The hydrogen peroxide (H2O2)-generating effects of 14 flavonoids were investigated. Seven out of 14 flavonoids tested were found to generate H2O2 in an acetate buffer of pH 7.4. The H2O2-generating abilities of flavonoids decrease in the order of myricetin>baicalein>quercetin>(-)-epicatechin>(+)-catechin>fisetin=7, 8-dihydroxy flavone. This ability was observed in flavonoids with either a pyrogallol or catechol structure, and the pyrogallol-type flavonoids generated more H2O2 than the catechol-types. The amount of H2O2 generated by myricetin (pyrogallol-type flavonoid) was proportional to its concentration and to the reaction time until about 4 h. In addition, H2O2 generation by myricetin was dependent on the amount of dissolved oxygen in the buffer, and it was inhibited by the addition of superoxide dismutase.These results suggest that the flavonoids generate H2O2 by donating a hydrogen from their pyrogallol or catechol structure to oxygen, through a superoxide anion radical.It was also found that flavonoids which generated more H2O2 were more powerful antioxidants in the NADPH-dependent lipid peroxidation of rat microsomes.
Estradiol benzoate (EB) and testosterone propionate (TP) were administered to male and female Wistar rats, and their effects on the zonal expression of hepatic phenol sulfotransferase (P-ST) activities were determined at pH 5.5 and 7.4. Cytosolic fractions from periportal (PP) and perivenous (PV) hepatocytes were prepared by the dual-digitonin-pulse perfusion technique. In control rats, P-ST activities assayed at pH 5.5 and 7.4 were higher in males than in females, and were higher in the PV fraction than in the PP fraction in both sexes. P-ST activities were increased by the administration of TP in the PP and PV fractions of females, whereas the same treatment diminished the enzyme activities in both fractions of the males. EB administration gave reduced P-ST activity at pH 5.5 of both fractions, irrespective of sex, but not a marked difference at pH 7.4. Chromatofocusing of PP and PV fractions revealed the presence of P-ST isoforms eluted at approx. pH 8.0 (peak 1), 7.5 (peak 2), 7.0 (peak 3 ) and 6.0 (peak 4). In male rats, peak 3, which showed high enzyme activity at pH 5.5 in the PP and PV fractions, was markedly decreased by EB treatment, whereas in females, peak 3 was present only in the PV fraction and was not affected by EB administration. TP treatment did not show remarkable changes in P-ST peaks in the males, while peaks 2 and 3 were increased in the females. Immunoblot analysis revealed the presence of multiple P-ST isoforms which showed different immunoreactivity and electrochemical properties.
Formation of DNA breaking hydroxyfuranone and hydroxypyranone derivatives in the Maillard reaction of glucose and bovine serum albumin (BSA) under physiological conditions was investigated. A mixture of glucose and BSA was incubated at 37 °C in water or in 1 M phophate buffer (pH 7.4). The ethyl acetate/2-propanol extract of the reaction mixtures showed significant DNA breaking activity against supercoiled DNA especially in the presence of Fe(III) ion. Gas chromatography/mass spectrometry analysis of the mixture revealed the formation of DNA breaking hydroxyfuranones (HMF and DMHF) and hydroxypyranone (DDMP).
We synthesized a new affinity gel (PKSI-Toyopearl) using a selective synthetic inhibitor of plasma kallikrein (PKSI-527) as an affinity ligand, and employed it for the rapid purification of plasma kallikrein from human plasma. Human plasma activated with kaolin after acid treatment was applied to a PKSI-Toyopearl column. Adsorbed protein was eluted with 50 mM glycine-hydrochloric acid buffer (pH 3.0). Plasma kallikrein was purified 181-fold with a yield of 85% from the kaolin-activated plasma. Further purification was performed by chromatography on a DEAE-Toyopearl 650M column. Plasma kallikrein was finally purified 1720-fold with a 63% yield by these procedures. On sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis, a band was observed at approximately 88 kDa. These findings indicate that PKSI-Toyopearl is a valuable tool for the purification of plasma kallikrein from human plasma.
In response to activation of phagocytic cells and during inflammatory disorders, some proteases and very reactive oxygen species are produced. These proteases and oxidants are involved in many diseases like tissue injury or atherosclerosis. We have shown in vitro that verapamil, a calcium channel blocker, had antielastase and antioxidant properties. This drug inhibited the release of elastase by neutrophils in a dose-dependent manner when these cells were stimulated by phorbol-myristate-acetate (PMA), by N-formyl-methionyl-leucylphenylala-nine (fMLP) and by the calicium ionophore A23187 (Ca.I). In addition, verapamil inhibited superoxide anion when human neutrophils were stimulated by PMA, fMLP, dioctanoylglycerol (DiC8), Ca.I or by opsonised zymosan (OZ). Verapamil did not act by scavenging elastase or oxidants as demonstrated in cell-free models which showed no direct antielastase or antioxidant effect involved by verapamil. Superoxide anion and elastase inhibition by verapamil has been considered to the mobilization of cytosolic calcium and inhibition of protein kinase C. The results suggest that verapamil might contribute help the development and progression of atheroma where oxidants and elastase are involved.
Effects of two types of stress, restraint and footshock, on plasma glucose level, insulin secretion, and glucose uptake by the liver, heart and femoral muscle were investigated in mice. Plasma glucose level gradually increased and reached maximum at 60 min after the onset of restraint stress, while footshock stress slightly increased plasma glucose level only 15 min after the onset of the stress, and this subsequently decreased significantly. The basal plasma insulin level and glucose-induced insulin secretion significantly decreased during both the stress stimuli. Glucose uptake by the femoral muscle was slightly increased during restraint stress, and was significantly increased during footshock stress. These results suggest that the transient increase in plasma glucose level during the early period of both stress stimuli might be caused by the inhibition of insulin secretion, and subsequent decrease in plasma glucose level during the latter phase of both stress stimuli was probably due to the increase in glucose uptake by the skeletal muscle. The present study confirmed that there was a difference in stress-induced glucoregulatory changes between restraint stress and footshock stress. This difference might be due to the degree of muscle movement during the stress stimuli.
The effects of Hange-shashin-to (TJ-14) on cholera toxin-induced intestinal fluid secretion were studied to elucidate the mechanism by which this kampo medicine manifests antidiarrheal effects. TJ-14 suppressed the intestinal fluid secretion induced by cholera toxin (1 μg/rat) in a dose-dependent manner at doses between 125 and 1000 mg/kg. It also inhibited the luminal prostaglandin E2 (PGE2) level. On the other hand, serotonin (5-HT) release was not affected by TJ-14. Subcutaneous injection of indomethacin at 10 mg/kg or ondansetron at 100 μg/kg significantly suppressed intestinal secretion. The luminal PGE2 level was also inhibited by indomethacin (10 mg/kg, s.c.). TJ-14, even at 10-4 g/ml, and little effect on the phasic contration of isolated guinea pig ileum induced by 5-HT (2×10-6 g/ml), while ondansetron suppressed the phasic contraction caused by 5-HT.These results indicate that TJ-14 is useful in suppressing cholera toxin-stimulated intestinal fluid secretion, and that this effect is partially due to its suppressive action on the PGE2 level.
We examined the mechanism of the protective effect of sodium malate on cis-diamminedichloroplatinum(II) (cisplatin, CDDP)-induced nephrotoxicity in mice and obtained the following findings : 1) Sodium malate showed a maximum reduction of toxicity when it was administered at the same time as CDDP or at 30 min before the administration of CDDP; the reduction was significantly decreased when sodium malate was given after the administration of CDDP.2) It is thought that diamminoplatinum(II) malate (DPM) is produced in the body following the administration of a combination of CDDP and sodium malate. DPM showed the same antitumor effect as CDDP and produced little nephrotoxicity.3) When CDDP was combined with sodium malate, the time necessary for the elimination of platinum from the blood was prolonged, showing a intermediate value between the times for the elimination of CDDP and DPM from the blood. When the drug clearance was calculated based on this result, it was found that about 40% of the CDDP administered in converted to DPM in the blood.4) In an experiment using L-[14C] malic acid, it was shown that sodium malate is distributed in the liver and the kidney at high concentrations, but in the tumor at only a low concentraiton.5) When sodium malate was administered in combination with CDDP, the amount of platinum which accumulated in the kidney after 24 h was decreased by about 55% compared with the uncombined group, but there was no change in the amount of platinum which accumulated in the tumor.These results suggest that the sodium malate administered may be distributed rapidly in the blood and the tissue, and about 40% is bound to CDDP and converted to DPM, thus reducing the nephrotoxicity of CDDP.
The effect of diesel exhaust particles (DEP) on the hemolytic activity of human serum complement was investigated. Previous treatment of human serum with DEP extracts at 37 °C decreased the hemolytic activity of human serum complement dose dependently, to 20% of its original value. This decrease in complement activity by DEP extracts was observed with previous incubation at 37 °C but not at 4 °C. A decrease in complement activity was observed after previous incubation at 37 °C in the presence of EGTA/Mg but not in the presence of EDTA, indicating that the alternative pathway of the complement system had been activated by DEP extracts. Activaion of the complement system by DEP extracts was further demonstrated by observation of the cleavage of the third component of the complement (C3) in serum to C3b with immunoelectrophoresis using goat anti-human C3 antiserum. This cleavage of C3 was similarly observed in the presence of EGTA/Mg, indicating the activation of the alternative pathway of the complement system by DEP extracts. These results indicate that DEP can activate the alternative pathway of the complement system, resulting in a decrease in the hemolytic activity of complement and in the production of biologically active degradation products of complement proteins such as C3a. The biological significance of the activation of the complement by DEP in the alveolus is discussed in relation to the influx of neutrophiles.
The effects of preparations of Chinese medicinal prescriptions on the activities of digestive enzymes were investigated. The starch dextrinizing activity of pancreatin was inhibited by Keishi-bukuryo-gan, Sho-seiryu-to, Hachimi-jio-gan, Unsei-in and Keishi-ka-shakuyaku-to to below 40% of the control activity. Hachimi-jio-gan and Sho-seiryu-to, in particular, lowered the activity to 4% and 24% of the control activity, respectively. The protein digestive activity of pancreatin was lowered by Keishi-bukuryo-gan, Oren-gedoku-to, Ryutan-shakan-to, Tokaku-joki-to, Yokuinin-to and Hange-shashin-to, to 56 to 70% of the control activity.To investigate the effects of the preparations of Chinese medicinal prescriptions on digestive enzymes in vivo, 600 mg (185 kBq) of 125I-egg white albumin were administered orally to rats 30 min before the administration of 100 mg of Tokaku-joki-to or Oren-gedoku-to. The radioactivity which transferred to the blood was less in the animals pre-fed these prescriptions than in the control animals, indicating that the digestion of egg white albumin was delayed in the presence of the prescriptions.
Aconitum alkaloids of the C20-diterpenoid type, kobusine (1) and pseudokobusine (2), and their acetyl, benzoyl, propionyl or cinnamoyl derivatives are examined for their peripheral vaso-activities by laster-flowmetrical measurement of the cutaneous blood flow in the hind foot of mice after intravenous administration. A dose-relationship of maximally increased blood flow after the administration of either of the Aconitum alkaloids existed. Kobusine 15-acetate (4), 11-benzoate (6) and 15-benzoate (7) were significantly effective at a low dose of 1 mg/kg, whereas the other kobusine derivatives were all inactive. Alkaloid 2, alone, and the 11-acetate (14), 15-acetate (15), 15-propionate (22) and 15-cinnamoate (25) were all active at 1 mg/kg and the effect of 14 at 5 mg/kg was remarkable. The activity of 2 was abolished by esterification of the hydroxyl group at C-6. Alkaloid 15 at 5 mg/kg showed a pattern of time course of blood flow in which the increase was repidly replaced with a decrease below the basal flow, probably suggesting the effect of excessive doses. Conclusively, it is considered that the hydroxyl groups of alkaloids, especially a free OH group of 2 at C-6, are important for action on the peripheral vasculature leading to dilatation, and these results indicated that esterification of the hydroxyl group at C-15 with either acetate or benzoate may contribute to enhancement of the activity of the parent alkaloids.
A macromolecular conjugate of mitomycin C (MMC) with transferrin (TF) which possessed binding ability for TF receptor was synthesized. The conjugate (TF-MMC) was internalized into the human leukemia cell line HL60 cells and distributed into intracellular fractions, then exocytosed into an incubation medium. Although these phenomena were similar to those of TF, part of the internalizen TF-MMC was degraded to a trichloroacetic acid (TCA)-soluble fraction. Therefore, the intracellular disposition of the conjugate was analyzed kinetically. The mean time of internalization of TF-MMC (7.14 min) was longer than that of TF (5.46 min). The mean exocytosis time of TF-MMC (22.1 min) was also longer than that of TF (13.3 min). Although elongation of both the internalization and exocytosis steps was responsible for the increase in recycling time of the conjugate, the binding process to the TF receptor in the internalization stage was found to be markedly retarded. The recycling times of TF-MMC and TF were 29.2 and 18.5 min, respectively. The mean decomposition time of TF-MMC was 76.3 min. Proliferation of HL60 cells was inhibited by TF-MMC in vitro. These results indicate that the TF-MMC was internalized via a TF receptor and a part of the internalized TF-MMC was degraded, so the released MMC might represent antitumor activity. TF-MMC was demonstrated to be a useful hybrid as a receptor-mediated targeting system.
The effects of Eurycoma longifolia JACK were studied on the orientation activities of sexually experienced male rats towards receptive females (mounting, licking, anogenital sniffing), environment (exploration, raring, climbing), themselves (genital grooming, non-genital grooming) and mobility (unrestricted, restricted) after dosing them with 200, 400 and 800 mg/kg body weight twice daily for 10 d prior to the test. The results showed that E. longifolia JACK modified the orientation activities of the treated male rats in that they significantly displayed more frequent and vigorous mounting, licking and anogenital sniffing towards the receptive females, and it further intensified self orientation as indicated by the increased grooming of the genitals compared to the controls (p<0.05). In addition, rats treated with 800 mg/kg of methanol, water and butanol extracts of E. longifolia JACK continued to show confinement to a particular area of the cage (around the female), thus showing restriction in movement as compared to the controls (p<0.05). However, the treated males possessed a lack of interest in the external environment as indicated by a reduction in exploration, raring and climbing on the cage wall. Hence, the present study further supports the folk use of E. longifolia JACK as an aphrodisiac.
The urinary excretion of D-serine (D-Ser) in human, rat and dog of various ages was studied. Great amounts of D-Ser were consistently excreted in human urine throughout life. No age-dependent changes were observed in urinary D-Ser/total-Ser ratios from the newborn to the aged. D-Ser/creatinine ratios in adult human urine were found to be relatively constant in individuals. The constant excretion of D-Ser in human urine was confirmed by analyzing the consecutive 24 h urine of three volunteers. High concentrations of D-Ser and D-alanine (D-Ala) were found in adult dog urine. The urinary D-Ser concentration was high in young rats at unweaned and weaned periods, and then declined with increasing age. In contrast, the urinary D-Ala concentration was very low in suckling rats, and increased rapidly after the weaned state and then declined with increasing age. The species- and age-related excretion of D-Ser in mammalian urine is considered to be due to the differences in the renal handing of D-Ser, because plasma D-Ser concentrations among the groups were not so different. Although free D-Ser has been detected in animal foods and human colostrum, the amount is insufficient to explain the concentration of D-Ser found in urine. These results indicate that urinary D-Ser in mammals may be mainly of endogenous origin.
Short-term cultured cells of rice (Oryza sativa) were found to be capable of regeneration, in contrast to those obtained from long-term cultures. For clarification of the mechanism of regeneration, it was first necessary to distinguish protein kinase activity in long-term and short-term cultured cells; this activity was found greater in the former than latter. The activity was dependent on calcium, not phospholipid, phorbol ester or calmodulin. The apparent Mr of both Ca2+-dependent protein kinases was 32 kDa according to gel phosphorylation. Phosphoserine was identified in serine residues in phosphorylated histone III-S by phosphoamino acid analysis. A Ca2+-dependent protein kinase having a relative Mr of 32 kDa is thus shown to be possibly essential to regeneration in rice cultured cells.
Human inter-alpha-trypsin-inhibitor (ITI) is a serine proteinase inhibitor with a molecular weight of 220 kDa which consists of 3 different polypeptides. The constitutive components are 2 heavy chains (H1 and H2 chains) and 1 light chain (L chain), and its inhibitory activity is considered to be derived from this L chain. It has also been reported that this L chain is almost identical to the trypsin inhibitor (UTI) occurring in human urine. We examined the gene expression of the ITI constitutive peptides in human tissues using the reverse transcription (RT) -PCR technique. As a result, the genes of the H1 chain were found to be expressed in various tissues, particularly strongly in the liver. On the other hand, the genes of the H2 chain were found to be strongly expressed in the adrenal glands, brain, kidneys, and lungs, as well as the liver. Further, the PCR amplification product of the L chain was strongly detected not only in the liver but also in the pancreas, kidneys, lungs, stomach and testes. These results suggest the possibility that the major tissue which produces ITI is the liver, and the H chains and L chain (UTI) are produced as a component of ITI-related proteins in other tissues as well as in the liver.
We previously cloned and sequenced the Acinetobacter baumannii dat and ddc genes encoding L-2, 4-diaminobutyrate : 2-ketoglutarate 4-aminotransferase (DABA AT) and DABA decarboxylase, respectively, involved in the 1, 3-diaminopropane (DAP) production pathway. Homology searches of the gene products provided an indication that a similar gene cluster is present in the genome of Haemophilus influenzae Rd. This was first verified by detection of the corresponding enzyme activities in and the production of DAP by all H. influenzae strains examined. Both of the crude enzymes from the representative strain of H. influenzae showed catalytic properties essentially similar to the A. baumannii DABA AT and DABA DC. An Escherichia coli clone carrying the dat homolog of H. influenzae Rd showed a high level of DABA AT activity, the enzyme protein responsible being detected by immunoblot analysis. These results specify the two H. influenzae genes involved in DAP production.
The effects of fatty acids, including oleate, on the interaction between furosemide and valproic acid in sera at respective serum therapeutic concentration levels were investigated using an ultrafiltration technique. The free fraction of furosemide was significantly increased in the presence of valproic acid. Mutual displacement experiments indicated that furosemide and valproic acid share a common high affinity binding site on human serum albumin (HSA). The serum free fraction of furosemide was increased by the presence of six or more fatty acid molecules per HSA molecule. This fatty acid-induced increase in the unbound fraction of furosemide was further increased by the binding of valproic acid. However, the inhibition of furosemide binding to serum for a fatty acid-valproic acid-furosemide system is nearly the same as the additive effect of fatty acid and valproic acid on the furosemide to serum. Thus, the mechanism for the displacement of HSA-bound furosemide by valproic acid was concluded to be different from that for fatty acid-catalyzed displacement.
Recombinant glycosaminoglycan-modified urinary thrombomodulin (GAG-UTM), which partially improved the amino acid sequence of human urinary thrombomodulin (UTM), was expressed in C127 cells. GAG-UTM accelerates protein C activation by thrombin and also thrombin inhibition by antithrombin III (ATIII) in the buffer system. Both accelerating activities of GAG-UTM are more potent than those of unmodified recombinant UTM (r-UTM) without a GAG chain. As ATIII in plasma also inhibits protein C activation by a thrombin-thrombomodulin complex, we studied whether GAG-UTM accelerates protein C activation in plasma. GAG-UTM suppressed the generation of thrombin in activating plasma protein C stronger than r-UTM. By Western blot analysis using anti-protein C antibody, activated protein C was generated by GAG-UTM more than by r-UTM. From these results, the acceleration of activated protein C formation by GAG-UTM was confirmed in plasma too.
The effects of various pentamethine trinuclear cyanine dyes, each of which has three alkyl chains, on ADP/Fe2+-induced lipid peroxidation in rat liver mitochondria were examined. Although the dye having the shortest -C2H5 chains (tri-S-C2(5)) did not show any appreciable effect, the dyes having -C4H9 (tri-S-C4(5)), -C7H15 (tri-S-C7(5)), and -C12H<25> (tri-S-C12(5)) chains significantly inhibited lipid peroxidation, the most potent inhibitory effect being observed with tri-S-C7(5). The mode of antiperoxidation effect of the dyes was dependent on the length of the alkyl chains. The relatively hydrophilic dye tri-S-C4(5) was suggested to scavenge radicals more efficiently at or near the membrane surface rather than in the interior of the lipid membrane, whereas the more hydrophobic dye tri-S-C7(5) was suggested to scavenge radicals efficiently in the membrane rather than at or near the membrane surface. The hydrophilic/hydrophobic balance of the dye was found to regulate the site of action of the dyes.
Twenty-eight mild hypercholesterolemic male and female adults were orally administered psyllium seed for 3 months. After psyllium treatment, the serum total sholesterol, low-density-lipoprotein-cholesterol and atherogenic index signigicantly decreased, but levels of high-density-lipoprotein-cholesterol, triglyceride and urea nitrogen did not. To determine the parameters associated with the cholesterol-lowering effect in the subjects' backgrounds, both biochemical and hematological parameters, we statistically examined the correlation between pretreatment parameters and the absolute change of total cholesterol level. The absolute change of total cholesterol level showed a direct correlation with the triglyceride level at pretreatment (r=0.41, P=0.03) and had an inverse correlation with urea nitrogen level (r=-0.46, P=0.01) but not with the total cholesterol level (r=-0.18). The change in urea nitrogen level had an inverse correlation with the urea nitrogen level itself at pretreatment (r=-0.82, P=7×10-8) and had a direct correlation with the triglyceride level (r=0.43, P=0.02). The change in triglyceride level had an inverse correlation with the urea nitrogen level (r=-0.48, P=0.008). Furthermore, the change in total cholesterol level had direct correlations with changes in the triglyceride level (r=0.56, P=0.002) and the urea nitrogen level (r=0.51, P=0.006), but these changes in triglyceride and urea nitrogen level did not correlate significantly. These findings suggest the close association of urea nitrogen and lipid metabolism in hyperlipidemia and psyllium seed treatment.
Biodegradable intravitreal rod-shaped implants containing dexamethasone sodium m-sulfobenzoate (DMSB) were prepared from blends of poly(DL-lactic acid) (PLA) with number-average molecular weight 2000 (PLA2000) and 4000 (PLA4000). The effect of the fraction of PLA2000 on the release of DMSB from the implant was investigated after implantation in the vitreous body of rabbit eyes. After the initial burst, the drug was released slowly from the blended PLA implants with a PLA2000 fraction of below 30 wt% in normal eyes within a period of 28 d. For the implants with a higher PLA2000 fraction of over 50 wt%, the drug was released following approximately first order kinetics. In the vitrectomized eyes, the release of DMASB from the PLA2000/PLA4000 (5/5) implant was 2.5 times more rapid than in normal eyes, and the clearance of drug was also appreciable accelerated as compared with that in normal eyes.
We studied the expression in Pseudomonas aeruginosa of an erythromycin-resistance (EMr) determinant that included the mphA gene for macrolide 2'-phosphotransferase I and originated in Escherichia colo. A recombinant plasmid, pTZ3609, that consisted of the EMr determinant and a broad-host-range vector RSF1010, endowed P. aeruginosa with high-level resistance to erythromycin. Furthermore, the EMr determinant on a self-transferable plasmid, RP1, was transferred from E. coli to P. aeruginosa by conjugation.
A new microdialysis method (Lipo-MD) using a lipid emulsion as the perfusate instead of Ringer's solution as in conventional microdialysis (MD) was designed. Recovery profiles of the alkylparabens (APBs) dissolved in Ringer's solution by Lipo-MD were compared with those by MD in vitro. Recovery of APBs in the perfusate in MD decreased with the increasing lipophilicity of APBs, whereas that in Lipo-MD increased. The enhancement of the relative recovery of APBs by Lipo-MD to MD for methyl-, ethyl-, propyl-, and butylparaben were 2.03, 5.24, 77.0, and 390.7, respectively. The utility of Lipo-MD for determination of lipophilic substances to perform pharmacokinetic study was suggested.