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R Sato

Publications and source records attributed to R Sato.

At least 163 records · Page 9Linked to original sources
PubMed1997

A novel metalloproteinase, almelysin, from a marine bacterium, Alteromonas sp. No. 3696: purification and characterization.

We have discovered a novel metalloproteinase, which has high activity at low temperatures, from the culture supernatant of a marine bacterium. The strain was identified as Alteromonas sp. No. 3696. The metalloproteinase, named almelysin, was purified to homogeneity from the cultured supernatant at 10 degrees C by two column chromatographies. About 20 mg of purified almelysin was obtained from 18.4 liters of the culture supernatant. The molecular mass of almelysin was estimated to be 28 kDa by SDS-PAGE and the isoelectric point was 4.3. The optimum pH for activity of almelysin was pH 8.5-9.0 and 6.5 using casein and (7-methoxycoumarin-4-yl)acetyl(MOCAc)-Pro-Leu-Gly-Leu-(N3- [2,4-dinitrophenyl]-L-2,3-diaminopropionyl) [A2pr(Dnp)]-Ala-Arg-NH2 as substrates, respectively. Almelysin was stable between pH 7.5-8.0 and below 40 degrees C. The optimum temperature for the activity was observed to be 40 degrees C using both casein and MOCAc-Pro-Leu-Gly-Leu-A2pr(Dnp)-Ala-Arg-NH2 as substrates. The activity of almelysin was inhibited by such metallo chelators as EDTA and o-phenanthroline, while talopeptin, phosphoramidon, and SMPI, typical metalloproteinase inhibitors, had no effect. Almelysin primarily cleaved the Ala14-Leu15 bond and Phe24-Phe25 bond, and secondarily the Tyr16-Leu17 bond in oxidized insulin B-chain. However, almelysin could not cleave the His5-Leu6, His10-Leu11, and Gly23-Phe24 bonds, which were cleaved by other metalloproteinases. These results indicate that the substrate specificity of almelysin is different from other metalloproteinases. Interestingly, Alteromonas sp. No. 3696 strain produced another proteinase as well as almelysin at 25 degrees C.

Amino Acid Sequence↗
PubMed1997

[Effects of intermittent wakening on sleep pattern of nighttime caregiver: an EEG study with a single subject].

In order to investigate the effects of night wakening on sleep pattern of caregiver, a woman aged 45 years was studied using electroencephalogram (EEG). After two adaptation nights, her all-night EEG readings were recorded for six nights. During the first three consecutive nights, the subject slept by the side of a patient at a private ward of hospital and wakened by herself several times a night to provide care for the patient (care nights). Of the later three nights, her sleep EEGs were recorded at her home (free nights). The first free night was following three care nights and the other two free nights were after 2 weeks. The results were summarized as follows; 1) Sleep period time (SPT) of care nights differed in different nights. In contrast to the SPT range of 356-367 min during free nights, it was 271-391 min during the care nights. 2) The rhythm of sleep cycles of care nights was not stable, though the subject wakened by herself, not using an alarm clock. 3) Although there were few changes occurring in sleep efficiency (SE) and percent each stage for SPT (%SPT) between the first and second care night, SE of the third care night was more similar to SE of free nights than SE of the first two care nights, and so was %SPT of the third care night. 4) Sleep latency (SL) decreased during the four consecutive nights, i.e. the three care nights and the first free night, and SL of the third care night and the first free night were shorter than SL of the last two free nights. The accumulation of fatigue and stress of nighttime care was suggested.

Caregivers↗
PubMed1997

Urine N-acetyl-beta-D-glucosaminidase activity in healthy cattle.

OBJECTIVE: To measure urine N-acetyl-beta-D-glucosaminidase (NAG) activity of healthy cattle, using 3 substrates (4-methylumbelliferyl-N-acetyl-beta-D-glucosaminide, sodio-m-cresolsulfonphthaleinyl-N-acetyl-beta-D-glucosaminid e, and p-nitrophenyl-N-acetyl-beta-D-glucosaminide), and to determine the relations between the obtained values and age and sex of cattle. ANIMALS: 50 healthy lactating Holstein-Friesian cows and 10 healthy Holstein-Friesian steers. PROCEDURE: Untimed urine samples were collected, and urine NAG activity was measured, using the 3 aforementioned methods. Urine creatinine concentration also was measured, and NAG activity was expressed as units per gram of creatinine (NAG index). Correlations between urine NAG activity and age and sex of cattle were investigated. Furthermore, correlations among data obtained by each of the 3 methods were determined. RESULTS: Urine NAG activity in cows measured by each of the 3 methods was < 3.0 U/L. Urine NAG activity in steers was significantly higher than that in cows. However, there was no significant difference between the sexes in NAG index. There were no significant differences in mean values of NAG activity and index among cows of various age groups. Individual values of urine NAG activity determined by each method correlated significantly with each other. CONCLUSIONS AND CLINICAL RELEVANCE: Urine NAG activity and NAG index of healthy cattle will be useful for determining diagnostic criteria of renal disease in cattle.

Acetylglucosaminidase↗
PubMed1997

Substances reactive with mannose-binding protein (MBP) in sera of patients with rheumatoid arthritis.

OBJECTIVES: In order to evaluate the role of mannose-binding protein (MBP) in rheumatoid arthritis, we characterized MBP-binding substances in sera of patients with this disease. METHODS: An enzyme linked immunosorbent assay (ELISA) was used to detect MBP-binding substances in sera of patients with RA. We applied the sera of two RA patients to an immobilized MBP column, and by means of both ELISA and molecular sieve chromatography, examined the substances that bound to MBP. MBP-MASP complexes were added to the fractions containing the binding substances, and C4-consuming activity was examined. RESULTS: Sera of patients with RA showed stronger MBP binding on ELISA than did those of normal controls. In the case of RA sera, both IgG was IgM-RF eluted from the MBP column, whereas with normal controls, only IgG was obtained. The results of molecular sieve chromatography showed that the binding substances of RA patients consisted of immune complexes containing IgG and IgM-RF. These substances were specifically bound to MBP, and once bound, the MBP-MASP complexes were then able to consume C4. CONCLUSION: MBP binds to immune complexes consisting of IgG and IgM-RF, and probably recognizes either the mannose moiety of IgM-RF or the N-acetyl-glucosamine of agalactosyl IgG. When this occurs in RA patients, the lectin pathway would then be activated.

Antigen-Antibody Complex↗
PubMed1996

Sterol-dependent transcriptional regulation of sterol regulatory element-binding protein-2.

We show in this manuscript that expression of the mRNA for sterol regulatory element-binding protein-2 (SREBP-2) is regulated by the cellular sterol level in cultured HeLa cells. We have cloned the 5'-flanking region of the gene encoding human SREBP-2. Characterization of this region shows the minimum 50-base pair segment, which contains a 10-base pair sterol regulatory element 1 (SRE-1) identical to the one in the human LDL receptor promoter, confers sterol responsiveness when fused to the luciferase reporter gene. Enforced expression of the truncated SREBP-2 protein (amino acid residues 1-481) also shows that this upstream segment contains the information required for transcriptional activation. The luciferase assays using mutant versions of the reporter genes reveal that the sterol-dependent transcriptional regulation is mediated by two nearby motifs, the SRE-1 and the NF-Y binding site (the inverted CCAAT box, ATTGGC); the latter is reported to play a critical role in sterol-dependent regulation of 3-hydroxy-3-methylglutaryl-coenzyme A synthase and farnesyl diphosphate synthase genes (Jackson, S. M., Ericsson, J., Osborne, T. F., and Edwards, P. A. (1995) J. Biol. Chem. 270, 21445-21448). Gel mobility shift assays demonstrate that the transcription factor NF-Y truly binds to the ATTGGC sequence. These findings suggest that the activity of SREBP-2 is controlled not only post-translationally by proteolytic activation of the precursor protein but also transcriptionally by itself together with NF-Y.

Binding Sites↗
PubMed1996

Amitriptyline inhibits the G protein and K+ channel in the cloned thyroid cell line.

We have reported that thyroid K+ channel is activated by extracellular application of the thyroid-stimulating hormone (TSH) using single channel recording method performed on cloned normal rat thyroid cell (FRTL-5) membrane. Treatment of dibutyryladenosine cyclic monophosphate (Bt2 cAMP) also activated the TSH-dependent K+ channel. These findings indicate that the thyroid K+ channel is activated through the TSH-adenosine cyclic monophosphate (cAMP)-protein kinase A system. We examined the effects of amitriptyline on TSH-guanosine triphosphate binding protein (G protein)-adenylate cyclase-cAMP-K+ channel system in the cloned normal rat thyroid cell line FRTL-5. Amitriptyline inhibited the cAMP production induced by TSH. Amitriptyline also inhibited the cAMP production induced by cholera toxin, indicating that amitriptyline inhibited the thyroid G protein. Amitriptyline had no effect on TSH-receptor binding and cAMP production by forskolin (adenylate cyclase stimulator). Amitriptyline inhibited the K+ channel activation by cAMP, indicating that the suppressing mechanism is not the inhibition of TSH receptor or G protein but the direct suppression of K+ channel. It was concluded that amitriptyline inhibited the thyroid G protein and K+ channel.

Amitriptyline↗
PubMed1996

Regulation of fatty acid synthase expression by cholesterol in human cultured cells.

The regulation of fatty acid synthase (FAS) expression by sterols in a cultured human hepatoblastoma cell line, Hep G2, was studied. When cells were treated with compactin in a medium containing lipoprotein deficient serum, FAS mRNA level increased 1.6-fold. A squalene synthase inhibitor, TAN1607A, decreased both free and esterified cholesterol contents in Hep G2 cells and increased mRNA levels for FAS, HMG-CoA reductase, squalene synthase and LDL receptor. However, for the increment of FAS mRNA, a 10-fold higher concentration of this inhibitor was needed. These results demonstrate that the concentration of cellular cholesterol which regulates FAS expression is necessarily lower than the levels which regulate other sterol sensitive genes. FAS mRNA was also increased by an inhibitor of SREBP degradation as well as chenodeoxycholic acid. These results indicate that FAS mRNA expression is regulated by cholesterol and is mediated through SREBPs. The implications of the different modes of sterol regulation of FAS and LDL receptor expression are discussed.

Bridged Bicyclo Compounds, Heterocyclic↗
PubMed1996

Inhibition of leukotriene synthesis by azelastine.

BACKGROUND: Azelastine, oxatomide, and ketotifen are used for patients with allergic diseases. These drugs inhibit the release of chemical mediators including the leukotrienes; however, the mechanism involved is unclear. OBJECTIVE: To clarify the mechanism of inhibition, we investigated the effects of three drugs on the function of phospholipase A2, 5-lipoxygenase, leukotriene C4 synthase, and leukotriene A4 hydrolase, which are all catabolic enzymes involved in synthesizing leukotriene C4 and leukotriene B4 in rat basophilic leukemia (RBL)-1 cells. METHODS AND RESULTS: The production of leukotriene C4 and leukotriene B4 was measured by high performance liquid chromatography (HPLC). All three drugs inhibited the production of leukotriene C4 and leukotriene B4 when cells were stimulated with A23187. All three drugs also inhibited the A23187-stimulated release of 3H-arachidonic acid from membrane phospholipids. Azelastine inhibited the production of leukotriene C4, but not leukotriene B4, when either arachidonic acid or leukotriene A4 free acid was used as the substrate in our cell free system. Oxatomide and ketotifen did not inhibit the synthesis of either leukotriene C4 or leukotriene B4 in the same cell free study. CONCLUSION: Results indicated that oxatomide and ketotifen inhibit the production of leukotriene C4 and leukotriene B4 by inhibiting phospholipase A2 activity, whereas, azelastine inhibits the leukotriene C4 production by inhibiting phospholipase A2 and leukotriene C4 synthase.

Animals↗
PubMed1996

RANTES and platelet-activating factor open Ca2+-activated K+ channels in eosinophils.

RANTES is a member of the low molecular cytokines and appears to be strictly a chemotactic and activating factor for eosinophils. We studied the effect of RANTES on the channel activity of the eosinophilic cell line (EoL-1 cells) using patch clamp techniques. Under cell-attached patch conditions, RANTES (20 ng/ml) activated channels in EoL-1 cells when applied to the recording pipette or to the bath. This channel was permeable to K+ with a unit conductance of 14 pS, and the reversal potential was close to the equilibrium potential for K+. The current-voltage relationship for this K+ channel was almost linear, showing little or no rectification. The open time and closed time histograms could he fitted to a single exponential function with time constants of 6.4 and 2.7 ms, respectively. Extracellular application of the calcium ionophore A23187 (1 microM) under cell-attached patch conditions or an increase in intracellular Ca2+ concentration under inside-out patch conditions increased K+ channel activity in a manner similar to that caused by RANTES, indicating that the RANTES-activated channel was the Ca2+ -activated K+ channel. Intracellular application of GTP gamma S (10 microM) opened the Ca2+V-activated K+ channels under inside-out patch conditions. Furthermore, RANTES failed to activate the K+ channels after the cells had been treated with pertussis toxin (100 ng/ml) for 2 h at 37 degrees C. These results suggest that RANTES opens the Ca2+ -activated K+ channels of EoL-1 cells through activation of G-proteins.

Calcium↗
PubMed1996

Interleukin-1 beta decreases sensitivity of guinea-pig airway to potassium chloride and isoproterenol by an epithelium-dependent mechanism.

Airway inflammation may cause alteration of airway responses in chronic airway diseases, such as bronchial asthma. The objective of this study was to examine whether interleukin-1beta (IL-1beta) [corrected], one of the pro-inflammatory cytokines, has a direct effect on airway functions. The effects of IL-1beta on carbachol, KCl and isoproterenol (ISO) responses of isolated guinea-pig tracheal strips were examined by measuring isometric tension in tissue bath. Responses of tracheal strips with or without epithelium to each agonist were compared before and after incubation with IL-1beta (25 ng or 250 ng/mL). Both 1 h and 5 h incubation of the strips with 250 ng/mL IL-1beta significantly decreased the sensitivity not only to KCl (P < 0.05; P < 0.01, respectively), but also to ISO (both P < 0.05) without affecting maximum contraction or relaxation. Response to carbachol was not affected by IL-1beta. Epithelial denudation abolished the effects of IL-1beta on KCl and ISO responses. Indomethacin (2 micromol/L) [corrected] reversed the effects of IL-1beta both on KCl and on ISO. These results suggest that IL-1 beta decreases the sensitivity of airway strips to KCl and ISO, possibly by stimulating prostaglandin production from the airway epithelium [corrected].

Animals↗
PubMed1996

Inhibition of leukotriene synthesis by honokiol in rat basophilic leukemia cells.

The effects of honokiol, a diphenyl compound extracted from a Chinese herbal medicine, on leukotriene (LT) synthesis were evaluated in rat basophilic leukemia (RBL) cells. The production of LTC4 and LTB4 stimulated by the Ca2+ ionophore A23187 was measured in RBL-1 cells by high-performance liquid chromatography. Honokiol inhibited the production of LTC4 and LTB4 stimulated by A23187 in RBL-1 cells. Honokiol did not inhibit either phospholipase A2 activity, measured by the release of 3H-arachidonic acid (AA), or LTC4 synthase and LTA4 hydrolase activities, measured with LTA4-free acid as substrate. The synthesis of LTC4 and LTB4 from AA in RBL-1 cell lysates in the presence of Ca2+ was inhibited by honokiol. These results indicate that honokiol blocks LT synthesis by inhibiting 5-lipoxygenase activity. Honokiol also inhibited immunoglobulin E-mediated production of these LTs in RBL-2H3 cells, which was measured by a specific radioimmunoassay (RIA). These results suggest that honokiol may exhibit antiallergic actions by inhibiting LT synthesis in immediate-type hyperreactivity.

Animals↗
PubMed1996

A new type of familial central diabetes insipidus caused by a single base substitution in the neurophysin II coding region of the vasopressin gene.

We studied the genetic basis of familial neurohypophyseal diabetes insipidus in a Japanese family. The members had polyuria and a deficiency of plasma vasopressin (AVP). Polymerase chain reaction (PCR) amplified exons of the AVP-neurophysin-II gene were subcloned and sequenced. Exons 1 and 3 were normal, but nucleotide 1884 Guanine (G) in exon 2 was substituted with Thymine (T), which induced a substitution of glycine (Gly) for valine (Val). To examine the presence of this mutation in the affected subjects, we designed two mutated primers. One of them induced a new endonuclease restriction site in the PCR fragments from normal, and the other induced a new endonuclease restriction site from patients with the mutation. DNA fragments from two affected members of this family were amplified with this primer, and the PCR products were digested by endonuclease and resolved by electrophoresis. The results indicated that these subjects had both normal and mutant alleles, indicating that the mutation was heterozygous. We concluded that this mutation caused neurohypophyseal diabetes insipidus in this family.

Arginine Vasopressin↗
PubMed1996

A novel alcohol resistant metalloproteinase, vimelysin, from vibrio sp. T1800: purification and characterization.

We found a novel metalloproteinase, which has high activity at low temperatures and in the presence of organic solvents, in the culture supernatant of a marine bacterium, Vibrio sp. T1800. The metalloproteinase, named vimelysin, was purified from the culture supernatant by three column chromatographies. About 150 mg of purified vimelysin was obtained from 3.3 liters of the culture supernatant with a high yield of 57%. The purified vimelysin showed a single protein band on SDS-PAGE with molecular weight of 38,000. The isoelectric point of vimelysin was 4.3 by isoelectric focusing. The optimum pH of vimelysin was pH 8.0 or pH 6.5 using casein or furylacryloyl-glycyl-leucine amide (FAGLA) as substrates, respectively. The optimum temperature of vimelysin was 50 degrees C when casein was used as a substrate, but it was 15 degrees C when FAGLA was used as a substrate. Interestingly, vimelysin activity was completely retained after 48 h of incubation at 25 degrees C in the presence of 50% ethanol. Moreover, vimelysin showed 40% activity of the control even in the presence of 10% ethanol, while thermolysin showed only 5% activity under the same conditions.

Amino Acid Sequence↗