Biomedical subjects
Publications and source records attributed to H Nishimura.
Separation of the immunosuppressive principles from Rehmanniae radix was done by monitoring hemolytic plaque-forming cells (HPFC) inhibitory activity to give two new phenethyl alcohol glycosides: jionosides A1 (4) and B1 (5), along with six known compounds: acetoside, isoacteoside, purpureaside C, echinacoside, and cistanosides A and F.
The accumulation of non-metabolized choline in isolated rat hepatocytes is concentrative in Na+ medium, whereas the accumulation does not exceed unity in a Li+ medium. Ouabain and 2,4-dinitrophenol inhibited the choline uptake. These results indicate that choline is taken up by rat hepatocytes via a Na+- and energy-dependent process, and choline oxidase is not directly connected with the choline transport system.
Two secretory glycoproteins of Saccharomyces cerevisiae, a soluble thiamin-binding protein and a thiamin-repressible acid phosphatase, were shown to be repressed to a similar extent by excess thiamin in the growth medium. Thiamin-repressible acid phosphatase was co-purified throughout the purification of the soluble thiamin-binding protein. Purified and deglycosylated soluble thiamin-binding proteins exhibited both thiamin-binding and acid phosphatase activity on non-denaturing polyacrylamide gel electrophoresis. Heat treatment of the purified soluble thiamin-binding protein caused a decrease in both activities with a similar inactivation profile. Furthermore, two thiamin-repressible acid phosphatase-defective mutants isolated had no and decreased soluble thiamin-binding activity, respectively. From the results, it was concluded that the soluble thiamin-binding protein is identical to the thiamin-repressible acid phosphatase in S. cerevisiae.
We have synthesized and characterized O-bromoacetylthiamine (BrAcThiamine), a new reagent for inactivating the thiamine transport system in Saccharomyces cerevisiae. A Lineweaver-Burk plot of data from the transport kinetic measurements showed that BrAcThiamine was a competitive inhibitor of thiamine transport in S. cerevisiae with a Ki value of 0.60 microM. Incubating BrAcThiamine with yeast cells at 40 degrees C in 0.05 M potassium phosphate buffer, pH 5.0, caused concentration- and time-dependently a remarkable loss of thiamine transport activity. The inactivating reaction of yeast thiamine transport by BrAcThiamine proceeded most effectively at pH 5.0, coinciding with the optimal pH of the transport activity. Thiamine and thiamine analogs (pyrithiamine and O-acetylthiamine) protected yeast thiamine transport activity against the inactivation by BrAcThiamine. In addition, it was found that a membrane fraction prepared from yeast cells treated with BrAcThiamine had a thiamine-binding activity only 20% of that from control cells without inactivating the binding activity of the soluble fraction. These results suggest that BrAcThiamine inactivates the uptake activity by irreversible binding to the binding site of carrier protein(s) in the thiamine transport system.
In the domestic fowl, angiotensin II (ANG II) causes an in vivo depressor response and in vitro relaxation of aortic rings which appear to be a direct action of ANG II on the blood vessels. Thus, we determined whether binding sites specific to ANG II exist in the membrane fraction of the fowl aorta. The particulate fraction of aortas from adult female fowl, Gallus gallus, exhibits high specific binding to ANG II ligand. 125I-[Ile5]ANG II (0.5 nM) binding to the above fraction (30 micrograms protein) in 50 mM Tris (pH 7.2), 10 mM MgCl2, and 0.2% bovine serum albumin at 12 degrees (1) is rapid, saturable, and reversible; (2) increases as a function of ligand or membrane concentration, time, and temperature; and (3) optimally fits to a two-site (high and low affinity) model. The equilibrium dissociation constant (0.15 +/- 0.03 nM) and binding site concentration (28.7 +/- 8.1 fmol/mg protein) of the high affinity site as well as association (0.055 nM-1.min-1) and dissociation (0.0122 min-1) rate constants are similar to those of mammalian vascular ANG II receptors. Both 125I-[Ile5]ANG II and 125I-[Val5]ANG II are competitively displaced by unlabeled ANG II. These results suggest that specific ANG II receptors exist in the fowl aorta.
We modified Porsolt's forced-swim test by suspending ropes or straws above the water in order to investigate a possible relationship between immobility and perceived escape responses from water. In this modified test, it was demonstrated clearly that rats reduced their duration of immobility and attempted to climb up the suspended ropes or straws. Most rats which had remained immobile during a 5-min test period in the forced-swim test, exhibited such climbing responses within 5-10 min of rope-suspension. Despite the suspension of ropes, however, some rats showed immobile postures and did not respond to the rope. On the other hand, straws were used in order to produce sliding and prevent climbing when the animals attempted to climb. There were no differences in immobility during either rope- or straw-suspension. It seems that the climbing behavior displayed by forced-swimming rats is due to a "pseudo-escape" effect produced by the suspension of an object above the water. The present findings were interpreted as further evidence for the notion that immobility in forced-swimming rats does not necessarily imply "behavioral despair," but rather an emotional reaction to an inescapable stressor.
Rats were forced to swim in a cistern until sinking in order to examine the possible relationship between sinking and immobility which has been reported to reflect "behavioral despair" in the forced swim test. Rats were classified into sinking and non-sinking groups, according to the appearance of sinking behavior over a 2 hr test. The sinking rats showed significantly shorter immobility times during the first 15 min as compared to the non-sinking rats. Therefore, sinking behavior seems to be a sign of emotional behavior such as fear and/or anxiety accompanied by defecation. Discriminant analysis showed that the immobility time during the first 15 min was a prediction of sinking. These findings suggest that the rapidly induced immobility in this forced swim test reflects the possibility of floating behavior in connection with the emotional reaction.
1. Ampicillin concentrations in human serum and periodontal membrane after a single oral administration of talampicillin (500 mg) were assayed by the agar diffusion (paper disc) method. 2. The peak times of serum and periodontal membrane were identical, being 150 min after administration. 3. The peak concentrations of serum and periodontal membrane were 7.81 micrograms/ml and 4.11 micrograms/g, respectively. 4. The mean ratio of periodontal membrane to serum concentration at the peak time was 0.53.
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To assess the abnormalities of insulin receptor in patients with Werner's syndrome, we established Epstein-Barr virus transformed lymphocytes and studied the binding as well as kinase activities of insulin receptor. The insulin binding to both intact cells and WGA-purified insulin receptor preparations was within normal range. Autophosphorylation of the beta-subunit was not altered in patients with Werner's syndrome, and the receptors of these patients phosphorylated an exogenous substrate to a degree comparable to that of the normal participants. Taken together, these findings indicate that insulin resistance in Werner's syndrome likely is caused by a defect that cannot be detected by means used in the present study.
A new trisaccharide sugar chain was identified in bovine blood coagulation factors VII and IX. A pentapeptide isolated from factor VII contained Ser-52, which could not be identified with a gas-phase sequencer, suggesting an unknown substituent on the serine residue (Takeya, H. et al. (1988) J. Biol. Chem., in press). The same results were obtained for a pentapeptide containing Ser-53 of factor IX. Component sugar analysis revealed that the peptide contained 1 mol of glucose and 2 mol of xylose. This sugar component was also confirmed by high-resolution fast atom bombardment mass spectrometric analysis of the pentapeptide. The trisaccharide was released from the peptides by means of beta-elimination reaction and its reducing end was coupled with 2-aminopyridine. The fluorescent pyridylamino (PA-) derivative of the trisaccharide was purified by gel-filtration and reversed-phase HPLC. The sugar composition of the PA-trisaccharide was found to be 2 mol of xylose and 1 mol of PA-glucose. These results indicate the existence of a (Xyl2)Glc-Ser structure in factors VII and IX.
Binding experiments with radioactively labelled influenza C virions were carried out to investigate the interaction of the virus with human erythrocytes. The erythrocytes from any of 35 different individuals were found to contain influenza C virus-binding sites though their number was variable among the individuals and was much less than that on mouse, rat and chicken erythrocytes. Attachment of influenza C virus to human erythrocytes was inhibited completely by prior treatment of the virus with anti-HE monoclonal antibody having a strong haemagglutination inhibition activity. Pretreatment of erythrocytes with neuraminidase or the neuraminate-O-acetylesterase of influenza C virus resulted in a marked reduction in the level of virus binding. Thus it appears that human erythrocytes have a low level of O-acetylated sialic acid-containing glycoconjugates that can interact specifically with the HE glycoprotein of influenza C virus. Proteolytic digestion of erythrocytes with ficin, bromelain or V-8 protease inhibited virus binding almost completely, suggesting that the erythrocyte receptor for influenza C virus is a glycoprotein. In contrast to these enzymes, trypsin treatment of erythrocytes reduced virus binding by only about 50%, and alpha-chymotrypsin treatment did not inhibit at all. It was also found that treatment of erythrocytes with monoclonal antibody to the M or N blood group antigen greatly inhibited virus binding to the cells. These results, taken together, suggest that most influenza C virus receptors on human erythrocytes, if not all, reside on glycophorin A which is known to possess the M or N blood group activity.
In our previous study, seven monoclonal antibodies specific for influenza C virus glycoprotein (gp88) were prepared and tentatively classified into two groups: group A (J14, J9, Q5, K16) has neutralization activity whereas group B (S16, J6, J15) does not. These antibodies were used to analyse the antigenic structure of gp88 and to examine the effect of glycosylation on the antigenicity of the glycoprotein. Operational analysis with a panel of antigenic variants selected with each of the group A antibodies identified two non-overlapping antigenic sites on the gp88 molecules, site A-1 recognized by J14, J9 and Q5 and site A-2 by K16. Sites A-1 and A-2 were shown, however, to be topographically overlapping by competitive binding assays. Competitive binding analysis with group B antibodies identified two additional non-over-lapping antigenic sites, site B-1 recognized by S16 and site B-2 by J6 and J15. It was found in radioimmunoprecipitation experiments that antibodies to sites B-1 and B-2 were reactive not only with gp88 but with its non-glycosylated form (T76) synthesized in the presence of tunicamycin. Antibodies to sites A-1 and A-2, in contrast, immunoprecipitated the T76 polypeptide in only trace amounts or not at all. Additionally, Western blot analysis showed that denatured gp88 blotted on nitrocellulose was reactive with antibodies to sites B-1 and B-2 but not with those to sites A-1 and A-2. These observations suggest that glycosylation of gp88 selectively influences the integrity of antigenic sites A-1 and A-2 which are composed of conformation-dependent epitopes.
Protein phosphatase C was purified 140-fold from bovine brain with 8% yield using histone H1 phosphorylated by the catalytic subunit of cyclic AMP-dependent protein kinase (cyclic AMP-kinase). Brain protein phosphatase C was considered to consist of 10 and 90%, respectively, of the catalytic subunits of protein phosphatases 1 and 2A on the basis of the effects of ATP and inhibitor-2. Protein phosphatase C dephosphorylated microtubule-associated protein 2 (MAP2), tau factor, and tubulin phosphorylated by a multifunctional Ca2+/calmodulin-dependent protein kinase (calmodulin-kinase) and the catalytic subunit of cyclic AMP-kinase. The properties of dephosphorylation of MAP2, tau factor, and tubulin were compared. The Km values were in the ranges of 1.6-2.7 microM for MAP2 and tau factor. The Km value for tubulin decreased from 25 to 10-12.5 microM in the presence of 1.0 mM Mn2+. No difference in kinetic properties of dephosphorylation was observed between the substrates phosphorylated by the two kinases. Protein phosphatase C did not dephosphorylate the native tubulin, but universally dephosphorylated tubulin phosphorylated by the two kinases. The holoenzyme of protein phosphatase 2A from porcine brain could also dephosphorylate MAP2, tau factor, and tubulin phosphorylated by the two kinases. The phosphorylation of MAP2 and tau factor by calmodulin-kinase separately induced the inhibition of microtubule assembly, and the dephosphorylation by protein phosphatase C removed its inhibitory effect. These data suggest that brain protein phosphatases 1 and 2A are involved in the switch-off mechanism of both Ca2+/calmodulin-dependent and cyclic AMP-dependent regulation of microtubule formation.
Ampicillin concentrations in human serum and dental granulomas of 31 patients were determined after a single oral dose of talampicillin (equivalent to 500 mg of ampicillin) was administered to each. The specimens were taken at 1.5, 2.0, 2.5, 3.0, and 3.5 h after the administration of talampicillin. The mean peak ampicillin concentrations in serum and dental granulomas occurred at identical times, 2.5 h, and were 8.29 micrograms/ml (range, 1.81 to 13.20 micrograms/ml) and 2.94 micrograms/g (range, 1.14 to 7.16 micrograms/g), respectively. The mean dental granuloma/serum ampicillin concentration ratio at the peak time (2.5 h) was 0.42 (range, 0.29 to 0.56). Ampicillin concentrations in dental granulomas exceeded most of the MICs for the bacteria commonly isolated from odontogenic infection.
To clarify the mechanism responsible for age-related changes in insulin action, the euglycemic clamp technique was performed with graded doses of insulin in conscious rats aged 2, 4, 10, and 20 mo. Insulin binding (IB) to muscle membranes was also studied. Maximal response of insulin-induced glucose disappearance rate (Rd) was decreased significantly between 2 and 4 mo of age. Dose-response curves shifted to the right progressively up to 20 mo of age. However, IB to the muscle membrane diminished between 1 and 4 mo of age without a decrease thereafter. When Rd was plotted against insulin bound to the membranes, the resulting curves shifted to the right with aging, suggesting a coupling defect between the binding and effector unit. In conclusion, insulin action alters in rats between 2 and 20 mo of age. The most pronounced impairment in IB and maximal response of insulin-induced Rd occurs during early life stage (through maturation) and then a coupling defect seems to be superimposed with further aging. However, we cannot exclude the possibility that these changes may be secondary to obesity or reduced physical activity, rather than aging per se.
Angiotensin II (ANG II) decreases blood pressure of fowl. To characterize the vasodilating action of ANG II and its underlying mechanisms, we examined the effect of [Asp1, Val5]ANG II (fowl ANG II) on isometric tension of fowl aortic rings. [Val5]ANG II (10(-8) to 10(-5) M) produced rapid, reversible, dose-dependent relaxation of aortas precontracted with phenylephrine. [Sar1,Ile8]ANG II blocked ANG II-induced relaxation; propranolol, atropine, methysergid, pyrilamine, and cimetidine did not. Endothelium removal abolished relaxation responses to ANG II and acetylcholine but not to isoproterenol or sodium nitroprusside. Inhibitors of phospholipase or arachidonic acid metabolism (quinacrine, indomethacin, 5,8,11,14-eicosatetraenoic acid, hydroquinone, metyrapone, SKF 525A) and a calcium channel blocker (verapamil) did not inhibit ANG II-induced relaxation, whereas indomethacin nearly completely blocked arachidonic acid-induced dilation of aortas with or without endothelia. Guanosine 3',5'-cyclic monophosphate (cGMP) levels in the aorta increased 15 s after ANG II application. Aortic relaxation was caused by 8-bromo-cGMP with or without intact endothelium. These results suggest that ANG II-induced relaxation of fowl aortas involves 1) an endothelium-dependent mechanism and 2) cGMP but not arachidonic acid metabolites.
A Philippine population was investigated for genetic polymorphism of orosomucoid 1 (ORM1), orosomucoid 2 (ORM2), and alpha-2-HS-glycoprotein (A2HS) by isoelectric focusing. The allele frequencies were: ORM1*1 = 0.7904, ORM1*2 = 0.1687, ORM1*2.1 = 0.0409, ORM2*1 = 1.0000; A2HS*1 = 0.6870, A2HS*2 = 0.3130. Compared with the Japanese population, the Philippine population has higher frequencies of ORM1*1 and A2HS*2, and lower frequencies of ORM1*2.1 and A2HS*1.