Role of STAT3/5 and Bcl-2/xL in 2-methoxyestradiol-induced endoreduplication of nasopharyngeal carcinoma cells.

2-methoxyestradiol (2ME2), an endogenous metabolite of 17-ß-estradiol, has been shown to induce apoptosis and cell cycle arrest in various tumor models. We have previously shown that 2ME2 induced endoreduplication in a well-differentiated nasopharyngeal carcinoma (NPC) HK-1 and a poorly differentiated C666-1 cell line. In the present study, we studied the survival factors involved in 2ME2-induced endoreduplicating NPC cells. In the HK-1 cells, knockdown of BcL-xL expression by siRNA resulted in the reduction of endoreduplication and an increase in the percentage of apoptosis. Further mechanistic study revealed that 2ME2 enhanced the expression of the phosphorylated form of STAT5 (p-STAT5-Y694), but not p-STAT3 (Y705) and p-STAT3 (S727), in the nucleus of HK-1 cells. Pre-treatment of cells with JAK/STAT inhibitor AG490 and STAT5 inhibitor resulted not only in the reduced expression of Bcl-xL, but also reduced the percentage of endoreduplicating cells. In contrast, 2ME2 enhanced the expression of p-STAT3 in the poorly differentiated C666-1 cells. Pharmacological inhibition of STAT3 or Bcl-2/xL resulted in a decrease in endoreduplication of C666-1 cells. Taken together, the expression of p-STAT5 and p-STAT3 was upregulated in 2ME2-induced endoreduplicating HK-1 and C666-1 cells, respectively. Combination of 2ME2 with Bcl-2/xL inhibitor is a novel strategy to reduce the formation of endoreduplicating cells during chemotherapeutic treatment of NPC. © 2011 Wiley Periodicals, Inc.

Ginsenoside-Rb1 promotes adipogenesis through regulation of PPARγ and microRNA-27b.

Ginsenoside-Rb1 (Rb1), one of the bioactive components in ginseng extract, is recently reported to be able to promote adipogenesis and peroxisome proliferator-activated receptor gamma (PPARγ) expression. Meanwhile, microRNA-27b (miR-27b) is also identified to regulate adipogenesis by targeting PPARγ2. In the present study, we attempted to link up the Rb1-promoted adipogenesis with PPARγ binding and miR-27b regulation. First, we demonstrated that GW9662, an antagonist of PPARγ, could block Rb1-induced 3T3-L1 differentiation with little toxicity towards cell proliferation. Then, expression levels for both of miR-27b and its primary transcript, pri-mir-27b, were found to decrease upon Rb1 treatment. Again, GW9662 could attenuate the inhibitory effect of Rb1 on both miR-27 and pri-mir-27b expression. Since Rb1 was demonstrated to have binding activity towards PPARγ, we thus speculate that Rb1 may act though PPARγ to downregulate mir-27b gene transcription and mature miR-27b activity, which in turn promotes PPARγ expression and adipogenesis. Enhancement on adipogenesis of adipose tissues is expected to prevent lipotoxicty in nonadipose tissues. Our data may give a better illustration to explain the antidiabetic effect of Rb1 and provide a hint on treatment of lipid related metabolic diseases in the future.

Role of mitogen-activated protein kinase in Zn-BC-AM PDT-induced apoptosis in nasopharyngeal carcinoma cells.

Photodynamic therapy (PDT) with a recently developed photosensitizer Zn-BC-AM was found to effectively induce apoptosis in a well-differentiated nasopharyngeal carcinoma (NPC) HK-1 cell line. Sustained activation of p38 mitogen-activated protein kinase (MAPK) and c-jun N-terminal kinase (JNK) as well as a transient increase in activation of extracellular signal-regulated kinase (ERK) were observed immediately after Zn-BC-AM PDT. A commonly used p38 MAPK/JNK pharmacological inhibitor PD169316 was found to reduce PDT-induced apoptosis of HK-1 cells. PD169316 also prevented the loss of Bcl-2 and Bcl-xL in PDT-treated HK-1 cells. However, inhibition of JNK with SP600125 had no effect on Zn-BC-AM PDT-induced apoptosis while inhibition of ERK with PD98059 or p38 MAPK with SB203580 significantly increased Zn-BC-AM PDT-induced apoptosis. Further study showed that knockdown of the p38beta isoform with siRNA also increased Zn-BC-AM PDT-induced apoptosis, indicating that the anti-apoptotic effect of PD169316 in PDT-treated HK-1 cells was probably independent of p38 MAPK or JNK activation. Taken together, the results suggest that inhibition of p38beta and ERK may enhance the therapeutic efficacy of Zn-BC-AM PDT on NPC cells. It should be noted that data only based on the use of PD169316 should be interpreted in caution.

Angiomodulatory and neurological effects of ginsenosides.

Panax ginseng C.A. Meyer, one of the most popular and valued herbs, has been used extensively in traditional Chinese medicine for thousands of years. More than thirty ginsenosides, the pharmacologically active ingredients in ginseng, have been identified with various sugar moieties attached at the C-3, C-6 and C-20 positions of the steroidal skeleton. We herein review the current literature on the pharmacological effects of ginsenosides on the modulation of angiogenesis, dysregulations of which contribute towards many pathological conditions. Regarding the adaptogenic property of ginseng, the effects of ginsenosides on central nervous system are also discussed. Recent researches have pointed to the steroid hormone receptors as the target molecules to elicit the diverse cellular and physiological activities of ginseng. We believe that understanding the interaction between ginsenosides and various steroid hormone receptors may provide clues to unravel the secret of ginseng.

Neuroprotective effects of ginsenoside-Rg1 in primary nigral neurons against rotenone toxicity.

Ginsenoside-Rg1, the pharmacologically active component isolated from ginseng, demonstrated neuroprotective effects on primary cultured rat nigral neurons against rotenone toxicity. Rotenone, a common household pesticide known for its specific and irreversible mitochondria complex I inhibition, has been suggested to be the causal agent of Parkinson's disease (PD) by inducing degeneration of cells in the substantial nigra. The present study demonstrated that co-treatment of rotenone and Rg1 could reduce rotenone-induced cell death by 58% (SEM=+/-5.60; N=3). Rotenone-induced mitochondria membrane potential (MMP, DeltaPsim) depletion was restored and elevated by at least 38% (SEM=+/-2.15; N=3) by Rg1. In addition, Rg1 prevented cytochrome c release from the mitochrondrial membrane and increased the phosphorylation inhibition of the pro-apoptotic protein Bad through activation of the PI3K/Akt pathway. The protective effects of Rg1 was blocked by glucocorticoid receptor antagonist RU486, indicating that the action of Rg1 is mediated through glucocorticoid receptor (GR). In conclusion, Rg1 inhibits the mitochondrial apoptotic pathway and increases the survival chance of the primary cultured nigral neurons against rotenone toxicity. Thus, Rg1 and its related compounds may be developed as protective agents against neurodegenerative diseases induced by mitochondrial toxins.

Analysis of the action of euxanthone, a plant-derived compound that stimulates neurite outgrowth.

We have investigated the neurite growth-stimulating properties of euxanthone, a xanthone derivative isolated from the Chinese medicinal plant Polygala caudata. Euxanthone was shown to exert a marked stimulatory action on neurite outgrowth from chick embryo dorsal root ganglia explanted in collagen gels, in the absence of added neurotrophins. It was also shown to promote cell survival in explanted chick embryo ganglia, and to stimulate neurite outgrowth from isolated adult rat primary sensory neurons in vitro. The further finding that euxanthone stimulates neurite outgrowth from explants of chick embryo retina and ventral spinal cord suggests an action on signaling pathways downstream of neuronal receptors for specific neurotrophic factors. Consistent with this, euxanthone did not promote neurite outgrowth from non-transfected PC12 cells, or from PC12 cells transfected with TrkB or TrkC, under conditions in which these cells extended neurites in response to, respectively, the neurotrophins nerve growth factor, brain-derived neurotrophic factor and neurotrophin 3. Western blot analysis of euxanthone-stimulated dorsal root ganglion explants showed that expression of phospho-mitogen-activated protein (MAP) kinase was up-regulated after 1 h of euxanthone-treatment. Inhibition of the MAP kinase pathway using PD98059, a specific inhibitor of MAP kinase kinase, blocked all euxanthone-stimulated neurite outgrowth. However, analysis of phospho-Akt expression indicated that the phosphatidylinositol-3 kinase-Akt pathway, another major signaling pathway engaged by neurotrophins, is not significantly activated by euxanthone. These results suggest that euxanthone promotes neurite outgrowth by selectively activating the MAP kinase pathway.

Ginsenoside Rb1 inhibits tube-like structure formation of endothelial cells by regulating pigment epithelium-derived factor through the oestrogen beta receptor.

BACKGROUND AND PURPOSE: Angiogenesis is a crucial step in tumour growth and metastasis. Ginsenoside-Rb1 (Rb1), the major active constituent of ginseng, potently inhibits angiogenesis in vivo and in vitro. However, the underlying mechanism remains unknown. We hypothesized that the potent anti-angiogenic protein, pigment epithelium-derived factor (PEDF), is involved in regulating the anti-angiogenic effects of Rb1. EXPERIMENTAL APPROACHES: Rb1-induced PEDF was determined by real-time PCR and western blot analysis. The anti-angiogenic effects of Rb1 were demonstrated using endothelial cell tube formation assay. Competitive ligand-binding and reporter gene assays were employed to indicate the interaction between Rb1 and the oestrogen receptor (ER). KEY RESULTS: Rb1 significantly increased the transcription, protein expression and secretion of PEDF. Targeted inhibition of PEDF completely prevented Rb1-induced inhibition of endothelial tube formation, suggesting that the anti-angiogenic effect of Rb1 was PEDF specific. Interestingly, the activation of PEDF occurred via a genomic pathway of ERbeta. Competitive ligand-binding assays indicated that Rb1 is a specific agonist of ERbeta, but not ERalpha. Rb1 effectively recruited transcriptional activators and activated an oestrogen-responsive reporter gene. Furthermore, Rb1-mediated PEDF activation and the subsequent inhibition of tube formation were blocked by the ER antagonist ICI 182,780 or transfection of ERbeta siRNA, indicating ERbeta dependence. CONCLUSIONS AND IMPLICATIONS: Here we show for the first time that the Rb1 suppressed the formation of endothelial tube-like structures through modulation of PEDF via ERbeta. These findings demonstrate a novel mechanism of the action of this ginsenoside that may have value in anti-cancer and anti-angiogenesis therapy.

Bifunctional modulating effects of an indigo dimer (bisindigotin) to CYP1A1 induction in H4IIE cells.

In this study, we measured and characterized the bifunctional effects of a newly identified natural compound-bisindigotin (SLY-1), isolated from leaf extracts of Isatis indigotica, to CYP1A1/EROD activities in H4IIE cells. The compound, SLY-1 (1muM) elicited a transitory and significant induction of CYP1A1 RNA/protein levels and EROD activities in the cells. Maximum levels of CYP1A1 expression and EROD induction were attained at 8 and 12h of post-treatment, respectively. Thereafter the induction decreased significantly. Similar profile of CYP1A2 and CYP1B1 mRNA induction was observed. In contrast TCDD elicited CYP1A1/EROD induction was persistent. The transitory effect by SLY-1 is most likely due to the clearance of SLY-1 by cellular metabolism. Taken together the observation indicated that SLY-1 is an Ah receptor agonist for CYP1A1/CYP1A2/CYP1B1/EROD induction. Interestingly in the TCDD/SLY-1 cotreatment study, although synergistic effects on CYP1A1 expression and EROD induction were observed at 4-8h, significant inhibitory effects to TCDD induced CYP1A1 protein and EROD activity were detected at 12-24h of post-treatment. Because there was no significant reduction of CYP1A1, CYP1A2 or CYP1B1 transcript levels between TCDD- and TCDD/SLY-1 treated cells, the data pointed to the translational and/or post-translational inhibitory effect. The cellular signal transduction system may be modulated following exposure to SLY-1. To investigate the possible mechanisms involved, various specific kinase inhibitors or activators (chelerythrin, PD98059, U0126, ZM336372, SB202190, PKA inhibitor PKI (6-22) amide, and dbcAMP) were used for the assessment. Chelerythrine, PD98059 or dbcAMP treatment in TCDD induced cells showed significant inhibitory effects on CYP1A1 mRNA/protein expressions and EROD activities. U0126 had no observable EROD inhibitory effect. ZM336372 or SB202190 showed inhibition only at EROD activities. The results indicated that the SLY-1 inhibitory effect was possibly not mediated by the cAMP/PKA, PKC or MEK pathways. Nevertheless our results indicate that SLY-1 is not only an inducer of the CYP1A1 system, but also a potent inhibitor of CYP1A1 enzyme.

Apoptosis and expression of cytokines triggered by pyropheophorbide-a methyl ester-mediated photodynamic therapy in nasopharyngeal carcinoma cells.

The photodynamic properties of pyropheophorbide-a methyl ester (MPPa), a semi-synthetic photosensitizer derived from chlorophyll a, were evaluated in a human nasopharyngeal carcinoma HONE-1 cell line. MPPa was non-toxic to the HONE-1. At the concentrations of 0.5-2µM, MPPa-mediated a drug dose-dependent photocytotoxicity in the HONE-1 cells. Confocal microscopy revealed a subcellular localization of MPPa in mitochondria and the Golgi apparatus. MPPa PDT-induced apoptosis was associated with the collapse of mitochondrial membrane potential, release of cytochrome c, the up-regulation of endoplasmic reticulum (ER) stress proteins (calnexin, Grp 94 and Grp78), and the activation of caspases-3 and -9. The photocytotoxicity was reduced by the corresponding specific caspase inhibitors. MPPa PDT-treated HONE-1 cells also up-regulated the gene expression of pro-inflammatory cytokines (IL-1ß, IL-6, and TNF-α) and beta-chemokines (MIP-1ß, MPIF-1, and MPIF-2). These results suggest that the MPPa may be developed as a chlorophyll-based photosensitizer for the treatment of nasopharyngeal carcinoma.

Comparative Analysis of Proteins with Stimulating Activity on Ovarian Estradiol Biosynthesis from Four Different Dioscorea Species in vitro Using Both Phenotypic and Target-based Approaches: Implication for Treating Menopause.

Rhizomes of Dioscorea species are traditionally used for relieving menopausal syndromes in Chinese medicine. The estrogen-stimulating bioactive principles have been demonstrated in our previous study. In this study, the estrogen-stimulating effects of proteins isolated from four Dioscorea species [D. alata L. (DA), D. zingiberensis C.H. Wright (DH), D. collettii var. hypoglauca (Palib.) S.J. Pei & C.T. Ting (DH), and D. oppositifolia L. (DO)] have been investigated and compared. Microscopic authentication of four Dioscorea species was performed by using paraffin and powder sections of the rhizomes. The potential bioactive proteins of four Dioscorea species have been rapidly isolated by using a DOI-antibody affinity column chromatography on immobilized antibodies against on estradiol-stimulating protein from DO (DOI), and their bioactivity has been rapidly confirmed and compared by phenotypic (i.e., estradiol-stimulating effect) and target-based (i.e., STAR, aromatase, estrogen receptors) screening approaches. The estrogen-stimulating activity of bioactive proteins from DO is the highest. In addition, bioactive proteins from DO upregulated the estradiol-metabolizing enzymes (aromatase and steroidogenic acute regulatory protein). Meanwhile, bioactive proteins from DA, DH and DO upregulated estrogen receptor ß (ERß). All bioactive proteins did not change the expression of estrogen receptor ß (ERα). The estrogen-stimulating bioactive proteins isolated from DO increased biosynthesis of estradiol and upregulated the protein expression of aromatase, steroidogenic acute regulatory protein, and ERß. The results scientifically support the traditional use of DO in Chinese medicine for relieving menopausal syndrome. Besides, proteins from DA and DZ could also upregulate the translational levels of ERß, and potentially reducing the risk of ovarian cancer, which also support the clinical use of them for treating female aging disorder. Graphical Abstract Comparative Analysis of DOI-like Proteins with Stimulating Activity on Ovarian Estradiol Biosynthesis from Four Different Dioscorea Species in vitro.

Depletion of glutamate GluR2 receptor-containing neurons in the rat neostriatum by specific immunotoxin.

In the present study, a novel GluR2 receptor-specific immunotoxin was produced. The immunotoxin was produced by conjugation of molecules of trichosanthin, a ribosome inactivating protein, with goat anti-mouse immunoglobulin molecules. The secondary antibody was then combined with a commercially available GluR2 specific primary antibody to form an immunotoxin. The immunotoxins were unilaterally injected either into the neostriatum or into the lateral ventricle of rats. After one week, ipsilateral turning movements were observed after apomorphine treatments in those animals injected by the striatal route. In perfuse-fixed sections of the neostriatum, immunoreactivity for GluR2 was found to decrease in the striatal-lesioned animals. Most of the GluR2-immunoreactive perikarya in the neostriatum, the presumed medium spiny neurons, were depleted. In addition, immunoreactivity for GluR2/3, GluR5/6/7 and NMDAR1 was found to decrease to a different extent in the lesioned neostriatum. The number of GluR1-immunoreactive perikarya in the neostriatum, a group of striatal interneurons, was not affected by the GluR2 lesion. Ventricular administration of the GluR2 immunotoxin however, was found to be less potent. These results demonstrate for the first time that an indirect immunotoxin is useful for immunolesioning. A difference in potency was also observed in different routes of administration. The depletion of GluR2-containing medium spiny neurons in the neostriatum may upset the balance of the output systems of the basal ganglia and has a profound effect in movement control of the animals.

Cellular localization of GluR1, GluR2/3 and GluR4 glutamate receptor subunits in neurons of the rat neostriatum.

Glutamate excitocytotoxicity is implied in the cause of neuronal degeneration in the neostriatum, in which the toxicity may be mediated by different families of glutamate receptors. The precise cellular localization of alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionate (AMPA)-type glutamate receptor subunits (GluR1-4), one of the major family that involves in the mechanisms of glutamate excitocytotoxicity, in different populations of striatal neurons is therefore of special interest. Immunoreactivity for GluR2/3 subunits was detected in the medium-sized spiny neurons. By double labelling experiments, immunoreactivity for GluR1 and GluR4 was detected only in aspiny striatal neurons that display parvalbumin immunoreactivity, but not in the other neuron populations that display choline acetyltransferase or muscarinic m2 receptor immunoreactivity, nor neurons that display nitric oxide synthase immunoreactivity or nicotinamide adenine dinucleotide phosphate-diaphorase activity. These results indicate that GluR1 and GluR4 immunoreactivity is displayed only in the GABAergic interneurons in the neostriatum. In addition, almost all of the GluR1-immunoreactive neurons were found to display GluR4 immunoreactivity. This finding indicates for the first time that the striatal GABAergic interneurons co-express GluR1 and GluR4 subunits. The results of the present study indicate that there is a differential localization of AMPA-type glutamate receptor subunits in different populations of striatal neurons and they may have a different susceptibility to glutamate excitocytotoxicity.

Immunolesioning of nerve growth factor p75 receptor-containing neurons in the rat brain by a novel immunotoxin: anti-p75-anti-mouse IgG-trichosanthin conjugates.

In the present study, a comparison of potency between a commercially available immunotoxin, 192-immunoglobulin-SAP (192-IgG), and a novel immunotoxin produced in our laboratory, anti-p75-anti-mouse IgG-trichosanthin conjugates (p75-TCS), was conducted. Both of the immunotoxins were specific for nerve growth factor p75 receptor. Cholinergic neurons in the rat basal forebrain and in the neostriatum were depleted after the injection of either 192-IgG or p75-TCS. These indicate that both types of immunotoxins are potent and useful in performing immunolesioning experiments. In addition, there were variations in potency among the two immunotoxins in different routes of administration. The 192-IgG was more potent than the p75-TCS in the case of ventricular injections. In case of striatal injections, 192-IgG caused serious tissue necrosis and considerable tissue damage in the brain region. In contrast, p75-TCS was potent and caused a selective and specific depletion of cholinergic neurons in the neostriatum. These results indicate that indirect immunotoxins may be more useful for performing immunolesioning experiments in case of brain parenchyma administration.

Role of TYR70 in the N-glycosidase activity of neo-trichosanthin.

Trichosanthin (TCS) is a type I ribosome-inactivating protein (RIP) which possesses rRNA N-glycosidase activity. TCS has long been used as an abortifacient in China. In recent years, its immunomodulatory, anti-tumor and anti-HIV properties have attracted more and more attention. An isoform of trichosanthin, neo-trichosanthin (n-TCS), has been cloned and expressed as recombinant protein. The biochemical studies revealed that n-TCS has virtually the same rRNA N-glycosidase activity as TCS. The crystal structure of n-TCS is similar to TCS. The crystal of Y70A n-TCS, the mutant of recombinant n-TCS, was soaked in sodium citrate buffer (pH 5.5) containing 25% KCl and AMP (10 mg/ml) prior to data collection. After structure determination and refinement, no electron density corresponding to adenine can be detected around the active pocket. Furthermore, the reaction products of Y70A n-TCS and AMP incubated at various reaction times were analyzed using HPLC. No adenine can be detected. These results suggest that Tyr70 is crucial to n-TCS for its substrate recognition, binding and perhaps N-glycosidase activity.

Induction of metallothionein in the Reuber H-35 rat hepatoma cell.

The feasibility of using the Reuber H-35 rat hepatoma cell (RH-35 cells) as model for studying metallothionein induction was examined. The RH-35 cells were treated with Cd, a toxic metal which is known to induce metallothionein. The LC50 after a 3-h treatment was 70 microM. The value was significantly higher (P < 0.05) if the cells were pre-treated with a sublethal dose of CdCl2 (5 microM) for 2 days, indicating that pre-treatment with a low dose of Cd can protect against a subsequent higher dose of the same metal. Both the mRNA and the gene product metallothionein can be identified in the cells 2 days after treatment with 5 microM Cd. In addition to Cd, Zn and Cu were also able to induce the expression of metallothionein to various degrees. The results indicate that the MT gene is present in RH-35 cells and is responsive to treatment with various metals. Thus, this cell line can be used as a model to study metallothionein induction.

Minireview: enzymatic properties of ribosome-inactivating proteins (RIPs) and related toxins.

Ribosome-inactivating proteins (RIPs) are a group of proteins that inhibit protein synthesis in eucaryotic cells. While the biological effects have been well characterized, the underlying enzymatic mechanisms have not been elucidated until recently. Two different mechanisms have been identified. Plant and bacterial RIPs act as N-glycosidases. They cleave a single N-glycosidic bond between adenine and ribose at a specific nucleotide A-4324 of the 28S rRNA of the 60S ribosomal subunit. On the other hand, the fungal RIPs act as ribonucleases and cleave a single phosphodiester bond between G-4325 and A-4326 of the same rRNA, just one nucleotide away from the site of action of plant/bacterial RIPs. Other protein synthesis inhibitory proteins act by their ADP-ribosyltransferase activity which modify and thus inactivate elongation factor-2. Recently, some toxins have been shown to possess deoxyribonuclease activity which may also account for their toxicity.

Protective effects of Danshensu from the aqueous extract of Salvia miltiorrhiza (Danshen) against homocysteine-induced endothelial dysfunction.

Homocysteine (Hcy) is a by-product of methionine metabolism. An imbalance of Hcy in the body may lead to hyperhomocysteinemia, a condition with elevated Hcy concentration in blood that may be one of the risk factors responsible for the development of several vascular diseases (thromboembolism, atherosclerosis, stroke, vascular diseases and dementia). Radix Salvia miltiorrhiza (Danshen), a well-known Chinese medicinal herb that can activate and improve blood microcirculation, is noticeable for its beneficial effect in treating cardiovascular diseases. The present study is to demonstrate the protective effect of Danshen extract against the homocysteine-induced adverse effect on human umbilical vein endothelial cell (HUVEC). Homocysteine (5 mM) not only decreased the cell viability but also caused the disruption of capillary-like structure formation in vitro. The protective effect of Danshen aqueous extract and its active compounds on endothelial cell function were demonstrated through an in vitro tube formation assay, which mimics the new blood vessel formation. To identify the active components in the aqueous extract of Danshen, the content was characterized by instrumental analysis using high performance liquid chromatography with diode array detector (DAD) and electrospray tandem mass spectrometry (ESI-MS/MS). Interestingly, Danshen extract and its pure compounds showed different effectiveness in protecting HUVEC against Hcy-induced injury according to the following descending order: Danshen aqueous extract, 3-(3,4-dihydroxy-phenyl)-2-hydroxy-propionic acid (Danshensu), protocatechuic acid, catechin and protocatechualdehyde. We believed that such findings might provide evidence in understanding the beneficial effects of Danshen on the cardiovascular system.

A highly efficient procedure for purifying the ribosome-inactivating proteins alpha- and beta-momorcharins from Momordica charantia seeds, N-terminal sequence comparison and establishment of their N-glycosidase activity.

A new purification scheme, involving two successive ion exchange chromatographic steps on DEAE-cellulose and Mono-S FPLC, was developed for the isolation of the ribosome-inactivating proteins, alpha- and beta-momorcharins, from the Chinese herb Kuquazi (seeds of Momordica charantia). This simple and rapid procedure yielded 3.1 and 1.7 mg of alpha- and beta-momorcharins, respectively, from 2.5 g of decorticated seeds in only two days. The N-terminal amino acid sequence of beta-momorcharin was found to be DVNFDLSTATAKTYTKFIED. It differed from that of alpha-momorcharin (DVSFRLSGADPRSYGMFIKD) in 10 out of the 20 positions investigated. Like other ribosome-inactivating proteins, the purified momorcharins showed specific N-glycosidase activity at nanomolar concentrations, when rRNA from rabbit reticulocyte lysate was used as substrate. The N-glycosidase activity of both momorcharins was optimal at pH7, not inhibited by K+ and not appreciably affected by NH4+. The activity of alpha-momorcharin was not drastically altered by Mn2+ but (1-10mM) Mn2+ inhibited the activity of beta-momorcharin by about 40%.

Effects of euxanthone on neuronal differentiation.

The growth inhibitory and differentiation inducing effects of euxanthone (1,7-dihydroxyxanthone) from the medicinal plant Polygala caudata on the neuroblastoma (Neural 2A, subclone BU-1) were investigated. At the concentration range of 0-100 microM, euxanthone inhibits the growth of BU-1 cells in a dose dependent manner. The 50% growth inhibitory concentration (IC50) was 41 microM. Significant induction of morphological differentiation and neurite growth was observed at the concentration of 100 microM. Frequency of proliferative neuroblastoma cells was determined after induction of differentiation. The frequency of proliferating BU-1 cells was markedly reduced from 1/1.1 to <1/99. Confocal microscopy also confirmed that the morphological differentiation of BU-1 was associated with the expression of neurite specific marker MAP-2 protein in neurites. These data suggest that euxanthone may be one of the neuropharmacological active compounds in the medicinal plant Polygala caudata.