Nitrotyrosine is considered a stable biomarker of reactive nitrogen species, including nitrogen dioxide (NO₂) and peroxynitrous acid (ONOOH) in biomaterials. There are inconsistent observations on the detection of free and protein-associated nitrotyrosine in normal human plasma. Human erythrocytes, differentiated from erythrocyte precursor cells in the bone marrow, circulating in the body for an average of 120 d, and finally removed by spleen macrophages, may be exposed to reactive nitrogen species. In the present study, membrane proteins and hemoglobin from the senescent erythrocyte population were compared with those from young erythrocytes separated from the same individuals in their nitrotyrosine presence using newly prepared rabbit polyclonal anti-nitrotyrosine-ribonuclease A and anti-nitro(N-butozycaronyl)tyrbosine-bovine serum albumin antibodies. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the membranes and hemoglobin, and subsequent Western blot analysis, showed that these antibodies only slightly bind to the bands of the proteins from both young and senescent erythrocytes, whereas these antibodies definitely bind to the protein bands of membranes and hemoglobin nitrated by NO₂ or ONOOH in vitro. This result indicates that nitrotyrosine is not detected in the membrane proteins and hemoglobin in human normal erythrocytes in circulation. However, this does not conclude that erythrocytes are not exposed to reactive nitrogen species in the circulation.

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This study was designed to identify a parathyroid hormone (PTH)/PTH-related peptide (PTHrP) receptor gene polymorphism in a healthy Japanese population. All Known 13 introns of this gene were amplified by PCR, except the 1st intron, which was amplified by the long-PCR method. No restriction fragment length polymorphisms (RFLPs) were detected by BsmI or XbaI in any of thses introns. Twenty-one other restriction enzymes (Hind III, Bgl II, Sty I, Pvu II, Eco81 I, Van91 I, BstX I, Sse8387 I, EcoR I, BamH I, Mbo II, Tth111 I, PshA I, Eam1105 I, Not I, Bgl I, Fok I, Sfi I, Apa I, Taq I) were tested on the 1st intron. Furthermore, digestion by Van91 I (CCANNNNNTGG) identified a single, two-allele polymorphism with a fragment of approximately 3.5 kb (V allele) or a fragment of 3.1 and 0.4 kb (υ allel). The frequency of the Van91 I polymorphism in 106 healthy Japanese bolunteers was 77.4% for type υυ, 19.8% for type Vυ and 2.8% for type VV. In addition, the urinary cAMP response to exogenous [1-34]PTH was studied in 17 healthy volunteers and found to be significantly greater in persons with type Vυ than type υυ (P<0.05). In conclusion, the Van91 I polymorphism of the PTH/PTHrP receptor gene can be used to study the role of polymorphism in various disorders involving PTH or PTHrP.

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Factors which regulate Intrinsic Factor (IF) content and IF mRNA level were examined with an abc-ELISA and Northern blot analysis in growing rats, and compared with pepsinogen (Pg) and in terms of the increased need for vitamin B₁₂ (V.B₁₂). Increases in IF content and IF mRNA level gradually occurred from day 13 after birth, whereas those of Pg and Pg mRNA strated from day 16. The effects of a few related hormones on the expression of IF mRNA were examined. The injection of hydrocortisone induced IF and Pg mRNA expression in 5-d-old postanatal rats. Furthermore, adrenalectomy-induced decreases in IF content and IF mRNA level in adult male rats were recovered with hydrocortisone administration.IF content and IF mRNA level were measured in the artificially and physiologically created needs for V.B₁₂, the first being regeneration of the liver, the V.B₁₂ storing tissue, following partial hepatectomy, and the second pregnancy or lactation. During regeneration of the liver, increases in IF content and IF mRNA level were marked, followed by reduction toward the original level after accomplishment of regeneration. Increases in IF content and IF mRNA level were also seen in lactating rats, but no increases were obtained in pregnant rats. These results suggest that the IF content and IF mRNA level are regulated not only by corticosteroids but also by the increased need for V.B₁₂.

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We previously identified and characterized a major lysosomal membrane glycoprotein, termed LGP85 (identical to LIMP II), in rat liver lysosomes. This study describes the distribution of the mRNA and protein of LGP85 in rat tissues. LGP85 protein and mRNA were detectable in all tissues when analyzed by Western and Northern blotting. The 4.2- and 2.2-kb transcripts of LGP85 were detected in all tissues. Liver and lung have the highest and lowest levels of LGP85 mRNA, respectively. A single protein band with an apparent molecular weight (M_r) of ∼85000 was detected in each tissue. The specific protein content of LGP85 in spleen was markedly higher than in other tissues. LGP85 protein is distributed in the tissues independently of LGP85 mRNA. Furthermore, there was a less significant relationship between LGP85 protein and another lysosomal membrane glycoprotein, lamp-1, in the tissue distribution (a regression coefficient of 0.086), which suggests that LGP85 may function in vivo independently of lamp-1.

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The Tumor necrosis factor (TNF)-resistant C12 cell line was established by continuous exposure of a toxic concentration of TNF to parental murine fibrosarcoma L929 cells. Introduction of the cytosilic phospholipase A2 (cPLA₂) gene to C12 cells resulted in restoration of the TNF sensitive phenotype (CPL4 cells). DNA ladder formation and nuclear condensation by TNF exposure suggested that TNF induced apoptotic cell death in L929, C12 and CPL4 cells. TNF-induced activation of transcription factor nuclear factor-κB (NF-κB) was observed in all 3 cell lines. The activation reached the maximum level at 30 min after the exposure to TNF and thereafter declined slowly. The amount of activated NF-κB in C12 cells was about twice as high as that of L929 cells with either dose of TNF tested in this study. It was found that C12 cells expressed latent NF-κB twice that of L929 cells. This abundance of latent NF-κB would provide a higher response of NF-κB in C12 cells. Pretreatment with the known potent NF-κB inhibitor pyrolidine dithiocarbamate (PDTC) profoundly suppressed the activation of NF-κB induced by TNF and potentiated TNF cytotoxity in all 3 cell lines. These results are consistent with the recently found anti-apoptotic action of NF-κB and suggest that NF-κB acts as an acquired TNF resistant factor in C12 cells.

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Zaldaride maleate (ZAL), a calmodulin inhibitor, that ameliorates secretory diarrhea in rodents, has a racemic structure. In this study, we compared the antidiarrheal and antisecretory effects of ZAL and its optical isomers, R(-)-isomer and S(+)-simer, in rats. In Ussing chamber experiments, the inhibitory action of ZAL on acetylcholine-induced ion transport in the rat colonic mucosa was equipotent for both optical isomers, with IC₅₀ values of apprpximately 3-4 μmol/l. In castor-oil-induced diarrhea, ZAL and its S(+)-isomer inhibited the incidence of diarrhea, whereas the R(-)-isomer had no effect. In 16, 16-dimethyl prostaglandin E₂-induced diarrhea, ZAL, the S(+)-isomer and the R(-)-isomer significantly ameliorated diarrhea at doses of 30, 10 and 30 mg./kg (p.o.), respectively; the ED₅₀ values were 25, 10 and above 30 mg/kg (p.o.), respectively. The pharmacokinetic parameters after administration of 30 mg/kg (p.o.) of each compound were as follows : ZAL (C_max : 378 ng/ml, AUC_0-12 : 1650 ng-h/ml); S(+)-isomer (C_max : 565 ng/ml, AUC_0-12 : 2230 ng-h/ml) and R(-)-isomer (C_max : 271 ng/ml, AUC_0-12 : 613 ng-h/ml) (mean, N=4). In conclusion, despite the fact that the antisecretory actions of ZAL and its optical isomers are the same, the antidiarrheal actions of ZAL and its S(+)-isomer are more potent than that of the R(-)-isomer. The antidiarrheal actions of ZAL and its optical isomers may be related to plasma levels.

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The present investigation was conduceted to examine whether a reversible inhibitor of monamine oxidase (MAO)-A, T-794, affects the shuttle-box escape deficit induced by transient middle cerebral artery (MCA) occulusion (MCAO). MCA-occluded and sham-operated rats (surgery on day 0) were subjected to daily shuttle-box session from day 7 to 9 (training series) and from day 13 to 15 (test series) and received twice daily administration of T-794 (10 mg/kg p.o., b.i.d.) or vehicle from the evening of day 9. In the final shuttle-box session of test series (day 15), while MCA-occluded-vehicle-treated rats showed significantly more escape failures than sham-operated-vehicle-treated rats, the failures made by MCA-occluded rats were significantly decreased by T-794 to the level of the sham-operated group. Additionally, biochemical examination was conducted after behavioral evaluation to examine possible involvement of the brain monoamine system in the observed behavioral syndrome. In occluded hemisphere of MCA-occluded rats, catecholamine levels were decreased and ratios of deaminated metabolite to corresponding monoamine were increased compared with the respective values of the sham-operated group, and these changes were reversed by T-794. Results are discussed in terms of possible relevance of the MCAO-induced escape deficit to post-stroke depression.

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It is well established that ginseng saponin has positive influences on various neural diseases, but little is known about its electrophysiological effects in the central nervous system. In this study, we examined the electrophysiological effects of ginseng saponin in rat hippocampal slices. Total saponin from ginseng root reduced the slope of fEPSPs (field excitatory postsynaptic potentials) in the CA1 area in a dose-dependent manner (9.1±5.4%, 48.4±12.1%, and 60.5±15.3% at 10, 50, and 100 μg/ml, respctively), which was reversed within 10 min of washout. Seven different ginsensosides resulted in varied degrees of fEPSPs reduction. The rank order of rduction was Rb1, Rg1>Rg2, Rh1, Rc>Rd, Re within a range of 5-64% reduction. No difference in the suppressive action between protopanaxadiol (Rb1, Rc, Rd) and protopanaxatriol (Rg1, Rg2, Re, Rh1) saponins was shown; the slope of fEPSPs was reduced by 38% and 40% on average, respectively. The possible role of γ-aminobutyric acid (GABA_A) receptor in the suppressive action of ginseng saponins was tested using whole cell patch recording in acutely isolated hippocampal neurons. Ginsenosides did not induce chloride current nor modified GABA-induced current. Also, the suppressive effect of ginsenosides on fEPSPs was still observed in the presence of the GABA_A receptor antagonist, bicuculline methiodide 50 μM. These results suggest that the suppressive effect is not attributable to regulation of GABA_A receptor activation.

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The effect of protoporphyrin (PP) on membrane fluidity was investigated by electron paramagnetic resonance spectroscopy using doxyl stearate spin probes in relation to the anti-lipidperoxidative effect of PP. PP decreased the membrane fluidity in rat liver microsomes at concentrations above 1 mM and also in phosphatidylcholine (PC)-choesterol (Cho) (100 : 8, a molar basis) liposomes. The lipid peroxidation stimulated by Fe²⁺ and L-ascorbic acid in those membrane preparations was attenuated along with the decrease in membrane fluidity by PP. Similar results were also found in Cho-rich PC (100 : 30 to 100) liposomes having less fluidity. These results suggest that the decrease in the membrane fluidity caused by PP may be involved in the antioxidative action of PP.

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Tumor necrosis factor alpha (TNFα) generates a potent cytotoxic effect, however many cancer cells are resistant to TNFα-Mediated Killing and the cause of the diferential sensitivity remains to be elucidated. In this study, we demonstrated that TNFα induced cell death in four different human colon cancer cell lines. The degree of cytotoxic effect was different in each cell line, in that HCT-15 was relatively sensitive, while DLD-1, HT-29 and WiDr were relatively resistant. TNFα induced apoptotic changes such as morphological changes, DNA fragmentation and activation of caspase-3 in HCT-15, but to a lesser degree in the others. Transcriptional expression of TNFR1(p55), as well as that of FLICE, Fas, FADD, DR3, FAF, TRADD, and RIP was similar in these cell lines, indicating that the susceptibility to TNFα-induced apoptosis may not be determined by the constitutive expression level of these factors. Interestingly, the cytotoxic effect of TNFα was well correlated with the DNA binding activity of NF-κB in the colon cancer cell lines. Further, the overexpression of a non-phosphorylated mutant form of IκBα enhanced the cytotoxicity of TNFα in the resistant cell line, DLD-1, indicating that NF-κB activity may determine the sensitivity of colon cancer cells to TNFα-induced apoptosis. Thus, our results indicate that modulation of NF-κB activity may provide a useful tool to sensitize colon cancer cells to TNFα treatment.

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We report here the antihypertensive effect of wheat germ (WG) hydrolysate and its dominant peptide, Ile-Val-Tyr (IVY), with potent angiotensin I-converting enzyme (ACE) inhibitory activity. The toxicity test of AG50W fraction purified from the WG hydrolysate and IVY in ddy mice recvealed that 1 week median lethal concentrations of AG fraction and IVY were less than 100 and 10 mg/kg, respectively. As a result of an intravenous administration test of both inihibitors in spontaneously hypertensive rat (SHR), the mean arterial blood pressure (MAP) significantly decreased with the dose; the MAP reduction of 10.3 and 19.2 mmHg was observed at a dose of 50 mg/kg of AG fraction and 5 mg/kg of IVY, respectively. In addition to this behavior, the MAP gradually decreased after the 5 mg/kg of IVY injection (time to give a maximum reduction; 8 min), and the reduction was held for 20 min. By using rat and human plasma, IVY was found to be metaboliazed by the action of aminopeptidase in plasma to form a subsequent ACE inhibitor, Val-Tyr (VY). Thus, the intake of OVY as a physiologically functional food would serve in the lowering of blood pressure (BP) by the combined depressor effect of itself and its metabolite after the absorption.

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An allergic dermatitis model was developed by repeated sensitization and challenge with antigen (ovalbumin, OA) over 7 months in mice. ddY mice were sensitized by i.p. injection of OA adsorbed in Al(OH)₃ (1 μg OA/2 mg Al(OH)₃/animal one every 3 weeks. Antigen challenge was conducted by injection of OA solution (0.1, 1 and 10 μg/site) into the skin of the hind paw instep 10 d after the respective sensitizations. At the 1st challenge, all the 3 groups showed an immediate edematous response with the peak at 30 min or 1 h after the challenge. The group challenged with the highest dose (10 μg/site) of the antigen developed a clear late-phase edema, which was observed at the 2nd challenge, increasing until the 3rd challenge, reaching a plateau at further challenges. On the other hand, such late phase edema scarcely developed in the group challenged with the lowest dose (0.1 μg/site) of the antigen. The amount of circulating specific IgE antibody increased following repeated sensitizations and challenges in all groups, but there were no significant differences in the levels among them. Mepyramine suppressed the early edema by approximately 50%, yet the late phase edema was unaffected. In conclusion, using Al(OH)₃+antigen for sensitization and an appropriate amount of antigen for challenge, reproducible biphasic edematous responses were observed long-term without desensitization. This model may by classified as an acute allergic dermatitis and can be useful for quantitatively evaluating the effects of anti-allergic drugs.

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Lactoferring (LF) from bovine colostrum was biochemically characterized as a glycyrrhizin (GL)-binding protein (gbP) in vitro. It was found that (i) bovine LF (bLF) and a synthetic bovine lactoferricin (bLFcin, the N'-terminal region of bLF at the positions 17-41) had a high affinity to a GL-affinity column; (ii) approximaterly 1.8 moles of GL were bound to a molecule of bLF with a binding constant of approx. 1.20×10⁴ M^-1 at pH 6.8; and (iii) GL, but not glucurrhetinic acid (GA), induced a conformational change of bLF. In addition, the glucuronic acid moiety of the GL molecule was found to be responsible for binding to bLF, because (i) no binding of GA and two glucoses-GA (Glc-Glc-GA) to bLF was detected; and (ii) a synthetic fluorinated GL (GlcA-GlcF-GA) and mono-glucuronyl-GA (mono-GlcA-GA) were bound significantly to bLF. A similar binding of GL to human LF (hLF) was also observed under the same experimental conditions. Data provided here suggest that (i) bLF contains plural GL-binding sites; and (ii) the specific binding of GL to bLF may modulate the physiological activity of bLF in vivo.

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The concentration of diethylcarbamazine in saliva was used to determine pharmacokinetic parameters, in comparison to plasma and urine concentrations. Six healthy adult male volunteers were administered 150 mg diethylcarbamazine with 400 ml of water. At seven different time intervals, blood, urine and saliva samples were taken, and different pharmacokinetic parameters measured.The plasma-saliva concentration ratio was calculated as 1.53 whereas the observed ratio was 3.82. The half lives, times to reach peak plasma concentration, and elimination rate constants did not show any significant difference in the different samples. The plasma peak concentration and areas under the curve were significantly (p<0.05) increased from those of the saliva. At 24 h, when diethylcarbamazine was absent in urine, the plasma and saliva concentrations were almost zero. Diethylcarbamazine is secreted in saliva, and its concentration in saliva can be used to monitor drug therapy.

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The augmentation by doxapram (DOP) of the reduction in viability and of the apoptosis of cells induced by acetaminophen (AA) was examined in mouse primary cultured hepatocytes. Loss of viability on exposure to AA and/or DOP in cultured hepatocytes was assessed by 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) assay and the apoptosis of cultured hepatocytes was detected by nuclear morphologic observation and from a ladder-like DNA fragmentation pattern. The combination of AA (5 mM) and DOP (10, 20, 50 or 100 μM) potentiated the reduction in cell viability and increased the oxidative stress. Hepatocytes exposed for 24 h to AA (5 mM) plus DOP (100 μM), showed atrophy of nuclei, including chromatin condensation and a ladder-like DNA fragmentation pattern, characteristic of apoptosis. Benzyl-oxycarbonyl-Asp-CH₂OC (O)-2, 6-dichlorobenzene (Z-Asp-CH₂-DCB, 50 μM), an inhibitor for caspases, improved the viability and ladder-like DNA fragmentation in cells exposed to DOP (200 or 500 μM) alone or AA (5 mM) plus DOP (100 μM). However, loss of viability on exposuer to a high concentration of AA (10 mM) and ladder-like DNA fragmentation were not affected by Z-Asp-CH₂-DCB. These results indicated that the synergistic increase in oxidative stress, activation of caspases and DNA fragmentation induced by DOP potentiated the hepatotoxicity of AA.

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Two flavonoids, quercetin-3-O-β-D-glucopyranoside (1) and quercetin-3, 7-di-O-β-D-glucopyranoside (2), were isolated from the leaves of Mours alba (Moraceae). These two flavonoids exerted a significant inhibitory effect on the growth of the human promyelocytic leukemia cell line (HL-60) at the concentration of 2×10^-4 M. Compound 2 also induced differentiation of the HL-60 cell line to express CD 66b and CD 14 antigens. These flavonoids exhibited significant free radical scavenging effects on 1, 1-diphenyl-2-picyl-hydrazyl radical.

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The MeOH extract of leaves of Combretum quadrangulare showed significant hepatoprotective effect on D-galactosamin (D-GalN)/lipopolysaccharide (LPS)induced experimental liver injury in mice and on D-GalN/tumor necrosis factor-α (TNF-α)-induced cell death in primary cultured mouse hepatocytes. Phytochemical investigation led to the isolation of thirty cycloartane-type triterpenes together with betulinic acid, β-stitosterol, β-sitosterol glucoside, 4 flavones (34-37), and 3 flavone C-glucosides (38-40). These compounds showed various potencies of hepatoprotective effect on D-GalN/TNF-α-induced cell death in primary cultured mouse hepatocytes. Quadrangularol B (29), methyl quadrangularate I (33), kamatakenin (34), 5, 7, 4'-trihydroxy-3, 3'-dimethoxyflavone (35), 5, 4'-dihydroxy-3, 7, 3'-trimethoxyflavone (36) and isokaempferide (37) showed strong inhibitory effect on TNF-α-induced cell death with IC₅₀ values of 34.3, 33.7, 13.3, 22.4, 13.4 and 22.8 μM, respectively, whereas clinically-used silibinin had an IC₅₀ value of 39.6 μM and glycyrrhizin showed very weak inhibitory effect. Methyl quadarangularates A (30) and N (32), norquadrangularic acid B (31) and vitexin (40) also showed potent inhibition on TNF-α-induced cell death with IC₅₀ values of 45.7, 89.3, 67.6 and 40.1 μM, respectively. The flavonoids and some of the cycloartane-type triterpenes appeared to be the hepatoprotective priniciples of the leaves of C. quadrangulare.

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After the local (subcutaneous) administration of tumor necrosis factor-alpha (TNF), two types of physiological change were clearly observed in dogs. One was a systemic change, the other was a local change at the injected site. Solution, negatively charged liposome and positively charged liposome were locally injected in dogs. Even with local administration, the increase of triglyceride in plasma and the decrease of blood pressure were the most serious after administration of the solution. These changes were typical systemic side effects of TNF. Consequently, liposomes suppressed these serious systemic side effects of TNF after a local administration. Another physiological change was irritation at the injected site. However, after administration of positively charged liposome, the lowest irritation at the injected site was observed, along with the highest local concentration of TNF. We reported the superior antitumor effect of positively charged liposomes in solution, as well as the lowest systemic circulation after an intratumor administration. Therefore, it was speculated that a positively charged liposome directly acted on the tumor cells without TNF release. These results exhibited the potency of liposomal delivery of TNF with local administration.

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We used low density lipoprotein (LDL) as a carrier of site-specific delivery of drugs to atherosclerotic lesions, prepared a dexamethasone palmitate (DP)-LDL complex, and examined the effect of the DP-LDL complex on foam cell formation of macrophages in vitro. LDL was isolated from human plasma and the DP-LDL complex was prepared by incubation in the presence of Celite 545. The complex contained about 50 mol of DP in 1 mol of LDL. When macrophages were incubated with LDL for 48 h, cholesterol ester was accumulated in the macrophages, indicating foam cell formation. This accumulation of cholesterol ester was significantly inhibited by incubation with the DP-LDL complex. The potency of the DP-LDL complex was similar to that of dexamethasone alone. The DP-LDL complex also significantly attenuated the accumulation of cholesterol ester induced by incubation with LDL prior to the incubation with the DP-LDL complex. These findings indicated that the DP-LDL complex showed similar characteristics to LDL, and the DP-LDL complex inhibited the foam cell formation of macrophages in in vitro experiments. This DP-LDL complex has a possibility as a drug-carrier complex for use in atherosclerosis.

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Microanalytical methods were developed for measuring galactosyl-β-cyclodextrin (Gal-βCD) and mannosyl (Man)-βCD in biological matrices of the rat by HPLC with pulsed amperometric detection. Then, using these methods, the absorption, distribution and excretion of intravenously and orally administered Gal-βCD and Man-βCD were determined in rats, and compared with those of glucosyl (Glc)-βCD. The pharmacokinetic behavior of Gal-βCD, Man-βCD and Glc-βCD after intravenous administration (50 mg/kg) was very similar. Within 6 h after intravenous administration, unchanged Gal-βCD and Man-βCD recovered in urine accounted for about 90% of each dose. After oral administration (500 mg/kg), 0.37% and 0.38% of Gal-βCD and Man-βCD, respectively, were excreted in urine. After intravenous and oral administration of Gal-βCD and Man-βCD, the decomposition of Gal-βCD and Man-βCD to βCD in the urine, kidney and liver was greater thn that of Glc-βCD. The sum of the molar concentrations of branced CDs and their decomposition product, βCD, in the liver at 4 h ater intravenous administration of Gal-βCD and Man-βCD was greater than that of Glc-βCD. Furthermore, the inclusion complexes of estriol and betamethasone with Gal-βCD, Man-βCD and Glc-βCD were prepared ant their absorption was evaluated after oral administration in rats. The plasma concentrations of the drugs after oral administration of drug-Gal-βCD and drug-Man-βCD complexes were the same as those after the administration of drug-Glc-βCD complexes.

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We have studied the influence of Gly-Ala-Arg peptide at the N-terminus and the oligosaccharide at Asn184 on the clearance of tissue plasminogen activator (t-PA). In order to intensify the influence of these structural features, Gln117 t-PA, which is a mutant tissue plasminogen activator (mt-PA) expressed in mouse C127 cells, was used for the investigation. It is altered to remove a high mannose type oligosaccharide by the mutation of an amino acid from Asn117 to Gln. We isolated 4 variants of Gln117 t-PA by cation-exchange chromatography, which are abbreviated as S-I, S-II, L-I and L-II. These variants originated from the heterogeneity of the peptide chains (S-chain, 527 amino acids, L-chain, 530 amino acids) and oligosaccharide (Type I, 2 oligosaccharides, Type II, 1 oligosaccharide). Pharmacolinetics of these variants were investigated after single intravenous administration to male rats at a dose of 250 μg/kg. Significant difference in pharmacokinetic parameters were observed among these variants, but there was no considerable difference in fibrin clot lysis time (FCLT) activity. Gly-Ala-Arg peptide at the N-terminus increased the CL_t, whereas the oligosaccharide at Asn184 decreased the CL_t. Moreover, the effects of the N-terminal peptide and the oligosaccharide on the CL_t were independent of each other. Our study with Gln117 t-PA revealed the role of the N-terminal peptide found in the L-chain produced during the processing of t-PA precursor.

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The pharmacokinetics of aniracetam (AP) and its main metabolites, 4-p-anisamidobutyric acid (ABA), 2-pyrrolidinone (PD) and p-anisic acid (AA), in 3 brain regions (cerebral cortex, hippocampus and thalamus) was investigated after single intravenous (i.v.) and oral administrations of AP to rats. AP, AA and PD were rapidly distributed into the 3 brain regions after i.v. administration of AP, but the amounts of AP were low. The concentrations of AP and AA in brain regions rapidly declined, whereas PD levels were higher and more sustained than those of AP and AA. ABA levels in the regions were below the detection limit. There were no significant differences in the distribution of these compounds in the 3 brain regions. The AUC_brain/AUC_plasma ratio of PD was 53-55%, in contrast to the low ratio of AP (2.4-3.2%) and AA (3.9-4.2%). On oral administration of AP, the AUC_brain/AUC_plasma ratio of PD was also higher than that of AA. When the transport of PD was tested using the in situ brain perfusion technique, it was clarified that PD was not transported across the blood-brain barrier (BBB) by a neutral amino acid carrier system. The high brain levels of PD and the low levels of AP suggest that the clinical efficacy of dosed AP may partly result from PD penetrating into the brain.

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SM-11355, cis[((1R, 2R)-1, 2-Cyclohexanediamine-N, N')bis(myristato)] platinum(II), is a lipophilic platinum complex.SM-11355 suspended in Lipiodol (SM-11355/Lipiodol) was shown to have antitumor activity against hepatic tumors after intra-hepatic arterial administration in animal models. In this study, the in vitro growth inhibitory activities of SM-11355 and ciSplatin (CDDP) following 7-d drug exposure were examined using rat ascite hepatoma AH-109A cells and various human tumor cell lines. In monolayer or suSpension cell cultures, SM-11355 did not inhibit the cell growth, whereas SM-11355/Lipiodol had dose-dependent growth inhibitory activities, as did CDDP suspended in Lipiodol (CdDP/Lipiodol). This was also the case in the colony formation assay in agarose gel. CDDP/Lipiodol released platinum compound into the culture medium rapidly, whereas SM-11355/Lipiodol released it slowly but constantly for 7 d. Furthermore, a significant amount of platinum was detected in the cells treated with CDDP/Lipiodol and SM-11355/Lipiodol. These results suggest that Lipiodol plays an important role in the in vitro cytotoxicity of SM-11355, and certain platinum compounds released from SM-11355/Lipiodol have growth inhibitory effects on these cells.

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Capillary zone electrophoretic separation of blueberry anthocyanins was studied using a Na-borate buffer containing trans-1, 2-diaminocyclohexane-N, N, N', N'-tetra acetic acid monohydrate (CyDTA) as the carrier buffer. The separation conditions were precisely examined using an aqueous extract of bilberry (wild type blueberry) as the separation sample which is rich in this type and amount of anthocyanins. Each separated peak was identified by comparing the mobility with that of anthocyanin standards after normalization against the mobility of malvidin 3-o-glucoside (Mv 3-Glc) added as an internal standard. As salt concentrations of the running buffer increased, the peak resolution was markedly improved over the whole range of separation, especially, among the fast moving components (petunidin 3-glucoside, cyanidin 3-glucoside and malvidin 3-galactoside). Inversely, the peak separtion both between petunidin 3-glucoside and peonidin 3-glucoside, and between delphinidin 3-glucoside and petunidin 3-galactoside, respectively, were decreased. The anthocyanins were, however, successfully separated by decreasing the buffer pH. Good separation of anthocyanins was finally achieved by 30 mM Na-borate (pH 8.78) containing 7.5 mM CyDTA within 10 min. Under this separation condition, anthocyanins from different blueberry sources were analyzed. The results revealed that different blueberry sources had their own patterns of anthocyanin distribution and amounts in the extracts, thus the pesent method is suitable for the quality control of anthocuanin-containing food materials.

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Effect of bisphenol A on drug-metabolizing enzyme activities by human hepatic cytochrome P450s (CYP) was investigated. We measured aminopyrine N-demethylation by eleven kinds of cDNA-expressed CYPs. CYP2C19 and CYP2B6 catalyzed most efficiently the aminopyring N-demethylation, followed by CYP2C8 and CYP2D6. Bisphenol A (1 mM) most efficiently inhibited aminopyrine N-demethylation by CYP2C8 and CYP2C19 by 82% and 85%, respectively, whereas inhibition of the activities by CYP 2B6 and 2D6 was less than 40%. Bisphenol A exhibited a noncompetitive-type inhibition of aminopyrine N-demethylase activity by CYP2C8 with K_i value of 97 μM. Additionally, we investigated the inhibitory effect of bisphenol A on CYP2C19-mediated S-mephenytoin 4-hydroxylation. Bisphenol A exhibited a mixed-type inhibition with K_i value of 113 μM. These results suggest that bisphenol A inhibits human hepatic CYP activities, especially CYP2C8 and CYP2C19.

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Inhibitory effects of thrombin inhibitors against clot-bound throumbin have been evaluated using clots prepared from human plasma as the source of clot-immobilized active enzyme, and the clot-bound thrombin has been reported to be protected from its inhibition by antithrombin. However, we found that the clot-bound thrombin was not intrinsically protected from inhibition by antithrombin, i.e., a large fraction of intially active clotbound thrombin mas inhibited by antithrombin present in human plasma time dependently, and only very small fractions (0.04-0.08%) of the thronbin retained their enzymatic activity after clot-aging. These results suggest that the extent of clot-aging determines the sensitivity of clot-bound thrombin to antithrombin, and that inhibitory effects of drugs against clot-bound thrombin in vitro must be interpreted with caution to estimate their effects in vivo.

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Internal acyl migration reactions of 1β-O-acyl glucuronides of 2-arylpropionic acids (profens) are of interest because of their possible role in covalent binding to serum proteins and consequent allergic reactions. The stereoselective degradation of 1β-O-acyl glucuronides of enantiomeric 2-phenylpropionic acids (PAs), the basic structures of profens, in phosphate buffer (pH 7.4) at 37°C, has been investigated using HPLC. Apparent first-order degradation of 1β-O-acyl glucuronide and the sequential appearance of 2-, 3- and 4-O-acyl isomers were observed for each enantiomer. Acyl migration was observed to predominate over hydrolysis as in the other profen glucuronides. All the positional isomers and anomers were characterized using NMR and HPLC-NMR. The overall degradation half-life of (R)- and (S)-PA glucuronides was 1.8 and 3.3 h, respectively. These results suggest that (R)-PA glucuronide could be more susceptible to covalent binding to proteins via acyl migration than the corresponding antipode. The lability of the (R)-diastereomer over the antipode is consistent with previous reports on other profen glucuronides. Thus, the diastereomeric PA glucuronides are considered to be the best model compounds for the computation of structural physicochemical parameters to control the stereoselective internal acyl migration of profen glucuronides because PA has the simplest chemical structure of all the profens.

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Three cytotoxic dihydroxanthone derivatives, nidulalin A(1), F380B(2), and F390C(3) were evaluated for inhibitory activity against DNA topoisomerases. Compounds 1 and 2 inhibited DNA topoisomerase II with IC₅₀ values of 2.2 μM and 16 μM, and 3 inhibited DNA topoisomerase I with an IC₅₀ value of 5.9 μM.

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Previous studies on the structural development of tumor necrosis factor α (TNF-α) production regulators derived from thalidomide (N-α-phthalimidoglutarimide) revealed that a hydrophobic substituent at the nitrogen atom of the phathalimide ring is critical for potent activity. We have designed and pepared phthalimide derivatives bearing a boron cluster, dicarba-closo-dodecaborane (carborane), which has a hydrophobic character and spherical geometry, as a novel candidate of biological response modifiers. These compounds were shown to regulate TNF-α production by HL-60 cells, as expected. The result provides a further example of the application of carborane as a hydrophobic pharmacophore of biologically active molecules.

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Two known diarylheptanoids, oregonin (1), (5S)-1, 7-bis-(3, 4-dihydroxyphenyl)-heptane-3-one-5-O-β-D-xylopyranoside and hirsutanonol (2), (5S)-1, 7-bis-(3, 4-dihydroxyphenyl)-5-hydroxyheptane-3-one isolated from the bark of Alnus hirsuta var. sibirica, showed significant inhibitory effects on 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced cyclooxygenase-2 (COX-2) expression in immortalized human breast epithelial MCDF10A cells.

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