Protective action of honokiol, administered orally, against oxidative stress in brain of mice challenged with NMDA.

Neuroprotective effect of honokiol (HK), orally administered, on oxidative damage in the brain of mice challenged with N-methyl-d-aspartic acid (NMDA) was examined. HK (1-100 mg/kg) was administered to Institute of Cancer Research (ICR) male mice through a gavage for 3 days consecutively, and on the third day, NMDA (150 mg/kg) was intraperitoneally (i.p.) administered. Administration of NMDA, causing a lethality of approximately 60%, resulted in a significant decrease of total glutathione (GSH) level and increase of thiobarbituric acid-reactive substances (TBARS) value in brain tissue. Meanwhile, oral administration of HK (> or = 3 mg/kg) for 3 days reduced the lethality (60%) in NMDA-treated group to 10% level, and alleviated the behavioral signs of NMDA neurotoxicity. Moreover, HK pretreatment restored the levels of total GSH and TBARS in the brain tissue to control levels (p<0.01). Additionally, GSH peroxidase activity in cytosolic portion of brain homogenate was also restored significantly (p<0.01), whereas GSH reductase activity was not. Separately, compared to vehicle-treated control, no significant changes in body and brain weight were observed in mice administered with HK. Based on these results, oral intake of HK is suggested to prevent oxidative stress in the brain of mice.

Neuroprotection by extract of Petasites japonicus leaves, a traditional vegetable, against oxidative stress in brain of mice challenged with kainic acid.

BACKGROUND: Reactive oxygen radicals have been implicated in the pathophysiology of many neurologic disorders and brain dysfunctions. Kainic acid has been used as a model agent for the study of neurotoxicity of various excitatory amino acids, since it induces neuronal damage through excessive production of reactive oxygen species. Petasites japonicus MAX (butterbur), cultivated as culinary vegetables in Eastern Asia, contains various kinds of phenolic compounds as well as sesquiterpenes, such as petasin. In European countries, the extracts from roots of Petasites species have been used in the therapy of headache or asthma. AIM OF THE STUDY: The objective of our study is to examine the neuroprotective action of the Petasites japonicus MAX (butterbur) extract against oxidative damage in the brain of mice treated with kainic acid. METHODS: Male ICR mice, 6-8 weeks of age, were administered orally the butanol fraction from methanol extract of Petasites japonicus (BMP) or its subfraction (BMP-I or BMP-II) for 5 consecutive days. Thirty min after the final administration, the animals were challenged s. c. with kainic acid (45 mg/kg), and neurobehavioral activities were monitored. In addition, biomarkers of oxidative stress and neuronal loss in the hippocampus for the biochemical, neurobehavioral,morphological evaluations were analyzed 2 days after the kainic acid challenge. RESULTS: During 5-day treatment with BMP or BMP-1, the body weight gain was not significantly different from that of vehicle- treated control animals. Administration of kainic acid alone induced severe epileptiform seizures, causing a lethality of approximately 50%, and injuries of pyramidal cells in the hippocampus of mice which survived the challenge. Kainic acid exposure also resulted in a remarkable decrease in total glutathione level and glutathione peroxidase activity, and an increase in the thiobarbituric acid-reactive substance (TBARS) value in brain tissues. In comparison, coadministration with BMP (400 mg/kg) reduced the 54% lethality of mice, administered with kainic acid alone, to 25 % (P <0.05). Moreover, BMP at the same dose restored the levels of reduced glutathione and TBARS to control values (P <0.05). In further studies, BMP-I (200 mg/kg) ameliorated significantly (P <0.05) the kainic acid-induced behavioral signs, such as seizure activity, and all mice administered with BMP-I (200 mg/kg) survived the kainic acid toxicity. Consistent with the above, the administration with BMP-1 remarkably attenuated the neurobehavioral signs and neuronal loss in hippocampal CA1 and CA3 regions. CONCLUSION: On the basis of these results, the butanol fraction, especially BMP-I, of Petasites japonicus MAX extract is possibly suggested to be a functional agent to prevent oxidative damage in the brain of mice.

Difference in susceptibility of tyrosine residue to oxidative lodination between a thioredoxin box region and a hormonogenic region.

Peptide fragments, isolated from proteolytic cleavage of thyroglobulin at specific sites, were examined for the iodination of tyrosine residues. The 50 kDa polypeptide, which was prepared from digestion of bovine thyroglobulin and continuous preparative SDS-PAGE, was subjected to reduction with DTT and alkylation with iodoacetic acid to generate S-carboxymethylated peptide derivative, which was further hydrolysed by endoproteinase-Asp N. Peptide products were separated by RP-HPLC, and each fraction was analyzed by LC/ ESI-MS and MALDI-MS analyses. Based on the specificity of endoproteinase Asp-N and the mass spectra data, a peptide fragment turned out to correspond to a peptide, DALGCVKCPEGSYFQ (1438-1452), characterized by the presence of a thioredoxin box (CVKC) and a tyrosine residue. In addition, another peptide fragment (1453-1465) containing a thioredoxin box (CIPC) and a tyrosine residue was also observed. However, any evidence of iodination of the tyrosine residue present in these peptides was not provided. Meanwhile, tyrosine residues in the peptides, DVEEALAGKYLAGRFA (1366-1381) and DYSGLLLAFQVFLL (1290-1303) were found to be iodinated; mono- or diiodinated tyrosine residues, characteristic of a hormogenic site, existed in both peptides. In addition, the tyrosine residue in the peptide (1218-1252), corresponding to a hormonogenic site was also iodinated. Thus, there was a sharp difference of the susceptibility to oxidative iodination between the tyrosine residue in a hormonogenic site and that in a thioredoxin region. From these results, it is suggested that polypeptide region adjacent to tyrosine residues may govern the susceptibility of tyrosine to oxidative iodination.

Oxidative inactivation of brain ecto-5'-nucleotidase by thiols/Fe2+ system.

5'-Nucleotidase, responsible for the conversion of adenosine-5'-monophosphate into adenosine, was purified from bovine brain membranes, and subjected to oxidative inactivation. The 5'-nucleotidase activity decreased slightly after the exposure to either glutathione or Fe2+. The glutathione-mediated inactivation of 5'-nucleotidase was potentiated remarkably by Fe2+, but not Cu2+, in a concentration-dependent manner. Similarly, glutathione exhibited a concentration-dependent enhancement of the Fe2+-mediated inactivation. In comparison, the glutathione/Fe2+ system was much more effective than the ascorbate/Fe2+ system in inactivating the enzyme. In support of an intermediary role of superoxide ions or H2O2 in the action of glutathione/Fe2+ system, superoxide dismutase and catalase expressed a substantial protection against the inactivation by the glutathione/Fe2+ system. Meanwhile, hydroxyl radical scavengers such as mannitol, benzoate or ethanol were incapable of preventing the inactivation, excluding the participation of extraneous hydroxyl radicals. Whereas adenosine 5'-monophosphate as substrate exhibited a modest protection against the glutathione/Fe2+ action, a remarkable protection was expressed by divalent metal ions such as Zn2+ or Mn2+. Structure-activity study with a variety of thiols indicates that the inactivating action of thiols in combination with Fe2+ resides in the free sulfhydryl group and amino group of thiols. Overall, thiols, expressing more inhibitory effect on the activity of 5'-nucleotidase, were found to be more effective in potentiating the Fe2+-mediated inactivation. Further, kinetic analyses indicate that Fe2+ and thiols inhibit the 5'-nucleotidase in a competitive or uncompetitive manner, respectively. These results suggest that ecto-5'-nucleotidase from brain membrane is one of proteins susceptible to thiols/Fe2+-catalyzed oxidation, and the oxidative inactivation may be related to the selective association of Fe2+ and thiols to the enzyme molecule.

Cu2+-catalyzed oxidative degradation of thyroglobulin.

Thyroglobulin (Tg) was subjected to metal-catalyzed oxidation, and the oxidative degradation was analyzed by SDS-polyacrylamide gel electrophoresis under reducing conditions. In contrast to no effect of hydrogen peroxide (H2O2) alone on the Tg degradation, the inclusion of Cu2+ (30 microM), in combination with 2 mM H2O2, caused a remarkable degradation of Tg, time- and concentration-dependent. The action of Cu2+ was not mimicked by Fe2+, suggesting that Tg may interact selectively with Cu2+. A similar degradation of Tg was also observed with Cu2+/ascorbate system, and the concentration of Cu2+ (5-10 microM), in combination with ascorbate, required for the effective degradation was smaller than that of Cu2+ (10-30 microM) in combination with H2O2. In support of involvement of H2O2 in the Cu2+/ascorbate action, catalase expressed a complete protection. However, hydroxyl radical scavengers such as dimethylsulfoxide or mannitol failed to prevent the oxidation of Tg whereas phenolic compounds, which can interact with Cu2+, diminished the oxidative degradation, presumably consistent with the mechanism for Cu2+-catalyzed oxidation of protein. Moreover, the amount of carbonyl groups in Tg was increased as the concentration (3-100 microM) of Cu2+ was enhanced, while the formation of acid-soluble peptides was not remarkable in the presence of Cu2+ up to 200 microM. In further studies, Tg pretreated with heat or trichloroacetic acid seemed to be somewhat resistant to Cu2+-catalyzed oxidation, implying a possible involvement of protein conformation in the susceptibility to the oxidation. Based on these observations, it is proposed that Tg could be degraded non-enzymatically by Cu2+-catalyzed oxidation.

Naphthazarin derivatives (VI): synthesis, inhibitory effect on DNA topoisomerase-I and antiproliferative activity of 2- or 6-(1-oxyiminoalkyl)-5,8-dimethoxy-1,4-naphthoquinones.

2- or 6-(1-Hydroxyiminoalkyl)-5,8-dimethoxy-1,4-naphthoquin-one (DMNQ) and 6-(1-propyloxyimino- alkyl)-DMNQ derivatives were synthesized, and their inhibitory effects on DNA topoisomerase-I (TOPO-I) and antiproliferative activities against L1210 cells were examined. In a comparison, it was found that 6-(1-hydroxyiminoalkyl)-DMNQ derivatives exhibited higher potencies in both bioactivities than 2-(1-hydroxyiminoalkyl)-DMNQ analogues, suggesting that the difference in bioactivities between two positional isomers might be due to the steric hindrance of the side chain. It is noteworthy that the optimal size of alkyl group for both bioactivities of 6-(1-hydroxyiminoalkyl)-DMNQ derivatives was pentyl to octyl (IC50, 22-29 microM) for the inhibition of TOPO-I and propyl to nonyl (ED50, 0.12-0.19 microM) for the antiproliferative activity. In addition, a similar potency of bioactivities was expressed by 6-(1-propyloxyiminoalkyl)-DMNQ derivatives, propylation products of the oximes.

Naphthazarin derivatives (IV): synthesis, inhibition of DNA topoisomerase I and cytotoxicity of 2- or 6-acyl-5,8-dimethoxy-1, 4-naphthoquinones.

Some 2- or 6-acyl-5,8-dimethoxy-1,4-naphthoquinone (DMNQ) derivatives were synthesized and evaluated for inhibition of DNA topoisomerase I and cytotoxicity against L1210 cells. Compared with 2-acyl-DMNQ derivatives, 6-acyl-DMNQ compounds, bearing a higher electrophilic quinone moiety, showed a higher potency in the inhibition of DNA topoisomerase I and the cytotoxicity, implying the possible participation of electrophilic arylation in their bioactivities. Time and temperature dependence of the enzyme inhibition suggests that the arylation occurs irreversibly. Among the 6-acyl-DMNQ derivatives, the ones possessing an acyl group of an intermediate size (C(5)-C(9)) showed higher potency in their bioactivities than other derivatives. Furthermore, for the effective inhibition of DNA topoisomerase I, the size of acyl moiety of 6-acylated derivatives seems to be limited to < 12 carbon atoms.

Potentiating effect of obacunone from Dictamnus dasycarpus on cytotoxicity of microtuble inhibitors, vincristine, vinblastine and taxol.

The limonoid triterpene, obacunone, was found to enhance the cytotoxicity of vincristine against L1210 cells by approximately 10-fold. Further, it was found that the cytotoxicity of other microtubule inhibitors such as vinblastine and taxol in drug-sensitive KB-3-1 cells as well as in multidrug-resistant KB-V1 cells was enhanced greatly in the presence of obacunone. On the other hand, there was no remarkable potentiating effect of obacunone on the cytotoxicity of other antineoplastic drugs such as adriamycin, cisplatin or 5-fluorouracil. From these results, it is implied that the potentiating action of obacunone may be limited to microtubule inhibitors.

Regulation of brain glycosylphosphatidylinositol-specific phospholipase D by natural amphiphiles.

Brain glycosylphosphatidylinositol-specific phospholipase D (GPI-PLD)-catalyzed conversion of amphiphilic form of Zn2+ -glycerophosphocholine cholinephosphodiesterase (Amp-GPC PDE) into hydrophilic form was investigated in the presence of natural amphiphiles. Monoacylglycerols enhanced considerably the conversion by GPI-PLD of Amp-GPC PDE to hydrophilic form, with the enhancing effect of monoacylglycerols being dependent on the size of acyl group (C8-C18). Whereas the maximal enhancement of GPI-PLD action was the greatest with monodecanoylglycerol, the concentration (EC50) required to achieve 50% maximal effect was the smallest for monomyristoyl- or monopalmitoylglycerol. In addition, monolaurylglycerol or its alkyl analogue, monododecylglycerol, showed a remarkable decrease in enhancing effect at high concentrations (>1 mM). Presence of double bond in acyl chain, as exemplified by monooleoylglycerol or mono-11-eicosenoin, further enhanced the conversion by GPI-PLD. Meanwhile, lysophosphatidylcholine (IC50, 25 microM) and phosphatidic acid (IC50, >100 microM), ionic amphiphiles, inhibited the GPI-PLD activity, which was determined in the presence of monooleoylglycerol as a detergent. From these results, it is suggested that the activity of GPI-PLD in vivo system may be regulated by natural amphiphiles.

Regulation and inactivation of brain phosphocholine-phosphatase activity.

Regulation of phosphocholine-hydrolyzing phosphatase (phosphocholine-phosphatase) activity, purified from bovine brain, was examined under physiological conditions. Various endogenous phosphomonoesters, which were utilized as substrate, inhibited the phosphocholine-phosphatase activity competitively (Ki, 5.5-82.0 microM); among phosphomonoesters tested, there was a similar order of capability between the binding affinity of substrate and the inhibitory potency. In addition, phosphate ions also inhibited the phosphatase activity competitively with a Ki value of approximately 167 microM. Although leucine or theophylline inhibited the phosphatase activity at pH 9.0, their inhibitory action decreased greatly at pH 7.4. The pH-Km and pH-Vm profiles indicate that ionizable amino acids are involved in substrate binding as well as catalysis, alluding that the phosphatase activity may be highly dependent on the intracellular pH. Amino acid modification study supports the existence of tyrosine, arginine or lysine residue in the active site, and the participation of tyrosine residue in the catalytic action may be suggested positively from the susceptibiliy to the action of tetranitromethane or HOI-generator. Separately, the oxidative inactivation of phosphocholine-phosphatase activity was investigated. Of oxidants tested, HOONO, HOCl, HOI and ascorbate/Cu2+ system were effective to inactivate the phosphatase activity. Noteworthy, a remarkable inactivation was accomplished by 30 microM HOCl in combination with 1 mM KI. In addition, Cu2+ (3 microM) in combination with ascorbate at concentrations as low as 0.1-0.3 mM reduced the phosphatase activity to a great extent. From these results, it is proposed that the phosphocholine-phosphatase activity may be regulated endogenously and susceptible to the various oxidant systems in vivo.

2- or 6-(1-azidoalkyl)-5,8-dimethoxy-1,4-naphthoquinone: synthesis, evaluation of cytotoxic activity, antitumor activity and inhibitory effect on DNA topoisomerase-I.

6-(1-azidoalkyl)-DMNQ derivatives compared to 2-(1-azidoalkyl)-DMNQ isomers, exhibited higher cytotoxic activity against L1210 mouse leukemia cells and stronger inhibition of DNA topoisomerase-I (TOPO-I), suggesting involvement of steric hindrance. However, similar antitumor activity against mice bearing S-180 cell was shown by 2- and 6-(1-azidoalkyl)-DMNQ derivatives.

Release of GPI-anchored Zn2+-glycerophosphocholine cholinephosphodiesterase as an amphiphilic form from bovine brain membranes by bee venom phospholipase A2.

Enzymatic release of Zn(2+)-glycerophosphocholine (GPC)cholinephosphodiesterase, as an amphiphilic form, from bovine brain membranes was examined. Of various membrane hydrolases, bee PLA2 was the most effective in the release of the GPC cholinephosphodiesterase (amphiphilic form, 63-70%) from membrane. Compared to pancreatic PLA2, bee PLA2 was more efficient in the release of GPC cholinephosphodiesterase. In pH-dependent release of GP1-anchored phosphodiesterase, there was a similar pH-release profile between PLA2-mediated release and spontaneous one, implying the involvement of membrane disruption in the PLA2 action. The PLA2-mediated release showed a limited time-dependence (until 45 min) and a limited dose dependence (up to 3 units/ml), characteristic of a receptor-type binding. An ionic binding of PLA2 to membrane may be alluded from the interfering effect of anionic phospholipids on the PLA2 action. In support of an interaction between PLA2 and membrane glycoproteins, the PLA2 action was found to be blocked by lectins, wheat germ agglutinin or concanavalin A. In combination with detergent, the PLA2-mediated release was found to be enhanced synergistically by saponin, a cholesterol-complexing agent. Meanwhile, an additive interaction between PLA2 and lysolecithin suggests that PLA2 action is independent of lysolecithin. It is suggested that the binding of PLA2 to specific sites of membranes, probably rich in GPI-anchored glycoproteins, may be related to the facilitated release of GPI-anchored proteins as amphiphilic form.

Naphthazarin derivatives (II): formation of glutathione conjugate, inhibition of DNA topoisomerase-I and cytotoxicity.

6-(1-Hydroxyalkyl)-5,8-dimethoxy-1,4-naphthoquinones, expressing a higher reactivity in conjugation with glutathione, showed a greater potency in the inhibition of DNA topoisomerase-I and the cytotoxicity against L1210 cells than 2-(1-hydroxyalkyl)-DMNQ derivatives, implying the participation of electrophilic arylation in the bioactivities. In further study 6-(1-Hydroxyalkyl)-5,8-dimethoxy-1,4-naphthoquinones with an alkyl group of shorter chain length (C2-C6) exerted a greater bioactivities than those with longer chain length(>C6).

Conversion of glycosylphosphatidylinositol (GPI)-anchored alkaline phosphatase by GPI-PLD.

Enzymatic conversion of brain glycosylphosphatidylinositol-linked alkaline phosphatase (GPI-AP), amphiphilic, was examined. When GPI-AP was incubated with glycosylphosphatidylinositol-specific phospholipase D (GPI-PLD), a negligible conversion of GPI-AP to hydrophilic form was observed. The inclusion of monoacylglycerols enhanced the enzymatic conversion, although the action of monoacylglycerols differed greatly according to the size of acyl group; the enzymatic conversion was enhanced considerably in the presence of monoacylglycerols possessing acyl group of longer chain length (C10-C18), while monoacylglycerols with acyl moiety of shorter length (C4-C8) did fail to augment the enzymatic conversion. Noteworthy, monooleoylglycerol was much more effective than the other monoacylglycerols in promoting the enzymatic conversion, indicating a beneficial role of the unsaturation in acyl chain. Meanwhile, ionic amphiphiles such as monohexadecyllysophosphatidylcholine and palmitoyl-carnitine decreased the enzymatic conversion of GPI-AP in a concentration-dependent manner, with monohexadecyllysophosphatidylcholine being more inhibitory than palmitoylcarnitine. Separately, when GPI-AP was exposed to various oxidants prior to the incubation with GPI-PLD, a remarkable decrease of the enzymatic conversion was observed with hypochlorite and peroxynitrite generators, but not H2O2. In further study, hypochlorite was found to inactivate GPI-PLD at low concentrations (3 to approximately 100 microM). From these results, it is suggested that the enzymatic conversion of GPI-AP by GPI-PLD may be regulated in vivo system.

Oxidative inactivation of brain alkaline phosphatase responsible for hydrolysis of phosphocholine.

Alkaline phosphatase, one of the enzymes responsible for the conversion of phosphocholine into choline, was purified from bovine brain membrane, where the phosphatase is bound as glycosylphosphatidylinositol-linked protein, and subjected to oxidative inactivation. The phosphatase activity, based on the hydrolysis of p-nitrophenyl phosphate and phosphocholine, decreased slightly after the exposure to H2O2. Inclusion of Cu2+ in the incubation with 1 mM H2O2 led to a rapid decrease of activity in a time- and concentration-dependent manner. In comparison, the H2O2/Cu2+ system was much more effective than the H2O2/Fe2+ system in inactivating brain phosphatase. In a further study, it was observed that the hydroxy radical scavengers mannitol, ethanol, or benzoate failed to prevent against H2O2/Cu2+-induced inactivation of the phosphatase, excluding the involvement of extraneous hydroxy radicals in metal-catalyzed oxidation. In addition, it was found that both substrates, p-nitrophenyl phosphate and phosphocholine, and an inhibitor, phosphate ion, at their saturating concentrations exhibited a remarkable, although incomplete, protection against the inactivating action of H2O2/Cu2+. A similar protection was also expressed by divalent metal ions such as Mg2+ or Mn2+. Separately, it was found that H2O2/Fe2+-induced inactivation was prevented by p-nitrophenyl phosphate or Mg2+ but not phosphate ions. Thus, it is implied that phosphocholine-hydrolyzing alkaline phosphatase in brain membrane might be one of enzymes susceptible to metal-catalyzed oxidation.

Organophosphate-induced brain injuries: delayed apoptosis mediated by nitric oxide.

The features of organophosphate-induced brain injuries were investigated. Rats were poisoned intraperitoneally with 9 mg/kg (1.8 LD(50)) of diisopropylfluorophosphate. Pyridostigmine bromide (0.1 mg/kg) and atropine methylnitrate (20 mg/kg), which are centrally inactive, were pre-treated intramuscularly to reduce the mortality and eliminate peripheral signs. Diisopropylfluorophosphate induced severe limbic seizures, and early necrotic and delayed apoptotic brain injuries. The necrotic brain injury was observed to be maximal as early as 1 h after diisopropylfluorophosphate treatment predominently in hippocampus and piriform/entorhinal cortices, showing a spongiform change (malacia) of neuropils in severe cases. In contrast, typical apoptotic (TUNEL-positive) cells started to appear at 12 h in thalamus, and a mixed type in amygdala. Separately, nitrite/nitrate content in cerebrospinal fluid was found to significantly increase after 2 h, reaching a maximal level at 6 h. Pre-treatment with l-N(G)-nitroarginine, an inhibitor of nitric oxide synthase, reduced nitrite/nitrate content and, noteworthy, attenuated only apoptotic brain injury in all four brain regions without affecting seizure intensity and necrotic injury. Taken together, the delayed apoptotic injury of brain induced by diisopropylfluorophosphate poisoning in rats might be mediated in part through nitric oxide production.

Active site of brain Zn2+-glycerophosphocholine cholinephosphodiesterase and regulation of enzyme activity.

Properties of active site of Zn2+-glycerophosphocholine cholinephosphodiesterase from ox brain were examined using substrates and inhibitors of the phosphodiesterase. The anionic binding site expressed a selectivity for a positively-charged group. Meanwhile, the glyceryl moiety-binding site appeared to be a narrow crevice of a limited size, excluding the entry of acylglycerophospholipids containing long acyl chains. While endogenous quaternary ammonium compounds such as phosphocholine, choline or carnitine inhibited the enzyme, divalent metal ions such as Co2+, Mn2+ or Zn2+ enhanced the activity by 1.5 to 2-folds. The pH dependence for the inhibition by phosphocholine or the hydrolysis of substrate implies the involvement of a basic amino acid residue with a pK value of 9.6-9.7, probably lysine, in the binding of phosphoryl group. In further support, the lysine modifiers such as trinitrobenzene sulfonic acid or diethylpyrocarbonate expressed some inactivation. The pH-rate profile indicates that an amino acid residue with a pK value of 10.2, presumably tyrosine, may participate as a nucleophile in the catalysis. This might be further supported by the inactivation of the enzyme by tyrosine modifiers such as tetranitromethane or HOI-generating system. Separately, the phosphodiesterase was observed to be susceptible to the action of hydrogen peroxide or peroxynitrite-generating system. From these results, it is implied that the phosphodiesterase may be affected by endogenous sources.

Inhibitory effect of bupleuri radix saponins on adhesion of some solid tumor cells and relation to hemolytic action: screening of 232 herbal drugs for anti-cell adhesion.

Anti-cell adhesive activity and hemolytic action of herbal drugs were investigated. Among 232 herbal drugs tested, six showed a remarkable anti-cell adhesive activity, and the extract from the roots of Bupleurum falcatum (Umbelliferae), the semen of Psorala corylifolia (Leguminosae), and the semen of Areca catechu (Palmae) showed an anti-cell adhesive action at non-cytotoxic concentrations. Saikosaponins-a, d and e, isolated from the roots of Bupleurum falcatum, exhibited a potent anti-cell adhesive activity and a strong hemolytic action. In a structure-activity relationship for both activities, it seems that a sugar moiety and an ether linkage between C-13 and C-28 are required for good bioactivities. In addition, saikosaponin d with a beta-hydroxy group at C-16 was more potent than saikosaponin a possessing an alpha-hydroxy group. Taken together, it is suggested that the mechanism for anti-cell adhesive activity of saikosaponin may resemble that for their hemolytic action.

Enzymatic release of Zn2+-glycerophosphocholine cholinephosphodiesterase from brain membranes by glycosylphosphatidylinositol-specific phospholipases and its regulation.

Enzymatic conversion of glycosylphosphatidylinositol (GPI)-linked Zn2+-glycerophosphocholine phosphodiesterase was investigated. The activity of glycosylphosphatidylinositol-specific phospholipase-D (GPI-PLD), based on the conversion of amphiphilic form of phosphodiesterase into hydrophilic form, showing an optimum pH of about pH 6.6, increased continuously until 60 min. The activity of membrane-bound GPI-PL, based on the formation of hydrophilic form of phosphodiesterase, exhibiting an optimum pH of 7.4, increased up to 30 min, and reached a plateau. Inhibition studies indicate that while GPI-PLD activity was generally sensitive to ionic bio-detergents, it was not inhibited by myristoyl glycerol, a neutral detergent. Meanwhile, the membrane-bound GPI-PL was not affected remarkably by these detergents except that myristoyl glycerol expressed a modest increase of activity of membrane bound GPI-PL. In addition, the membrane-bound GPI-PL appeared to be enhanced by by suramin or oleic acid, which strongly inhibited GPI-PLD. From this results, it is suggested that in brain there may be two phospholipases responsible for the conversion of membrane-bound GPI-anchors to hydrophilic forms, and that this conversion might be regulated by endogenous lipids.

Ascorbate-induced oxidative inactivation of Zn2+-glycerophosphocholine cholinephosphodiesterase.

Zn2+-glycerophosphocholine cholinephosphodiesterase, responsible for the conversion of glycerophosphocholine into glycerol and phosphocholine, was inactivated during incubation with ascorbic acid at 38 degrees C. The inclusion of copper ions or Fe2+ accelerated the ascorbate-induced inactivation, with Cu2+ or Cu+ being much more effective than Fe2+, suggestive of ascorbate-mediated oxidation. Dehydroascorbic acid had no effect on the phosphodiesterase, but H2O2 inactivated the enzyme in a concentration-dependent manner. Also, the enzyme was inactivated partially by a superoxide anion-generating system but not an HOCl generator. In support of involvement of H2O2 in the ascorbate action, catalase and superoxide dismutase expressed a complete and a partial protection, respectively. However, hydroxy radical scavengers such as mannitol, benzoate, or dimethyl sulfoxide were incapable of preventing the ascorbate action, excluding the participation of extraneous .OH. Although p-nitrophenylphosphocholine exhibited a modest protection against the ascorbate action, a remarkable protection was expressed by amino acids, especially by histidine. In addition, imidazole, an electron donor, showed a partial protection. Separately, when Cu2+-induced inactivation of the phosphodiesterase was compared with the ascorbate-mediated one, the protection and pH studies indicate that the mechanism for the ascorbate action is different from that for the Cu2+ action. Here, it is proposed that Zn2+-glycerophosphocholine cholinephosphodiesterase is one of brain membrane proteins susceptible to oxidative inactivation.