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Y Hashimoto

Publications and source records attributed to Y Hashimoto.

At least 55 records · Page 3Linked to original sources
PubMed1999

Construction of a screening system for selecting lysozyme mutants unable to form a stable structure from random mutants.

To collect folding information, we screened and analyzed the recombinant hen lysozyme mutants which were not secreted from yeast. As model mutants, Leu8Arg, Ala10Gly, and Met12Arg were prepared by site-directed mutagenesis and analyzed as to whether they were secreted from yeast or not. Consequently, Ala10Gly was found to be secreted from yeast, but Leu8Arg and Met12Arg were not. Next, these mutants were expressed in Escherichia coli and refolded in vitro. As a result, Ala10Gly folded as the wild-type did. Leu8Arg efficiently refolded in renaturation buffer containing glycerol. Met12Arg did not refold even in the presence of glycerol. These results show that the Ala10Gly mutation does not affect folding or stability, that Leu8Arg is too unstable to be secreted from yeast, and that Met12Arg may be very unstable or the mutation affects the folding pathway. We screened the mutants that were not secreted by yeast from a randomly mutated lysozyme library, and obtained Asp18His/Leu25Arg and Ala42Val/Ser50Ile/Leu56Gln. These two mutants were expressed in E. coli and then refolded in the presence of urea or glycerol. These mutants were refolded only in the presence of glycerol. Each single mutant of Asp18His/Leu25Arg and Ala42Val/Ser50Ile/Leu56Gln was independently prepared and folded in vitro. The results showed that Leu25Arg and Leu56Gln were the dominant mutations, respectively, which cause destabilization. These results show that the mutant lysozymes which were not secreted from yeast may be unstable or have a defect in the folding pathway. Thus, we established a screening system for selecting mutants which are unable to form a stable structure from random mutants.

Amino Acids↗
PubMed1999

Analysis of a catalytic pathway via a covalent adduct of D52E hen egg white mutant lysozyme by further mutation.

We previously demonstrated by X-ray crystallography and electrospray mass spectrometry that D52E mutant hen lysozyme formed a covalent enzyme-substrate adduct on reaction with N-acetylglucosamine oligomer. This observation indicates that D52E lysozyme may acquire a catalytic pathway via a covalent adduct. To explain this pathway, the formation and hydrolysis reactions of the covalent adduct were investigated. Kinetic analysis indicated that the hydrolysis step was the rate-limiting step, 60-fold slower than the formation reaction. In the formation reaction, the pH dependence was bell-shaped, which was plausibly explained by the functions of the two catalytic pKas of Glu35 and Glu52. On the other hand, the pH dependence in the hydrolysis was sigmoidal with a transition at pH 4. 5, which was identical with the experimentally determined pKa of Glu35 in the covalent adduct, indicating that Glu35 functions as a general base to hydrolyze the adduct. To improve the turnover rate of D52E lysozyme, the mutation of N46D was designed and introduced to D52E lysozyme. This mutation reduced the activation energy in the hydrolysis reaction of the covalent adduct by 1.8 kcal/mol at pH 5.0 and 40 degrees C but did not affect the formation reaction. Our data may provide a useful approach to understanding the precise mechanism of the function of natural glycosidases, which catalyze via a covalent adduct.

Animals↗
PubMed1999

The relaxant effect of propofol on guinea pig tracheal muscle is independent of airway epithelial function and beta-adrenoceptor activity.

UNLABELLED: Airway epithelium and vascular endothelium modulate the tension of the underlying smooth muscle by releasing relaxing factors such as prostanoids and nitric oxide (NO). We investigated whether the relaxant effect of propofol on airway smooth muscle is dependent on airway epithelial function. Tracheal spirals of female guinea pigs were mounted in water-jacketed organ baths filled with Krebs-bicarbonate buffer aerated with 95% O2 and 5% CO2 at 37 degrees C. Changes in isometric tension of the specimens were measured with a force-displacement transducer and recorded with a polygraph. Propofol (10(-4) to 10(-3) M) inhibited carbachol (CCh)-, histamine (HA)-, or endothelin-1-induced contractions of the muscles in a dose-dependent manner. Neither mechanical removal of the epithelial layer, chemical inhibition of epithelial synthesis of prostanoids, nor NO affected the relaxant effect of propofol on CCh- or HA-induced tracheal contraction. Furthermore, the blockade of beta-adrenoceptors did not change the relaxant effect of propofol. These results indicate that the relaxant effect of propofol on the airway smooth muscle is independent of the epithelial function or beta-adrenoceptor activity. Propofol is an excellent anesthetic for patients with hyperreactive airways in which the epithelial layer is damaged. IMPLICATIONS: Airway epithelium, as well as vascular endothelium, plays an important role in modulating the baseline tone and reactivity of underlying smooth muscle. We investigated, in vitro, whether the relaxant effect of propofol on airway smooth muscle is dependent on airway epithelial function. We suggest that propofol relaxes airway smooth muscle independently of the epithelial function.

Anesthetics, Intravenous↗
PubMed1999

Induction of apoptotic cell death in human hepatocellular carcinoma SK-HEP-1 cells by a polyamine synthesis inhibitor, methylglyoxal bis(cyclopentylamidinohydrazone).

The antitumor effects of a polyamine biosynthetic pathway inhibitor methylglyoxal bis(cyclopentylamidinohydrazone) (MGBCP) on the human hepatocellular carcinoma SK-HEP-1 cell line have been investigated. The growth of these cultured hepatocellular carcinoma cells was inhibited by MGBCP in a dose-dependent manner. Spermidine and spermine levels were dose-dependently depressed, and morphological changes due to programmed cell death (apoptosis) were observed in these MGBCP-treated hepatocellular carcinoma cells. These results suggest that in addition to reducing the growth rates, MGBCP can induce apoptotic cell death in this human hepatocellular carcinoma cell line.

Antineoplastic Agents↗
PubMed1999

Relaxant effect of magnesium and zinc on histamine-induced bronchoconstriction in dogs.

OBJECTIVE: Magnesium sulfate (MgSO4) has been reported to produce bronchodilation in asthmatic patients. In vitro studies have suggested that divalent cations inhibit L-type voltage-sensitive calcium ion (Ca2+) channels in cardiac and smooth muscles. In this study, we evaluated the in vitro and in vivo effects of magnesium ion (Mg2+) and zinc ion (Zn2+) on the airway contracted by histamine. SETTING: A university research laboratory. SUBJECTS IN VITRO: Tracheal smooth muscle from guinea pigs. IN VIVO: Mongrel dogs. MEASUREMENTS AND MAIN RESULTS IN VITRO STUDY: The tension of isolated guinea pig tracheal strips was measured isometrically with a force displacement transducer. The specimen was contracted with histamine (10 microM). Then, MgSO4 (n = 6), zinc sulfate (ZnSO4, n = 6), or sodium sulfate (Na2SO4, n = 6) was cumulatively added to the organ bath. IN VIVO STUDY: The bronchial cross-sectional area of mongrel dogs was measured by a direct visualization method demonstrated previously. The dogs were randomly assigned to three groups: group Mg (n = 7), group Zn (n = 7), and group Na (n = 7). Bronchoconstriction was elicited with histamine (10 microg/kg plus 500 microg/kg/hr iv). Thirty minutes after the start of histamine infusion, 0 (saline), 1, 10, and 100 micromol/kg ZnSO4 or 1, 10, 100, and 1000 micromol/kg MgSO4 or Na2SO4 were administered intravenously in group Zn, Mg, or Na, respectively. The bronchial cross-sectional area was assessed before (basal) and 30 mins after the start of histamine infusion and 5 mins after each dose of ZnSO4, MgSO4, or Na2SO4. Arterial blood was also obtained to measure plasma levels of epinephrine and norepinephrine by gas chromatography-mass spectrometry. All data are expressed as mean +/- SEM. The doses of the divalent cations that reversed histamine-induced contraction by 50% were calculated by GraphPad Prism. MgSO4 and ZnSO4 (9.38+/-0.28 and 1.84+/-0.30 mM, respectively) relaxed histamine-contracted tracheal strip in a concentration-dependent manner, whereas Na2SO4 did not. Similarly, the in vivo study showed that MgSO4 and ZnSO4 dose-dependently reversed histamine-induced bronchoconstriction (potency, ZnSO4 > MgSO4), whereas Na2SO4 did not. In groups Mg and Zn, the plasma catecholamine levels also dose-dependently increased except when 1000 micromol/kg MgSO4 was administered. CONCLUSION: Because the divalent cations tested produced a spasmolytic effect on the contracted airway, infusion of divalent cations might be effective against asthmatic attack. However, high concentrations of these cations produce significant toxicity, so dosage will be an important concern in development of these agents.

Animals↗
PubMed1999

Functional and binding studies of insurmountable antagonism of 606A, a novel AT1-receptor antagonist in rabbit tissues.

Angiotensin AT1-receptor antagonists can be classified into two types, surmountable and insurmountable ones, based on the way they inhibit angiotensin II (AII)-induced vasoconstriction. To elucidate the causes of the difference, we studied how several antagonists associate with and dissociate from AT1-receptor sites by using rabbit adrenal cortical membrane. Four antagonists, 606A, EXP3174, CV11974, and E4177, showed equipotent competitive antagonism when they were added simultaneous with [125I]-AII in binding experiments. However, in AII-induced contraction studies with rabbit aorta, 606A, EXP3174, and CV11974 inhibited the contraction noncompetitively, whereas E4177 inhibited competitively. The longer the pretreatment period with EXP3174 or CV11974, the more effectively the antagonists suppressed AII-induced contraction. However, the suppression of contraction by 606A and E4177 changed little with the length of the pretreatment period. AII-induced contraction of 606A- or E4177-treated aorta recovered easily by washout, but that of CV11974-treated aorta was hard to recover by washout. These results obtained in the aorta were consistent with their characteristics observed in the AII binding study in the rabbit adrenal cortical membrane in cases of EXP3174 and CV11974. The differences between association rate with and dissociation rate from the AT1 receptor of E4177 and 606A were slight, in spite of the clear difference between their action in the contraction study. Because of the variations observed with the four compounds, mechanisms of insurmountable antagonism may not be uniform among AT1-receptor antagonists.

Angiotensin II↗
PubMed1999

ADP-ribosylation as an intermediate step in inactivation of rifampin by a mycobacterial gene.

Mycobacterium smegmatis DSM43756 inactivates rifampin, and the inactivated antibiotic product recovered from culture medium was ribosylated on the 23-OH group. To study this process, the gene responsible for the inactivation was expressed at high levels by the lac promoter in Escherichia coli conferring resistance to >500 microg of antibiotic per ml. Cell homogenates generated a novel derivative designated RIP-TAs; in this study, we determined that RIP-TAs is 23-(O-ADP-ribosyl)rifampin. Our results indicated that RIP-TAs is an intermediate in the pathway leading to ribosylated rifampin and that the previously characterized gene encodes a mono(ADP-ribosyl)transferase which, however, shows no sequence similarity to other enzymes of this class.

ADP Ribose Transferases↗
PubMed1999

Functional characteristics of basolateral peptide transporter in the human intestinal cell line Caco-2.

The apical H+-coupled peptide transporter (PEPT1) and basolateral peptide transporter in human intestinal Caco-2 cells were functionally compared by the characterization of [14C]glycylsarcosine transport. The glycylsarcosine uptake via the basolateral peptide transporter was less sensitive to medium pH than uptake via PEPT1 and was not transported against the concentration gradient. Kinetic analysis indicated that glycylsarcosine uptake across the basolateral membranes was apparently mediated by a single peptide transporter. Small peptides and beta-lactam antibiotics inhibited glycylsarcosine uptake by the basolateral peptide transporter, and these inhibitions were revealed to be competitive. Comparison of the inhibition constant values of various beta-lactam antibiotics between PEPT1 and the basolateral peptide transporter suggested that the former had a higher affinity than the latter. A histidine residue modifier, diethyl pyrocarbonate, inhibited glycylsarcosine uptake by both transporters, although the inhibitory effect was greater on PEPT1. These findings suggest that a single facilitative peptide transporter is expressed at the basolateral membranes of Caco-2 cells and that PEPT1 and the basolateral peptide transporter cooperate in the efficient transepithelial transport of small peptides and peptidelike drugs.

Caco-2 Cells↗
PubMed1999

CREB-independent regulation by CBP is a novel mechanism of human growth hormone gene expression.

Hypothalamic growth hormone-releasing hormone (GHRH) stimulates growth hormone (GH) gene expression in anterior pituitary somatotrophs by binding to the GHRH receptor, a G-protein-coupled transmembrane receptor, and by mediating a cAMP-mediated protein kinase A (PKA) signal-transduction pathway. Two nonclassical cAMP-response element motifs (CGTCA) are located at nucleotides -187/-183 (distal cAMP-response element; dCRE) and -99/-95 (proximal cAMP-response element; pCRE) of the human GH promoter and are required for cAMP responsiveness, along with the pituitary-specific transcription factor Pit-1 (official nomenclature, POU1F1). Although a role for cAMP-response element binding protein (CREB) in GH stimulation by PKA has been suggested, it is unclear how the effect may be mediated. CREB binding protein (CBP) is a nuclear cofactor named for its ability to bind CREB. However, CBP also binds other nuclear proteins. We determined that CBP interacts with Pit-1 and is a cofactor for Pit-1-dependent activation of the human GH promoter. This pathway appears to be independent of CREB, with CPB being the likely target of phosphorylation by PKA.

Base Sequence↗
PubMed1999

Serum concentration of 20K human growth hormone (20K hGH) measured by a specific enzyme-linked immunosorbent assay. Study Group of 20K hGH.

Several GH isoforms have been identified in pituitary and serum, the most abundant of which is the 22K human GH (hGH) isoform. The 20K hGH isoform is produced by alternative splicing of GH messenger ribonucleic acid and comprises approximately 10% of all GH in the pituitary. The physiological role of 20K hGH remains to be determined, partly because of the lack of a simple and specific assay. We have established sensitive enzyme-linked immunosorbent assays (ELISAs) specific to 20K and 22K hGH. To determine whether regulation of 20K hGH secretion is the same as that for 22K hGH, we measured serum concentrations of both species of hGH in normal subjects and patients with a variety of endocrine disorders. The serum levels of 20K hGH after overnight fasting was 118 +/- 178 pg/mL (n = 282) in normal women, significantly higher than that in normal men (64 +/- 170 pg/mL; n = 226). However, there was no difference in the proportion of 20K hGH to 20K plus 22K hGH between men (6.3 +/- 2.6%, mean +/- SD; n = 176) and women (6.3 +/- 2.1%; n = 263). No correlation was detected between the ratio of 20K hGH and age, body height, body weight, or body fat mass in normal subjects. The proportion of 20K hGH was significantly (P < 0.001) higher in patients with active acromegaly (9.2 +/- 2.2%; n = 33) and patients with anorexia nervosa (9.0 +/- 1.9; n = 8), both of which are characterized by chronic elevation of circulating GH levels. The proportion of 20K hGH in successfully treated acromegalic patients did not differ from that in normal subjects, suggesting that GH-producing pituitary tumors secrete a higher proportion of 20K hGH, or that a chronic excess of 22K hGH alters the MCR of 20K hGH. The values in patients with adult GH deficiency, hyperthyroidism, primary hypothyroidism, or GH-independent short stature did not differ from those in normal subjects. The 20K ratio did not change after acute GH provocative tests, such as the insulin tolerance test and the GHRH test. These results suggest that secretion of 20K hGH from the pituitary is under the same control as that of 22K hGH. This new assay may provide a tool for understanding the physiological or pathophysiological role of the 20K hGH isoform.

Acromegaly↗
PubMed1999

Serum levels of 20-kilodalton human growth hormone (GH) are parallel those of 22-kilodalton human GH in normal and short children.

Twenty-kilodalton human GH (20K), which is one of the human GH (hGH) variants, is thought to be produced by alternative premessenger ribonucleic acid splicing. However, its physiological role is still unclear due to the lack of a specific assay. We have measured serum 20K and 22-kDa hGH (22K) by specific ELISAs to investigate the physiological role of 20K in children. The subjects were 162 normal children, aged 1 month to 20 yr; 12 patients with GH deficiency (GHD), aged 11 months to 13 yr; 57 children with non-GHD short stature, aged 2-17 yr; and 13 girls with Turner's syndrome, aged 5 months to 15 yr. Samples were collected at random from normal children and were collected after hGH provocative tests and 3-h nocturnal sleep from GHD, non-GHD short stature, and Turner's syndrome children. The mean basal serum concentrations of 22K and 20K were 2.4 +/- 2.8 ng/mL and 152.3 +/- 184.0 pg/mL in normal boys and 2.5 +/- 3.1 ng/mL and 130.6 +/- 171.5 pg/mL in normal girls, respectively. The percentages of 20K (%20K) were 5.8 +/- 2.1% and 6.0 +/- 3.2% in 83 normal boys and 79 normal girls, respectively. There was no significant difference in %20K either among ages or between the prepubertal stage and the pubertal stage in normal boys and girls. The mean %20K values in basal samples of provocative tests in 12 patients with GHD, non-GHD short stature, and Turner's syndrome were 6.5 +/- 2.4%, 6.5 +/- 3.8%, and 5.9 +/- 3.2%, respectively. There was no significant difference in %20K among normal children and these growth disorders, and there was no significant difference in %20K throughout the hGH provocative tests and 3-h nocturnal sleep in these growth disorders. There was also no significant correlation between the percentage of 20K and the height SD score or body mass index in either normal children or subjects with these growth disorders. In conclusion, the %20K is constant, regardless of age, sex, puberty, height SD score, body mass index, and GH secretion status. The regulation of serum 20K levels remains to be established.

Adolescent↗
PubMed1999

Effects of fosfomycin and imipenem-cilastatin on the nephrotoxicity of vancomycin and cisplatin in rats.

The nephrotoxicity of vancomycin and cisplatin and the protective effects of fosfomycin and imipenem-cilastatin on renal function have been studied in rats. The renal clearance of vancomycin after the induction of renal dysfunction was also evaluated by calculating the glomerular filtration rate (GFR) and its secretory clearance. Plasma concentrations of creatinine and urea nitrogen increased dose-dependently after vancomycin injection. No such increases were observed after co-treatment with fosfomycin or imipenem-cilastatin. Changes of N-acetyl-beta-D-glucosaminidase activity in the urine of vancomycin-treated rats were not remarkable compared with those in cisplatin-treated animals. The reduced renal clearance of vancomycin in rats with acute renal failure induced by vancomycin was because of a decrease in both GFR and secretory clearance. However, the changes in GFR and secretory clearance were not proportional-the change in GFR was more pronounced than that of secretory clearance in the experimental groups. In addition, the renal clearance of vancomycin was maintained at the control level after co-administration of fosfomycin or imipenem-cilastatin with vancomycin. These results suggest that vancomycin impairs glomerular filtration more markedly than renal tubular function as compared with cisplatin. Co-administration with fosfomycin or imipenem-cilastatin confers significant protection against the nephrotoxic effects of vancomycin.

Acetylglucosaminidase↗
PubMed1999

Identification and immunological characterization of a novel 40-kDa protein linked to CD98 antigen.

Monoclonal antibodies (mAbs) were obtained from hybridoma clones established by cell fusion between mouse myeloma cells and spleen cells from a mouse immunized against an affinity-purified 40-kDa component of rat 125-kDa glycoprotein (GP125). Two mAbs designated as 3F2 and 6B4 detected a 40-kDa and a 125-kDa band under reducing and nonreducing conditions, respectively, in extracts prepared from rat, mouse and human tumor cells. Association of the 40-kDa protein with CD98 was revealed by sandwich-type enzyme-linked immunosorbent assay. The two mAbs were strongly reactive with various tumor cells and activated lymphocytes, but were only weakly reactive with resting lymphocytes. Confocal microscopy indicated colocalization of CD98 and the 40-kDa protein defined with 3F2 and 6B4 at the cell surface and perinuclear regions. On immunohistochemical analysis of frozen sections of rat tongue, the anti-rat CD98 mAb B3 selectively stained the basal layer and 3F2 stained the upper epithelial part in addition to the basal layer, indicating the existence of CD98-unlinked 40-kDa protein.

Animals↗
PubMed1999

Potent homophthalimide-type inhibitors of B16F10/L5 mouse melanoma cell invasion.

Recently, we developed a series of novel and potent aminopeptidase inhibitors with a homophthalimide skeleton. Among them, N-(2,6-diethylphenyl)homophthalimide (PIQ-22) possesses a specific aminopeptidase-inhibiting activity more potent than that of bestatin or actinonin, as assayed in terms of hydrolysis of L-alanine 4-methylcoumaryl-7-amide (Ala-AMC) by human acute lymphoblastic leukemia MOLT-4 cells. We show here that PIQ-22 and its 2,6-dimethylphenyl derivative (PIQ-11) are more potent inhibitors of tumor cell invasion than bestatin and actinonin in a Matrigel assay using mouse melanoma B16F10/L5 cells.

Aminopeptidases↗
PubMed1999

Modification by heme oxygenase inhibitor, tin protoporphyrin, of cellular differentiation of human myeloid leukemia K562 cell line.

Human myeloid leukemia K562 cells can be induced to differentiate to mature cells bidirectionally, i.e., hemin induces erythroid differentiation, while 12-O-tetradecanoylphorbol 13-acetate (TPA) induces differentiation to monocytes. TPA is also a potent inducer of heme oxygenase (HO), which catabolizes heme to biliverdin. We show here that TPA suppresses hemin-induced erythroid differentiation of K562 cells, while retinoids augment it. Further, an HO inhibitor, tin protoporphyrin (SnPP), suppresses TPA-induced K562 cell differentiation to monocytes. It was also found that co-treatment of K562 cells with SnPP and TPA induces erythroid differentiation of K562 cells, though SnPP alone or TPA alone does not induce erythroid differentiation, suggesting a role of HO in the directional switch of differentiation.

Cell Differentiation↗
PubMed1999

[Molecular identification of cytokinin-specific binding protein].

Synthetic phenylurea derivatives such as N-phenyl-N'-(4-pyridyl)urea (4PU) and N-(2-chloro-4-pyridyl)-N'-phenylurea (4PU30) have strong cytokinin activities. Using tritiated 4PU30 as a probe, we found the presence of a cytokinin-specific binding protein (CSBP) with high affinity for 4PU30 (Ka for 4PU30 = 4 x 10(10) M-1) in the soluble fraction of etiolated mung bean seedlings. We purified CSBP by the use of 4PU-Sepharose 4B, an affinity gel ligated with 4PU. Analysis of its cDNA revealed that CSBP was a novel member of a major pollen allergen/pathogenesis-related protein family with a calculated molecular weight of 17 kDa. Recombinant CSBP was expressed in Escherichia coli was confirmed to bind specifically to cytokinins.

Amino Acid Sequence↗
PubMed1999

Diverse effects of AT1 receptor antagonists on normal blood pressure and regulatory system.

An AT1 receptor antagonist, losartan, has been reported to improve survival and quality of life in patients with congestive heart failure as angiotensin converting enzyme inhibitors do. Since many of the patients are normotensive, it may be a drawback if the compound decreases normal blood pressure. In this study, we investigated whether a novel AT1 receptor antagonist, TA-606, which is more potent than losartan, affects normal blood pressure and its regulatory system in comparison with losartan. TA-606 (30 and 100 mg/kg, p.o.) did not change normal blood pressure, whereas losartan (100 mg/kg, p.o.) tended to decrease it. Although EXP3174 (1 and 10 mg/kg, i.v.), an active metabolite of losartan, suppressed the baroreceptor-heart rate (HR) reflex, 606A (1 and 10 mg/kg, i.v.), an active metabolite of TA-606, did not affect it. Since losartan is known to affect the L-glutamate receptor which is part of the central blood pressure regulatory system, we also investigated whether 606A affects L-glutamate receptor binding. We found that 606A did not affect the binding of the L-glutamate receptor, but EXP3174 inhibited the binding with IC50 values of 13.3 microM. These findings suggest that, even having the same AT1 receptor antagonist properties as losartan and EXP3174, TA-606 and its active metabolite do not influence normal blood pressure or its regulatory system.

Angiotensin Receptor Antagonists↗