Secoiridoid Glucosides and Anti-Inflammatory Constituents from the Stem Bark of Fraxinus chinensis.

Qin Pi (Fraxinus chinensis Roxb.) is commercially used in healthcare products for the improvement of intestinal function and gouty arthritis in many countries. Three new secoiridoid glucosides, (8E)-4''-O-methylligstroside (1), (8E)-4''-O-methyldemethylligstroside (2), and 3'',4''-di-O-methyl-demethyloleuropein (3), have been isolated from the stem bark of Fraxinus chinensis, together with 23 known compounds (4-26). The structures of the new compounds were established by spectroscopic analyses (1D, 2D NMR, IR, UV, and HRESIMS). Among the isolated compounds, (8E)-4''-O-methylligstroside (1), (8E)-4''-O-methyldemethylligstroside (2), 3'',4''-di-O-methyldemethyloleuropein (3), oleuropein (6), aesculetin (9), isoscopoletin (11), aesculetin dimethyl ester (12), fraxetin (14), tyrosol (21), 4-hydroxyphenethyl acetate (22), and (+)-pinoresinol (24) exhibited inhibition (IC50 ≤ 7.65 µg/mL) of superoxide anion generation by human neutrophils in response to formyl-L-methionyl-L-leuckyl-L-phenylalanine/cytochalasin B (fMLP/CB). Compounds 1, 9, 11, 14, 21, and 22 inhibited fMLP/CB-induced elastase release with IC50 ≤ 3.23 µg/mL. In addition, compounds 2, 9, 11, 14, and 21 showed potent inhibition with IC50 values ≤ 27.11 µM, against lipopolysaccharide (LPS)-induced nitric oxide (NO) generation. The well-known proinflammatory cytokines, tumor necrosis factor-alpha (TNF-α) and interleukin 6 (IL-6), were also inhibited by compounds 1, 9, and 14. Compounds 1, 9, and 14 displayed an anti-inflammatory effect against NO, TNF-α, and IL-6 through the inhibition of activation of MAPKs and IκBα in LPS-activated macrophages. In addition, compounds 1, 9, and 14 stimulated anti-inflammatory M2 phenotype by elevating the expression of arginase 1 and Krüppel-like factor 4 (KLF4). The above results suggested that compounds 1, 9, and 14 could be considered as potential compounds for further development of NO production-targeted anti-inflammatory agents.

Chemical Constituents from the Stems of Tinospora sinensis and Their Bioactivity.

Fifty-seven compounds were purified from the stems of Tinospora sinensis, including three new compounds characterized as a lignan (1), a pyrrole alkaloid (11), and a benzenoid (17), respectively. Their structures were elucidated and established by various spectroscopic and spectrometric analytical methods. Among the isolates, fifteen compounds were examined for their anti-inflammatory potential in vitro. The results showed that several compounds displayed moderate inhibition of N-formyl-methionyl-leucyl-phenylalanine/cytochalasin B (fMLP/CB)-induced superoxide anion generation and elastase release.

Spindle tubulin and MTOC asymmetries may explain meiotic drive in oocytes.

In the first meiotic division (MI) of oocytes, the cortically positioned spindle causes bivalent segregation in which only the centre-facing homologue pairs are retained. 'Selfish' chromosomes are known to exist, which bias their spindle orientation and hence retention in the egg, a process known as 'meiotic drive'. Here we report on this phenomenon in oocytes from F1 hybrid mice, where parental strain differences in centromere size allows distinction of the two homologue pairs of a bivalent. Bivalents with centromere and kinetochore asymmetry show meiotic drive by rotating during prometaphase, in a process dependent on aurora kinase activity. Cortically positioned homologue pairs appear to be under greater stretch than their centre-facing partners. Additionally the cortex spindle-half contain a greater density of tubulin and microtubule organising centres. A model is presented in which meiotic drive is explained by the impact of microtubule force asymmetry on chromosomes with different sized centromeres and kinetochores.

Evaluation of the genotoxicity of zerumbone in cultured human peripheral blood lymphocytes.

The chromosomal aberrations (CA) assay and micronucleus (MN) test were employed to investigate the effect in vitro of zerumbone (ZER) on human chromosomes. ZER is a sesquiterpene compound isolated from the rhizomes of wild ginger, Zingiber zerumbet Smith. The rhizomes of the plant are employed as a traditional medicine for some ailments and as condiments. ZER has been shown to have anti-cancer and apoptosis-inducing properties against various human tumour cells. It has also been shown to be active in vivo against a number of induced malignancies. Studies on ZER genotoxicity in cultured human peripheral blood lymphocytes (PBL) have not been reported so far. Therefore, the present study was undertaken to investigate the ability of ZER to induce chromosomal aberrations and micronuclei formation in human lymphocytes in vitro. Human blood samples were obtained from four healthy, non-smoking males aged 25-35years. Cultures were exposed to the drug for 48h at four final concentrations: 10, 20, 40 and 80 microM. Mitomycin C (MMC) was used as a positive control. The results of chromosomal aberrations assay showed that ZER was not clastogenic, when compared to untreated control, meanwhile MN test results showed a dose-dependent increase in MN formation. The overall clastogenic effect of ZER on human PBL was statistically not significant. In conclusion, ZER is a cytotoxic but not a clastogenic substance in human PBL.

Relative importance of enhanced glucose uptake versus attenuation of long-chain acyl carnitines in protecting ischemic myocardium.

BACKGROUND: A number of experimental studies have shown that increasing glucose use or decreasing accumulation of long-chain acyl carnitines (LCAC) protect ischemic hearts. METHODS: To evaluate the relative importance of these two strategies in protecting ischemic myocardium, isolated rat hearts (n = 6 in each group) were paced at 300 bpm and subjected to 50 min of low-flow ischemia followed by 60 min of reperfusion. Buffer contained 0.4 m mol/l albumin, 0.4 m mol/l palmitate, and 70 mU/l insulin, and either normal glucose (5 m mol/l) (CON), high glucose (10 m mol/l total) (HG, known to increase glucose use), 5 m mol/l glucose and niacin (10 micromol/l) (NIA, known to increase glucose use and decrease LCAC) or carnitine (10 m mol/l) (CAR, known to increase glucose use and decrease LCAC). Separate groups of hearts were perfused in the presence of 10 micromol/l cytochalasin-B (CB), an inhibitor of insulin-sensitive glucose transporters. RESULTS: Ischemic injury, as assessed by creatine kinase (CK) release was diminished by an average of 50% in HG, NIA, and CAR hearts, and the percentage recovery of left ventricular (LV) function with reperfusion was enhanced by approximately 20% compared with CON hearts (P < 0.05 for each comparison). Cytochalasin-B abolished all of the salutary effects. Long-chain acyl carnitines levels were higher in HG hearts compared with NIA- and CAR-treated hearts ( P < 0.05), but ischemic protection and functional recovery was greater in HG hearts. CONCLUSIONS: The data support the adjunctive use of agents that promote glucose uptake during ischemia and suggest that increasing glucose use is more important than decreasing LCAC in the protection against ischemic injury or in the recovery of contractile function.

Nicotinamide is not a substrate of the facilitative hexose transporter GLUT1.

It has been proposed that GLUT1, a membrane protein that transports hexoses and the oxidized form of vitamin C, dehydroascorbic acid, is also a transporter of nicotinamide (Sofue, M., Yoshimura, Y., Nishida, M., and Kawada, J. (1992) Biochem. J. 288, 669-674). To ascertain this, we studied the transport of 2-deoxy-D-glucose, 3-O-methyl-D-glucose, and nicotinamide in human erythrocytes and right-side-out and inside-out erythrocyte membrane vesicles. The transport of nicotinamide was saturable, with a K(M) for influx and efflux of 6.1 and 6.2 mM, respectively. We found that transport of the hexoses was not competed by nicotinamide in both the erythrocytes and the erythrocyte vesicles. Likewise, the transport of nicotinamide was not affected by hexoses or by inhibitors of glucose transport such as cytochalasin B, genistein, and myricetin. On the other hand, nicotinamide blocked the binding of cytochalasin B to human erythrocyte membranes but did so in a noncompetitive manner. Using GLUT1-transfected CHO cells, we demonstrated that increased expression of GLUT1 was paralleled by a corresponding increase in hexose transport but that there were no changes in nicotinamide transport. Moreover, nicotinamide failed to affect the transport of hexoses in both control and GLUT1-transfected CHO cells. Therefore, our results indicates that GLUT1 does not transport nicotinamide, and we propose instead the existence of other systems for the translocation of nicotinamide across cell membranes.

Genotoxic effects of N-nitrosodicyclohexylamine in isolated human lymphocytes.

Dicyclohexylamine x nitrite is classified as an "experimental equivocal tumorigenic agent" by the National Toxicology Program. Since no genotoxic effects of the substance itself are known, the reported tumorigenic potential of dicyclohexylamine x nitrite could be due to generation of N-nitrosodicyclohexylamine (N-NO-DCHA), which occurs under conditions of use and can be detected in foils that contain dicyclohexylamine x nitrite. Therefore, we investigated possible mutagenic properties of N-NO-DCHA in the Ames test and the cytokinesis-block micronucleus assay with human lymphocytes. Since N-NO-DCHA is not commercially available, the substance was synthesized and purified by thin-layer chromatography. Identity was confirmed by gas chromatography/mass spectroscopy (GC/MS) and 1H- and 13C-NMR. More than 97% purity was achieved. Stability and availability in the solvent were checked by GC/MS. N-NO-DCHA induced micronuclei in isolated human lymphocytes at a dose range of 15-100 micrograms/ml (= 71.4-476.2 microM), exceeding the base rate significantly at one or two nontoxic concentrations in four out of six experiments. For the Ames test, arochlor-1254-, beta-naphthoflavone/phenobarbital- and pyrazole-induced S9-fractions were used with Salmonella typhimurium TA100, TA1535, TA98 and TA104. No effects were seen in the Ames test, with the exception of microcolony induction at doses higher than 250 micrograms (= 1.2 mmol) N-NO-DCHA/plate using TA104 and 20% arochlor-1254 induced S9 at pH 6.5. In conclusion, N-NO-DCHA was negative in the Ames test using TA98, TA100 and TA1535, inconclusive using TA104, and weakly genotoxic in the in vitro micronucleus test with isolated human lymphocytes. With regard to the tumorigenicity of the majority of nitrosamines, our data underline the necessity of further studies on possible genotoxic effects of N-NO-DCHA.

A synthetic peptide containing a predominant protein kinase C site within p47phox inhibits the NADPH oxidase in intact neutrophils.

In vivo loading of a synthetic peptide (peptide 4) corresponding to residues 314-331 (RSRKRLSQDAYRRNSVRF) consistently diminished the oxidative burst in response to either phorbol 12-myristate 13-acetate (PMA) or formylmethionyl-leucyl-phenylalanine and cytochalasin B (fMLP/CB) compared to other synthetic peptides derived from the p47phox sequence. The effects of peptide 4 were concentration dependent with respect to both PMA and fMLP/CB. In contrast, peptide 4 enhanced the oxidative burst in response to fMLP alone. Peptide 4 inhibited the PMA and fMLP-mediated phosphorylation of endogenous neutrophil cytosolic proteins including p47phox. The PMA-induced translocation of p47phox to the plasma membrane was diminished in neutrophils loaded with peptide 4. These data represent the first report of a synthetic peptide derived from p47phox that inhibits the NADPH oxidase in intact neutrophils and inhibits the protein kinase C-mediated phosphorylation of endogenous p47phox.

Phalloidin inhibits epinephrine's and cytochalasin B's facilitation of aqueous outflow.

OBJECTIVE: To determine whether phalloidin, a fungal peptide that inhibits actin filament depolymerization, could inhibit the ability of cytochalasin B and epinephrine to increase the facility of aqueous outflow in the eyes of living cynomolgus monkeys. METHODS: Outflow facility was determined by two-level constant-pressure perfusion of the anterior chamber. After measurement of baseline facility in both eyes, one eye of each animal received intracameral phalloidin (1.3, 13, or 130 mumol/L); the opposite eye received vehicle. Both eyes then received either epinephrine (0.3 mmol/L) or cytochalasin B (0.2 mmol/L), and facility was again measured. RESULTS: Cytochalasin B and epinephrine increased facility by 120% to 190% and 100% to 180%, respectively (uncorrected for 15% resistance washout caused by perfusion itself). Phalloidin itself (13 or 130 mumol/L) did not affect facility, but it inhibited up to 50% of the facility-increasing effect of cytochalasin B and epinephrine. CONCLUSIONS: We conclude that (1) the aqueous humor outflow facilitating effects of cytochalasin B or epinephrine depend in some manner on depolymerization of actin filaments within trabecular meshwork cells, and (2) actin filaments may help regulate aqueous outflow.

Thyroid cell spreading and focal adhesion formation depend upon protein tyrosine phosphorylation and actin microfilaments.

Adhesion to proteins of the extracellular matrix exerts a profound influence upon cell function and behavior. Similar adhesive interactions mediate the spreading of cultured cells upon artificial substrata. Recently we observed that thyrotropin (TSH) and intercellular contact regulated thyroid cell-substrate adhesion to inhibit cell spreading, but not initial attachment. This is a mechanism which preserves thyroid follicular differentiation in culture. In the present study we have investigated the role of cytoplasmic components in mediating thyroid cell adhesion to collagen. The earliest change associated with cell spreading was the accumulation of vinculin and phosphotyrosine in developing focal adhesions, which was followed by stress fiber and microtubule assembly. Genistein, an inhibitor of tyrosine kinases, and cytochalasin B inhibited cell spreading and focal adhesion formation without affecting initial attachment to substrate. In contrast microtubule disorganization by colchicine did not alter any parameter of thyroid cell-substrate adhesion. These observations indicate that protein tyrosine phosphorylation and dynamic microfilament integrity are essential for attached thyroid cells to spread upon substrate. They are therefore potential intracellular loci at which TSH and intercellular contact may regulate cell adhesion to extracellular matrix and influence thyroid cell behavior.

Cytochalasin B induces cellular DNA fragmentation.

Cellular DNA fragmentation can be induced in many biological instances without plasma membrane damage. The fungal metabolite, cytochalasin B, is capable of modifying numerous cellular functions related to DNA synthesis. In this work it is demonstrated that cytochalasin B is capable of inducing DNA fragmentation in a number of cells lines. This DNA fragmentation occurs before plasma membrane lysis and over a period of hours. Cytochalasin E and villin, agents that act on the microfilaments, also induce DNA fragmentation. Phorbol dibutyrate, a diacylglyceral analog, is able to inhibit cytochalasin B-induced DNA fragmentation in a dose-dependent fashion. These findings support the interpretation that cytochalasin B is inducing DNA fragmentation via its effect on the actin filaments.

Microtubule inhibitors block the morphological changes induced in Drosophila blood cells by a parasitoid wasp factor.

The shape change of Drosophila melanogaster blood cells (lamellocytes) from discoidal to bipolar that is caused by a factor from the female parasitoid Leptopilina heterotoma is blocked by the tubulin inhibitors vinblastine and vincristine in vitro. The actin inhibitor, cytochalasin B, causes arborization of Drosophila lamellocytes and acts synergistically with the wasp factor to alter lamellocyte morphology. Lamellocyte aborization induced by cytochalasin B is blocked by simultaneous treatment with vinblastine. These observations indicate that the changes in lamellocyte shape induced by both the wasp factor and cytochalasin B require microtubule assembly.

Direct interaction of phenylarsine oxide with hexose transporters in isolated rat adipocytes.

It has previously been shown that phenylarsine oxide (PhAsO), an inhibitor of protein internalization, also inhibits stereospecific uptake of D-glucose and 2-deoxyglucose in both basal and insulin-stimulated rat adipocytes. This inhibition of hexose uptake was found to be dose-dependent. PhAsO rapidly inhibited sugar transport into insulin-stimulated adipocytes, but at low concentrations inhibition was transient. Low doses of PhAsO (1 microM) transiently inhibit stereospecific hexose uptake and near total (approx. 90%) recovery of transport activity occurs within 20 min. Interestingly, once recovered, the adipocytes can again undergo rapid inhibition and recovery of transport activity upon further treatment with PhAsO (1 microM). In addition, PhAsO is shown to inhibit cytochalasin B binding to plasma membranes from insulin-stimulated adipocytes in a concentration-dependent manner which parallels the dose-response inhibition of hexose transport by PhAsO. The data presented suggest a direct interaction between the D-glucose transporter and PhAsO, resulting in inhibition of transport. The results are consistent with the current recruitment hypothesis of insulin activation of sugar transport and indicate that a considerable reserve of intracellular glucose carriers exists within fat cells.

Inverse effects of colchicine on the induction of long microvillous-like processes by cytochalasin B in cultured chick embryonic duodena.

Previously, the author reported that long microvillous-like processes (long processes) containing core actin filaments seemed to be related to the formation of previllous ridges in the chick embryonic duodenum and that similar long processes were induced by cytochalasin B (CB) treatment. The appearance of long processes by CB (polymerization of core actin filaments) seemed to be related to the ability of cell proliferation. In this study, the effect of colchicine on the induction of long processes by CB was examined in 11-day-old chick embryonic duodena in the culture system. The results were as follows: The induction of long processes by CB was inversely influenced by the concentration of colchicine; the appearance of long processes by CB was differently influenced by local parts of the epithelial cells in the presence of colchicine; and alteration of the arrangement of intracellular actin elicited by CB and colchicine was confirmed by immunohistochemistory using actin antiserum. It was also suggested that sudden intracellular alterations elicited by higher concentrations of colchicine may act as a trigger of cell division differing from normal conditions.

[Inhibitory effect of nafamostat mesilate (FUT-175) on O2- production in rat polymorphonuclear leucocytes].

Effect of nafamostat mesilate (FUT-175), a serine protease inhibitor, having anti-inflammatory effects was studied on superoxide (O2-) production in rat polymorphonuclear leucocytes (PMN) and compared with those of other serine protease inhibitors and typical anti-inflammatory agents. 1) O2- productions in rat PMN stimulated with concanavalin A (Con A) and cytochalasin B (Cyt B) were too weak to observe. With NADH, however, strong O2- production was induced by Con A and Cyt B. 2) FUT-175 at 10(-6) and 10(-5) M inhibited O2- production in rat PMN induced by Con A and Cyt B with NADH in a concentration-dependent manner. 3) The serine protease inhibitor L-tosylamido-2-phenylethyl-chloromethyl ketone (TPCK) and soybean trypsin inhibitor (SBTI) inhibited O2- production at 10(-5) M and 10(-4) M, respectively, while aprotinin, chymostatin and leupeptin did not. 4) Neither indomethacin nor dexamethasone, typical anti-inflammatory agents, inhibited O2- production. Mepacrine, a phospholipase A2 inhibitor, strongly inhibited it. 5) O2- production in PMN prepared from the rat administered FUT-175, 200 mg/kg, p.o., was significantly decreased in comparison with that of the control rat. 6) FUT-175 had no effect on O2- production by hypoxanthine-xanthine oxidase. These results showed FUT-175 had a strong inhibitory effect on O2- production in rat PMN which other typical anti-inflammatory agents did not have.

A new model of reticulopodial motility and shape: evidence for a microtubule-based motor and an actin skeleton.

Cytoskeletal inhibitors were used as probes to test the involvement of microtubules and actin microfilaments in the development, motility, and shape maintenance of the pseudopodial networks (i e, reticulopodia) of the foraminifers Allogromia sp strain NF and Allogromia laticollaris. Agents that disassemble cytoplasmic microtubules (cold, colchicine, and nocodazole) arrest all movement but have variable effects on reticulopodial shape. Electron microscopy reveals a granulofibrillar matrix but few, if any, microtubules in these motility-arrested reticulopods. Allogromiids treated with cytochalasin B or D lose substrate adhesion and undergo dramatic changes in shape and motile behavior, highlighted by the coalescence of reticulopodial cytoplasm into irregularly shaped bodies with chaotic motility. Serial semithick sections of such preparations, viewed by high-voltage electron microscopy, document a striking rearrangement of microtubules within these cytochalasin-induced bodies. All aspects of cytochalasin-altered motility are completely inhibited by colchicine. Actin is present in reticulopodia, as determined by staining with rhodamine-phalloidin; this staining is not observed in cytochalasin-treated organisms. These data provide compelling evidence that microtubules are required for reticulopodial motility. An actin-based cytoskeleton is thought to play a role in maintaining shape, mediating pseudopod/substrate adhesion, and coordinating the various microtubule-dependent processes.

[Separation of the virotoxins of the mushroom Amanita virosa and comparative study of their interaction on actin in vitro]. / Séparation des virotoxines du champignon Amanita virosa et étude comparative de leur interaction sur l'actine in vitro.

Cyclic peptides have been extracted with methanol from Amanita virosa and separated by preparative HPLC. Six peptides were obtained: phalloidin and five new peptides recently identified by Faulstich as virotoxins. We have compared the interaction of these six peptides with actin in vitro. They increased the rate of polymerization of actin and protected F-actin against several denaturating agents: proteases, heat, chaotropic ions, cytochalasin B, and DNAse I. The five virotoxins have therefore the same biological properties as phalloidin. However, the differential spectra of interaction between actin and the five virotoxins are different than the differential spectra between actin and phalloidin, thus it appears that the molecular interaction of actin with virotoxins is different than with phalloidin. The five virotoxins have the same activity. Although these virotoxins have different functional groups on amino acids 1 and 7, it is concluded that these two amino acids are of minor importance in the interaction of these peptides with actin.

Cytochalasin B facilitates the inhibition of human polymorphonuclear leukocyte generation of superoxide by verapamil.

Several functions of the human neutrophil are dependent upon extracellular calcium for optimal activation. We studied the calcium dependency of human polymorphonuclear leukocyte (PMN) superoxide generation in response to opsonized zymosan particles and its modulation by the calcium channel antagonist verapamil. PMNs were isolated from anticoagulated blood after Ficoll-Hypaque density centrifugation. The isolated PMNs were incubated with opsonized zymosan particles. The superoxide anion generated was measured by a cytochrome C reduction assay. When PMNs were incubated with opsonized zymosan in a calcium-free buffer, there was 0.45 +/- 0.06 nmol of cytochrome C reduced per 1 X 10(6) PMN per minute. This increased to 0.76 +/- 0.12 nmol per 1 X 10(6) PMN per minute when 0.6 mmol/L Ca++ was present. Moreover, if the PMNs were incubated with cytochalasin B (5 micrograms/ml), the generation of superoxide anion was further enhanced. If the same experiments were conducted in the presence of verapamil, 100 mumol/L, superoxide generation was inhibited by 51.5% +/- 8.4%. For verapamil to inhibit superoxide generation, cytochalasin B was necessary. Our results suggest that verapamil-sensitive calcium channels may exist in human PMNs, and the demonstration of their presence is cytochalasin B-dependent.

Identification of the D-glucose-inhibitable cytochalasin B binding site as the glucose transporter in rat diaphragm plasma and microsomal membranes.

[3H]Cytochalasin B binding and its competitive inhibition by D-glucose have been used to identify, the glucose transporter in plasma and microsomal membranes prepared from intact rat diaphragm. Scatchard plot analysis of [3H]cytochalasin B binding yields a binding site with a dissociation constant of roughly 110 nM. Since the inhibition constant of cytochalasin B for D-glucose uptake by diaphragm plasma membranes is similar to this value, this site is identified as the glucose transporter. Plasma membranes prepared from diaphragms bind approx. 17 pmol of cytochalasin B/mg of membrane protein to the D-glucose-inhibitable site. If 280 nM (40000 microunits/ml) insulin is present during incubation, cytochalasin B binding is increased roughly 2-fold without alteration in the dissociation constant of this site. In addition, membranes in the microsomal fraction contain 21 pmol of D-glucose-inhibitable cytochalasin B binding sites/mg of membrane protein. In the presence of insulin during incubation the number of these sites in the microsomal fraction is decreased to 9 pmol/mg of membrane protein. These results suggest that rat diaphragm contain glucose transporters with characteristics identical to those observed for the rat adipose cell glucose transporter. In addition, insulin stimulates glucose transport in rat diaphragm through a translocation of functionally identical glucose transporters from an intracellular membrane pool to the plasma membrane without an alteration in the characteristics of these sites.