It was confirmed that the flexible arm region of HUα forms an antiparallel β-sheet and that all of the residues of phenylalanines, together with some of leucines and/or valines, form a hydrophobic core within the dimer of HUα. HUα protein alone is thermally labile and melts at 38°C, but it becomes remarkably stabilized and melts at 59°C in the presence of DNA. Several resonances from both HUα and DNA perturbed by their complex formation, notably those of His C-2 and C-4 protons, downfield shifted Cα protons in the antiparallel β-sheet, as well as Arg Cδ and Lys Cε protons. The results indicated that a β-sheet region of HUα binds to DNA, and also showed that rapid equilibrium occurs on the NMR time scale between bound and unbound states of HUα. A few intermolecular nuclear Overhauser effects (NOEs) were also observed between the protein and H1' protons of DNA in the complex, suggesting that HUα binds primarily to the minor groove of DNA.
The water-soluble fraction containing bone-inductive activity was purified from guanidine-hydrochloride extracts of bovine demineralized bone. The purification steps include ultrafiltration, dialysis, affinity chromatography on heparin-Sepharose and gel chromatography on Sephacryl S-200. Combination of these steps was proven to be an effective and rapid method for the purification of this protein.Subcutaneous implantation of the water-soluble protein with type I collagen was carried out in the thorax of rats. When alkaline phosphatase activity and calcium content in implants were used as indices for purification, the water-soluble bone-inductive protein was purified >600-fold according to the enzyme activity and 64-fold according to the calcium content. A morphological examination revealed that many chondrocyte and osteoblast cells were seen in the location of the implanted material.Sodium dodecyl sulfate/gel electrophoresis of the protein produced in this way under non-reducing conditions revealed four protein bands of 18, 16, 14 and 11 kDa. None of the separated bands had any biological activity. This result suggests that the water-soluble bone-inductive activity depends on an associated form of various proteins in the range of 18 to 11 kDa.
The final stage in a series of blood coagulating reactions is fibrinogen-fibrin conversion by thrombin. This reaction consists of fibrinopeptide A and fibrinopeptide B release, polymerization of fibrin monomer, and stabilized fibrin formation by factor XIII. The latter two reactions require calcium. In the present study there was no difference in the rate of thrombin-induced fibrinopeptide release between fibrinogen and asialofibrinogen where sialic acid in the terminal end of carbohydrate moiety of fibrinogen was removed by neuraminidase, but turbidity associated with asialofibrin clot formation was increased more rapidly. In asialo-derivatives, the dissolution time of the clots in high concentrated urea solution tended to be shortened and rigidity as a gel tended to be decreased. In measurement by thromboelastography there was no difference in the reaction time (r) between fibrinogen and asialofibrinogen, but the maximum amplitude (ma) was obviously decreased in asialofibrinogen. Furthermore, when the rate of cross-link formation between γ chains by F-XIII was compared, the production of γ-dimer in the same reaction time was found to be lower and formation of stabilized fibrin tended to be retarded in asialofibrinogen. Sialic acid in fibrinogen thus may clearly influence the polymerization of fibrin-monomer and the formation of cross-linked fibrin in a series of reactions for fibrinogen-fibrin conversion. This may be consistent with the theory that fibrinogen sialic acid residues are low affinity calcium-binding sites and influence fibrin assembly.
A protein was purified from rabbit seminal plasma using preparative acrylamide disc electrophoresis, ammonium sulfate precipitation and gel filtration. The protein inhibited the in vitro fertilization of mouse ova inseminated with epididymal sperm and with capacitated sperm. The inhibition was not observed, however, when only ova were exposed to the protein prior to mixing with sperm. The partial protein sequence analysis revealed the strong homology of the rabbit fertilization inhibitory protein to human annexin V.
The influence of phosphatidylcholine liposomes on the tryptic digestion of insulin was studied to obtain basic information on the interaction between liposomes and peptides or proteins. Protection of insulin from tryptic digestion by liposomes depended on the encapsulation efficiency and the method of preparation of liposomes. REV (reverse phase evaporation vesicles) were most effective for the protection of insulin from tryptic digestion. Tryptic digestion of insulin was accelerated by negatively-charged liposomes (containing 10% phosphatidylserine, phosphatidylinositol, or phosphatidic acid) or by neutral empty liposomes, this digestion being enhanced by progressively smaller liposome size (from 2 to 0.1 μm) in the case of neutral empty liposomes. These results suggest that this enhancement had occurred on the surface of the empty neutral or negatively-charged liposomes. Positively-charged empty liposomes (containing 10% of stearylamine) weakly suppressed the tryptic digestion of insulin. The size of the positively-charged liposomes increased after the addition of insulin, this size increase suggesting that these liposomes were fused by insulin. Presumably, insulin was protected by the lipids around the insulin molecule after the insulin-induced fusion or aggregation of positively-charged liposomes
Effects of the pretreatment of murine peritoneal macrophages with several polysaccharides on the production of H2O2 induced with unopsonized zymosan were examined. Pretreament with most of (1→3)-β-D-glucans for 6 h at 37°C inhibited the zymosan-mediated H2O2 production by macrophages. The phorbol myristate acetate (PMA)-mediated H2O2 production was not affected by the pretreatment. The pretreatment of macrophages with (1→3)-β-D-glucans decreased the ability to ingest unopsonized zymosan, but did not affect the ingestion of IgG-coated sheep red blood cells (IgG-SRBC). These results suggested that the pretreatment with (1→3)-β-D-glucans interfered with the interaction of macrophages to zymosan and that the occupation of the receptor for the (1→3)-β-D-glucans inhibited zymosan-mediated production of H2O2 by macrophages. Chemical modification by substitution with carboxymethyl groups or hydroxyethyl groups of a (1→6)-branched (1→3)-β-D-glucan reduced the inhibitory effect of pretreatment on zymosan-mediated H2O2 production. The above results indicated the possibility that murine peritoneal macrophages possess certain receptors for β-anomeric glucans, and one ligand specificity of the receptors is to restrict the intact (1→3)-β-D-glucosyl back bone.
Streptozotocin-induced diabetic rats (LZ-DM rats) developed nephrocalcinosis after being fed a low zinc diet for 4 weeks. However, administration of 1α-hydroxy vitamin D3 (1-OHD3) or insulin inhibited the formation of nephrocalcinosis.The urinary calcium level increased when 1-OHD3 was administered to these rats, but decreased in the group receiving insulin. It was also observed that the femur recovered its hardness when insulin was administered, although the hardness in the LZ-DM rats remained lower than that of the control rats. Rat femur hardness, however, did not completely recover with 1-OHD3 treatment.These results indicate that the effects of insulin and 1-OHD3 differ, even though nephrocalcinosis was reduced by administration of either insulin or 1-OHD3. It appears that insulin assists bone formation, and the 1-OHD3 hastens calcium excretion.
Cardiovascular effects of intravenously administered denopamine and its derivatives were investigated in anesthetized dogs and their positive inotropic and hypotensive effects were compared. Structure-activity relationships were examined by modifying the methoxy group in ring B and the hydroxy group in ring A in structure II, a ring-fissioned product of trimetoquinol. Almost all test compounds demonstrated positive inotropic and chronotropic effects as well as hypotensive effects which were mediated by β-adrenoceptors. With modification of the methoxy group in ring B, only 3, 4-dimethoxy, 2, 3, 4-trimethoxy and 3, 4, 5-trimethoxy derivatives exhibited β1-adrenoceptor selectivity. The 3, 4-dimethoxy derivative showed the most potent positive inotropic effect and the highest selectivity to β1-adrenoceptor. By structural modification of the hydroxyl group in ring A of the 3, 4-dimethoxy derivatives, the potency of positive inotropic effect was affected, while β1-adrenoceptor selectivity of the derivatives with 3, 5-dihydroxy, 3- and 4-monohydroxy groups were essentially maintained. Among β1-adrenoceptor selective compounds, the dose ratios between intravenous and intraduodenal administrations of catechol derivatives like isoproterenol were higher than those of non-catechol derivatives. The 4-monohydroxy derivative (racemic denopamine) exhibited the smallest dose ratio with a long-lasting action. Thus, we could identify selective β1-adrenoceptor agonists by the structural modification of a selective β2-adrenoceptor agonist, trimetoquinol. In this group of compounds, β-hydroxy moiety was suggested to be requisite to potent β-adrenoceptor stimulating action and 3, 4-dimethoxyphenethyl structure was important for manifestation of β1-adrenoceptor selectivity.
The effect of benidipine on experimental cerebral ischemia was investigated in rats subjected to occlusion of the bilateral common carotid arteries. Benidipine (30 μg/kg, i.p.) improved neurological symptoms such as ataxia, convulsion and loss of righting reflex, and prolonged survival time after occlusion of the bilateral common carotid arteries. In the nicardipine (100 μg/kg, i.p.)-treated group, a similar effect was observed, whereas nifedipine (100, 300 μg/kg, i.p.) and verapamil (300 μg/kg, i.p.) did not show any beneficial effect in this model. Furthermore, pretreatment with benidipine (30 μg/kg, i.p.) suppressed the increase in cerebral water content 3 h after the occlusion. Nicardipine (100 μg/kg, i.p.) showed a tendency to reduce the increase in cerebral water content, though the effect was not statistically significant. Nifedipine (100μg/kg, i.p.) produced no improvement. After occlusion of the bilateral common carotid arteries, depletion of adenosine triphosphate (ATP) and phosphocreatine (CP) and an accumulation of lactate occurred in a time-dependent manner. Prophylactic administration of benidipine (30 μg/kg, i.p.), 20 min before occlusion, attenuated the depletion of ATP and CP and the accumulation of lactate 3 h after the occlusion. Furthermore, post-treatment with benidipine 30 min after occlusion also suppressed these metabolic disorders. In conclusion, the beneficial effects of benidipine in this severe cerebral ischemia model show that the compound has advantages over nicardipine, nifedipine and verapamil. Thus, these results suggest that benidipine may be useful in the treatment of acute ischemic cerebral damage.
The effects of chronic treatment with bunazosin, atenolol, ketanserin and verapamil on the myocardium of the spontaneously hypertensive rat (SHR) were examined by the radioligand binding assay method using [3H]prazosin, [<125>I]iodocyanopindolol and [3H]nitrendipine binding to α1- and β1-adrenergic receptors and Ca2+ binding sites. The norepinephrine concentration in the rat myocardium was also determined. (1) All of these drugs lowered the elevated blood pressure of the SHR. (2) Only ketanserin administration increased the Kd value of the α1-adrenoceptor in the SHR. (3) Administration of atenolol and ketanserin to the SHR decreased the Bmax value of the β1-adrenoceptor. (4) Verapamil, bunazosin and atenolol also lowered the Bmax values of the Ca2+ binding sites of SHRs. (5) All drugs except for atenolol lowered the norepinephrine concentration in the myocardium of the SHR. These findings suggest that the SHR has an abnormality of the α1 and β1-adrenoceptors and Ca2+ binding site of the myocardium, that the drugs had a beneficial effect on these receptors, that the drugs could also lower the high norepinephrine content in the myocardium of the SHR and that some of these drugs also affected the binding characteristics of other types of membrane receptors.
In immature female rats, the secretion of ovarian inhibin and estradiol is greatly accelerated by equine chorionic gonadotropin (eCG) treatment. The present study has been carried out to determine whether or not the levels of the two hormones are inhibited by a single s.c.-injection of indomethacin (INDO) 24 h after eCG administration. The levels of ovarian hormones and gonadotropins were measured by double-antibody radioimmunoassay using 125I-labeled radioligands. The serum levels of inhibin and estradiol were considerably inhibited within 24 and 12 h, respectively, after INDO injection. In addition, the serum levels of follicle-stimulating hormone (FSH) after INDO injection remained lower than the basal levels before eCG treatment. The luteinizing hormone (LH) levels were significantly reduced within 12 h after INDO treatment. The results demonstrate that the levels of inhibin and estradiol, even in the situation where the production of both hormones is already accelerated by eCG pretreatment, are suppressed by an inhibitor of prostaglandin (PG) synthesis, suggesting that locally produced PGs may play a role in the regulation of the production of both hormones in the ovary.
The preventive effect of β-alanyl-L-histidinato zinc (AHZ) on the deterioration of bone metabolism was investigated in the femoral diaphysis of ovariectomized rats. AHZ (10, 30 and 100 mg/kg body weight/d) was orally administered to ovariectomized rats for 6 weeks. Ovariectomy produced a significant decrease in estradiol, calcitonin, calcium and inorganic phosphorus concentrations in the serum as compared with those from sham-operated rats. The dose of 30 and 100 mg AHZ/kg prevented any decrease in serum inorganic phosphorus concentration caused by ovariectomy. Alkaline phosphatase activity, deoxyribonucleic acid (DNA) and calcium contents in the femoral diaphysis of ovariectomized rats significantly decreased in comparison with those from sham-operated rats. These decreases were completely prevented by the dose of AHZ (10, 30 and 100 mg/kg). Electron microscopical analysis showed a rough alteration of bone matrix in the femoral diaphysis of ovariectomized rats. This alteration was clearly modified by the doses of AHZ (10, 30 and 100 mg/kg). Also, dosages of AHZ (30 and 100 mg/kg) restored the atrophy of osteoblasts and cartilage cells caused by ovariectomy. The present study suggests that oral administration of AHZ can prevent the deterioration of bone metabolism by ovariectomy. AHZ may have a therapeutic role in the treatment of osteoporosis.
A series of monoclonal antibodies (mAbs) that react with methamphetamine-bovine serum albumin (MA-BSA) were established by intrasplenic immunization method. Among established 36 clones, two typical mAbs, designated NK-1 and NK-2, were described. The inhibition assay of enzyme-linked immunosorbent assay (ELISA) analysis using methamphetamine analogs indicated that NK-1 showed considerable reactivity not only MA-BSA but also methamphetamine and its major metabolite, para-hydroxymethamphetamine (p-hydroxymethamphetamine). The cross-reactivity between NK-1 and the methamphetamine analogs with modified alkyl side chain, indicates that methyl groups of R5 and R7 in the methamphetamine molecules are important for the maximum affinity. The length of alkyl side chain on methamphetamine significantly affected the binding affinity of NK-1. The results may suggest that NK-1 will recognize not only methamphetamine but also the bridge part of the methamphetamine that binds the methamphetamine molecules to a carrier protein.
Xenopus laevis oocytes were used as an expression system to prove and characterize the carrier-mediated transport of uridine in the small intestine. Significant Na+ dependency was observed for the uptake of [3H]uridine by Xenopus laevis oocytes injected with poly(A)+RNA prepared from rabbit small intestinal mucosa. By contrast, the uptake of [3H]uridine was negligible in water-injected oocytes. There was no significant difference in the Na+ dependent uptake rates of [3H]uridine among oocytes expressed by using mRNA prepared by three different methods. The uptake of [3H]uridine by mRNA-injected oocytes was enhanced by increasing the culturing time after mRNA injection. Concentration dependency for uridine transport was observed with the Michaelis constant of 8.27 μM, which was comparable to that reported in the study using the brush-border membrane vesicles from rabbit small intestine (6.4 μM). Furthermore, the uptake of [3H]uridine was significantly inhibited by adenosine and thymidine, but not by adenine and uracil. Consequently, the transport system of uridine expressed in mRNA-injected oocytes is clarified to be similar to that functioning in the brush-border membrane of the small intestine.
In order to elucidate the uptake mechanism of fractionated heparin by rat parenchymal hepatocytes, the concentration dependent uptake was kinetically analyzed in primary culture of rat parenchymal hepatocytes, and the effects of established transport inhibitors and heparin analogues on the uptake were also examined. The uptake rate of heparin measured over an extended period of 60 min was saturable, with the maximum uptake velocity (Vmax) of 0.36±0.05 pmol/min/mg protein and the Michaelis constant (Km) of 21.2±5.4 nM. The uptake was inhibited by the addition of 4, 4'-diisothiocyanatostilbene-2, 2'-disulfonic acid (DIDS), and inhibitor of the anion transport system, and rose bengal, an organic anion. Heparin analogues (pentosan polysulphate, or heparan sulphate) also inhibited the uptake of fractionated heparin. However, the uptake was not inhibited by the inhibitors of receptor-mediated endocytosis (phenylarsine oxide). These results suggest that fractionated heparin may be taken up by anion transport system, rather than by receptor-mediated endocytosis, though the fractionated [3H]heparin is a compound with the high molecular weight of about 20000 Da. At least the negative charge or sulphate group in the drug structure is supposed to play an important role in the uptake of fractionated heparin by parenchymal hepatocytes.
The population pharmacokinetics of theophylline were studied in 55 patients with stable chronic airway obstruction. Two hundred and seventy six theophylline serum concentrations after intravenous short infusion were analyzed using a nonlinear mixed-effect model. The influence of hepatic dysfunction, smoking habit, age and the measurement of arterial blood gases (oxygen tension : PaO2, carbon dioxide tension : PaCO2, blood pH) and clinical laboratory tests (serum albumin concentration, haematocrit) on the pharmacokinetic parameters of theophylline was examined by the likelihood ratio test. Assessment of each factor was made by a forward selection method. In the final regression model, the total body clearance (CL, l/h/kg) was related to the value of PaCO2 as well as to the presence of hepatic dysfunction, and the volume of distribution (Vd, l/kg) was related with the PaCO2 value as expressed in the following equations : CL=exp(-3.78-0.525·HF+0.0233·PaCO2) and Vd=exp(-1.12+0.00934·PaCO2), where HF is a categorical variable with a value of unity if a patient has hepatic dysfunction otherwise zero. The interactions among blood gas measurements were observed and the CL and Vd of theophylline would be inversely correlated with PaO2 or pH, if we selected PaO2 or blood pH to be a more important factor than PaCO2. The inter-individual variabilities in CL and Vd were 38.5% and 12.5%, respectively, and the residual variability in theophylline serum concentrations was 10.6% as a coefficient of variation. This final model and the population parameters of theophylline will be useful for individualization of a drug dosage regimen by means of the Bayesian method. Significant correlation between arterial blood gas measurements and pharmacokinetic parameters observed in this study indicates that the pharmacokinetics of theophylline can vary in association with the severity of respiratory diseases.
A mechanism of hemolytic hole formation during rapid hemolysis in a hypotonic medium has been investigated using eosin-5-maleimide (EMI) as a probe. The EMI-labeled erythrocytes revealed a distinct cluster and/or ring of intense fluorescence staining in a hypotonic 5 mM Hepes buffer (pH 7.4), but not in an isotonic buffer containing 150 mM KCl. This EMI cluster indicates an association of band 3 proteins, which correspond to a hemolytic hole. The hole was confirmed by an atomic force microscopy image. The erythrocytes showed a single large hole in the membrane. By the use of EMI-labeled ghosts, it was observed that the lateral clustering of band 3 was accompanied by a biphasic change of fluorescence intensity of EMI. This biphasic change is interpreted as the hemolytic hole formation by band 3, followed by a disappearance of the hole accompanied by band 3 diffusion or distribution within membrane. The latter event corresponds to a spontaneous membrane seal. When a cytoplasmic domain of band 3 was digested with trypsin, or when SH groups in the cytoplasm-facing components of the membrane were also labeled by EMI, no fluorescence change was observed. These results suggest that the association and/or dissociation of band 3 proteins in a hypotonic medium are strongly influenced by cytoplasmic domains. The appearent biphasic change of the fluorescence intensity in the hypotonic medium was well explained by assuming three events : swelling, clustering of band 3, and sealing accompanied by band 3 redistribution.
[125I]2'-Iododiazepam (IDZ) was prepared and its application in a benzodiazepine receptor binding assay was studied. [125I]2'-IDZ binds to the rat cortical membrane with a high affinity (Kd, 0.66 nM). Various benzodiazepines showed competition with [125I]2'-IDZ for the binding sites in the rat cortical membrane, and the specificity of its binding correlated well with that of [3H]diazepam (r=0.992, p<0.001). These findings suggested that [125I]2'-IDZ binds to the same sites as [3H]diazepam and indicated that [125I]2'-IDZ can be used in a benzodiazepine receptor assay.
The immunoreactivity (specific binding) of three Gly(A1)-sulfobenzoxadiazole-labeled insulins which have different spacer groups, Gly(A1)-[3-(4-sulfobenzoxadiazol-7-ylthio)propanoyl]-insulin, Gly(A1)-[2-(4-sulfobenzoxadiazol-7-ylthio)acetyl]-insulin, Gly(A1)-[3-carboxy-2(or 3)-(4-sulfobenzoxadiazol-7-ylthio)propanoyl]-insulin, toward an antiporcine insulin monoclonal antibody was investigated, where solid-phase fluoroimmunoassay technique was utilized. The immunoreactivities of three labeled insulins were 2.1, 1.8 and 1 in that order.
Rats were fed corn oil-free or corn oil-containing diets for 4 weeks to determine their effect on the competition between glucuronidation and sulfation in phenol and its para substituents. The activities of uridine diphosphate-glucuronyltransferase (UDPGT) on chemicals were enhanced with a 12% corn oil diet, whereas sulfotransferase activity showed no significant change. Following the intravenous injection of p-ethylphenol and p-tert-butylphenol, an increase of glucuronide and a decrease of sulfate were observed in the rats fed the corn oil diet. In contrast, no significant changes were observed in phenol and p-phenylphenol. These results are discussed in association with van der Waals volume (Vw) of substituent and phospholipid dependence of UDPGT.
Hinokitiol (I) and tropolone (II) showed characteristic cytotoxic effects in vitro on five kinds of human and murine cell lines and blastic lymphocytes from mouse splenocytes. The cytotoxic effect of I on the growth of murine and human tumor cell lines, including RL♂-1, MH134, HL60, K562 and KATO-III was definite when examined by thymidine incorporation into DNA and its 50% inhibitory concentration (IC50) on all cells was 0.3-0.6 μg/ml. Compound II also showed comparable cytotoxic effects on these cell lines, indicating a little lower activity when compared to I. Furthermore, I and II also completely suppressed the [3H]thymidine ([3H]TdR) incorporation of mitogen-induced blastic lymphocytes. The suppressive activity on mouse lymphocyte proliferative response to concanavaline-A was also found with both compounds at a low concentration of 0.32 μg/ml. As compound I is known to be of fairly low toxicity (LD50 : 453+24 mg/kg in mice), the antitumor and immuno-suppressive effect of hinokitiol (I) should be further investigated.