A truncated actin with an N-terminus of Met-44 is known to be selectively increased in neutrophils of patients with Behçet’s disease and to be generated proteolytically by PMN-elastase (Yamashita S. et al., Biol. Pharm. Bull., 23, 519—522 (2000); Biol. Pharm. Bull., 24, 119—122 (2001)). In this study, the functions of the N-terminal peptide consisting of Asp-2 to Val-43 of β-actin (42-merP) and the truncated actin with an N-terminus of Met-44 were examined. We first confirmed that the 42-merP existed in the patient plasma. The motility of human peripheral blood neutrophils and neutrophilic granulocytes differentiated from HL-60 cells was suppressed by the 42-merP. Furthermore, when neutrophil-like cells from HL-60 cells were preincubated with 10 nM 42-merP, migration of the cells induced by chemotactic factors such as fMLP and IL-8 was suppressed. The release of PMN-elastase, which is a neutrophil granular enzyme that is responsible for the production of the 42-merP and truncated actin, was suppressed by pretreating the neutrophils with 42-merP before fMLP-stimulation. The truncated actin was unable to polymerize in 0.1 M KCl, suggesting that the increase of truncated actin damages the reconstitution capacity of actin in neutrophils of the patients. These results suggest that the increase of 42-merP and truncated actin in patients with Behçet’s disease changes functions of neutrophils

View full abstract

A rapid and highly sensitive LC-MS-MS method for simultaneous determination of 25-hydroxyvitamin D₂ [25(OH)D₂] and 25-hydroxyvitamin D₃ [25(OH)D₃] in human plasma has been developed using derivatization with a Cookson-type reagent, 4-[2-(6,7-dimethoxy-4-methyl-3-oxo-3,4-dihydroquinoxalyl)ethyl]-1,2,4-triazoline-3,5-dione (DMEQTAD). The derivatization with DMEQTAD significantly improved the ionization efficiencies of 25(OH)D₂ and 25(OH)D₃ with detection limits of 20 and 12.5 fmol (8 and 5 pg) per injection, respectively. The method employed two steps of solvent extraction but did not require chromatographic purifications for sample pretreatment. The determination was carried out by mass chromatography of the protonated molecular ions formed by atmospheric pressure chemical ionization operating in the positive-ion mode after the derivatization, and 25-hydroxyvitamin D₄ was used as an internal standard. The intra-assay coefficients of variation were below 4.02 and 3.24% for 25(OH)D₂ and 25(OH)D₃, respectively, and the analytical recoveries of both compounds were quantitative. Assay linearity was obtained in the range of 0.05—1 ng per tube and the determination limit was 3 ng/ml for a 20 μl plasma aliquot, for each compound. The developed method was applied to plasma samples obtained from volunteers, two of whom had received vitamin D₂ supplementation, and gave satisfactory results.

View full abstract

In the mouse Ba/F3-hGHR cell line, which stably expresses human growth hormone receptors (hGHRs), the hGHRs were rapidly degraded in the absence of the ligand. Human growth hormone-binding protein (hGH-BP), a soluble form of hGHR, was released from Ba/F3-hGHR cells, but the hGH-BP release was less than 1% of total hGHRs in the cells. Therefore, the hGH-BP release does not markedly contribute to hGHR degradation in Ba/F3-hGHR cells. The constitutive degradation of hGHRs was inhibited by the proteasome inhibitors MG-132 and clasto-lactacystin β-lactone, or the vacuolar H⁺-ATPase inhibitor, bafilomycin A₁. hGH-enhanced degradation of hGHRs was also inhibited by MG-132. Moreover, MG-132 inhibited the internalization of hGHRs as assessed by ¹²⁵I-hGH binding to the cell surfaces. Ubiquitinated hGHRs were detected in the cell lysate and increased by hGH-treatment. Furthermore, MG-132 accumulated the ubiquitinated hGHRs induced by hGH. However, the ratio of ubiquitinated hGHRs to unubiquitinated hGHRs was very small, even with treatment involving both hGH and MG-132. In the hGH-untreated cells, the ubiquitinated hGHRs were weakly detected. However, the ubiquitination of hGHR was not enhanced by MG-132 as a result of immunoblotting. Thus, the ubiquitination of hGHR is unlikely to be involved, at least in the constitutive degradation. Taken together, both the proteasome pathway and endosome/lysosome pathway are involved in the constitutive degradation of hGHRs. Our results also suggest that ubiquitination of the hGHR itself is unlikely to be the trigger of the proteasome-dependent degradation.

View full abstract

Treatment with the triester of S-(1,2-dicarboxyethyl)glutathione (DCE-GS) prevented the hepatotoxicity induced by acetaminophen via elevation of the glutathione (GSH) level in rat hepatocytes. This elevation of the GSH level in rat hepatocytes by DCE-GS triester was dose- and time-dependent (2.1-fold in 24 h with 0.5 mM). DCE-GS triester increased the GSH level much more effectively than GSH, DCE-GS, and DCE-GS monoester and diester. Furthermore, the activity of γ-glutamylcysteine synthetase (γ-GCS), the rate-limiting enzyme in GSH biosynthesis, was also increased by DCE-GS triester treatment (1.4-fold in 24 h with 1.0 mM). In contrast, with a rat liver homogenate, DCE-GS increased the γ-GCS activity, whereas DCE-GS triester had no effect on this activity. These results suggested that DCE-GS triester, which is transported into hepatocytes much more effectively than DCE-GS and other DCE-GS esters due to its greater lipophilicity, was hydrolyzed to DCE-GS, and then the DCE-GS produced increased the GSH level via activation of γ-GCS in rat hepatocytes.

View full abstract

Damage to the vascular endothelium by reactive oxygen species causes many cardiovascular diseases including atherosclerosis. Such damage can be prevented by selenium (Se), which is thought to exert its actions mainly through the expression of selenoproteins. Se deficiency increased the susceptibility to tert-butylhydroperoxide (t-BuOOH) and enhanced lipid peroxidation in bovine arterial endothelial cells (BAEC). We investigated the effects of Se deficiency on the expression of the selenoproteins in BAEC. ⁷⁵Se metabolic labeling analysis and RT-PCR analysis revealed that BAEC expressed two glutathione peroxidase (GPx) isozymes, cytosolic GPx (cGPx) and phospholipid hydroperoxide GPx (PHGPx), three thioredoxin reductase (TrxR) isozymes, TrxR1, TrxR2 and TrxR3, and selenoprotein P (SelP). Se deficiency reduced both enzyme activity and mRNA level of cGPx, but did not affect those of PHGPx. SelP mRNA level was also reduced by Se deficiency, although the extent of reduction was much smaller than that of cGPx mRNA. We further found that TrxR activity was also decreased by Se deficiency but none of the mRNA levels of TrxR isozymes were reduced. These results indicate that vascular endothelial cells express several selenoproteins including cGPx, PHGPx, TrxR isozymes and SelP which might play important roles in the defense system against oxidative stresses and that the expressions of these selenoproteins are differently regulated by Se status.

View full abstract

A base-nonspecific and acid ribonuclease (RNase Os) belonging to the RNase T2 family was purified from rice bran to a homogeneous state by SDS-PAGE. The primary structure of RNase Os was determined by protein chemistry and molecular cloning. The RNase Os was a simple protein and consisted of 205 amino acid residues. Its molecular weight was 22578 and its amino acid sequence showed that it was most similar to barley RNase among the known RNase T2 family enzymes having 157 amino acid residues indentical with barley RNase. However, its N-terminus was blocked by a γ-pyroglutamyl residue. The optimal pH of RNase Os was around 5.5. The base preference at the B1 and B2 site of RNase Os was estimated from the rates of hydrolysis of 16 dinucleoside phosphates, to be guanine as the case of RNase LE from tomato. RNase Os was successfully expressed from yeast cells using the E. coli/yeast expression vector pYE-RNAP.

View full abstract

A neutrophil chemoattractant has been purified from the conditioned medium of granulation tissue obtained from carrageenan-induced inflammation in rats. The purified chemoattractant was a basic protein with a molecular mass of 10 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing and non-reducing conditions. NH₂-terminal amino acid sequence of the purified 10-kDa protein was identical with the sequence of rat fibronectin starting from the residue Thr⁵⁸⁵, indicating that the purified 10-kDa chemoattractant is a fragment derived from the NH₂-terminal type III domain of rat fibronectin.

View full abstract

The aim of this study was to determine the relationship between the neuroprotective effect of SM-20220 (N-(aminoiminomethyl)-1-methyl-1H-indole-2-carboxamide methanesulfonate) and the timing of its administration in an experimental stroke model. Two hours of occlusion followed by 22 h of perfusion of the left middle cerebral artery (MCA) was performed by inserting a nylon thread into the MCA to occlude it, and pulling the thread to initiate reperfusion. Intravenous infusion of SM-20220 for 1 h reduced the infarct volume at doses of 0.2—0.8 mg/kg in a dose-dependent manner without causing changes in the systemic arterial blood pressure or blood gases, when SM-20220 administration was started 1 h after the onset of occlusion. Administration of SM-20220 at a dose of 0.4 mg/kg also reduced the edema formation induced by ischemia. In contrast, SM-20220 failed to reduce the infarction, even at 1.6 mg/kg, when administration was started 2 h after the onset of occlusion. Thus, the therapeutic time window of SM-20220 for this transient MCA occlusion model is 1 h. Daily administration of SM-20220 (0.4 mg/kg) for the 7 d following 1.5 h of middle cerebral artery occlusion reduced the infarct volume with statistical significance (p<0.05), showing that SM-20220 did not merely delay but prevented ischemic damage.

View full abstract

Our previous study has demonstrated that the exposure of male BALB/c mice to social isolation stress caused a suppressed immune response and enhanced liver metastasis of colon 26-L5 carcinoma cells. To more precisely understand the influence of psychosocial factors on the metastatic process, here we have investigated the effect of social isolation stress on the vulnerability of the host to develop liver metastasis of colon 26-L5 cells, including the time span and incidence of metastatic formation, survival time and chemotherapy response. Isolation stress decreased the time period required for the metastasis formation relative to that in controls. On day 7 after the tumor injection, the 75% incidence of tumor metastasis in the stressed mice was 5 times the 15% incidence in the unstressed mice. When exposed to the challenge of lower cell numbers (0.025, 0.05, 0.1×10⁴/mouse) of colon 26-L5 cells, mice subjected to isolation stress developed an elevated incidence of metastasis (33.3, 66.6, and 100%, respectively) as compared with the controls (0, 33.3 and 50%, respectively). The survival time following the tumor inoculation was also shorter in the stressed mice (21.83±1.59 d) than in the control mice(24.08±1.68 d). Futhermore, the response of liver metastasis to chemotherapy consisting of 2 mg/kg cisplatin (CDDP) was worse in the stressed mice than that in unstressed mice. These findings suggested that social isolation stress could significantly impair the resistance of mice to the development of metastasis.

View full abstract

The effects of benzylisoquinoline compounds such as ethaverine, laudanosine, and tetrahydropapaverine on monoamine oxidase (MAO, EC 1.4.3.4) activity in mouse brain were investigated. Ethaverine showed an inhibition of MAO activity in a concentration-dependent manner (57.6% inhibition at 40 μM). Papaverine also inhibited MAO activity (38.1% inhibition at 40 μM). However, laudanosine and tetrahydropapaverine did not inhibit MAO activity. The IC₅₀ value of ethaverine for MAO was 25.5 μM. Ethaverine non-competitively inhibited MAO activity with a substrate kynuramine. The K_i value for ethaverine was 11.9 μM. In addition, ethaverine proved to preferentially inhibit type B MAO activity in a concentration-dependent manner, with an IC₅₀ value of 32.8 μM. These results suggest that ethaverine partially contributes to the regulation of catecholamine content.

View full abstract

Rikkunshi-to, a traditional Chinese (Kampo) medicine, has been used to treat chronic hypofunctions of the gastrointestinal tract. The effects of Rikkunshi-to on the plasma levels of gut-regulated peptide (somatostatin, motilin, gastrin, and vasoactive intestinal peptide (VIP)) levels were studied in healthy subjects. A single oral administration of Rikkunshi-to caused significant increases in plasma somatostatin and gastrin levels at 60 to 240 min compared with a placebo group. On the other hand, this medicine showed no effects on motilin and VIP levels. In conclusion, these results might indicate that the pharmacological action of Rikkunshi-to is closely related to changes in somatostatin- and gastrin-immunoreactive substance levels.

View full abstract

The aim of this paper was to study the influence of Fraxetin (7,8-dihydroxy-6-methoxy coumarin) treatment in a Drosophila melanogaster experimental model, analyzing several parameters in normal situations and instances of induced oxidative stress. Fraxetin treatment was introduced at different ages. Antigravity capacity and survival parameters were evaluated as in vivo assays, and levels of oxidative status, glutathione and lipid peroxidation, as ex vivo assays. The stress situation was induced by negative geotaxis, so physical exercise enhanced its basal metabolism, generating free radicals, which are probably implicated in the molecular damage related to the aging process. In our study, all treatment groups demonstrated a beneficial effect on the evaluated parameters. So, in vivo Fraxetin protects fruit flies against oxidative stress and improves the survival parameters. Moreover, Fraxetin prevents oxidative stress by an important increase in antioxidant reserves of GSH, and peroxidative damage is preserved by Fraxetin treatments.

View full abstract

The fruiting bodies of Isaria fungi have been traditionally used in Korea to treat cancer. An apoptosis-inducing compound, 4-acetyl-12,13-epoxyl-9-trichothecene-3,15-diol, was isolated from the methanol extract of fruiting bodies of Isaria japonica YASUDA by bioassay-guided fractionation. The apoptosis of the human leukemia cells (HL-60) by the compound was accessed by propidium iodide-staining flow cytometric analysis, and apoptosis-inducing activity at IC₅₀ concentration (10 nmol/l) was further confirmed by a nuclear morphological change, a ladder pattern of internucleosomal DNA fragmentation, and an activation of caspase-3.

View full abstract

Gallic acid (GA) derivatives, 3,4-methylenedioxyphenyl 3,4,5-trihydroxybenzoate (GD-1) and S-(3,4-methylenedioxyphenyl)3,4,5-trihydroxythiobenzoate (GD-3), were previously reported to induce apoptosis in tumor cells with IC₅₀s of 14.5 μM and 3.9 μM, respectively. To elucidate the mechanism by which these gallic acid derivatives (GDs) induce apoptosis, we studied whether GD-1 and GD-3 can activate caspases. When promyelocytic leukemia HL-60RG cells were treated with GD-1 and GD-3, poly(ADP-ribose)polymerase (PARP), a substrate of caspase-3, was cleaved into 85 kDa of degradative product with increasing incubation time. GA also activated PARP cleavage, which was inhibited by catalase, N-acetyl-L-cysteine (NAC), and intracellular Ca²⁺ chelator 1,2-bis(2-aminophenoxyethane)-N, N, N, N'-tetraacetic acid tetrakis (acetoxymethyl ester) (BAPTA-AM), in addition to a caspase inhibitor, Z-VAD-FMK. Its inhibitory pattern was identical with that of hypoxanthine/xanthine oxidase. On the other hand, GD-1 and GD-3-induced PARP cleavage was not suppressed by catalase or NAC, but by BAPTA-AM. This suggested that the GD-elicited signaling pathway is different from GA’s. Taken together, GDs activated caspase-3 following intracellular Ca²⁺ elevation independent of reactive oxygen species. Thus, it became evident that the signaling pathway leading to apoptosis was regulated by GDs in a different manner from GA.

View full abstract

An extract of the leaves of Apocynum venetum L. (Apocynaceae) markedly shortened the immobility time of male rats in a forced swimming test (FST) in a dose range of 30—125 mg/kg, indicating a possible antidepressant activity. This effect was comparable to that of the tricyclic antidepressant imipramine (20 mg/kg). Neither imipramine (20 mg/kg) nor the Apocynum extract in various doses (30, 60, 125 mg/kg) produced any overt behavioural change or motor dysfunction in the open field test. This result confirms the assumption that the antidepressant effect of an Apocynum extract in the FST is specific. Further, it can be speculated that this effect might be related to hyperoside and isoquercitrin which are major flavonoids in the extract.

View full abstract

The pharmacokinetics of epinastine (EPN), an anti-allergic agent, was investigated in rats. The plasma concentration-time profile of EPN after intravenous (i.v.) administration was triexponential. After oral administration of EPN (7.5 and 20 mg/kg), the drug was rapidly absorbed, and C_max was reached 2 h after dosing. A minor secondary peak was observed in EPN plasma concentration-time profiles at both doses. The bioavailability of EPN after oral dosing was 41 and 40%. The kinetic parameters (T_1/2, AUC and MRT) for unlabeled EPN were much smaller than those for ¹⁴C-EPN, which has already been reported. The total biliary excretion of EPN at a 7.5 mg/kg dose was 15.5% of the dose, but the percentage of conjugates in bile was extremely low and about 11% of the total biliary excretion. The increase in the plasma concentration in bile duct-linked rats after oral administration of EPN (20 mg/kg) was not observed, indicating that a secondary increase in drug concentration based on enterohepatic circulation was ruled out. When the gastrointestinal (GI)-transit of phenol red (PR) after oral administration of EPN (20 mg/kg) was estimated, the GI-transit of PR was significantly delayed, and at 3—4 h after dosing half of the PR dose reached the jejunum. The remaining EPN in the small intestine after oral administration (7.5 mg/kg) reached peak levels 2 h after dosing, but then partly increased again at 4 h. As a result, it was clarified that the double peaks observed after oral doses are mainly due to the delayed absorption of a part of EPN, based on the reduction in gastric motility caused by the drug.

View full abstract

The purpose of this study was to reveal the effectiveness of the polymer coated liposomes as a carrier of the anticancer drug doxorubicin in intravenous administration. The size controlled doxorubicin-loaded liposomes (egg phosphatidylcholine: cholesterol=1 : 1 in molar ratio) were coated with hydrophilic polymers (polyvinyl alcohol; PVA and hydroxypropylmethylcellulose; HPMC) having a hydrophobic moiety in the molecules (PVA-R, HPMC-R). The existence of a thick polymer layer on the surface of the polymer coated liposomes was confirmed by measuring the change in particle size and the amount of polymer on the liposomal surface. The polymer coating effects on the tumor accumulation of the drug encapsulated in the liposomes were evaluated in Walker rat carcinoma 256 cell bearing rats. The doxorubicin-loaded liposomes coated with PVA-R and HPMC-R showed higher drug accumulation into the tumor site by prolonging the systemic circulation in tumor-bearing rats. The targeting efficiency of the polymer coated liposomes calculated with the total and tumorous clearance of the drug was ca. 5 times larger than that of non-coated liposomes. We ascertained that polymers having a hydrophobic moiety in the molecule such as PVA-R and HPMC-R are suitable materials for modifying the surfaces of the doxorubicin-loaded liposome to improve its targeting properties.

View full abstract

In vitro protein binding of KE-298 and its plasma metabolites, deacetyl-KE-298 (M-1) and S-methyl-KE-298 (M-2), was high in rat (>97%), dog (>89%) and human plasma (>99%), respectively. Human serum albumin (>93%) was the main protein involved in the binding to plasma proteins, while the binding to human serum globulins was low (16—33%). The binding of KE-298 and its metabolites in all species of plasma was stereoselective. The (+)-(S)-enantiomers of these compounds bound rat, dog and human plasma proteins to a greater extent than did the (−)-(R)-enantiomers, except that the case of KE-298 was opposite in rat plasma. The stereoselective plasma levels of these compounds in rats, dogs, or humans would likely be due to stereoselective differences in binding to plasma albumin. The protein binding of M-1 in adjuvant-induced arthritis rat plasma was >97%, and the stereoselectivity was similar to the case of normal rat plasma. KE-298 and its metabolites remarkably displaced [¹⁴C]warfarin, which bound on albumin in a solution of diluted rat serum albumin. Similarly, there was a displacement of [¹⁴C]warfarin in solutions of dog and human serum albumin, and concomitantly the displacement of [¹⁴C]diazepam. [³H]Digitoxin was not displaced by any of the enantiomers in each albumin solution. No stereoselectivity was found in displacement by enantiomers of the three compounds. These results suggest that stereoselective protein binding can be attributed to quantitative differences in binding to albumin rather than to the different binding sites.

View full abstract

We have estimated the pharmacokinetic and pharmacodynamic interactions of verapamil (VP) enantiomers and also the interaction between VP and its metabolite, norverapamil (NVP). ECGs of conscious rabbits were studied to determine the pharmacokinetics of VP enantiomers and racemic NVP in relation to their prolongation effect on PR intervals, which were used as an index of VP’s antiarrhythmic effect. Plasma free fractions of VP enantiomers showed constant values at concentrations ranging from 0.022 to 1.10 μM. There were no interactions between enantiomers or between VP and NVP. The pharmacological effect of the S-enantiomer (S-VP), which was determined by linear regression analysis, showed it was about 20 times more potent than that of the R-enantiomer (R-VP). The effect of racemic VP was the simple sum of those elicited by both enantiomers. These relationships were not significantly different between intravenous infusion and bolus injection. Simultaneous intravenous infusion of NVP had no influence on the PR intervals. In conclusion, we demonstrated that the relationship between plasma unbound concentration of VP enantiomers and their pharmacological effect was the simple sum of two enantiomers.

View full abstract

The stereoselective acyl migration of diastereomeric 1β-O-acyl glucuronides of (R)- and (S)-2-phenylpropionic acid [(R)-1PG and (S)-1PG, respectively] in phosphate buffer (pH 7.4) at 310 K was investigated using HPLC. The disappearance of (R)-1PG was faster than that of (S)-1PG according to pseudo first-order kinetics. A kinetic model describing the degradation reactions was constructed. The rate constant for acyl migration from the 1β-O-isomer to the 2-O-acyl isomer (k₁₂) was about one order magnitude larger than that for hydrolysis from 1β-O-acyl isomer to aglycone (k₁₀). The k₁₂ of (R)-1PG (0.377±0.005 h^-1) was about two times larger than that of (S)-1PG (0.184±0.003 h^-1). The results indicated that the stereoselectivity in the degradation of 1PG was apparently governed by the acyl migration from 1-isomer to 2-isomer. The kinetic parameters for acyl migration from 1-isomer to 2-isomer were estimated from temperature-dependent experiments using the transition state theory. The value of the free energy of activation at 310 K for (R)-1PG (99.67 kJ/mol) was smaller than that of (S)-1PG (101.60 kJ/mol), suggesting that (R)-1PG showed thermodynamically higher reactivity in acyl migration than (S)-1PG.

View full abstract

The immunomodulating effects of various gel-forming (1→3)-β-glucans, grifolan (GRN), SSG, sonifilan (SPG) and alkaline-treated SPG (SPG-OH), on balancing helper T cell activity were examined in a murine model. Plasma from mice that were injected with GRN or SPG-OH and trinitrophenyl ovalbumin (TNP-OVA) contained TNP-specific antibodies of both IgG1 and IgG2a isotypes. Administration of SSG and TNP-OVA significantly augmented the synthesis of IgG2a antibodies, while the synthesis of IgG1 was reduced. However, SPG did not enhance the antibody response. In the culture supernatants of splenocytes obtained from GRN- or SPG-OH-administered mice, high levels of IgG1 and low levels of IgG2a and IFN γ were detected. In contrast, high levels of IgG2a and IFN γ and low levels of IgG1 were detected in the case of administration of SSG. Furthermore, it was shown by intracellular cytokine staining that the proportion of IFN γ⁺CD4⁺ double-positive cells among the CD4⁺ cells from mice administered SSG was most strongly increased by addition of PMA and A23187. On the other hand, the expression of IL-12 p40 mRNA was more markedly elevated in splenocytes after combined administration of TNP-OVA plus SSG than after administration of TNP-OVA alone. The highest IFN γ production was observed when adherent cells of mice administered TNP-OVA and SSG were cultured with TNP-primed lymphocytes. This effect of administration of SSG on IFN-γ production was completely inhibited by addition of anti-IL-12 mAb. In conclusion, our study showed that β-glucans have various effects on the Th1 or Th2-dependent antibody subclasses, in particular. SSG induces the development of Th1 cells via the IL-12 pathway.

View full abstract

Agaricus blazei is a medically important mushroom widely eaten and prescribed in Japan. Polysaccharide fractions were prepared from cultured A. blazei by repeated extraction with hot water (AgHWE), cold NaOH (AgCA), and then hot NaOH (AgHA). By chemical, enzymic, and NMR analyses, the primary structures of AgHWE, AgCA, and AgHA were mainly composed of 1,6-β-glucan. Among these fractions, the NaOH extracts showed antitumor activity against the solid form of Sarcoma 180 in ICR mice. To demonstrate the active component in these fractions, several chemical and enzymic treatments were applied. These fractions were found to be i) neutral β-glucan passing DEAE-Sephadex A-25, ii) resistant to periodate oxidation (I/B) and subsequent partial acid hydrolysis (I/B/H), iii) resistant to a 1,3-β-glucanase, zymolyase, before I/B, but sensitive after I/B/H. In addition, after I/B/H treatment of the neutral fraction of AgCAE, a signal around 86 ppm attributable to 1,3-β-glucosidic linkage was detectable in the ¹³C-NMR spectrum. These facts strongly suggest that a highly branched 1,3-β-glucan segment forms the active center of the antitumor activity.

View full abstract

Immunoglobulin E (IgE)-dependent histamine release from purified rat peritoneal mast cells (PMC) is very low in comparison to that from a non-purified preparation (PEC). The reduced histamine release from PMC is recovered or potentiated by reconstitution with separated non-mast cells (NMC). In the present study, further characterization was undertaken to elucidate the mechanisms involved. Sensitized mast cells were recovered from peritoneal cavities of rats, and purified by density gradient centrifugation with Percoll. Effects of NMC reconstitution, membrane fraction of NMC, NMC incubation supernatant, adhesion molecules and extracellular matrix proteins on IgE-dependent histamine release from PMC were examined. IgE-dependent histamine release was significantly potentiated by NMC reconstitution to PMC. The potentiation was dependent on the concentration of NMC reconstituted and reached a plateau after 30 min incubation. Increasing concentration of PMC did not affect the histamine relase. Membrane fraction prepared from NMC also potentiated PMC histamine release in a dose-dependent manner. The potentiation reached a plateau in 5 min. Furthermore, incubation supernatant of NMC potentiated PMC histamine release. Antibodies against intercellular adhesion molecule (ICAM)-1, lymphocyte function-associated antigen (LFA)-1, very late activation antigen (VLA)-1, VLA-4 and VLA-6, and fibronectin did not affect the potentiation of PMC histamine release by NMC reconstitution. Fibronectin, laminin and collagen failed to potentiate PMC histamine release. These results indicate that the membrane component(s) of NMC in the rat peritoneal cavity seems to modulate IgE-dependent histamine release from peritoneal mast cells of rats, and that the active molecule(s) may be released from NMC. Adhesion molecules such as ICAM-1, LFA-1 and VLA are not involved.

View full abstract

Drug-reducing ability of monkey liver cytosol was examined in this study. Monkey liver cytosol exhibited significant reductase activities toward zonisamide, sulindac and imipramine N-oxide in the presence of 2-hydroxy-pyrimidine or benzaldehyde, an electron donor to aldehyde oxidase. These activities were abolished by inhibitors of aldehyde oxidase, such as menadione. These reductase activities in monkeys were extremely high compared to those in other animals. The zonisamide reductase activity of monkey liver cytosol was about 40-fold higher than that of the liver microsomes. It appears that the high levels of aldehyde oxidase exists in monkey liver, and zonisamide, sulindac and imipramine N-oxide are mainly reduced by this enzyme, not by cytochrome P450.

View full abstract

Several N-phenylhomophthalimide derivatives were prepared and their inhibitory activity on thymidine phosphorylase/platelet-derived endothelial cell growth factor (TP/PD-ECGF) was assessed. Among them, 2-(2,6-diethylphenyl)-7-nitro-1,2,3,4-tetrahydroisoquinoline-1,3-dione (9) was found to be a more potent inhibitor than the classical inhibitor, 5-nitrouracil (1). Lineweaver-Burk plot analysis indicated that 9 shows mixed-type competitive inhibition TP/PD-ECGF, while 1 is a competitive inhibitor.

View full abstract