Search PubMed⌕ Search

Biomedical subjects

J Sakurai

Publications and source records attributed to J Sakurai.

At least 37 records · Page 2Linked to original sources

Strict dietary sodium reduction worsens insulin sensitivity by increasing sympathetic nervous activity in patients with primary hypertension.

To assess the effects of sodium reduction on insulin sensitivity in hypertension, we examined the change of insulin sensitivity after two degrees of dietary sodium restriction by the euglycemic hyperinsulinemic glucose clamp method in 12 subjects with primary hypertension. A controlled period of 1 week, when the subjects were taking a normal sodium diet, was followed by a randomized crossover study in which the subjects were placed on either moderate or strict reduced sodium diets for 1 week. The result of the 1-week moderate dietary sodium reduction from 200 to 100 mmol/day showed significant decreases in systolic and diastolic blood pressure by 6.5 and 5.0 mm Hg, respectively. Strict dietary sodium reduction to 30 mmol/day for 1 week resulted in no further decrease in blood pressure, but it increased plasma insulin by 40.6% without changing plasma glucose. There were no changes in glucose infusion rate (GIR) or insulin sensitivity index (ISI), which is a measure of GIR divided by plasma insulin, after moderate dietary sodium reduction. However, strict dietary sodium reduction induced decreases in GIR by 19.8% (from 1318+/-189 to 1057+/-173 micromol/m2/ min; P < .01), and ISI by 20.5% (from 16.6+/-2.1 to 13.2+/-1.9 micromol/m2/min/microU/mL; P < .01) with a paralleled increase of plasma norepinephrine by 90.0% (from 150.5+/-61.6 to 287.3+/-114.9 pg/mL; P < .01). These results indicate that dietary sodium restriction leads to a deterioration of insulin sensitivity when plasma norepinephrine levels increase, and suggest that moderate dietary sodium reduction may lower blood pressure without a distinct adverse effect on glucose metabolism in subjects with primary hypertension.

Adult↗

Mechanism of membrane damage by Clostridium perfringens alpha-toxin.

The effect of Clostridium perfringens alpha-toxin on liposomes prepared from phosphatidylcholine (PC) containing the fatty acyl residues of 18 carbon atoms was investigated. The toxin-induced carboxyfluorescein (CF) leakage and phosphorylcholine release from multilamellar liposomes increased as the phase transition temperature of the phosphatidylcholines containing unsaturated fatty acyl residues decreased. However, there was no difference between the sensitivity of the different phosphatidylcholines solubilized by deoxycholate to the phospholipase C (PLC) activity of the toxin. However, the toxin did not hydrolyze solubilized distearoyl-L-alpha-phosphatidylcholine (DSPC) or phosphatidylcholine containing saturated fatty acyl residue, and caused no effect on liposomes composed of DSPC. These results suggest that the activity of the toxin is closely related to the membrane fluidity and double bond in PC. The N-terminal domain of alpha-toxin (AT1-246) and variant H148G did not induce CF leakage from liposomes composed of dioleoyl-L-alpha-phosphatidylcholine (DOPC). H148G bound to the liposomes, but AT1-246 did not. However, the C-terminal domain (AT251-370) conferred binding to liposomes and the membrane-damaging activity on AT1-246. These observations suggest that the membrane-damaging action of alpha-toxin is due to the binding of the C-terminal domain of the toxin to the double bond in the PC in the bilayer and hydrolysis of the PC by the N-terminal domain.

Bacterial Toxins↗

[Relationship between aging and morphological bone changes of the condyle in temporomandibular disorders].

In order to clarify the clinical significance of morphological bone changes of the mandibular condyle in temporomandibular disorders, the relationship between radiographic bone changes and aging was clinically studied. The subjects of the study were 1,120 patients (812 females and 308 males). The average age was 33 years +/- 15 years (from 10 to 81 years of age). The 1,120 patients were divided into six age brackets (10 to 19 years, 20 to 29 years, 30 to 39 years, 40 to 49 years, 50 to 59 years, 60 to 81 years). According to radiograms taken by lateral oblique transcranial projection, orbitcondyle projection and orthopantomography, the morphological bone changes were classified into seven groups, erosion, osteophyte, eburnation, flattening, deformity, concavity and sclerosis. The results were as follows: 1. The incidence of the patients with radiographic bone changes of the mandibular condyle increased with aging and showed significantly different age distribution from that without bone changes (Mann-Whitney U test: p < 0.01). 2. The incidence of erosion did not increase with aging and showed significantly different age distribution from osteophyte or eburnation (Mann-Whitney U test: p < 0.01, 0.05). The incidence of flattening also did not increase with aging and showed significantly different age distribution from osteophyte, eburnation or deformity (Mann-Whitney U test: p < 0.01, 0.05, 0.05).

Adolescent↗

Assembly of Clostridium perfringens epsilon-toxin on MDCK cell membrane.

Clostridium perfringens epsilon-toxin bound to the Madin Darby canine kidney (MDCK) cells and aggregated. The complex of the toxin was formed in a dose- and a time-dependent manner. The formation of the complex increased with a decrease in viable counts of MDCK cells and with increasing K+ release from the cells. The inactivated toxin heated at 100 degrees C did not aggregate under the condition. In addition, the prototoxin dose-dependently bound to the cells, but did not form the complex. Incubation of the toxin with MDCK cell membranes also showed the formation of the complex, but that with membrane preparations prepared from Vero cells or sheep erythrocytes, which are insensitive for the toxin, showed no formation of the complex. Incubation of the toxin with mouse brain homogenates resulted in formation of the complex, but that with brain homogenates heated at 80 degrees C or mouse liver homogenates showed no formation of the complex. These observations show that the complex formation of epsilon-toxin is essential for toxicity of the toxin.

Animals↗

Evaluation of anti-hepatitis B virus (HBV) drugs using the HBV transgenic mouse: application of the semiquantitative polymerase chain reaction (PCR) for serum HBV DNA to monitor the drug efficacy.

For evaluation of anti-hepatitis B virus (HBV) drugs, we have employed the HBV transgenic mouse in which virion-like particles can be assayed in the serum. Bispivaloyloxymethyl-9-(2-phosphonylmethoxyethyl)-adenine [bis (POM) PMEA] 100 mg/kg/day, 2',3'-dideoxy-3'-thiacytidine [(+-)-BCH189] 200 mg/kg/day and a placebo were orally administered to mice twice a day for 14 days. Anti-viral effects were monitored by checking the levels of serum HBV DNA by the semiquantitative polymerase chain reaction, HBsAg and HBeAg by enzyme immunoassay, and replicative intermediates in the liver by Southern blotting. As expected, decrease from the 10(0.5) to 10(3) copies of HBV DNA per microl of sera detected before the treatment to the undetectable level was evident for all five animals treated with bis(POM) PMEA 100 mg/kg/day. However (+-)-BCH189 200 mg/kg/day, which is known to act as the inhibitor of reverse transcriptase for HBV or HIV in vivo and in vitro, did not suppress HBV DNA levels in the transgenic mouse. Thus, we were able to detect the effects of anti-HBV drugs semi-quantitatively, and confirm differences in drug efficacy.

Adenine↗

Site-specific mutagenesis of Clostridium perfringens alpha-toxin: replacement of Asp-56, Asp-130, or Glu-152 causes loss of enzymatic and hemolytic activities.

The current study has investigated the role of D-56, D-130, and E-152 in zinc ion binding properties, as well as the hemolytic, phospholipase C (PLC), and sphingomyelinase (SMase) activities of Clostridium perfringens alpha-toxin, based upon crystallography studies of the Bacillus cereus PLC, which had suggested these residues might be important for these functional activities. The replacement of D-56 in alpha-toxin resulted in complete loss of hemolytic, PLC, and SMase activities. The variant toxins at D-130 showed an approximately 100-fold reduction of biological activities compared to that of the wild-type toxin. The substitution of glutamine or glycine for E-152 caused complete loss of these activities, but substitution of aspartic acid for E-152 reduced but did not completely inhibit these activities. The variant toxins at D-56 and D-130, as well as the wild-type toxin, possessed approximately 2 mol of zinc atoms per mol of the protein, but E152G and E152Q contained approximately 1 mol of zinc metal per mol of the protein. On the other hand, the zinc content in E152D was calculated as about 1.4 mol in the toxin molecule. The replacement of D-56, D-130, or E-152 had no effect on binding to sheep erythrocytes and uptake of free zinc ion from the solution. The variant toxins at D-130 showed partial antigenic identity with the wild-type toxin on a double gel diffusion test. These observations suggest that D-56 in alpha-toxin is required for catalytic activity of alpha-toxin, D-130 is essential for maintenance of structure, and the carboxyl group of E-152 tightly ligands one zinc ion, which is essential for catalytic activity of the toxin.

Animals↗

Vacuolar protein sorting in fission yeast: cloning, biosynthesis, transport, and processing of carboxypeptidase Y from Schizosaccharomyces pombe.

PCR was used to isolate a carboxypeptidase Y (CPY) homolog gene from the fission yeast Schizosaccharomyces pombe. The cloned S. pombe cpy1+ gene has a single open reading frame, which encodes 950 amino acids with one potential N-glycosylation site. It appears to be synthesized as an inactive pre-pro protein that likely undergoes processing following translocation into appropriate intracellular organelles. The C-terminal mature region is highly conserved in other serine carboxypeptidases. In contrast, the N-terminal pro region containing the vacuolar sorting signal in CPY from Saccharomyces cerevisiae shows fewer identical residues. The pro region contains two unusual repeating sequences; repeating sequence I consists of seven contiguous repeating segments of 13 amino acids each, and repeating sequence II consists of seven contiguous repeating segments of 9 amino acids each. Pulse-chase radiolabeling analysis revealed that Cpy1p was initially synthesized in a 110-kDa pro-precursor form and via the 51-kDa single-polypeptide-chain intermediate form which has had its pro segment removed is finally converted to a heterodimer, the mature form, which is detected as a 32-kDa protein on sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions. Like S. cerevisiae CPY, S. pombe Cpy1p does not require the N-linked oligosaccharide moiety for vacuolar delivery. To investigate the vacuolar sorting signal of S. pombe Cpy1p, we have constructed cpy1+-SUC2 gene fusions that direct the synthesis of hybrid proteins consisting of N-terminal segments of various lengths of S. pombe Cpy1p fused to the secreted enzyme S. cerevisiae invertase. The N-terminal 478 amino acids of Cpy1 are sufficient to direct delivery of a Cpy1-Inv hybrid protein to the vacuole. These results showed that the pro peptide of Cpy1 contains the putative vacuolar sorting signal.

Base Sequence↗

Production and purification of Clostridium perfringens alpha-toxin using a protein-hyperproducing strain, Bacillus brevis 47.

Clostridium perfringens alpha-toxin was produced in a protein-hyperproducing strain, Bacillus brevis 47, by cloning the gene into the constructed expression-secretion vector which has the multiple promoters and the signal peptide coding region of an outer cell wall protein gene. The amount of alpha-toxin produced by the B. brevis 47 transformant carrying the gene was approximately 10 times greater than that produced by a B. subtilis transformant carrying the toxin gene. Biological activities and the N-terminal amino acid sequence of the toxin secreted by the B. brevis 47 transformant were identical to those of wild-type alpha-toxin.

Amino Acid Sequence↗

Maintenance and activation of Cyp2e-1 gene expression in mouse hepatocytes in primary culture.

The expression of Cyp2e-1 mRNA and protein was investigated in the C57BL/6NCrj mouse hepatocytes in primary culture, as well as liver and kidney. The mRNA and protein expression in the liver was in the same range in both sexes and was not affected by orchiectomy or ovariectomy. The mRNA expression was enhanced in the kidney of ovariectomized mice, in which the protein contents were not influenced. Orchiectomy decreased the expression of both mRNA and protein. When the hepatocytes were transferred to primary culture, the amounts of the mRNA were not changed within 24 h and about half remained by day 3. However, the expression was low thereafter. The expression of the protein gradually decreased after the start of culture. Dexamethasone showed a potential as an inducer at more than 10(-8) M. Sex hormones increased the expression of this P-450 species a little in culture, but growth hormone did not. These observations indicated that glucocorticoid hormone plays a role in modifying expression of Cyp2e-1 and that the mouse hepatocyte culture is useful for examining its regulation mechanism.

Animals↗

Membrane-damaging action of Clostridium perfringens alpha-toxin on phospholipid liposomes.

The effect of Clostridium perfringens alpha-toxin on multilamellar liposomes prepared from various phospholipids and cholesterol was investigated. The toxin induced carboxyfluorescein leakage from liposomes composed of the choline-containing phospholipids such as egg-yolk phosphatidylcholine and bovine brain sphingomyelin in dose-dependent manner, but did not induce leakage from those liposomes composed of bovine brain phosphatidylethanolamine, egg-yolk phosphatidylserine or phosphatidylglycerol. The toxin-induced carboxyfluorescein leakage from egg-yolk phosphatidylcholine liposomes was increased by addition of divalent cations. The toxin induced carboxyfluorescein release from liposomes composed of phosphatidylcholine containing unsaturated fatty acyl residues or shorter chain length saturated fatty acyl residues (12 or 14 carbon atoms), but did not induce such release from liposomes composed of phosphatidylcholine containing saturated fatty acyl residues of between 16 and 20 carbon atoms. Furthermore, the toxin-induced carboxyfluorescein release decreased with increasing chain length of acyl residues of phosphatidylcholine used. The toxin bound to liposomes composed of phospholipids which are hydrolyzed by the toxin, but did not bind to those composed of phospholipids which are not attacked by the toxin. The toxin-induced carboxyfluorescein release from liposomes composed of dipalmitoleoyl-L-alpha-phosphatidylcholine and cholesterol and the toxin binding to the liposomes decreased with decreasing cholesterol contents. These observations suggest that the specific binding site formed by the choline-containing phospholipids and cholesterol, and membrane fluidity in liposomes are essential for the membrane-damaging activity of alpha-toxin.

Bacterial Toxins↗

Threonine-74 is a key site for the activity of Clostridium perfringens alpha-toxin.

A mutant toxin (MT) that abolished almost 99% of the hemolytic activity of alpha-toxin was isolated by random polymerase chain reaction (PCR) mutagenesis of the gene for Clostridium perfringens alpha-toxin. In the mutant toxin, the amino acids at Tyr (Y)-62, Thr (T)-74 and Ile (I)-345 were substituted with His, Ile and Met, respectively. Replacement of T-74 with Ile by site-directed mutagenesis resulted in the loss of hemolytic, phospholipase C and sphingomyelinase activities by 1/250-fold of that of the wild-type. The replacement of Y-62 with Ile or I-345 with Met alone did not affect the activities of the toxin. T74I mutant bound to sheep erythrocyte membranes and specifically bound [65Zn]2+ in Tris-buffered saline, in the same manner as the wild-type, and contained 2 mol of zinc ions per mol of protein. These results suggest that the T-74 residue plays a key role in these biological activities of C. perfringens alpha-toxin.

Bacterial Toxins↗

Acute thrombosis in a contralateral kidney after radical nephrectomy: successful treatment by thrombolytic therapy using a tissue-type plasminogen activator.

A 67-year-old male became anuric immediately after a right radical nephrectomy for renal cell carcinoma. The patient was diagnosed with an acute arterial thrombosis of the remaining kidney within 4 hours after surgery by both CT scan and angiography. Thrombolytic therapy was started by a transcatheteral infusion of tissue-type plasminogen activator (TPA) resulting in a complete recanalization. Hydration and systemic administration of heparin followed, and renal function recovered within 3 weeks. This is the first report of acute thrombosis in a contralateral renal artery immediately after a radical nephrectomy which was successfully treated with TPA. It is probable that compression of the contralateral renal artery by the retractor for an extended period of time during surgery led to this unfavorable condition.

Aged↗

Phospholipid metabolism induced by Clostridium perfringens alpha-toxin elicits a hot-cold type of hemolysis in rabbit erythrocytes.

GTP and AIF4- significantly stimulated the late phosphatidic acid (PA) formation induced by Clostridium perfringens alpha-toxin in rabbit erythrocyte lysates. Pertussis toxin blocked the PA production. AIF4- markedly enhanced phosphatidylethanol production induced by alpha-toxin in the presence of ethanol. GTP[gamma S] stimulated the PA formation and hemolysis induced by alpha-toxin, and GDP[beta S] inhibited them. An H-to-G mutation at position 126 (H126G) induced the PA formation and hemolysis in a Co2+ concentration-dependent manner. H148G induced neither the PA formation nor hemolysis. These results suggest that the toxin-induced hemolysis is due to activation of phospholipid metabolism systems through GTP-binding protein.

Aluminum Compounds↗

Improvement of insulin sensitivity contributes to blood pressure reduction after weight loss in hypertensive subjects with obesity.

To access the role of insulin resistance in obesity hypertension, we examined the change of insulin sensitivity after weight loss in 24 obese hypertensive subjects by the euglycemic hyperinsulinemic glucose clamp method. The results of the 4-week calorie-restricted diet were a weight loss of 10.2% (from 74 +/- 12 to 67 +/- 11 kg, P < .01) and a decrease in mean blood pressure of 13.1% (from 124 +/- 7 to 107 +/- 9 mm Hg, P < .01). A decrease in plasma norepinephrine (from 208 +/- 74 to 142 +/- 52 pg/mL, P < .01) was associated with decreases in plasma renin activity (from 1.06 +/- 0.98 to 0.62 +/- 0.63 ng/mL per hour, P < .01) and serum aldosterone (from 70 +/- 28 to 57 +/- 24 pg/mL, P < .05). Glucose infusion rate increased significantly (42.9%), from 809 +/- to 1155 +/- 251 mumol/m2 per minute. The insulin sensitivity index, which is a measure of the glucose infusion rate divided by plasma insulin, increased significantly (42.6%), from 10.8 +/- 3.5 to 15.4 +/- 4.4 (mumol/m2 per minute)/(microU/mL). Stepwise multiple linear regression analysis showed that changes of plasma norepinephrine, insulin sensitivity index, plasma renin activity, and age were significant predictive factors for the change of mean blood pressure after weight loss. These results indicate a distinct relation between an improvement of insulin sensitivity and a decrease in blood pressure after weight loss in obese hypertensive subjects. The decrease in blood pressure after weight loss is probably related to the suppression of sympathetic nervous activity.

Anthropometry↗

Glucocorticoid and sex hormones as activating or modulating factors for expression of Cyp2b-9 and Cyp2b-10 in the mouse liver and hepatocytes.

The effects of male and female hormones as well as glucocorticoid upon the expression of Cyp2b-9 and Cyp2b-10 were investigated in C57BL/6 mouse liver and hepatocytes in primary culture. Their expression in the untreated female liver was higher than that in the male liver. More Cyp2b-9 mRNA than Cyp2b-10 was present in the female than in the male liver, whereas the levels of Cyp2b-10 were higher in the male. Phenobarbital increased Cyp2b-10 expression more than that of Cyp2b-9 in both sexes. Treatment with beta-estradiol also induced both in the male liver, with more Cyp2b-10 than Cyp2b-9 being found. In the female liver beta-estradiol and testosterone slightly increased the levels of Cyp2b-10, whereas the expression of Cyp2b-9 was reduced. Ovarectomy did not reduce the expression of the two mRNAs, but rather increased them. Using the spheroid culture system, in which mouse hepatocytes were cultured as multicellular aggregates and in which both mRNAs were expressed for a number of days, beta-estradiol as well as phenobarbital potently induced mRNA in hepatocytes from either sex and the inducibility at 10(-5) M corresponded to 10(-3) M phenobarbital. The expression level of Cyp2b-10 mRNA by phenobarbital or beta-estradiol was higher than that of Cyp2b-9, as observed in vivo, and > 10(-7) M beta-estradiol induced both in vitro. Dexamethasone induced the expression of more Cyp2b-10 mRNA than Cyp2b-9, and it was necessary for the expression by either phenobarbital or beta-estradiol. The expression was not enhanced in vitro by concomitant exposure of beta-estradiol and phenobarbital. Among the other female hormones tested, estrone induced Cyp2b-9 and Cyp2b-10 mRNAs at levels equivalent to those induced by beta-estradiol, and estriol and progesterone had a lower potential for the induction. Male hormones, such as androstenedione and testosterone, also had inducing potency in vitro. However, testosterone concentration dependently reduced the expression in cells cultured without dexamethasone. Tamoxifen or cryproterone, having antiestrogenic or antiandrogenic effects, respectively, also induced Cyp2b-9 and Cyp2b-10 mRNA. The suppressive effect of growth hormone on the expression of the mRNA was slight. The observation that endogenous hormones altered the expression of Cyp2b-9 and Cyp2b-10 in vivo and in vitro suggested that male and female hormones, as well as glucocorticoid, play activating or modulating roles in the constitutive expression of P450s.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

Maintenance of phenobarbital-inducible Cyp2b gene expression in C57BL/6 mouse hepatocytes in primary culture as spheroids.

Expression of the Cyp2b-9 and Cyp2b-10 genes was investigated in primary cultured adult C57BL/6NCrj mouse hepatocytes in monolayers or during the formation of spheroids (multicellular aggregates). Both the constitutive and phenobarbital-inducible expression of Cyp2b-9 and Cyp2b-10 mRNA decreased rapidly after transferring the hepatocytes to monolayer culture. The decrease was dependent on cell-density, and became more rapid in more densely seeded cells. However, in spheroid culture, the Cyp2b-9 and Cyp2b-10 mRNAs were induced by phenobarbital at high levels for at least 4 days either with continuous exposure from the start of cultivation or for 24 h before harvesting. The expression of both Cyp2b-9 and Cyp2b-10 species was confirmed by either reverse transcriptase-polymerase chain reaction or Western blotting. Although more Cyp2b-9 than Cyp2b-10 was expressed in the liver of control mice, the amounts of the latter became relatively overwhelming in untreated hepatocytes because of a faster decline of Cyp2b-9 species in culture. The level of mRNA induced by phenobarbital was concentration-dependent, the highest being at 2 or 4 mM, which was equivalent to in vivo treatment levels. There was more Cyp2b-10 species than those of Cyp2b-9 after exposure to phenobarbital both in vivo and in vitro. Phenobarbital also induced CYP1A2 mRNA, which was again peculiar to spheroid culture. Although the expression levels of both Cyp2b-9 and Cyp2b-10 species was very low in hepatocytes cultured without dexamethasone even in the presence of phenobarbital, the addition of 10(-7) or 10(-6) M dexamethasone caused an increase in the mRNA. When given concomitantly with phenobarbital, the expression was greatly enhanced. Nicotinamide or isonicotinamide similarly enhanced the expression of the two mRNA species, but the levels in monolayer cultures were still far lower than those in vivo. In contrast to the findings for mRNA expression, the protein levels in the presence of nicotinamide were similar to those in vivo under both monolayer and spheroid conditions. Since previous efforts to maintain expression of the CYP2B1 and CYP2B2 genes in primary cultured rat hepatocytes, orthologous to mouse Cyp2b-9 and Cyp2b-10 genes, required special cell attachment factors, our spheroid culture system, in which mouse hepatocytes are simply seeded onto noncoated dishes, has advantages for mechanistic studies.

Animals↗