halothane increased the apparent activation energy. We propose that the adsorption of halothane on the vesicle surface produces two effects: (1) destruction of the hydration shell, and (2) increase in the binding of electrolytes to the vesicle surface. In the absence of electrolytes, the first effect predominates and the apparent activation energy is decreased. In the presence of electrolytes, the latter effect predominates and the apparent activation energy is increased.

1,2-Dipalmitoylphosphatidylcholine↗
PubMed1992

Sulfated alkyl oligosaccharides with potent inhibitory effects on human immunodeficiency virus infection.

Compounds with medium relative molecular masses active against human immunodeficiency virus (HIV) were synthesized. Sulfated alkyl oligosaccharides such as sulfated octadecyl maltohexaoside, sulfated dodecyl laminaripentaoside and sulfated dodecyl laminari-oligomer caused 50% inhibition of virus infection in the EC50 range of 0.4-0.7 microgram/mL in vitro using the MT-4 cell line and HIV-1HTLV-IIIB virus isolate, though sulfated oligosaccharides without alkyl groups showed low anti-HIV activities. This anti-HIV activity was close to the EC50 of 0.43 microgram/mL for a highly active sulfated polysaccharide curdlan sulfate which was reported to inhibit completely the HIV infection at a concentration as low as 3.3 micrograms/mL. These compounds were also active against HIV-2 and a clinically isolated HIV-1 with reduced AZT sensitivity. For such sulfated alkyl oligosaccharides, the mechanism of inhibition of HIV infection was assumed to be the inhibition of HIV binding to the cell and to some extent the interaction of the alkyl portion with the lipid bilayer of the virus.

Amino Acid Sequence↗
PubMed1992

Crystallization and preliminary diffraction studies of hydroxypyruvate reductase (D-glycerate dehydrogenase) from Hyphomicrobium methylovorum.

Two crystal forms of hydroxypyruvate reductase (D-glycerate dehydrogenase) from the methylotrophic bacterium Hyphomicrobium methylovorum have been grown from ammonium sulphate solutions. One crystal form is triclinic, with unit cell parameters a = 60.4 A, b = 60.5 A, c = 66.3 A, alpha = 102.3 degrees, beta = 113.7 degrees and gamma = 102.7 degrees, suggesting that a dimer (monomer M(r) 38,000) occupies the unit cell. This crystal form diffracts to beyond 2.4 A resolution and is suitable for crystallographic structure analysis.

Alcohol Oxidoreductases↗
PubMed1992

Delivery of an accessory signal for cell activation by exogenous phosphatidylinositol-specific phospholipase C.

Digestion of phosphatidylinositol (PI) or glycosylphosphatidylinositol (GPI) anchors of membrane proteins on the external cell surface with exogenous PI-specific phospholipase C (PIPLC) from Bacillus thuringiensis was shown to transmit a signal into the thymocyte to modulate the TCR/CD3 complex-induced signal delivery for cell activation. This was demonstrated for very early protein tyrosine phosphorylation, early c-fos transcription and late DNA synthesis. For this effect preincubation of the cells with PIPLC was required, but there was no evidence of involvement of any soluble products released from the cell surface by PIPLC in the signaling, suggesting a crucial role of the membrane-bound counterpart (diacylglycerol or diradylglycerol) of the PI/GPI hydrolysate. A possible role for this accessory signal in the microorganism-linked control of the (diacylglycerol or diradylglycerol) of the PI/GPI hydrolysate. A possible role for this accessory signal in the microorganism-linked control of the T cell receptor function is discussed.

Animals↗
PubMed1992

Induction of hepatic microsomal cytochrome P450 and drug-metabolizing enzymes by 4-benzylpyridine and its structurally related compounds in rats. Dose- and sex-related differential induction of cytochrome P450 species.

We examined the abilities of 4-, 3- and 2-benzylpyridine and 4-tert-butylpyridine to induce hepatic microsomal cytochrome P450 and drug-metabolizing enzymes in male and female rats in order to define the effects of pyridine-containing compounds on drug metabolism. 4-Benzylpyridine (0.4 mmol/kg, for 2 consecutive days) induced total cytochrome P450 to about three times that of the controls at 24 hr, and its inducing effect was sustained for 120 hr after the treatment in male and female rats. 4-Benzylpyridine was a more potent inducer of cytochrome P450 than 3- and 2-benzylpyridine, which induced the cytochrome to 71.4 and 43.9%, respectively, of that produced by the 4-substituted isomer. 4-tert-Butylpyridine also induced cytochrome P450. Immunoblot analysis revealed that a single treatment of male rats with 4-benzylpyridine at doses ranging from 0.05 to 0.80 mmol/kg induced cytochrome P450b/e and caused a maximum increase in the level of the isozyme at the 0.2 mmol/kg dose. 4-Benzylpyridine at doses from 0.40 to 0.80 mmol/kg also induced cytochrome P450c/d in male rats. In female rats, 4-benzylpyridine induced cytochrome P450b at doses ranging from 0.1 to 0.80 mmol/kg and produced a maximum increase in the level of this isozyme at 0.40 to 0.60 mmol/kg. Induction of cytochrome P450c/d by 4-benzylpyridine in female rats was observed at a dose of 0.20 mmol/kg, and the magnitude of the induction of the isozyme was increased in a dose-dependent manner. Both 3- and 2-benzylpyridine induced cytochrome P450b/e and/or c/d depending on the increase of total cytochrome P450 without changing the induction patterns of the isozymes. 4-tert-Butylpyridine induced cytochrome P450b at doses ranging from 0.20 to 0.60 mmol/kg and slightly induced P450c/d at doses ranging from 0.10 to 0.40 mmol/kg in male rats. These results and our previous report (Matsuura et al., Biochem Pharmacol 41: 1949-1956, 1991) clearly show that the pyridine compounds having lipophilic groups at the 4- or 3-position of the ring could be inducers of cytochrome P450. The present results also revealed that 4-benzylpyridine shows dose- and sex-related differences in the induction of cytochrome P450b/e and c/d in rats.

Aminopyrine N-Demethylase↗
PubMed1992

Induction and identification of mast cells from long-term culture of mouse spleen cells without conditioned medium.

A simple method is described for the preparation of large numbers of mast cells from mouse spleen cells in vitro. Mouse spleen cells were cultured with RPMI 1640 medium supplemented with 10% FCS and 2-ME. Half of the total volume of the medium was changed every 4-5 days. Mast cell numbers increased with the culture time and reached a peak between 16 and 20 days. Using this method, 2 x 10(6) mast cells could be induced from 1 x 10(7) nucleated normal spleen cells. T cells and supernatant derived from ConA-stimulated T cells were unnecessary for mast cell induction. Phenotype analysis by FACS showed that Thy1,2, L3T4, Ly-2, Ig, B220, Asialo GM1, and WGA receptors were all negative but functional IgE receptors were positive. The granules in the cells could be stained by alcian blue but not by safranin. There was 1.632 +/- 0.024 micrograms stored histamine in 1 x 10(6) of the cells. Histamine was released from the cells in an antigen-induced and IgE-mediated process. Compound 48/80 and A23187 induced degranulation of the cells, and the mast cells were able to respond to ConA.

Animals↗
PubMed1992

Sodium ion and proline binding sites in the Na+/proline symport carrier of Escherichia coli.

Proline binding activity of the Escherichia coli Na+/proline symport carrier is inhibited by a sulfhydryl reagent, N-ethylmaleimide (NEM). Proline and its analogs protected the carrier against the NEM-inactivation in a Na+ (or Li+)-dependent manner. Na+ alone, even in the absence of proline, partially protected it from the NEM-inactivation. Mutant proline carriers, CS281, CS344 and CS349, which have a serine residue in place of Cys-281, Cys-344 and Cys-349, respectively (Yamato, I. and Anraku, Y. (1988) J. Biol. Chem. 263, 16055-16057) were also analyzed for cation-dependent proline binding and NEM-sensitivity. Proline binding activities of CS281 and CS344 were almost completely resistant to NEM, whereas that of CS349 was not. Furthermore, the proline binding activity of CS344 was remarkably lower than those of the wild-type, CS281 and CS349 carriers. These results indicate that Cys-344, which is located in the putative eighth membrane-spanning domain in the carrier, is a cysteine residue functionally involved in the high-affinity binding for sodium ion and proline.

Amino Acid Sequence↗
PubMed1992

Importance of histidine residue 25 of rat heme oxygenase for its catalytic activity.

A truncated, soluble, and enzymatically active rat heme oxygenase lacking its membrane-associative, C-terminal segment was expressed in E. coli strain JM109. The roles of its four histidine residues were examined by determining the enzymatic activities of mutant enzymes in which each of these residues in turn was replaced by alanine. Mutation of histidine residue 25 to alanine resulted in marked decrease in activity for heme breakdown, indicating that this histidine residue has an important role in the heme oxygenase reaction.

Alanine↗
PubMed1992

Occurrence of differentiated keratin peptide(K1) in cultured human squamous cell carcinomas.

To date, the largest keratin peptide(K1, 68 KD) has been absent in cultured human squamous cell carcinomas. Using a low salt aqueous solution, not containing high salt and Triton X-100, as a washing buffer for keratin extraction, followed by two dimensional polyacrylamide gel electrophoresis, immunological techniques and Northern blot analysis, we demonstrated K1 peptide in two kinds of cultured human squamous cell carcinomas. Until now keratin extraction has been done using high salt/Triton X-100 solution during which K1 peptide may be removed together developed an affinity with the buffer. Many investigators may have therefore overlooked K1.

Amino Acid Sequence↗
PubMed1992

Cortisol secretion induced by substance P from bovine adrenocortical cells and its inhibition by calmodulin inhibitors.

When primary cultured bovine adrenocortical cells were treated with substance P (SP) at concentrations higher than 10 pM, cortisol output increased in a dose-dependent fashion. Although other neurokinins, such as neurokinin A (NKA) and neurokinin B (NKB), were also effective in secreting cortisol, SP was the most potent among the tested neurokinins, the potency order being SP greater than NKA much greater than NKB. This suggests that the NK-1 type receptor on adrenocortical cells may be the site of action of SP on cortisol secretion. The maximal response in SP-induced cortisol secretion was comparable to that elicited by adrenocorticotropic hormone (ACTH). SP-induced cortisol secretion was dependent upon extracellular Ca2+ concentrations, and 45Ca2+ uptake into adrenocortical cells treated with SP was long-lasting. While, in the case of ACTH, 45Ca2+ uptake proceeded transiently, the increase in intracellular cAMP content was much greater compared with that of SP. Although KT-5720, an inhibitor of protein kinase A, inhibited potently ACTH-induced cortisol secretion, SP-induced secretin was not affected by this inhibitor at all. On the other hand, calmodulin inhibitors, such as calmidazolium, trifluoperazine and N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide, were not more effective in inhibiting SP-induced cortisol secretion than secretion induced by ACTH. The present study indicates that SP may be one of the physiological stimulants of cortisol secretion and that an increase in intracellular Ca2+ concentration and the subsequent activation of calmodulin may precede SP-induced cortisol secretion.

Adrenal Cortex↗
PubMed1992

Expression of high affinity binding sites for erythropoietin on L8057 cells, a mouse megakaryoblastic cell line, associated with cell differentiation.

In this study, specific binding sites were examined for erythropoietin (EPO) on the mouse leukemic cell line, L8057. This cell line is megakaryoblastic in origin as evidenced by an enlargement of cell size, multinuclearity, intense activity of acetylcholinesterase, more expression of glycoprotein IIb and IIIa antigen, and higher ploidy distribution after the treatment with 12-o-tetradecanoylphorbor-13-acetate (TPA). The original undifferentiated cells possessed a single class of low affinity binding sites for recombinant human (rh) EPO with a Kd of 3.5 nM. Following the treatment with TPA, high affinity binding sites (Kd; 440 pM) were expressed in addition to the low affinity sites. EPO stimulated the incorporation of 3H-leucine into TPA-treated L8057 cells, and the maximal effect of EPO was observed at the same order as the Kd value of high affinity sites. The present data demonstrates that the expression of high affinity binding sites for EPO is associated with the differentiation of L8057 cells which have megakaryocytic characteristics. Furthermore, protein synthesis stimulated by EPO may be mediated through the high affinity sites.

Acetylcholinesterase↗
PubMed1992

Heme oxygenase is a heat shock protein and PEST protein in rat astroglial cells.

Cultured rat forebrain astrocytes contained significant amounts of immunostainable heme oxygenase-1 (HO-1) isozyme, whereas HO-1 was undetectable in spontaneously transformed rat astroglial cells (ATs). HO-1 was inducible in both cell types by heat shock and by submicromolar amounts of H2O2. Inhibition of RNA synthesis with actinomycin D or protein synthesis with cycloheximide resulted in the rapid loss of immunostainable heme oxygenase in astrocytes. Analysis of the primary structure of heme oxygenase suggests that it is a PEST protein, i.e., targeted for rapid turnover.

Animals↗