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A highly diastereoselective synthesis of 2-amino alcohol derivatives bearing a difluoromethylphosphonothioate group at the 3-position was achieved through LiAlH(O-t-Bu)(3)-mediated reduction of the corresponding alpha-amino ketones. The phosphonothioate moiety of the product was readily converted into the corresponding phosphonate by oxidation with m-CPBA, followed by aqueous workup. The developed methods should be useful for SAR studies of SMA-7, a potent inhibitor of SMases.

Prosaposin triggers G-protein-coupled receptor (GPCR)-mediated protein kinase B (Akt)/extracellular signal-regulated kinase (ERK) phosphorylation cascades to exert its neurotrophic and myelinotrophic activity capable of preventing neural cell death and promoting neural proliferation and glial differentiation. In the present study, we investigated the down-stream neurotrophic signaling mechanism of prosaposin by which rat pheochromocytoma (PC-12) cells are protected from cell death induced by oxidative stress. When PC-12 cells were exposed to H2O2, the cells underwent abrupt shrinkage followed by apoptosis. Prosaposin treatment at as low as 1 nM protected PC-12 cells from cell death by the oxidative stress with the activation of an ERK phosphorylation cascade. Simultaneously, prosaposin blocked the oxidative stress induced-Akt phosphorylation that acts on the down-stream of caspase-3 activation. A MEK inhibitor, PD98059, or a phosphatidylinositol 3-kinase (PI3K) inhibitor, LY294002, abolished the survival effect of prosaposin on the oxidative stress-induced cell death. Furthermore, prosaposin blocked the oxidative stress-induced phosphorylations of c-Jun N-terminal kinase (JNK) and p38 stress-activated protein kinase. We further investigated the effect of prosaposin treatment on the phosphorylation of activating protein-1 (AP-1) complex components, c-Jun and activating transcription factor (ATF)-3. Western blot analysis demonstrated that prosaposin treatment at 100 ng/ml decreased the levels of c-Jun and ATF-3 induced by H2O2 stimulation. Our results suggest that prosaposin aids survival of PC-12 cells from oxidative stress not only by reducing the phosphorylation levels of JNK and p38, but also by regulating the c-Jun/AP-1 pathway.

Astrocytes are thought to be critical to neurons' surviving damage caused by ischemic stroke or other injury. Plasminogen activator inhibitor-1 is one of the active soluble factors released by astrocytes and regulates plasminogen activator-plasmin proteolytic sequence in the CNS as a serpin. In this study, we show that plasminogen activator inhibitor-1 can promote neurite outgrowth and survival of rat pheochromocytoma cells in serum-deprived conditions, and that this neuroprotective activity is correlated with enhanced activation of both extracellular signal-regulated kinases following a direct phosphorylation of nerve growth factor receptor, Trk A, and of c-Jun. Our results suggest that plasminogen activator inhibitor-1 can act as a neurotrophic factor, protecting neurons from serum deprivation-induced neuron death not only by compensating for nerve growth factor functions, but also by activating the c-Jun/activating protein-1 pathway.

A sphingomyelin analogue 2 , in which the long alkenyl chain and the phosphodiester moiety of sphingomyelin were replaced by a phenyl and an isosteric difluoromethylenephosphonic acid, was prepared to evaluate its inhibitory potency to sphingomyelinase. The analogue non-competitively inhibited the neutral sphingomyelinase in bovine brain microsomes with an IC 50 of 400 μM. The compound had the ability to suppress tumor necrosis factor α-induced apoptosis of PC-12 neurons at a low concentration of 0.1 μM.

The present study underscores a regulatory role of intracellular ceramide in astrocytes for the release of an extracellular serine protease, tissue-type plasminogen activator (t-PA), and its inhibitor, plasminogen activator inhibitor-1 (PAI-1). Treatment of cultured human astrocytes with N-acetylsphingosine, a cell-permeable short-chain ceramide analogue or daunorubicin that could increase intracellular ceramide via activation of ceramide synthase or sphingomyelin hydrolysis increased the release of t-PA and conversely decreased the PAI-1 release. Interestingly, treatment of the astrocytes with tumor necrosis factor (TNF)-alpha also increased the intracellular ceramide levels but caused the elevation of PAI-1 release without altering the t-PA release. These data suggest that the generation of ceramide in astrocytes is linked at least with the regulation of PAI-1 release. We also demonstrate that the suppression of PAI-1 release with daunorubicin accelerates the cell death of neuronally differentiated PC12 cells and suggest an antiapoptotic role of PAI-1 in the nervous system.

1. A fever-induced model in rat was created by repeated injection of interleukin 1 beta (IL-1 beta) in the cerebroventricle and the influence of fever on hepatic drug metabolism was investigated. Fever apparently decreased the content of cytochrome P450 (CYP) and the activities of NADPH-ferrihaemoprotein reductase (fp2), aminopyrine N-demethylase, aniline hydroxylase, FAD-monooxygenase, p-nitrophenol UDP-glucuronosyl-transferase and glutathione S-transferase. Immunoblot analysis of the CYP isozymes indicated that CYP2C11 and CYP3A were extensively decreased in the IL-1 beta-induced fevered rat. 2. Repeated administration (5 days) of mefenamic acid in the fevered rat could not restore the activities of fp2, aminopyrine N-demethylase and aniline hydroxylase to control levels, although their hyperthermic state had been improved. The CYP content in the mefenamic acid-treated fevered rat was also lower than that in the control. 3. These findings suggest that fever impairs the hepatic drug-metabolizing capacity (both oxidation and some conjugations) and that the fever-induced impairments are partially retained, even if the hyperthermia has been offset by the administration of antipyretics.

We have previously shown that the naturally occurring polyamines, spermidine and spermine, reverse effectively the in vitro inhibition of prolyl oligopeptidase (POPase) by its endogenous inhibitor by forming a kinetically significant complex (Soeda et al., J. Neurochem. (1986) 46, 1304-1307). In this study, we examined changes in the activities of POPase and its endogenous inhibitor and in the concentrations of polyamines during the regeneration of rat liver. POPase activity in the liver cytosol peaked 2 days after partial hepatectomy and then decreased near to control activity by 9 days, without its altered synthetic levels. Total polyamine concentrations also peaked at 2 days and remained elevated by 9 days, while cytosolic POPase inhibitor activity was minimal (56% of control) at 2 days. Treatment of the animals with a synthetic POPase inhibitor, Z-Gly-Pro-CHN2 (4 mg/kg), resulted in an obvious suppression of the liver regeneration. These results imply that the activity of POPase involved in nonlysosomal proteolytic pathway is exquisitely regulated by changes not only in its endogenous inhibitor levels but also in intracellular cationic potentials such as polyamines, and that POPase plays a crucial role for the growth and differentiation of liver cell.

Fucoidan, a sulfated poly (L-fucopyranose), is an effective anticoagulant in vitro and in vivo. In the present study, an aminated derivative of fucoidan was prepared and examined for its fibrinolytic and anticoagulant activities. The aminated derivative was more potent than native fucoidan as a stimulator of tissue plasminogen activator-induced plasma clot lysis, and its effectiveness was comparable to that of oversulfated fucoidan reported previously. Furthermore, the ability of aminated fucoidan to accelerate heparin cofactor II-mediated thrombin inhibition was 2.3 times more potent than that of native fucoidan. Aminated fucoidan effectively prevented endotoxin-induced hepatic vein thrombosis in hyperlipemic rats and decreased the elevated levels of serum cholesterol and triglyceride. The present results that the anticoagulant and antilipemic potency of fucoidan can be improved by charge modification may provide useful clues for the development of an ideal anticoagulant and antilipemic drug.

Two diabetes-inducible forms of cytochrome P-450, named P-450ST-1 and -ST-2, were purified from the liver microsomes of streptozotocin-diabetic male rats by sodium cholate solubilization, octylamino-Sepharose 4B chromatography and high-performance liquid chromatography with DEAE-5PW and hydroxyapatite columns. The purified P-450 forms gave a single band each on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, with an apparent molecular weight of 48,500 for P-450ST-1 or 48,000 for P-450ST-2. The CO-reduced spectral maxima of P-450ST-1 and -ST-2 were at 451 nm. The two cytochromes had the low-spin state of heme in the oxidized form. Both P-450ST-1 and -ST-2 catalyzed the metabolism of aniline, benzphetamine, p-nitroanisole, testosterone and aminopyrine. However, the catalytic activity of P-450ST-2 for these substrates was apparently higher than that of ST-1. Analyses of the NH2-terminal amino-acid sequence and Western immunoblot showed that P-450ST-1 and -ST-2 differed structurally from each other. The catalytic activities, molecular weights, NH2-terminal sequences and/or immunochemical properties of P-450ST-1 and -ST-2 did not agree with those of the other cytochrome P-450 forms purified from diabetic rats previously.

SummaryPlasminogen activator inhibitor-1 (PAI-1), a member of the ser-pin gene family, is the primary inhibitor of urokinase-type and tissue-type PA s.PA I-1 plays an important role in the process of peripheral tissue remodeling and fibrinolysis through the regulation of PA activity. This serpin is also produced in brain tissues and may regulate the neural protease sequence in the central nervous system (CNS), as it does in peripheral tissues. In fact, PA I-1 mRNA is up-regulated in mouse brain after stroke.The serpin activity of PA I-1 helps to prevent tissue-type PA -induced neuron death.However, we have previously found that PAI-1 has a novel biological function in the CNS: the contribution to survival of neurites on neurons. In neuronally differentiated rat pheochromocytoma (PC-12) cells, a deficiency of PA I-1 in vitro caused a significant reduction in Bcl-2 and Bcl-XL mRNAs and an increase in Bcl-XS and Bax mRNAs.The change in the balance between mRNA expressions of the anti- and pro-apoptotic Bcl-2 family proteins promoted the apoptotic sequence: cas-pase-3 activation, cytochrome c release from mitochondria and DNA fragmentation. Our results indicate that PA I-1 has an antiapoptotic role in neurons.PAI-1 prevented the disintegration of the formed neuronal networks by maintaining or promoting neuroprotective signaling through the MAPK/ ERK pathway, suggesting that the neuroprotective effect of PAI-1 is independent of its action as a protease inhibitor. This review discusses the neuroprotective effects of PA I-1 in vitro, together with the relevant data from other laboratories. Special emphasis is placed on its action on PC-12 cells.

Hepatic drug metabolism (flavin-containing monooxygenase (FMO), glutathione related enzymes, phase II conjugation reactions) and the hepatic contents of glutathione were investigated in normal rats, alloxan induced diabetic rats and streptozotocin (STZ) induced diabetic rats. The hepatic content of reduced or oxidized glutathione, the activities of glutathione related enzymes (glutathione reductase and glutathione peroxidase) and several enzymes (p-nitrophenol glucuronosyltransferase, aryl sulphotransferase I and II) involved in conjugation reactions were lower in alloxan- and STZ-induced diabetic rats than those in normal rats. In contrast, the activities of FMO and aryl sulphotransferase IV were significantly higher in alloxan- and STZ-induced diabetic rats than those in normal rats. Glutathione S-transferase (GST) activity also was remarkably higher in STZ-induced diabetic rats than that in normal rats. Insulin administered to STZ-induced diabetic rats prevented the hyperglycemia indicative of STZ-induced diabetes, but had no effect on the increased activities of GST. Another diabetogenic agent, alloxan, did not alter the activities of GST. On the other hand, the fluctuations in the enzymatic activities of FMO, UDP-glucuronosyltransferase, aryl sulphotransferase and glutathione related enzymes were restored to normal level by treatment with insulin in both diabetic rats. These results show that STZ may be directly increasing activities of GST, and not as a result of the diabetic state the diabetogenic agent induces. However, the fluctuations of the activities of FMO, glutathione related enzymes and some phase II reactions were dependent on diabetic states.

Crocus sativus L. (saffron) has been used as a spice for flavoring and coloring food preparations, and in Chinese traditional medicine as an anodyne or tranquilizer. Our previous study demonstrated that crocin, a carotenoid pigment of saffron, can suppress the serum deprivation-induced death of PC12 cells by increasing glutathione (GSH) synthesis and thus inhibiting neutral sphingomyelinase (nSMase) activity and ceramide formation. The carotenoid pigments of saffron consist of crocetin di-(beta-d-glucosyl)-ester [dicrocin], crocetin-(beta-d-gentiobiosyl)-(beta-d-glucosyl)-ester [tricrocin] and crocetin-di-(beta-d-gentiobiosyl)-ester [crocin]. Saffron also contains picrocrocin, the substance causing saffron's bitter taste. In this study, to confirm whether neuroprotective effects of saffron are caused solely by crocin, we examined the antioxidant and GSH-synthetic activities of these crocins in PC12 cells under serum-free and hypoxic conditions. Measurements of cell viability, peroxidized membrane lipids and caspase-3 activity showed that the rank order of the neuroprotective potency at a concentration of 10 muM was crocin>tricrocin>dicrocin and picrocrocin (the latter two crocins had a little or no potency). In addition, we show that among these saffron's constituents, crocin most effectively promotes mRNA expression of gamma-glutamylcysteinyl synthase (gamma-GCS), which contributes to GSH synthesis as the rate-limiting enzyme, and that the carotenoid can significantly reduce infarcted areas caused by occlusion of the middle cerebral artery (MCA) in mice.

Plasminogen activator inhibitor-1 is a serpin that regulates the activities of plasminogen activators. However, its physiological roles in the CNS are incompletely understood. We have found that plasminogen activator inhibitor-1 has a novel biological function in the CNS: the contribution to survival of neurites on neurons. PC-12 cells treated with nerve growth factor differentiated into neurons and formed a network of neurites. In a serum-free culture medium, these neurites disappeared within 24 h. The addition of plasminogen activator inhibitor-1 prevented the disintegration of the neuronal networks, while the addition of the serpin inhibitors aprotinin and antipain did not. The plasminogen activator inhibitor-1 maintained or promoted the phosphorylated state of extracellular signal-regulated kinase (ERK), but not of protein kinase B (Akt). These results are the first evidence that plasminogen activator inhibitor-1 in the CNS acts to maintain the morphology of neurites via activation of the ERK-related pathway in the neurons.

Crocin is a pharmacologically active component of Crocus sativus L. (saffron) used in traditional Chinese medicine. We report here the effects of crocin on neuronally differentiated pheochromocytoma (PC-12) cells deprived of serum/glucose. Depriving the PC-12 cells of serum/glucose caused peroxidation of their cell membrane lipids and decreased intercellular superoxide dismutase (SOD) activity. Treating the PC-12 cells with 10 microM crocin inhibited the formation of peroxidized lipids, partly restored the SOD activity, and maintained the neuron's morphology. These antioxidant effects of crocin were more effective than those of alpha-tocopherol at the same concentration. Crocin also suppressed the activation of caspase-8 caused by serum/glucose deprivation. These results together with our previous data suggest that crocin is a unique and potent antioxidant that combats oxidative stress in neurons.

Magnesium-dependent neutral sphingomyelinase (N-SMase) present in plasma membranes is an enzyme that can be activated by stress in the form of inflammatory cytokines, serum deprivation, and hypoxia. The design of small molecule N-SMase inhibitors may offer new therapies for the treatment of inflammation, ischemic injury, and cerebral infarction. Recently, we synthesized a series of difluoromethylene analogues (SMAs) of sphingomyelin. We report here the effects of SMAs on the serum/glucose deprivation-induced death of neuronally differentiated pheochromocytoma (PC-12) cells and on cerebral infarction in mice. SMAs inhibited the enhanced N-SMase activity in the serum/glucose-deprived PC-12 cells, and thereby suppressed the apoptotic sequence: ceramide formation, c-Jun N-terminal kinase phosphorylation, caspase-3 activation, and DNA fragmentation in the nuclei. Administration of SMA-7 (10 mg/kg i.v.) with IC50= 3.3 microM to mice whose middle cerebral arteries were occluded reduced significantly the size of the cerebral infarcts, compared to the control mice. These results suggest that N-SMase is a key component of the signaling pathways in cytokine- and other stress-induced cellular responses, and that inhibiting or stopping N-SMase activity is an important strategy to prevent neuron death from ischemia.

Crocin is a pharmacologically active component of Crocus sativus L. (saffron) that has been used in traditional Chinese medicine. In a previous study, we demonstrated that crocin inhibits apoptosis in PC-12 cells by affecting the function of tumor necrosis factor-alpha. In this study, we found that depriving cultured PC-12 cells of serum/glucose causes a rapid increase in cellular ceramide levels, followed by an increase in the phosphorylation of c-jun kinase (JNK). The accumulation of ceramide was found to depend on the activation of magnesium-dependent neutral sphingomyelinase (N-SMase), but not on de novo synthesis. The serum/glucose-deprived PC-12 cells also decreased the cellular levels of glutathione (GSH), which is the potent inhibitor of N-SMase. Treating the PC-12 cells with crocin prevented N-SMase activation, ceramide production, and JNK phosphorylation. We also found that the chemical can enhance the activities of GSH reductase and gamma-glutamylcysteinyl synthase (gamma-GCS), contributing to a stable GSH supply that blocks the activation of N-SMase. Thus our data suggest that crocin combats the serum/glucose deprivation-induced ceramide formation in PC-12 cells by increasing GSH levels and prevents the activation of JNK pathway, which is reported to have a role of the signaling cascade downstream ceramide for neuronal cell death.

Sphingomyelin (SM) pathway, where sphingomyelinase (SMase) hydrolyzes SM to produce ceramide, has recently been suggested to link to the signaling pathway that determines cell death. Therefore, elucidation of the mechanism by which SMase is activated and the regulation of SMase activity will be an important therapeutic strategy for various cytokine-related and ischemic diseases. We have synthesized nine difluoromethylene analogues of SM as SMase inhibitors and evaluated their inhibitory potencies. In this study, we show that the inhibitor suppresses ceramide production and cell death of PC-12 neurons that have been induced by deprivation of serum from the culture medium. The SMase inhibitor could also suppress neuronal cell death in a mouse model of brain ischemia. These results suggest a possibility that the prevention of various extracellular stress-induced cell death signalings is accomplished by inhibiting the cell membrane SMase.

Plasminogen activator inhibitor-1 (PAI-1) is a serpin that suppresses fibrinolysis by inhibiting the activity of plasminogen activator (PA). Together with PA, PAI-1 is expressed in the central nervous system and may play a role in the regulation of PA activity. Our present study has demonstrated that, in cultures of PC-12 neurons, depletion of PAI-1 from the culture medium induces disappearance of the cell's neurites and the cell death. Aprotinin and antipain, the inhibitors of PA, were not counterparts of PAI-1 in the protection of neurite disappearance. We also found that PAI-1 had the abilities to promote release of the survival factors of neurons, IL-6 and VEGF and activation of a survival serine/threonine kinase Akt. These results suggest that PAI-1 has physiological functions other than its role as PA inhibitor for the survival of neurons.

Leukotriene C 4 synthase (LTC 4 S) is considered a pivotal enzyme for generation of potent proinflammatory mediators, cysteinyl-leukotrienes (cysLTs). LTC 4 S cDNA was cloned in rat basophilic leukemia-1 (RBL-1) cells, and exhibited 84.8% and 94.5% identity with the reported human and mouse LTC 4 S cDNA sequences, respectively. Homology between the rat LTC 4 S amino acid sequence and the corresponding sequences from the other species was 86.5% and 95.3% with human and mouse sequences, respectively. Rat LTC 4 S thus showed extensive homology with both mouse and human cDNA sequences. The active enzyme as assessed by LTC 4 S activity was expressed in COS-7 cells. While RBL-1 cells after the culture for 48h in the presence of 0.1μg/ml all trans -retinoic acid (RA) exhibited 27 times higher LTC 4 S activity than control cells, Northern-blot analysis of RA-treated cells showed upregulation of LTC 4 S mRNA. Polyclonal antibody was raised against the synthesized peptide deduced from the nucleotide sequence. Thus, Western-blot analysis of RBL-1 cells treated with RA and COS-7 cells transfected with pcDNA-LTC 4 S commonly showed a band at approximately 18kDa in each solubilized enzyme solution, but either control cells did not. This cDNA probe and antibody may be useful for investigating the roles of cysLTs in various experimental models of rats.

Crocus sativus L. is used in Chinese traditional medicine to treat some disorders of the central nervous system. Crocin is an ethanol-extractable component of Crocus sativus L.; it is reported to prevent ethanol-induced impairment of learning and memory in mice. In this study, we demonstrate that crocin suppresses the effect of tumor necrosis factor (TNF)-alpha on neuronally differentiated PC-12 cells. PC-12 cells dead from exposure to TNF-alpha show apoptotic morphological changes and DNA fragmentation. These hallmark features of cell death did not appear in cells treated in the co-presence of 10 microM crocin. Moreover, crocin suppressed the TNF-alpha-induced expression of Bcl-Xs and LICE mRNAs and simultaneously restored the cytokine-induced reduction of Bcl-X(L) mRNA expression. The modulating effects of crocin on the expression of Bcl-2 family proteins led to a marked reduction of a TNF-alpha-induced release of cytochrome c from the mitochondria. Crocin also blocked the cytochrome c-induced activation of caspase-3. To learn how crocin exhibits these anti-apoptotic actions in PC-12 cells, we tested the effect of crocin on PC-12 cell death induced by daunorubicin. We found that crocin inhibited the effect of daunorubicin as well. Our findings suggest that crocin inhibits neuronal cell death induced by both internal and external apoptotic stimuli.

Plasminogen activator inhibitor-1 (PAI-1) plays an important role in the processes of peripheral tissue remodeling and fibrinolysis through the regulation of plasminogen activation. We found that cultured human astrocytes efficiently released PAI-1, and that both mRNA expression and protein release of PAI-1 were suppressed by pretreatment of the cells with daunorubicin. To examine the role of PAI-1 in the nervous system, neuronally differentiated PC-12 cells (PC-12 neurons) were maintained in a PAI-1-deficient culture medium derived from daunorubicin-pretreated astrocytes. The deficiency of PAI-1 in the medium caused a significant reduction in Bcl-2 and Bcl-XL mRNAs and an increase in Bcl-XS and Bax mRNAs in PC-12 neurons at 3 h. The changes in balance between mRNA expressions of the anti- and pro-apoptotic Bcl-2 family proteins caused caspase-3 activation following the release of cytochrome c from mitochondria. Apoptotic morphological change and DNA fragmentation were also observed in the neuronal cells at 24 h. Addition of exogenous PAI-1 protein to the inhibitor-deficient medium blocked the apoptotic changes in PC-12 neurons. However, addition of PAI-1 antibodies to control medium caused similar apoptotic changes in PC-12 neurons. During the apoptotic processes, plasminogen activator (PA) activity in the PAI-1-deficient medium was as low as the control level. The present data suggest that PAI-1 has physiological functions other than its role as PA inhibitor for the survival of neurons.

1. Adjuvant-induced arthritic (AA) rats show a striking decrease in the level of cytochrome P450, a key microsomal haemoprotein involved in electron transport and drug metabolism in the liver. In the present study, we examined the relationship between the reduction of P450 content and haem metabolism in the liver of AA rats. 2. The activities of many enzymes catalyzing the biosynthesis of haem in the liver were significantly higher in AA rats than in normal rats, whereas only coproporphyrinogen oxidase activity in AA rats was markedly lower than that in normal rats. Furthermore, the activity of haem oxygenase, a key enzyme in the haem degradative pathway, increased significantly in AA rats. In addition, the degree of increase in the activity of this enzyme was clearly higher than that in the activity of 5-aminolevulinate synthase, a key enzyme in the haem synthetic pathway. 3. These results suggest that the reduction of live P450 content in AA rats is based on the lowering of liver haem content due to the combined action of the increased haem oxygenase activity and the decreased coproporphyrinogen oxidase activity. The changes in these enzyme activities were apparently suppressed by the continuous administration of indomethacin, which improved the arthritic states of the animals.

1. Hepatic drug metabolism was investigated in normal, adjuvant-induced arthritic (AA), indomethacin-treated AA and prednisolone-treated AA rats. The contents of P450 and b5 and the activities of NADH-b5 reductase (fp2), NADPH-ferrihaemoprotein reductase, P450 mixed function oxidase, FAD-monooxygenase and several enzymes involved in conjugation were remarkably lower in AA than in normal rats. 2. Many of the decreased enzyme activities were restored to normal levels by the continuous administration (3 weeks) of indomethacin or prednisolone, which improved the arthritic states of the animals. However, the restoration of FAD-monooxygenase activity by the administration of indomethacin or prednisolone was incomplete. The P450 and b5 contents and the fp2 activity in prednisolone-treated AA rats were also significantly lower than those in normal rats. 3. These findings indicate that the ability of the liver to metabolize drugs (both oxidation and conjugation) in AA rats is greatly decreased and that a long series of the treatment of AA rats with anti-inflammatory drugs is required to restore several enzyme activities.

Oversulfated fucoidan fragments (20-40 and 40-60 kDa) were prepared, and their fibrinolytic and anticoagulant activities were compared with those of oversulfated fucoidan (100-130 kDa) reported previously [Soeda et al., Biochem. Pharmacol. 43, 1853-1858, 1992]. The results of these experiments indicated that the in vitro abilities of oversulfated fucoidan to stimulate tissue plasminogen activator (t-PA)-catalyzed plasminogen activation and to potentiate thrombin inhibition by antithrombin III or heparin cofactor II decreased with a decrease in its molecular size. However, the preventive effects of both fucoidan fragments on endotoxin-induced hepatic vein thrombosis in hyperlipemic rats were almost the same as that of oversulfated fucoidan (100-130 kDa). We also found that, unlike heparin treatment, the concentrations of serum and vascular endothelium t-PA in rats treated with oversulfated fucoidan or its fragments (1 mg each/kg/week) were maintained at normal levels. The 20-40 and 40-60 kDa fragments had an ability to decrease the elevated levels of serum cholesterol in hyperlipemic rats, whereas the 100-130 kDa fucoidan derivative did not. These results suggest that oversulfated fucoidan and its fragments have another function(s), besides the regulation of blood coagulation and fibrinolysis, and are of therapeutic benefit for the prevention of thrombus formation in hyperlipemia.

A series of fucoidan [sulfated poly( l -fucopyranose)] derivatives were prepared by chemical sulfation and desulfation, and they were tested for their abilities to stimulate tissue plasminogen activator (t-PA)-catalyzed plasminogen activation, clot lysis, and the inhibition of fibrin polymer formation. The magnitude of their activities was dependent upon the degree of sulfation. A striking feature of the sulfated fucoidan was that, unlike heparin, it stimulated t-PA-induced plasma clot lysis by protecting plasmin activity from α2-plasmin inhibitor and decreased the rate of fibrin polymer formation. The inhibition of hyaluronic acid-mediated enhancement of fibrin clot formation was also observed with the fucoidan derivative. We also showed that highly sulfated fucoidan prevents significantly endotoxin-induced hepatic vein thrombosis in the hyperlipemic rat model. The present results are the first to describe the fibrinolytic and anticoagulant activities of fucoidan, and thus may provide useful clues for the development of an ideal thrombolytic agent.

Coenzyme A (CoA), its related compounds and acylcarnitine non-competitively inhibited the activity of proline endopeptidase (PEPase) purified from rat liver cytosol. The degree of inhibition was in the order of acyl-CoA greater than CoA greater than dephospho-CoA greater than or equal to acylcarnitine. However, carnitine did not inhibit the enzyme activity. Among the compounds examined, n-decanoyl-CoA showed the highest inhibitory activity (Ki = 9 microM). These results suggest that both the acyl group and CoA contribute to the inhibition of PEPase by acyl-CoA. The abilities of n-decanoyl-CoA and its related compounds to quench the intrinsic fluorescence at 332 nm from PEPase excited at 280 nm, was used as a probe for the binding affinity of the enzyme for these compounds. The quenching of fluorescence by CoA was nearly equal to that by n-decanoyl-CoA. n-Decanoylcarnitine and carnitine were unable to quench the fluorescence. These results indicate that n-decanoyl-CoA at least binds to PEPase through its CoA portion.

A proline endopeptidase inhibitor was purified from rat brain cytosol by ion exchange chromatography on SP-Sephadex C-25 and repeated gel filtrations on Sephadex G-50. The purified inhibitor appeared to be homogeneous on polyacrylamide gel electrophoresis with and without sodium dodecyl sulfate and displayed no multiple forms. The inhibitor showed Mr=7000 by gel filtration and by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and contained 61.6% polar amino acid residues, 3.3% aromatic amino acid residues and no tryptophan. The inhibitor was highly specific for proline endopeptidase from rat brain and hog kidney, and inhibited the enzyme competitively. It did not act on proline-specific exopeptidase such as dipeptidyl aminopeptidase IV, or on usual endopeptidases such as trypsin, elastase and plasmin, or thiol proteases such as papain and ficin.

The administration of DL-3-pyridylalanine (DL-3-PA, DL-α-amino-3-pyridinepropanoic acid) or L-3-PA significantly increased brain serotonin (5-HT) concentration without affecting the other tissue 5-HT concentrations in male Wistar rats. The increase in brain 5-HT upon the administration of DL or L-3-PA was maintained for a long time (at least 96h). DL and L-3-PA decreased tryptophan pyrrolase activity in the liver and increased free tryptophan concentration in the serum. These effects found in rats given a 100mg/kg dose of DL-3-PA were not statistically different from those in rats given a 50mg/kg dose of L-3-PA. DL and L-3-PA hardly affected the activities of L-tryptophan 5-hydroxylase, 5-hydroxy-L-tryptophan decarboxylase and monoamine oxidase in the brain. D-3-PA had no effect on 5-HT concentration or tryptophan-5-HT metabolizing enzymes in rats. These results suggest that the action of DL-3-PA is due to L-3-PA, not D-3-PA.

Single doses of DL-alpha-amino-beta-(2-pyridine)propanoic acid (2-PA, 100 mg/kg) significantly decreased the holoenzyme and apoenzyme activities of rat liver tryptophan pyrrolase (TP) and increased brain tryptophan, serotonin (5-HT) and 5-hydroxyindole-3-ylacetic acid concentrations. 2-PA had no inhibitory effect on either of the enzyme activities in vitro, but its expected metabolites were effective. Single doses of DL-alpha-amino-beta-(3-pyridine)propanoic acid (3-PA, 100 mg/kg) decreased only the holoenzyme activity and elevated brain tryptophan and its metabolites levels in rats. 3-PA and its metabolite, 3-pyridylpyruvate, inhibited only the holoenzyme activity in vitro. DL-alpha-Amino-beta-(4-pyridine)propanoic acid (4-PA) caused significant changes in liver TP (holo- and apoenzyme forms) activity and brain tryptophan concentration only after repeated administration (100 mg/kg/day). 4-PA was a weak inhibitor of the holoenzyme, but its metabolites apparently inhibited the holo- and apoenzyme activities in vitro. These findings suggest that PA analogs (and/or their metabolites) increased brain tryptophan (and hence 5-HT synthesis) by directly inhibiting liver TP activity.

Our previous investigations disclosed that the heat-stable and heat-labile factors obtained from brain cytosol are required for alpha-hydroxylation and oxidation of lignoceric acid by rat brain particulate fraction. The heat-stable factor was recently found to contain glucose-6-phosphate, N- acetylaspartate , glutamate, aspartate, glutamine, inorganic phosphate and low levels of adenosine nucleotide as active components. A combination of these compounds was as effective as the crude heat-stable factor for enzymic activity. Using these compounds, we reinvestigated the requirement for the heat-labile factor. With crude heat-stable factor there was an absolute requirement for the heat-labile factor; however, with various combinations of the individual components of the heat-stable factor, some degree of activity was obtained without the heat-labile factor. When aspartate or one of its derivatives, N- acetylaspartate or oxaloacetate, was used in place of the heat-stable factor, the activity was relatively low but highly stimulated by the addition of heat-labile factor. On the other hand, higher activity was obtained when glutamate or one of its derivatives, glutamine or alpha-ketoglutarate, was used without heat-labile factor. The addition of heat-labile factor to this system did not stimulate the activity. When studying the aspartate family, we discovered that the requirement for the heat-labile factor varied in a descending order: N- acetylaspartate greater than aspartate greater than oxaloacetate. Lignoceric acid oxidation was further characterized with rat brain particulate fraction, NADPH, Mg2+, glutamate, inorganic phosphate, and AMP without heat-stable and heat-labile factors. It was found that the requirement for NADPH was also partially eliminated with glutamate but not aspartate. The effects of various inhibitors, such as inhibitors of the electron transfer system, oxidative phosphorylation, the enzymes involved in citric acid cycle, and glycolysis, suggest that the heat-stable factor is involved in producing ATP or other high energy compounds to be used for the activation of lignoceric acid. ATP added to the system in place of heat-stable factor resulted in less than one-half of the lignoceric acid oxidation.

1. A new support system has been used which provides long-term maintenance of rat liver parenchymal cells in monolayer cultures. The cells, maintained on collagen gel/polychlorinated vinylidene film, expressed aminopyrine metabolizing activity for up to 5 days. This culture system was utilized to study the metabolism of aminopyrine in the liver cells isolated from normal, alloxan- and streptozotocin-diabetic rats. 2. Aminopyrine was metabolized at a slower rate in both types of cultured diabetic rat hepatocytes than in cultured normal rat hepatocytes, as judged from higher levels of the unchanged drug in the culture medium. 3. The formation of the metabolites 4-monoaminoantipyrine, 4-acetylaminoantipyrine and 4-formylaminoantipyrine decreased in the cultured diabetic rat hepatocytes, while that of 4-aminoantipyrine was at the same levels as controls. In contrast, 3-hydroxymethyl-2-methyl-4-dimethylamino-1-phenyl-3-pyrazolin-5-on e (AM-CH2OH) formation in the cultured diabetic rat hepatocytes increased over control value. These findings agree with in vivo results which have been reported by the authors. 4. The increase in AM-CH2OH was prevented by insulin in a dose-dependent manner. However, insulin did not affect the formation of other metabolites. These findings indicate that the amount of cytochrome P-450 isozyme involved in the oxidation of 3-methyl group may be regulated by insulin. 5. The present results, indicate that this primary culture system is a useful tool for the study of drug metabolism in diabetes.

1. The metabolism of aminopyrine has been investigated in normal, alloxan- and streptozotocin (STZ)-diabetic rats. The drug was administered i.p. and the serum concentrations of the unchanged aminopyrine and its main metabolites were measured using h.p.l.c. 2. Aminopyrine was metabolized at a slower rate in both diabetic rats, as judged from higher serum levels of the unchanged drug. Pharmacokinetic studies of aminopyrine in diabetic rats also showed a decrease in serum clearance of the drug and an increase in its serum half-life. 3. The serum concentrations of the metabolites 4-monomethylaminoantipyrine, 4-acetylaminoantipyrine and 4-formylaminoantipyrine decreased in diabetic rats. In contrast, serum levels of 3-hydroxymethyl-2-methyl-4-dimethylamino-1-phenyl-3-pyrazolin-5-on e increased over control values. Serum concentrations of 4-aminoantipyrine remained unaltered by the induction of diabetes. 4. The magnitudes of changes in serum levels of these metabolites were larger in alloxan-diabetes than in STZ-diabetes. 5. Additional support for changed metabolism of aminopyrine was obtained from the investigation of microsomal preparations from diabetic and normal rats. 6. These findings indicate that it is important to use intact animals for evaluation of the metabolism of drugs in pathological states.

The conversion of lignoceric acid to cerebronic acid, ceramides, cerebrosides, and glutamic acid is catalyzed by a rat brain particulate preparation. The heat-stable factor, prepared from calf cerebellum, together with the heat-labile factor, a pyridine nucleotide, and Mg2+ are essential to all of these metabolic pathways. Our previous work showed that the heat-stable factor is composed of at least two components, HSF-1 and HSF-2, and identified HSF-2 as d-glucose-6-phosphate. In the current investigation, HSF-1 was further purified and found to be N-acetyl-l-aspartic acid. In addition, it was discovered that a third component, HSF-3, is also required for heat-stable factor activity. A reconstituted system composed of N-acetylaspartic acid, glucose-6-phosphate, and HSF-3 fully replaced the heat-stable factor essential for the conversion of lignoceric acid to cerebronic acid and glutamic acid. The reconstituted heat-stable factor did not show the initial time lag always observed with the crude heat-stable factor.

The in vitro effects of polyamines on the activity of proline endopeptidase (PEPase) in rat brain cytosol, which contains an endogenous PEPase inhibitor, have been studied. Of the three amines tested (spermine, spermidine, and putrescine), spermine and spermidine markedly enhanced the enzyme activity in brain cytosol. At 6.25 mM spermine or 25 mM spermidine, a 13- or 14-fold enhancement of the enzyme activity was observed. When Mg2+ was used, an approximately fourfold enhancement of the enzyme activity was observed at 50 mM. The enhancement produced by spermine or spermidine was unaffected by Mg2+ up to 50 mM. The activity of purified PEPase was only slightly affected by each polyamine, but it was inhibited 50% by 50 mM Mg2+. On the other hand, 50% inhibition of the enzyme produced by the purified PEPase inhibitor (Mr 7,000: Ki 0.67 mM) was completely restored by addition of 0.7 mM spermine, 3.5 mM spermidine, or 28 mM putrescine. This restoration of inhibition by polyamines was reversed by increasing the inhibitor concentration. These data suggest that polyamines effectively reverse the inhibition of PEPase by its endogenous inhibitor by the reversible formation of a kinetically significant complex. The possible functions of polyamines in the regulation of PEPase in vivo are discussed.

1) Single administration of tryptophan (25-200 mg / kg) to Wistar male rats resulted in a dose-related increase in liver tryptophan pyrrolase (TP) activity. The increase in brain serotonin (5-HT) concentration was not proportional to the dose given, and brain 5-HT concentration rapidly fell to the control (saline-treated) level or below. 2) The tryptophan-induced increase in liver TP activity was clearly prevented by DL-3-pyridylalanine (3-PA, 100 mg/kg). Serum free tryptophan and brain 5-HT concentrations increased more markedly and maintained higher levels after the combined administration of tryptophan with 3-PA than after a single administration of tryptophan. 3) Although allopurinol (20 mg/kg) reduced liver TP activity under control and tryptophan-treated conditions, it did not increase serum free tryptophan and brain 5-HT concentrations. 4) Nicotinamide also failed to alter the catabolism of tryptophan and the concentration of brain 5-HT in control or tryptophan-treated rats. 5) These data suggest that 3-PA would increase the therapeutic effect of tryptophan given to treat depressive illness, but that allopurinol and nicotinamide would not be suitable drugs for this purpose.