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Tumor necrosis factor-alpha (TNF-alpha) is a major cytokine of host immune reaction by foreign agents. Phytohemagglutinin (PHA) is a dynamic contributor to mitogenic stimulation and augmentation of host immune defense. Interferon-gamma (IFN-gamma) induces induction of cytokines in macrophages and lymphocytes. The aim of this study was to examine the synergistic effects of PHA plus low dose IFN-gamma on TNF-alpha mRNA production, cytosolic levels, and secretion in RAW 264.7 cells. The cells were stimulated with PHA or IFN-gamma using various concentrations for various times. The effects of PHA on TNF-alpha expression appeared in dose- and time-dependent manners. The maximum doses of PHA and IFN-gamma to produce them were 300 microg/ml PHA and 10 ng/ml IFN-gamma. The optimum time of PHA for the TNF-alpha mRNA production and release were 6 and 7 h after stimulation, respectively, whereas the time of IFN-gamma on them was achieved at 3 and 8 h. Although the TNF-alpha mRNA production, cytosolic levels, and secretion from the cells were slightly detected under 10 microg/ml PHA and 1 ng/ml IFN-gamma, the combination of PHA (10 microg/ml) and IFN-gamma (1 ng/ml) greatly increased them, indicating the synergistic effect of PHA plus low dose IFN-gamma on TNF-alpha expression.

Phospholipase D (PLD) is activated via receptors in a wide variety of cells where it is thought to regulate intracellular signaling processes and functions such as membrane trafficking, cytoskeletal organization, and degranulation of mast cells (reviewed in references 15, 25, and 31). PLD catalyzes the hydrolysis of phosphatidylcholine to form phosphatidic acid, which is rapidly converted to other biologically active molecules, namely, lysophosphatidic acid and diacylglycerol. In the presence of relatively low concentrations of primary alcohols, the production of phosphatidic acid is diverted to more metabolically inert phosphatidylalcohols by transphosphatidylation, a reaction that is unique to PLD and one that is utilized in the assay of PLD in vivo (39) and to unmask the physiologic roles of phosphatidic acid (62). Two isoforms of PLD have been cloned, PLD1 and PLD2, with PLD1 existing as two variants, PLD1a and PLD1b (11, 21). PLD1 is activated in vitro by small GTPases such as ARF and Rho and protein kinase C (PKC) α in the presence of phosphatidylinositol 1,4-bisphosphate (PIP2) (4, 21, 37, 43, 55). There is also evidence that PLD1 can be regulated in vivo by Rho kinase (48), Ca2+/calmodulin-dependent kinase II (35), and PKC in a catalytically dependent or independent manner (21, 26, 63). PLD2, in contrast, is activated in vitro by PIP2 alone, and this activity is minimally affected by the small GTPases or PKCα (11, 32, 54). However, the mechanisms regulating PLD2 activity in vivo are unclear. There are reports of tyrosine phosphorylation of PLD1 (33, 36) and PLD2 (1, 44, 51) and indications from pharmacological studies that tyrosine phosphorylation may regulate PLD activity (6, 27, 36, 44). In addition, PLD2 was shown to associate with, and be phosphorylated by, the tyrosine kinase receptor for epidermal growth factor (EGF) (51) and by Src kinase (1, 42). Nevertheless, the role of such phosphorylation is uncertain. Although tyrosine-11 was identified as the specific residue phosphorylated in PLD2, mutation of this site enhanced basal PLD2 activity but had no effect on the magnitude of the PLD2 response to EGF (51). Mast cells and blood basophils are responsible for a variety of allergic disorders (5, 59). These cells respond to immunoglobulin E (IgE)-directed antigens via the high-affinity receptor for IgE, namely, FcɛRI, by release of granules that contain preformed inflammatory mediators and the generation of inflammatory lipids and cytokines. PLD is thought to play an essential role in mast cell degranulation (7, 10, 58). PLD is activated in isolated mast cells (12) and cultured mast cell lines (10, 28, 30) by a variety of stimulants, including antigen. Cross-linking of the IgE/FcɛRI complex with antigen results in the recruitment and activation of Src kinases and subsequently other tyrosine kinases. The function of the individual PLD isoforms in mast cells has been studied in the RBL-2H3 cell line, which is now known to be an analog of rat mucosal mast cells (49). Studies with transiently expressed forms of both PLDs in RBL-2H3 cells indicate that PLD1b and PLD2 associate with granule membranes and the plasma membrane, respectively (7, 9), and that both isoforms are activated upon antigen stimulation (8, 40). The mechanisms of activation of these PLDs by antigen are unknown. However, the location of PLD2 at the plasma membrane makes this isoform particularly accessible to FcɛRI-associated tyrosine kinases. As reported here, activation of PLD and degranulation in antigen-stimulated RBL-2H3 cells is inhibited by low concentrations of the Src kinase inhibitor PP2. We investigated whether Src kinases regulate PLD directly by tyrosine phosphorylation and, if so, whether this phosphorylation is essential for degranulation. We show by coexpression studies, site-directed mutagenesis, and the use of small interfering RNAs (siRNAs) directed against Src kinases that Fyn and Fgr phosphorylate PLD2 but not PLD1b in vitro and in vivo and that this phosphorylation is required for the activation of PLD2 in vivo. Furthermore, suppression of this phosphorylation or the activation of PLD2 itself by various strategies also results in suppression of degranulation in stimulated RBL-2H3 cells.

The effects of various extracts from oriental medicinal herbs on mast cell-mediated allergic reactions have been investigated. Among the extracts, Arecae semen was the most potent inhibitor of antigen-induced degranulation in RBL-2H3 mast cells. A. semen inhibited DNP-BSA- and compound 48/80-induced degranulation in RBL-2H3 mast cells with IC50 values of approximately 53 and 52 μg mL−1, respectively, and inhibited compound 48/80-induced systemic anaphylaxis by 46% at 300 mg kg−1 in mice. A. semen also inhibited the expression of TNF-α and the activation of mitogen activated protein kinase, ERK1/2, which is critical for the production of inflammatory cytokines in mast cells, as indicated by the suppression of the activating phosphorylation of ERK1/2. These results suggest that A. semen may be useful for the treatment of various immediate and delayed allergic diseases.

To define the mechanism of arsenite-induced tumor promotion, we examined the role of reactive oxygen species (ROS) in the signaling pathways of cells exposed to arsenite. Arsenite treatment resulted in the persistent activation of p70(s6k) and extracellular signal-regulated kinase 1/2 (ERK1/2) which was accompanied by an increase in intracellular ROS production. The predominant produced appeared to be H(2)O(2), because the arsenite-induced increase in dichlorofluorescein (DCF) fluorescence was completely abolished by pretreatment with catalase but not with heat-inactivated catalase. Elimination of H(2)O(2) by catalase or N-acetyl-L-cysteine inhibited the arsenite-induced activation of p70(s6k) and ERK1/2, indicating the possible role of H(2)O(2) in the arsenite activation of the p70(s6k) and the ERK1/2 signaling pathways. A specific inhibitor of p70(s6k), rapamycin, and calcium chelators significantly blocked the activation of p70(s6k) induced by arsenite. While the phosphatidylinositol 3-kinase (PI3K) inhibitors wortmannin and LY294002 completely abrogated arsenite activation of p70(s6k), ERK1/2 activation by arsenite was not affected by these inhibitors, indicating that H(2)O(2) might act as an upstream molecule of PI3K as well as ERK1/2. Consistent with these results, none of the inhibitors impaired H(2)O(2) production by arsenite. DNA binding activity of AP-1, downstream of ERK1/2, was also inhibited by catalase, N-acetyl-L-cysteine, and the MEK inhibitor PD98059, which significantly blocked arsenite activation of ERK1/2. Taken together, these studies provide insight into mechanisms of arsenite-induced tumor promotion and suggest that H(2)O(2) plays a critical role in tumor promotion by arsenite through activation of the ERK1/2 and p70(s6k) signaling pathways.

We previously reported the presence of nitric oxide synthase (NOS) in Staphylococcus aureus ATCC6538P whose activity was induced by methanol. In the present study, the methanol-induced NOS was purified 900-fold from S. aureus by means of Mono Q ion exchange column, 2',5'-ADP-agarose affinity column, and Superdex 200HR gel permeation column chromatography. The purified bacterial NOS showed two protein bands with 67 and 64 kDa molecular mass on SDS-PAGE. However, the molecular mass of the NOS was 135 kDa on Superdex 200HR gel permeation column chromatography, indicating that the native enzyme exists as a heterodimer. This bacterial NOS had K(m) value of 13.4x10(-6) M for L-arginine and V(max) of 35.3 nmol min(-1) mg(-1) protein. In addition, reduced nicotinamide adenine dinucleotide phosphate, flavin adenine dinucleotide, flavin mononucleotide, tetrahydrobiopterin, calmodulin and Ca(2+) were required as cofactors in the conversion of L-arginine to L-citrulline, and NOS inhibitors selectively inhibited the activity of the purified NOS.

Background Complementary and alternative herbal medicines are recently considered as a promising approach for treating various diseases. We screened approximately 100 plant extracts for anti-allergic activity. Rhamnus davurica leaf extract showed the most potent inhibitory effect on the activation of RBL-2H3 mast cells. Although Rhamnus davurica extract has been used to treat pruritus, dysuresia, and constipation as a traditional herbal medicine in some Asian countries, an anti-allergic effect of Rhamnus davurica has not yet been demonstrated. We aimed to investigate the effect and mechanism of the leaf extract of Rhamnus davurica (LERD) on mast cells in vitro and allergic responses in vivo. Methods The effects of LERD on the activation of mast cells and mast cell-mediated passive cutaneous anaphylaxis (PCA) were measured in mice and two types of mast cells, mouse bone marrow-derived mast cells (BMMCs) and RBL-2H3 cells in vitro. A mechanistic study of its inhibitory effect was performed by using degranulation assay, reverse transcriptase-polymerase chain reaction, enzyme-linked immunosorbent assay, and western blotting analysis. Results LERD reversibly suppressed antigen-stimulated degranulation in BMMCs and RBL-2H3 cells, and also inhibited mRNA expression and secretion of TNF-α and IL-4 in a dose-dependent manner. In a PCA animal model, LERD significantly inhibited antigen-induced allergic response and degranulation of ear tissue mast cells. As for the mechanism of action, LERD inhibited the activation of Syk, which is the pivotal signaling protein for mast cell activation by antigen. Furthermore, LERD also impeded the activations of well-known downstream proteins such as LAT, Akt and three MAP kinases (Erk, p38 and JNK). In an in vitro kinase assay, LERD suppressed the activation of Fyn in antigen-stimulated mast cells. Conclusion This study demonstrated for the first time that LERD has anti-allergic effects through inhibiting the Fyn/Syk pathway in mast cells. Therefore, this study provides scientific evidence for LERD to be used as an herbal medicine or health food for patients with allergic diseases.

The molecular mechanism underlying the selective vulnerability of neurons to oxidative damage caused by ischemia-reperfusion (I/R) injury remains unknown. We sought to determine the role of NADPH oxidase 1 (Nox1) in cerebral I/R-induced brain injury and survival of newborn cells in the ischemic injured region. Male Wistar rats were subjected to 90 min middle cerebral artery occlusion (MCAO) followed by reperfusion. After reperfusion, infarction size, level of superoxide and 8-hydroxy-2'-deoxyguanosine (8-oxo-2dG), and Nox1 immunoreactivity were determined. RNAi-mediated knockdown of Nox1 was used to investigate the role of Nox1 in I/R-induced oxidative damage, neuronal death, motor function recovery, and ischemic neurogenesis. After I/R, Nox1 expression and 8-oxo-2dG immunoreactivity was increased in cortical neurons of the peri-infarct regions. Both infarction size and neuronal death in I/R injury were significantly reduced by adeno-associated virus (AAV)-mediated transduction of Nox1 short hairpin RNA (shRNA). AAV-mediated Nox1 knockdown enhanced functional recovery after MCAO. The level of survival and differentiation of newborn cells in the peri-infarct regions were increased by Nox1 inhibition. Our data suggest that Nox-1 may be responsible for oxidative damage to DNA, subsequent cortical neuronal degeneration, functional recovery, and regulation of ischemic neurogenesis in the peri-infarct regions after stroke.

Functions attributed to phospholipase (PL) D include the regulation of intracellular trafficking of Golgi-derived vesicles and secretion of granules from mast cells. We have reported that activation of PLD and secretion in a rat mast cell (RBL-2H3) line is substantially enhanced by cholera toxin, a known activator of protein kinase (PK) A. Here we review the evidence that (1) the synergistic interactions of cholera toxin and other pharmacological agents on mast cell secretion are attributable to the synergistic activation of PLD via PKA, CaM kinase II, and PKC and (2) both PLD1 and PLD2 participate in this process. For example, treatment with cholera toxin, thapsigargin, and phorbol 12-myristate 13-acetate (which activate PKA, CaM kinase II, and PKC, respectively) exhibit synergy in the stimulation of both PLD and secretion. These kinases and PLD are likely confined to membrane components, as similar synergistic interactions could be demonstrated in permeabilized cells. The regulation of PLD and secretion by these kinases is also apparent from studies of inhibitors of PKA and other kinases. Also, by overexpression of either PLD1 or PLD2 it is apparent that both isoforms respond to the same stimuli as endogenous PLD, although PLD1 is largely associated with secretory granules and PLD2 with plasma membrane. The studies reveal interesting differences in the regulation of the translocation of granules (regulated by PKA) and the fusion of these granules with the plasma membrane (regulated by Ca(2+) and PKC). The pathological/physiological implications of the regulation of PLD by PKA require further evaluation in other cell systems.

Mast cells, constituents of virtually all organs and tissues, are critical cells in IgE-mediated allergic responses. The aim of this study was to investigate the effect and mechanism of an indoxyl chromogenic compound, 5-bromo-4-chloro-3-indolyl 1,3-diacetate, CAC-0982, on IgE-mediated mast cell activation and allergic responses in mice. CAC-0982 reversibly suppressed antigen-stimulated degranulation in murine mast cells (IC 50 , ~ 3.8 μM) and human mast cells (IC 50 , ~ 3.0 μM). CAC-0982 also inhibited the expression and secretion of IL-4 and TNF-α in mast cells. Furthermore, CAC-0982 suppressed the mast cell-mediated allergic responses in mice in a dose-dependent manner (ED 50 27.9 mg/kg). As for the mechanism, CAC-0982 largely suppressed the phosphorylation of Syk and its downstream signaling molecules, including LAT, Akt, Erk1/2, p38, and JNK. Notably, the tyrosine kinase assay of antigen-stimulated mast cells showed that CAC-0982 inhibited Fyn kinase, one of the upstream tyrosine kinases for Syk activation in mast cells. Taken together, these results suggest that CAC-0982 may be used as a new treatment for regulating IgE-mediated allergic diseases through the inhibition of the Fyn/Syk pathway in mast cells.

Background DJ-1 protein plays multifunctional roles including transcriptional regulation and scavenging oxidative stress; thus, it may be associated with the development of renal disorders. We investigated whether DJ-1 protein regulates the expression of (pro)renin receptor (PRR), a newly identified member of renin–angiotensin system. Methods The levels of mRNA and protein were determined by real-time PCR and western blot, respectively. H 2 O 2 production was tested by using fluorescence probe. Histone modification was determined by chromatin immunoprecipitation. Results The expression of PRR was significantly higher in the kidney from DJ-1 knockout mice (DJ-1 −/− ) compared with wild-type mice (DJ-1 +/+ ). Histone deacetylase 1 recruitment at the PRR promoter was lower, and histone H3 acetylation and RNA polymerase II recruitment were higher in DJ-1 −/− than in DJ-1 +/+ . Knockdown or inhibition of histone deacetylase 1 restored PRR expression in mesangial cells from DJ-1 +/+ . H 2 O 2 production was greater in DJ-1 −/− cells compared with DJ-1 +/+ cells. These changes in PRR expression and epigenetic modification in DJ-1 −/− cells were induced by H 2 O 2 treatment and reversed completely by addition of an antioxidant reagent. Prorenin-stimulated ERK1/2 phosphorylation was greater in DJ-1 −/− than in DJ-1 +/+ cells and this was inhibited by a PRR-inhibitory peptide, and by AT1 and AT2 receptor inhibitors. The expression of renal fibrotic genes was higher in DJ-1 −/− than in DJ-1 +/+ cells and decreased in PRR-knockdown DJ-1 −/− cells. Conclusions We conclude that DJ-1 protein regulates the expression of renal PRR through H 2 O 2 -mediated epigenetic modification. General significance We suggest that renal DJ-1 protein may be an important molecule in the acceleration of renal pathogenesis through PRR regulation.

Plasminogen activator inhibitor-1 (PAI-1) is an endogenous inhibitor of tissue plasminogen activator (tPA) that acts as a neuromodulator in various neurophysiological and pathological conditions. Several researchers including us reported the induction of PAI-1 during inflammatory condition; however, the mechanism regulating PAI-1 induction is not yet clear. In this study, we investigated the role of non-receptor tyrosine kinase Fyn in the regulation of lipopolysaccharide (LPS)-induced upregulation of PAI-1 in rat primary astrocyte. The activation of toll-like receptor 4 (TLR4) signaling, induced by its ligand LPS, stimulated a physical interaction between TLR4 and Fyn along with phosphorylation of tyrosine residue in both molecules as determined by co-immunoprecipitation experiments. Immunofluorescence staining also showed increased co-localization of TLR4-Fyn on cultured rat primary astrocytes after LPS treatment. The increased TRLR4-Fyn interaction induced expression of PAI-1 through the activation of PI3k/Akt/NFĸB pathway. Treatment with Src kinase inhibitor (PP2) or transfection of Fyn small interfering RNA (siRNA) into cultured rat primary astrocytes inhibited phosphorylation of tyrosine residue of TLR4 and blocked the interaction between TLR4 and Fyn resulting to the inhibition of LPS-induced expression of PAI-1. The activation of PI3K/Akt/NFĸB signaling cascades was also inhibited by Fyn knockdown in rat primary astrocytes. The induction of PAI-1 in rat primary astrocytes, which resulted in downregulation of tPA activity in culture supernatants, inhibited neurite outgrowth in cultured rat primary cortical neuron. The inhibition of neurite extension was prevented by PP2 or Fyn siRNA treatment in rat primary astrocytes. These results suggest the critical physiological role of TRL4-Fyn interaction in the modulation of PAI-1-tPA axis in astrocytes during neuroinflammatory responses such as ischemia/reperfusion injuries.

A facile one-step affinity chromatographic purification of the recombinant catalytic subunit (PDPc) of bovine pyruvate dehydrogenase phosphatase (PDP) to near homogeneity is described. PDPc binds in the presence of Ca(2+) to the inner lipoyl domain (L2) of the dihydrolipoamide acetyltransferase component (E2) of the mammalian pyruvate dehydrogenase complex. The affinity column consists of a glutathione S-transferase (GST)-L2 fusion protein bound to glutathione-Sepharose 4B beads. An extract of transformed Escherichia coli cells containing 50 mM Tris buffer (pH 7.5), 2 mM CaCl(2), 5 mM MgCl(2,) 150 mM NaCl, 0.5 mM dithiothreitol, 1% Triton X-100, and l M urea was passed through the affinity column, and the column was washed extensively with this buffer mixture. PDPc was eluted with 50 mM Tris buffer (pH 7.5) containing 5 mM MgCl(2), 0.5 mM dithiothreitol, and 1 mM EGTA. Approximately 22 mg of highly purified PDPc was obtained from 10 g (wet weight) of transformed cells. The preparation contained a small amount of a "nicked" form of PDPc. The cleavage is between Arg-394 and Arg-395.

We have investigated the effect of the newly synthesized proton-pump inhibitor YJA20379-8, 3-butyryl-4-[R-1-methylbenzylamino]-8-ethoxy-1,7-naphthyridine, on gastric mucosal proton pump (H+/K+-ATPase) activity, gastric acid secretion and gastric lesions in experimental animals. In lyophilized pig gastric microsomes, YJA20379-8 was shown to inhibit H+/K+-ATPase activity; the inhibitory effect was not affected by pH, the IC50 (dose resulting in 50% inhibition) being 28.0 μM and 30.0 μM at pH 6.4 and pH 7.4, respectively. The effect was fully reversed by dilution and subsequent washing of the incubation mixtures of H+/K+-ATPase and YJA20379-8, suggesting the reversible nature of the enzyme inhibition. In pylorus-ligated rats, YJA20379-8 administered by different routes (intraduodenal, subcutaneous, intravenous or oral) resulted in dose-dependent suppression of basal gastric acid secretion. The duration of antisecretory action of 30 mg kg−1 YJA20379-8 given intraduodenally was very brief (less than 7 h). Pretreatment with YJA20379-8 also dose-dependently prevented gastric lesions induced by absolute ethanol and water-immersion stress in rats. These results suggest that YJA20379-8 might exert its antiulcer activity partly by reversible suppression of acid secretion and partly by protecting the gastric mucosa against ulcerative stimuli.

The biochemical and pharmacological characteristics of a newly synthesized H+-K+ATPase inhibitor, 2-amino-4,5-dihydro-8-phenylimidazole[2,1-b]thiazolo[5,4-g]benzothiazole (YJA20379-1), were investigated. In the pig gastric microsomes, YJA20379-1 inhibited the gastric H+-K+ATPase regardless of pH condition, IC50values being 21 and 24 µM at pH 6.4 and 7.4, respectively. The inhibitory activity of YJA20379-1 was antagonized by dithiothreitol treatment but could not be reversed by dilution and washing of the enzyme preparation. In Sprague-Dawley rats, YJA20379-1, administered i.d., p.o, i.v., or s.c., significantly inhibited basal gastric acid secretion, with ED50values of 4.7, 20.2, 6.3, and 13.4 mg/kg, respectively. The antisecretory action of YJA20379-1 was short lasting (less than 7 h at an oral dosing of 30 mg/kg). Oral administration of YJA20379-1 also prevented the formation of ethanol, indomethacin, and water immersion stress induced gastric lesions and mepirizole-induced duodenal ulcers in rats. Furthermore, YJA20379-1 accelerated the healing of acetic acid induced chronic gastric ulcers in rats. In conclusion, these results suggest that YJA20379-1 has a potent inhibitory activity on the gastric H+-K+ATPase but much shorter duration of antisecretory action than omeprazole, thereby exerting its anti-ulcer effects partly with cytoprotective activity.Key words: proton pump inhibitor, acid secretion, anti-ulcer effects, cytoprotective activity.

This study was designed to determine biochemical and pharmacological properties of a newly synthesized benzimidazole derivative, 2-amino-4,5-dihydropyrido [1,2-a] thiazolo [5,4-g] benzimidazole (YJA20379-5) in vitro and in vivo. In the leaky membrane vesicles of pig gastric mucosa, YJA20379-5 inhibited the K(+)-stimulated H+,K(+)-ATPase activity in a concentration- and time-dependent manner, with IC50 values being 43 microM and 31 microM at pH 6.4 and 7.4, respectively. YJA20379-5, given intraduodenally, had a potent inhibitory effect on the gastric acid secretion in pylorus-ligated rats. The ED50 value for acid secretion was 15.4 mg/kg. YJA20379-5, administered orally, also suppressed gastric damages induced by water-immersion stress, indomethacin and ethanol, and duodenal damage induced by mepirizole in rats; the ED50 values were 17.6, 4.7, 3.0 and 18.7 mg/kg, respectively. Furthermore, repeated oral administration of YJA20379-5 accelerated the spontaneous healing of acetic acid-induced gastric ulcers in rats. It is concluded that the antisecretory activity of YJA20379-5 appears to be associated with inhibition of H+,K(+)-ATPase, while its antigastric and antiduodenal lesion activities are primarily related to the antisecretory effect.

The presence of L-arginine methylester (AME), L-arginine ethylester (AEE), or N-nitro-L-arginine methylester (NAME) in the growth media of Staphylococcus aureus increased the nitric oxide synthase (NOS) activity approximately 5- to 14-fold. The increase of NOS activity was confirmed by two assay methods, namely assaying the formation of L-[3H] citrulline from L-[3H] arginine and NO formation. The increase of NOS activity was most likely due to increased de novo synthesis, demonstrated by Western immunoblot analysis. The addition of methanol to the culture medium also increased the NOS activity as much as that found with the above three compounds. Evidence is presented to show that AME, AEE, or NAME gave rise to the formation of methanol in vivo by the action of intracellular esterase(s) and that methanol is subsequently involved in the induction of NOS in this bacterial system.

The activities of two types of antiulcer agents against 9 strains of Helicobacter pylori (H. pylori) were determined by the agar dilution method. The antiulcer agents were YJA20379, a newly synthesized proton pump inhibitor developed by Yung-Jin Pharmaceutical company, and omeprazole. Both compounds were found to have significant activities against this organism. The MIC values of YJA20379 and omeprazole were 11.7 and 31.25 micrograms/ml, respectively. In addition, the inhibitory potency of both compounds was investigated on H. pylori urease which is believed to be an important colonization and virulence factor in the pathogenesis of gastritis and peptic ulcers. These compounds dose-dependently inhibited urease extracted with distilled water and their IC50 values were 16.4 x 10(-5) M and 14.3 x 10(-5) M, respectively. In addition, a pH-dependent study to determine whether inhibitory potency would be activated by acid condition was performed. It was found that unlike omeprazole, YJA20379 was not affected by acid condition. To determine the inhibition pattern and optimal concentration of substrate, kinetics were evaluated at various pH levels (pH 5.0, 7.0, and 8.5). The data show that YJA20379 noncompetitively inhibited H. pylori urease and KM/Ki values were 0.96 mM/60 microM (pH 5.0), 0.56 mM/141.5 microM (pH 7.0), and 1.94 mM/34 microM (pH 8.5), respectively. Based on data obtained, it is concluded that YJA20379 is a significant inhibitor of H. pylori growth and urease and therefore, taking these results into consideration, YJA20379 might be a beneficial therapy for gastritis and peptic ulcers induced by H. pylori.

Effects of a newly synthesized antiulcer agent, YJA20379-4, on gastric proton pump (H+/K+-ATPase) activity, Helicobacter pylori (H. pylori) growth, gastric acid secretion, and gastro-duodenal lesions, were examined in comparison with those of omeprazole. YJA20379-4 markedly inhibited the H+/K+-ATPase activity in a concentration-dependent manner and the inhibitory effect was increased under a weak acidic condition; the IC50 values were 32 and 81 microM at pH 6.4 and 7.4, respectively. The inhibition was completely antagonized by 0.5 mM dithiothreitol (DTT). In addition, YJA20379-4 showed a significant anti-H. pylori activity determined by the agar dilution method. The value of minimum inhibitory concentration (MIC, 3.9-11.7 microg/ml) was at least 3 times more potent than that of omeprazole. In pylorus ligated rats, YJA20379-4 inhibited basal gastric acid secretion when administered by the intraduodenal route (ED50: 23.6 mg/kg). In experimental ulcer models, YJA20379-4 administered by the oral route dose-dependently prevented the development of gastro-duodenal lesions in rats. Moreover, repeated administration of YJA20379-4 promoted the healing of gastric ulcers induced by acetic acid. On the basis of the data obtained, it is suggested that YJA20379-4 has a wide spectrum of antiulcer activities, and its mode of antiulcer actions is dependent on the inhibition of H+/K+-ATPase activity and H. pylori growth and the enhancement of a mucosal defense. Thus, YJA20379-4 might prove to be a beneficial therapy for gastritis and peptic ulcer diseases.

The protein prohibitin (PHB) is implicated in diverse cellular processes, including cell signaling, transcriptional control, and mitochondrial function. We found that PHB was abundant in the intracellular granules of mast cells, which are critical for allergic responses to antigens. Thus, we investigated whether PHB played a role in signaling mediated by the high-affinity receptor for antigen-bound immunoglobulin E (IgE), FcεRI. PHB-specific small interfering RNAs (siRNAs) inhibited antigen-mediated signaling, degranulation, and cytokine secretion by mast cells in vitro. Knockdown of PHB inhibited the antigen-dependent association of the tyrosine kinase Syk with FcεRI and inhibited the activation of Syk. Fractionation studies revealed that PHB translocated from intracellular granules to plasma membrane lipid rafts in response to antigen, and knockdown of PHB suppressed the movement of FcεRIγ and Syk into lipid rafts. Tyrosine phosphorylation of PHB by Lyn was observed early after exposure to antigen, and point mutations in PHB indicated that Tyr(114) and Tyr(259) were required for the recruitment of Syk to FcεRIγ and mast cell activation. In mice, PHB-specific siRNAs inhibited antigen-initiated mast cell degranulation, passive cutaneous anaphylaxis, and passive systemic anaphylaxis. Together, these results suggest that PHB is essential for FcεRI-mediated mast cell activation and allergic responses in vivo, raising the possibility that PHB might serve as a therapeutic target for the treatment of allergic diseases.

New potential chemotherapeutic strategies are required to overcome multidrug resistance (MDR) in cancer. This study investigated the anticancer effect of a novel anthracene derivative MHY412 on doxorubicin-resistant human breast cancer (MCF-7/Adr) cells. We measured cell viability and the expression of apoptosis-related genes; in addition, the antitumor activity of MHY412 was confirmed using an in vivo tumor xenograft model. MHY412 significantly inhibited the proliferation of MCF-7/Adr and MCF-7 cells in a concentration-dependent manner. Notably, the half-maximal inhibitory concentration (IC50) values of MHY412 in MCF-7/Adr (0.15 µM) and MCF-7 (0.26 µM) cells were lower than those of doxorubicin (MCF-7/Adr, 13.6 µM and MCF-7, 1.26 µM) after treatment for 48 h. MHY412 at low concentrations induced S phase arrest, but at high concentrations, the number of MCF-7/Adr cells in the sub-G1 phase significantly increased. MHY412-induced sub-G1 phase arrest was associated with inhibition of cyclin, cyclin-dependent kinase 2 (CDK2) and p21 expression in MCF-7/Adr cells. MHY412 markedly reduced P-glycoprotein (P-gp) expression and increased apoptotic cell death in MCF-7/Adr cells. Cleavage of poly-ADP ribose polymerase, reduced Bcl-2 expression, and increased in cytochrome c release in MCF-7/Adr cells confirmed the above results. In addition, MHY412 markedly inhibited tumor growth in a tumor xenograft model of MCF-7/Adr cells. Our data suggest that MHY412 exerts antitumor effects by selectively modulating the genes related to cell cycle arrest and apoptosis. In particular, MHY412 is a new candidate agent for the treatment of Bcl-2 overexpressed doxorubicin-resistant human breast cancer.

Background Multidrug resistance is a major problem in the treatment of breast cancer, and a number of studies have attempted to find an efficient strategy with which to overcome it. In this study, we investigate the synergistic anticancer effects of resveratrol (RSV) and doxorubicin (Dox) against human breast cancer cell lines. Methods The synergistic effects of RSV on chemosensitivity were examined in Dox-resistant breast cancer (MCF-7/adr) and MDA-MB-231 cells. In vivo experiments were performed using a nude mouse xenograft model to investigate the combined sensitization effect of RSV and Dox. Results and conclusion RSV markedly enhanced Dox-induced cytotoxicity in MCF-7/adr and MDA-MB-231 cells. Treatment with a combination of RSV and Dox significantly increased the cellular accumulation of Dox by down-regulating the expression levels of ATP-binding cassette (ABC) transporter genes, MDR1, and MRP1. Further in vivo experiments in the xenograft model revealed that treatment with a combination of RSV and Dox significantly inhibited tumor volume by 60%, relative to the control group. General significance These results suggest that treatment with a combination of RSV and Dox would be a helpful strategy for increasing the efficacy of Dox by promoting an intracellular accumulation of Dox and decreasing multi-drug resistance in human breast cancer cells.

Aims DJ-1/park7 is a ubiquitously expressed multifunctional protein that plays essential roles in a variety of cells. However, its function in the vascular system has not been determined. We investigated the protective roles of DJ-1/park7 in vascular disorders, especially in neointimal hyperplasia. Methods and results DJ-1/park7 was strongly expressed in the neointimal layer, in which its oxidized form was predominant. Treatment of vascular smooth muscle cells (VSMCs) from the mouse aorta with H2O2 increased the oxidation of DJ-1/park7 visualized on two-dimensional electrophoresis gels. The growth of VSMCs in FBS-containing media and the release of H2O2 were significantly increased in DJ-1/park7–/– knockout mice compared with DJ-1/park7+/+ wild-type mice. The expression of cyclin D1 and the phosphorylation of extracellular signal-regulated kinase (ERK) 1/2 were greater in VSMCs from the DJ-1/park7–/– aorta than from the DJ-1/park7+/+ aorta. Both of these measures were inhibited by treatment with an ERK1/2 inhibitor or antioxidants and in DJ-1/park7-overexpressing cells. VSMC proliferation, cyclin D1 expression, and ERK1/2 phosphorylation in response to platelet-derived growth factor-BB were upregulated in DJ-1/park7–/– compared with DJ-1/park7+/+ mice. VSMCs of DJ-1/park7–/– mice exhibited higher levels of sprout outgrowth of aortic strips and neointimal plaque formation elicited by carotid artery ligation compared with those of DJ-1/park7+/+ mice. Conclusion These results indicate that DJ-1/park7 is involved in the growth of VSMCs, thereby inhibiting neointimal hyperplasia, and suggest that it might play protective roles in vascular remodelling.

Ethnopharmacological relevance Morus bombycis Koidzumi (MB) is widely distributed throughout Korea, where it is used as a traditional folk remedy for the treatment of allergic diseases including asthma. However, the pharmacological effect and the mechanistic study of MB have not been investigated. We aimed to investigate the anti-allergic activity of MB in vitro and in vivo and the mechanism of its action on mast cells. Materials and methods The anti-allergic activity of MB extract (MBE) was assessed using passive cutaneous anaphylaxis (PCA) in mice and mouse bone marrow-derived mast cells (BMMCs) in vitro. The effects of MBE on mast cell activation were evaluated by using the β-hexosaminidase release assay, reverse transcriptase-polymerase chain reaction, enzyme-linked immunosorbent assay, and western blotting analysis. Results MBE reversibly inhibited degranulation and generation of cytokines (TNF-α and IL-4) in antigen-stimulated mast cells. With regard to its mechanism of action, MBE inhibited the activation of Lyn and Syk, which have essential roles in degranulation and the production of various inflammatory cytokines. MBE also inhibited the activating phosphorylation of mitogen-activated protein (MAP) kinases, Erk1/2, p38, JNK, and Akt. In agreement with its in vitro effect, MBE significantly inhibited mast cell-mediated PCA reactions in IgE-sensitized mice. Conclusions The present results strongly suggest that MBE exerts an anti-allergic effect, both in vitro and in vivo by inhibiting the Lyn and Syk pathways in mast cells. Therefore, MBE may be useful for the treatment of allergic diseases, including atopic dermatitis and allergic asthma.

Sirtuins (SIRTs), NAD+-dependent class III histone deacetylases (HDACs), play an important role in the regulation of cell division, survival and senescence. Although a number of effective SIRT inhibitors have been developed, little is known about the specific mechanisms of their anticancer activity. In this study, we investigated the anticancer effects of sirtinol, a SIRT inhibitor, on MCF-7 human breast cancer cells. Apoptotic and autophagic cell death were measured. Sirtinol significantly inhibited the proliferation of MCF-7 cells in a concentration-dependent manner. The IC50 values of sirtinol were 48.6 µM (24 h) and 43.5 µM (48 h) in MCF-7 cells. As expected, sirtinol significantly increased the acetylation of p53, which has been reported to be a target of SIRT1/2. Flow cyto-metry analysis revealed that sirtinol significantly increased the G1 phase of the cell cycle. The upregulation of Bax, downregulation of Bcl-2 and cytochrome c release into the cytoplasm, which are considered as mechanisms of apoptotic cell death, were observed in the MCF-7 cells treated with sirtinol. The annexin V-FITC assay was used to confirm sirtinol-induced apoptotic cell death. Furthermore, the expression of LC3-II, an autophagy-related molecule, was significantly increased in MCF-7 cells after sirtinol treatment. Autophagic cell death was confirmed by acridine orange and monodansylcadaverine (MDC) staining. Of note, pre-treatment with 3-methyladenine (3-MA) increased the sirtinol-induced MCF-7 cell cytotoxicity, which is associated with blocking autophagic cell death and increasing apoptotic cell death. Based on our results, the downregulation of SIRT1/2 expression may play an important role in the regulation of breast cancer cell death; thus, SIRT1/2 may be a novel molecular target for cancer therapy and these findings may provide a molecular basis for targeting SIRT1/2 in future cancer therapy.

Objectives We aimed to determine the anti-arthritis effect and its mechanism of a combination of herbal extracts from Trachelospermi caulis (TC) and Moutan cortex radicis (MC) (TCMC). Methods The anti-arthritis activity of TCMC was assessed using a mouse model of type II collagen-induced arthritis (CIA). Reverse transcriptase-polymerase chain reaction (RT-PCR), enzyme-linked immunosorbent assay (ELISA), electrophoretic mobility shift assay (EMSA) and other biological assays were performed. Key findings TCMC significantly ameliorated various inflammatory parameters, such as clinical arthritis index, histological deformation of joints, serum levels of rheumatoid arthritis biomarkers (cartilage oligomeric matrix protein, serum amyloid P and anti-collagen type II IgG antibody), and Th1-related responses (T cell proliferation, and production of Interferon-γ and interleukin (IL)-2 in splenocytes isolated from CIA mice). The production of matrix metalloproteinases (MMPs), pro-inflammatory cytokines (tumour necrosis factor-α, IL-1β and IL-6) and chemokines (macrophage inflammatory protein-1, monocyte chemoattractant protein-1, and Regulated upon Activation, Normal T-cell Expressed, and Secreted) was suppressed by TCMC in CIA mice. In addition, the number of osteoclasts in the hind tibia was significantly decreased. With regard to the mechanism, TCMC suppressed the activation of the transcription factors nuclear factor (NF)-κB and activator protein (AP)-1. Conclusions TCMC exerts an anti-arthritis effect in CIA mice by suppression of the production of various inflammatory factors and the formation of osteoclasts through the inhibition of NF-κB and AP-1 activation.

Upon dimerization by oxidation, Hsp33 functions as a molecular chaperone in prokaryotes. Previously published structures of both the inactive and active species are of doubtful relevance to the solution conformations since the inactive (reduced) crystal structure was dimeric, while the active (oxidized) species was crystallized with a truncation of its regulation domain. The interdomain contact site of the inactive monomer, identified in this work, is consistent with that previously observed in the reduced dimer crystal. In contrast, fluorescence quenching of the active dimer contradicted the results expected from the domain-swapped fold observed in the truncated dimer crystal. The results of this study provide important new information concerning controversial issues in the activation process of Hsp33.

In this study, specific oral tolerance induction using interferon-gamma (IFN-γ) could successfully treat food allergies. Allergen-specific IL-10-producing regulatory B cell (Br1) responses are characteristic in immune tolerance of food allergies. The in-vivo effects of IFN-γ on allergen-induced changes in Br1 proportion and numbers in food allergies were investigated. Oral food challenges were conducted and 20 allergic patients to cow’s milk were selected. Of these 20 patients, five were treated with IFN-γ and milk (SOTI group), five were treated with only milk, five were treated with only IFN-γ, and five did not receive any treatment. In addition, 10 milk-tolerant subjects were involved in this study. Peripheral blood mononuclear cells (PBMCs) were stimulated using casein and stained for CD5, CD19, annexin V, and IL-10 before and after treatment. Allergy tolerance was induced only in the SOTI group along with induction of allergen-induced Br1 changes. Thus, IFN-γ can show tolerogenic effects in vivo when introduced with an allergen, which may be at least partly due to its effect on allergen-induced Br1 responses.

Polybrominated diphenyl ethers (PBDEs), commonly used flame retardants, have been reported as potential endocrine disruptor and neurodevelopmental toxicants, thus giving rise to the public health concern. The goal of this study was to investigate the relationship between umbilical cord blood, maternal blood, and breast milk concentrations of PBDEs in South Korean. We assessed PBDE levels in paired samples of umbilical cord blood, maternal blood, and breast milk. The levels of seven PBDE congeners were measured in 21 paired samples collected from the Cheil Woman’s Hospital (Seoul, Korea) in 2008. We also measured thyroid hormones levels in maternal and cord blood to assess the association between PBDEs exposure and thyroid hormone levels. However, there was no correlation between serum thyroxin (T4) and total PBDEs concentrations. The total PBDEs concentrations in the umbilical cord blood, maternal blood, and breast milk were 10.7 ± 5.1 ng g −1 lipid, 7.7 ± 4.2 ng g −1 lipid, and 3.0 ± 1.8 ng g −1 lipid, respectively. The ranges of total PBDE concentrations observed were 2.28–30.94 ng g −1 lipid in umbilical cord blood, 1.8–17.66 ng g −1 lipid in maternal blood, and 1.08–8.66 ng g −1 lipid in breast milk. BDE-47 (45–73% of total PBDEs) was observed to be present dominantly in all samples, followed by BDE-153. A strong correlation was found for major BDE-congeners between breast milk and cord blood or maternal blood and cord blood samples. The measurement of PBDEs concentrations in maternal blood or breast milk may help to determine the concentration of PBDEs in infant.

CD19(+)CD5(+) regulatory B cells produce transforming growth factor β (TGF-β) in both mouse and human B-cell leukemias. In this study, TGF-β was uniquely produced by normal human regulatory B cells. TGF-β-producing regulatory B-cell (Br3) responses were characterized through allergic responses to cow's milk. In total, 10 subjects allergic to milk and 13 milk-tolerant subjects were selected following double-blinded, placebo-controlled food challenges. Their peripheral blood mononuclear cells were stimulated in vitro with casein. Following allergen stimulation, the percentage of Br3s among CD5(+) B cells decreased from 11.5% ± 13.7% to 8.0% ± 9.6% (P = 0.042, n = 5) in the milk-allergy group and increased from 14.7% ± 15.6% to 18.9% ± 20.1% (P = 0.006, n = 7) in the milk-tolerant group. However, the numbers of Br3s increased only in the milk-tolerant group, from 1,954 ± 1,058 to 4,548 ± 1,846 per well (P = 0.026), whereas the numbers of Br3s in the milk-allergy group were unchanged [2,596 ± 823 to 2,777 ± 802 per well (P = 0.734)]. The numbers of apoptotic events were similar to the numbers of total Br3 responses. The percentage of non-TGF-β-producing CD5(+) B cells with apoptotic changes increased from 13.4% ± 17.1% to 16.4% ± 20.3% (P = 0.047, n = 5) in the milk-allergy group and remained unchanged [from 9.9% ± 11.9% to 9.3% ± 11.4% (P = 0.099, n = 7)] in the milk-tolerant group. Using carboxyfluorescein succinimidyl ester labeling, we observed that the percentage of proliferating Br3s among CD5(+) B cells was unchanged [from 6.1% ± 2.8% to 6.4% ± 2.9% (P = 0.145)] in the milk-allergy group and increased from 6.8% ± 3.9% to 10.2% ± 5.3% (P = 0.024) in the milk-tolerant group. In conclusion, Br3s proliferated in response to allergen stimulation in the milk-tolerant group and not in the milk-allergy group. TGF-β-producing regulatory B cells (Br3) may be involved in allergy tolerance by negatively regulating the immune system with TGF-β, and this negative regulation may be controlled by apoptosis.

Based on the turbidimetric response of protein with 50%-trichloroacetic acid (TCA), this study aims to introduce an assay method for protein in solution. The standard procedure consists of mixing equal volume of sample solution (standard or unknown) with 50%-TCA solution and measuring the absorbance at 450 nm after 20 min. The absorbances of the solutions were almost stable over 120 min at room temperature. This assay method is simple, reproducible, and tolerant to many interfering substances. It can detect less amount than 10 μg/ml of bovine serum albumin. The assay method has low protein-to-protein variability over wide range of molecular weight.

Foxp3 is a transcript factor for regulatory T cell development. Interestingly, Foxp3-expressing cells were identified in B cells, especially in CD19(+)CD5(+) B cells, while those were not examined in CD19(+)CD5(-) B cells. Foxp3-expressing CD5(+) B cells in this study were identified in human PBMCs and were found to consist of 8.5±3.5% of CD19(+)CD5(+) B cells. CD19(+)CD5(+)Foxp3(+) B cells showed spontaneous apoptosis. Rare CD19(+)CD5(+) Foxp3(+) regulatory B cell (Breg) population was unveiled in human peripheral blood mononuclear cells and suggested as possible regulatory B cells (Breg) as regulatory T cells (Treg). The immunologic and the clinical relevant of Breg needs to be further investigated.

While natural antimicrobial peptides are potential therapeutic agents, their physicochemical properties and bioactivity generally need to be enhanced for clinical and commercial development. We have previously developed a cationic, amphipathic α-helical, 11-residue peptide (named herein GA-W2: FLGWLFKWASK-NH2) with potent antimicrobial and hemolytic activity, which was derived from a 24-residue, natural antimicrobial peptide isolated from frog skin. Here, we attempted to optimize peptide bioactivity by a rational approach to sequence modification. Seven analogues were generated from GA-W2, and their activities were compared with that of a 12-residue peptide, omiganan, which is being developed for clinical and commercial applications. Most of the modifications reported here improved antimicrobial activity. Among them, the GA-K4AL (FAKWAFKWLKK-NH2) peptide displayed the most potent antimicrobial activity with negligible hemolytic activity, superior to that of omiganan. The therapeutic index of GA-K4AL was improved more than 53- and more than 31-fold against Gram-negative and Gram-positive bacteria, respectively, compared to that of the starting peptide, GA-W2. Given its relatively shorter length and simpler amino acid composition, our sequence-optimized GA-K4AL peptide may thus be a potentially useful antimicrobial peptide agent.

Aim of the study Gastrodia elata (GE) Blume (Orchidaceae) has been traditionally used as a folk medicine in Oriental countries since centuries for their variety of therapeutic benefits. This study is an attempt to investigate the protective effects of GE extract against MPP + -induced cytotoxicity in human dopaminergic SH-SY5Y cells and explore the neuroprotective mechanisms involved. Materials and methods Human dopaminergic SH-SY5Y cells were used to demonstrate the protective effects of GE against multiple parameters such as MPP + -induced cell viability, oxidative damage, expression of Bcl-2 and Bax, caspase-3 and poly(ADP-ribose) polymerase proteolysis. Results GE effectively attenuated the cytotoxicity and improved cell viability in a dose-dependent manner. GE was effective in inhibiting both, the increased production of reactive oxygen species (ROS) and increase in Bax/Bcl-2 ratio, cleaved caspase-3 and PARP proteolysis. Conclusion Data from this study suggests the protective effects of GE on MPP + -induced cytotoxicity in dopaminergic cells, which may be ascribed to its significant anti-oxidative and anti-apoptotic properties, thus, GE might prove to be a valuable therapeutic agent for the treatment of various neurodegenerative diseases including progressive Parkinson's disease (PD).

Histone deacetylase (HDAC) inhibitors are a new class of anticancer agents that act by inhibiting cancer cell proliferation and inducing apoptosis both in vitro and in vivo. This study examined the anti-tumor effect of apicidin on human endometrial cancer Ishikawa cells in an animal model by inhibiting specific HDAC expression. Nude mice were injected subcutaneously (s.c.) with Ishikawa cells, and the levels of cell proliferation and apoptosis were measured in the tumor tissues after an apicidin treatment. The expression patterns of a specific HDAC class by apicidin were measured in Ishikawa endometrial cancer both in vitro and in vivo. The tumor volume and weight were measured after the apicidin treatment. Apicidin significantly increased the acetylated histone H3 levels in an Ishikawa cells in vitro culture but the levels of HDAC3 and HDAC4 expression were significantly decreased. Apicidin suppressed the tumor growth of transplanted Ishikawa cells, the expression of proliferative cell nuclear antigen (PCNA) and vascular endothelial growth factor (VEGF) in tumor xenograft model, respectively. The inhibitory effect of apicidin on tumor growth was mediated in part through the down-regulation of HDAC3 and HDAC4. We suggest that apicidin is an effective anti-tumor agent on human endometrial cancer cells, and acts by regulating cell proliferation and apoptosis through the down-regulation of HDAC3 and HDAC4.

Histone deacetylase (HDAC) inhibitors are a new class of anticancer agents that act by inhibiting cancer cell proliferation and inducing apoptosis both in vitro and in vivo. This study examined the anti-tumor effect of apicidin on human endometrial cancer Ishikawa cells in an animal model by inhibiting specific HDAC expression. Nude mice were injected subcutaneously (s.c.) with Ishikawa cells, and the levels of cell proliferation and apoptosis were measured in the tumor tissues after an apicidin treatment. The expression patterns of a specific HDAC class by apicidin were measured in Ishikawa endometrial cancer both in vitro and in vivo. The tumor volume and weight were measured after the apicidin treatment. Apicidin significantly increased the acetylated histone H3 levels in an Ishikawa cells in vitro culture but the levels of HDAC3 and HDAC4 expression were significantly decreased. Apicidin suppressed the tumor growth of transplanted Ishikawa cells, the expression of proliferative cell nuclear antigen (PCNA) and vascular endothelial growth factor (VEGF) in tumor xenograft model, respectively. The inhibitory effect of apicidin on tumor growth was mediated in part through the down-regulation of HDAC3 and HDAC4. We suggest that apicidin is an effective anti-tumor agent on human endometrial cancer cells, and acts by regulating cell proliferation and apoptosis through the down-regulation of HDAC3 and HDAC4.

We investigated whether oral administration of curcumin suppressed type II collagen–induced arthritis (CIA) in mice and its effect and mechanism on matrix metalloproteinase (MMP)-1 and MMP-3 production in CIA mice, RA fibroblast-like synoviocytes (FLS), and chondrocytes. CIA in mice was suppressed by oral administration of curcumin in a dose-dependent manner. Macroscopic observations were confirmed by histological examinations. Histological changes including infiltration of immune cells, synovial hyperplasia, cartilage destruction, and bone erosion in the hind paw sections were extensively suppressed by curcumin. The histological scores were consistent with clinical arthritis indexes. Production of MMP-1 and MMP-3 were inhibited by curcumin in CIA hind paw sections and tumor necrosis factor (TNF)-α–stimulated FLS and chondrocytes in a dose-dependent manner. As for the mechanism, curcumin inhibited activating phosphorylation of protein kinase Cδ (PKCδ) in CIA, FLS, and chondrocytes. Curcumin also suppressed the JNK and c-Jun activation in those cells. This study suggests that the suppression of MMP-1 and MMP-3 production by curcumin in CIA is mediated through the inhibition of PKCδ and the JNK/c-Jun signaling pathway. Keywords:: curcumin, matrix metalloproteinase (MMP)-1, MMP-3, protein kinase Cδ (PKCδ), collagen-induced arthritis

B cells have regulatory functions in immune responses. Antigen-specific responses of B cell subsets by allergen stimulation ex vivo were examined in milk allergy of late eczematous reactions. Eight milk allergy subjects and 13 milk tolerant subjects were selected by DBPCFC. PBMCs were stimulated by casein ex vivo and stained for B cell subsets using monoclonal antibodies. CD19+ B cells unchanged from 8.7 ± 3.8% to 8.0 ± 5.1% (p = 0.504, n = 8) in the milk allergy group and decreased in the milk tolerant group from 8.5 ± 3.2% to 5.0 ± 1.6% (p = 0.001, n = 13). The fraction of apoptotic B cells in B cells significantly decreased 4.4 ± 3.1% to 1.3 ± 0.4% (p = 0.027, n = 4) in the allergy group and insignificantly increased from 2.8 ± 0.6% to 5.4 ± 2.6% (p = 0.059, n = 6) in the milk tolerant group. CD5+ regulatory B1 cell% in B cells decreased in milk allergy subjects from 36.2 ± 5.0% to 31.0 ± 5.7% (p = 0.010) and unchanged in milk tolerant subjects from 41.6 ± 10.2% to 43.8 ± 10.0% (p = 0.413). IL-10 producing CD19+CD5+ regulatory B cell% in CD19+CD5+ regulatory B cells significantly decreased from 24.9 ± 6.5% to 13.8 ± 5.6% (p = 0.002, n = 5) by casein stimulation in milk allergy group and unchanged from 44.8 ± 11.3% to 43.9 ± 10.0% (p = 0.297, n = 5) in the milk tolerant group. B cell subset responses to IL-4 and IL-5 were also similar in both groups. B cell subset changes seemed to have diagnostic value. Exact immunologic roles of regulatory CD5+ B1 cells need further investigation.

Objectives The aim was to investigate the anti-inflammatory effects of Artemisia princeps extract on the activity of anti-CD3/CD28-stimulated CD4+CD25- T cells and antigen-expanded regulatory T cells. Methods CD4+CD25- T cells were activated with coated anti-CD3 and anti-CD28 and cultured in the presence or absence of various concentrations of A. princeps extract. The cultures were pulsed on Day 6 with [3H]thymidine and, after harvesting the cells, [3H] thymidine incorporation was measured. For analysis of interleukin-2 and interferon-γ secreted from CD4+CD25- T cells, culture supernatants were collected on Days 2 and 6. For the analysis of interleukin-10 secreted from the CD4+CD25- T cells and expanded regulatory T cells, supernatants were collected after 2 and 7 days, respectively. Cytokine levels were determined using an enzyme-linked immunosorbent assay. Potential medicinal components of the A. princeps extract were determined using gas chromatography–mass spectrometry. Key findings A. princeps (30 μg/ml) effectively suppressed proliferation of CD4+CD25- T cells that were stimulated with anti-CD3/CD28 without causing cytotoxicity in spleen cells incubated under conditions lacking antigen stimulation. A. princeps inhibited production of the pro-inflammatory cytokines interleukin-2 and interferon-γ in anti-CD3/CD28-stimulated CD4+CD25- T cells. Also, the extract slightly increased production of the anti-inflammatory cytokine interleukin-10 in these cells. In regulatory T cells expanded by anti-CD3/CD28, A. princeps increased production of interleukin-10 and Foxp3. Conclusions The results suggest that A. princeps may be useful in the treatment of autoimmune diseases and organ transplantation rejection by inhibiting proliferation of inflammatory T cells, suppressing inflammatory processes in antigen-stimulated CD4+CD25- T cells and increasing activity of expanded regulatory T cells.

Ethnopharmacological relevance Chrysanthemum morifolium Ramat (Asteraceae) has (CM) long been used in Korean and Chinese traditional herbal medicines with numerous therapeutic applications. Aim of the study To evaluate the neuroprotective activities of Chrysanthemum morifolium (CM) extract against 1-methyl-4-phenylpridinium ions (MPP+), Parkinsonian toxin through oxidative stress and impaired energy metabolism, in human SH-SY5Y neuroblastoma cells and the underlying mechanisms. Materials and methods The effects of CM against MPP+-induced cytotoxicity and neuronal cell viability, oxidative damage, the expression of Bcl-2 and Bax, caspase-3 and poly(ADP-ribose) polymerase (PARP) proteolysis were evaluated by using SH-SY5Y neuroblastoma cells. Results CM effectively inhibited the cytotoxicity and improved cell viability. CM also attenuated the elevation of reactive oxygen species (ROS) level, increase in Bax/Bcl-2 ratio, cleavage of caspase-3 and PARP proteolysis. Conclusion These results demonstrate that CM possesses potent neuroprotective activity and therefore, might be a potential candidate in neurodegenerative diseases such as Parkinson's disease.

Objective Interleukin-32 (IL-32) is a recently discovered cytokine that appears to play a critical role in human rheumatoid arthritis (RA). It is highly expressed in synovium and fibroblast-like synoviocytes (FLS) from RA patients, but not in patients with osteoarthritis (OA). This study was undertaken to assess IL-32 levels in RA synovial fluid (SF) and to investigate the secretion and regulation of IL-32 in RA FLS. Methods FLS and SF were obtained from the joints of RA patients. The secretion and expression of IL-32 and activation of signaling molecules were examined by enzyme-linked immunosorbent assay, immunoblotting, immunoprecipitation, reverse transcriptase–polymerase chain reaction, and small interfering RNA (siRNA) transfection. Results IL-32 levels were high in RA SF compared with OA SF. Furthermore, RA FLS expressed and secreted IL-32 when stimulated with tumor necrosis factor α (TNFα). TNFα-induced expression of IL-32 was significantly suppressed, in a dose-dependent manner, by inhibitors of Syk, protein kinase Cδ (PKCδ), and JNK and by knockdown of these kinases and c-Jun with siRNA. We also observed that PKCδ mediated the activation of JNK and c-Jun, and experiments using specific inhibitors and siRNA demonstrated that Syk was the upstream kinase for the activation of PKCδ. Conclusion The present findings suggest that IL-32 may be a newly identified prognostic biomarker in RA, thereby adding valuable knowledge to the understanding of this disease. The results also demonstrate that the production of IL-32 in RA FLS is regulated by Syk/PKCδ-mediated signaling events.

Mast cells are responsible for IgE-mediated allergic responses. Although dietary flavonoid morin has been known to suppress mast cell activation, its in vivo anti-allergic activity and the underlying mechanisms remain are largely unknown. In this study, we determine whether morin suppresses IgE-mediated allergic responses in an animal model and its mechanism of action. Morin suppressed IgE-mediated PCA in mice (ED50 23.9 mg/kg) and inhibited degranulation and production of tumor necrosis factor-alpha (TNF-α) and interleukin (IL)-4 in antigen (Ag)-stimulated mast cells. The mechanism of action was a follows. Morin inhibited the activating phosphorylation of spleen tyrosine kinase (Syk) and linker for activation of T cells (LAT) in rat basophilic leukemia (RBL)-2H3 cells and bone marrow-derived mast cells (BMMCs). Akt and the mitogen-activated protein (MAP) kinases, p38, extracellular signal-regulated kinase (ERK)1/2, and c-Jun N-terminal kinase (JNK) were inhibited as well. In vitro kinase assay indicated that Fyn kinase, not Lyn and Syk, was inhibited by morin in a dose-dependent manner (IC50 5.7 μM). In conclusion, the results suggest that morin suppresses the IgE-mediated allergic response by primarily inhibiting Fyn kinase in mast cells.

This study examined the mechanism for the anti-cancer effects of histone deacetylase (HDAC) inhibitor trichostatin A (TsA) in H-ras-transformed human breast epithelial (MCF10A-ras) cells. The effects of TsA on anti-cancer effects of MCF10A-ras cells were determined by measuring the level of cell cycle regulator expression and apoptotic cell death using Western blotting and flow cytometry analysis, respectively. TsA induced morphological changes, apoptotic cell death and modulation of the cell cycle regulatory proteins in the MCF10A-ras cells. TsA increased the levels of acetylated histone H3 and H4 in MCF10A-ras cells. In addition, TsA markedly down-regulated the expression of cyclin D1 and CDK4, up-regulated the expression of p21WAF1 and p53 and induced cell cycle arrest at the G1 phase in MCF10A-ras cells. The levels of hyperphosphorylation of the Rb protein were lower in MCF10A-ras cells after the TsA treatment. Furthermore, the up-regulation of p53 promoted Bax expression, which led to the activation of pro-caspase-3 and eventually to apoptosis in MCF10A-ras cells. TsA significantly increased the levels of ERK1/2 phosphorylation in MCF10A-ras cells. Overall, the TsA-activated ERK pathway plays an important role in cell cycle arrest and apoptosis through the ERK-dependent induction of p21 in Ras-related human cancer cells.

This study examined whether an extract of Cudrania tricuspidata shows anti-proliferative effects in anti-CD3/CD28-mediated spleen and CD4+CD25− T cells and decreases the production of the pro-inflammatory cytokines interleukin-2 (IL-2) and interferon-γ (IFN-γ) in anti-CD3/CD28-mediated CD4+CD25− T cells. The proliferation of anti-CD3/CD28-mediated spleen cells and CD4+CD25− T cells was effectively suppressed by C. tricuspidata. This extract, however, did not show cytotoxicity in spleen cells under conditions where the antigen was not stimulated using CCK-8 analysis. C. tricuspidata also decreased the production of the pro-inflammatory cytokines IL-2 and IFN-γ by selective inhibition of this extract on proliferating cells in anti-CD3/CD28-mediated CD4+CD25− T cells. These results suggest that C. tricuspidata may be useful in the treatment of autoimmune diseases and organ transplantation through the inhibitory action of T cells in inflammation.

Complementary and alternative medicines are considered as a promising direction for the development of anti-allergic therapies in oriental countries. We screened approximately 100 oriental herbal medicines for anti-allergic activity. Sophorae flos exhibited the most potent effect on degranulation in antigen-stimulated mast cells. We further investigated the effect of Sophorae flos on the IgE-mediated allergic response in vivo and its mechanism of action in mast cells. Sophorae flos exhibited a significant inhibitory effect on degranulation in antigen-stimulated mast cells with IC50 values of ~31.6 μg/mL (RBL-2H3 mast cells) and ~47.8 μg/mL (bone marrow-derived mast cells). Sophorae flos also suppressed the expression and secretion of TNF-α and IL-4 in the cells and IgE-mediated passive cutaneous anaphylaxis (PCA) in mice. Sophorae flos inhibited the activating phosphorylation of Syk and LAT in mast cells. Further downstream, activating phosphorylation of Akt and the prototypic MAP kinases, namely, p38, ERK1/2, and JNK, were also inhibited. These results suggest that Sophorae flos inhibits the Src family kinase-dependent signaling cascades in mast cells and may thus exert anti-allergic activity.

Cyclic AMP receptor protein (CRP) is allosterically activated by cAMP and functions as a global transcription regulator in enteric bacteria. Structural information on CRP in the absence of cAMP (apo-CRP) is essential to fully understand its allosteric behavior. In this study we demonstrated interdomain interactions in apo-CRP, using a comparative thermodynamic approach to the intact protein and its isolated domains, which were prepared either by limited proteolysis or using recombinant DNA. Thermal denaturation of the intact apo-CRP, monitored by differential scanning calorimetry, revealed an apparently single cooperative transition with a slight asymmetry. Combined with circular dichroism and fluorescence analysis, the thermal denaturation of apo-CRP could be interpreted as a coupled process involving two individual transitions, each attributable to a structural domain. When isolated individually, both of the domains exhibited significantly altered thermal behavior, thus pointing to the existence of non-covalent interdomain interactions in the intact apo-CRP. These observations suggest that the allosteric conformational change of CRP upon binding to cAMP is achieved by perturbing or modifying pre-existing interdomain interactions. They also underline the effectiveness of a comparative approach using calorimetric and structural probes for studying the thermodynamics of a protein.

Two new guaiane sesquiterpene lactones ( 1 and 2) and seven new amino acid-sesquiterpene lactone conjugates ( 3- 9), together with six known sesquiterpene lactones ( 10- 15), were isolated from the methanol extract of the aerial parts of Saussurea pulchella. Their structures were determined on the basis of spectroscopic and chemical methods to be 8alpha- O-(3'-hydroxy-3'-methylbutyryl)desacylcynaropicrin ( 1), 8alpha- O-(2', 3'-dihydroxyisobutyryl)11beta,13-dihydrodesacylcynaropicrin ( 2), and pulchellamines A, B, C, D, E, F, and G ( 3- 9). The structures of the new amino acid-sesquiterpene lactone conjugates, pulchellamines A, B, C, D, E, F, and G ( 3- 9), were confirmed by synthesis. The isolated compounds were evaluated for cytotoxic activity against four human tumor cell lines. Compounds 11 and 12 exhibited cytotoxicity against skin melanoma (SK-MEL-2) and ovary malignant ascites (SK-OV-3) human tumor cell lines with ED 50 values of 1.53 and 4.07 microM, and 2.49 and 7.42 microM, respectively.

The cellular susceptibility of cancer cells to histone deacetylase (HDAC) inhibitors is increased by the etopic expression of oncogenic Ras. However, the ability of HDAC inhibitors to regulate the apoptotic pathway in human breast cancer cells is still not completely understood. In this study, the anti-proliferative effects of apicidin were compared in H-ras-transformed human breast epithelial (MCF10A-ras) and non-transformed epithelial (MCF10A) cells. MCF10A-ras cells showed a significantly higher growth rate than MCF10A cells. Apicidin significantly increased the levels of acetylated histone H3 and H4 in both cell lines. Western blot analysis and flow cytometry were used to determine if the anti-proliferative effects of apicidin in MCF10A and MCF10A-ras cells could be mediated by modulating the cell cycle. Apicidin attenuated the expression of cyclin E and CDK2 in MCF10A cells, decreased cyclin D1 and cyclin E levels in MCF10A-ras cells, and increased the levels of CDK inhibitors, p21WAF1/Cip1 and p27Kip1, in both cell lines. Notably, the levels of hyperphosphorylation of the Rb protein levels were lower in the MCF10A-ras cells after apicidin treatment. Studies on the regulation of apoptosis showed that apicidin induces the up-regulation of p53 and the downstream activation of ERK in MCF10A-ras cells. The up-regulation of p53 promoted Bax expression leading to activation of caspases-9 and -6, and eventually to apoptosis in MCF10A-ras cells. In addition, apicidin significantly increased the levels of ERK1/2 phosphorylation in MCF10A-ras cells. Therefore, the apicidin-mediated ERK pathway appears to play an important role in modulating the pro-apoptotic pathway in MCF10A-ras cells.