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Marked Inhibition (marked + inhibition)
Selected AbstractsBSc2118 is a novel proteasome inhibitor with activity against multiple myelomaEUROPEAN JOURNAL OF HAEMATOLOGY, Issue 2 2010Jan Sterz Abstract Objectives:, The ubiquitin,proteasome system emerged as a new therapeutic target in cancer treatment. The purpose of this study was to elucidate the effects of the novel proteasome inhibitor BSc2118 on t(4;14) positive and negative multiple myeloma (MM) cells and normal peripheral blood mononuclear cells (PBMNC). Methods:, Human MM cell lines OPM-2, RPMI-8226, and U266 and primary MM cells from bone marrow aspirates were exposed to BSc2118. Cytotoxicity levels were evaluated using the MTT-test. BSc2118-induced apoptosis was analyzed by annexin-V assay. Further methods used included proteasomal activity determination, cell cycle analysis, western blot, and transcription factor assays. Results:, In OPM-2, RPMI-8226, U266 cell lines and primary MM cells, BSc2118 caused dose-dependent growth inhibitory effects. After 48 h, dose-dependent apoptosis occurred both in cell lines and primary myeloma cells irrespective of t(4;14). A significant G2-M cell cycle arrest occurred after 24 h. Furthermore, we observed a marked inhibition of intracellular proteasome activity, an increase in intracellular p21 levels, and an inhibition of NF-,B activation. The toxicity against PBMNC remained low, suggesting a broad therapeutic range of this agent. Conclusion:, Taken together, BSc2118 shows significant antimyeloma activity and may be considered as a promising agent in cancer drug development. [source] Proliferation and interleukin,5 production by CD8hiCD57+ T cellsEUROPEAN JOURNAL OF IMMUNOLOGY, Issue 4 2008Abstract CD8hiCD57+ T cells have previously been described as effector memory T cells with minimal expansion capacity and high susceptibility to activation-induced cell death. In contrast, we demonstrate here that CD8hiCD57+ T cells are capable of rapid expansion using multiple techniques including [3H]thymidine uptake, flow cytometric bead-based enumeration and standard haemocytometer counting. Previous reports can be explained by marked inhibition of activation-induced expansion and increased 7-amino-actinomycin,D uptake by CD8hiCD57+ T cells following treatment with CFSE, a dye previously used to measure their proliferation, combined with specific media requirements for the growth of this cell subset. The ability of CD8hiCD57+ T cells to further differentiate is highlighted by a distinct cytokine profile late after activation that includes the unexpected release of high levels of interleukin,5. These data indicate that CD8hiCD57+ T cells should not be considered as "end-stage" effector T cells incapable of proliferation, but represent a highly differentiated subset capable of rapid division and exhibiting novel functions separate from their previously described cytotoxic and IFN-, responses. [source] Resveratrol inhibits proliferation of human epidermoid carcinoma A431 cells by modulating MEK1 and AP-1 signalling pathwaysEXPERIMENTAL DERMATOLOGY, Issue 7 2006Arianna L. Kim Abstract:, Resveratrol (trans -3,4,,5-trihydroxystilbene) is a naturally occurring polyphenolic phytoalexin found in grapes, and has been shown to inhibit the growth of various types of cancer cells. We investigated the mechanism of the antiproliferative effect of resveratrol in A431-transformed keratinocytes harbouring mutant p53, and show that it is accompanied by G1 cell cycle arrest, which coincides with a marked inhibition of G1 cell cycle regulatory proteins, including cyclins A and D1 and cyclin-dependent kinase (CDK)6 and p53-independent induction of p21WAF1. Cell cycle arrest was also associated with the accumulation of hypophosphorylated Rb and p27KIP1. Resveratrol inhibited mitogen-activated protein kinase/extracellular signal-regulated kinase kinase (MEK)1 > extracellular signal-regulated protein kinase (ERK)1/2 signalling, downregulated c-Jun, and suppressed activating protein (AP)-1 DNA-binding and promoter activity. In addition, the inhibition of MEK1 > ERK1/2 signalling appears to be independent of retinoblastoma protein (pRb) hypophosphorylation in A431 cells, as PD098059 did not suppress pRb phosphorylation. Our results demonstrate that resveratrol affects multiple cellular targets in A431 cells, and that the downregulation of both AP-1 and pRb contributes to its antiproliferative activity in these cells. [source] Bicarbonate-rich choleresis induced by secretin in normal rat is taurocholate-dependent and involves AE2 anion exchanger,HEPATOLOGY, Issue 2 2006Jesús M. Banales Canalicular bile is modified along bile ducts through reabsorptive and secretory processes regulated by nerves, bile salts, and hormones such as secretin. Secretin stimulates ductular cystic fibrosis transmembrane conductance regulator (CFTR),dependent Cl, efflux and subsequent biliary HCO3, secretion, possibly via Cl,/HCO3, anion exchange (AE). However, the contribution of secretin to bile regulation in the normal rat, the significance of choleretic bile salts in secretin effects, and the role of Cl,/HCO3, exchange in secretin-stimulated HCO3, secretion all remain unclear. Here, secretin was administered to normal rats with maintained bile acid pool via continuous taurocholate infusion. Bile flow and biliary HCO3, and Cl, excretion were monitored following intrabiliary retrograde fluxes of saline solutions with and without the Cl, channel inhibitor 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB) or the Cl,/HCO3, exchange inhibitor 4,4,-diisothiocyanatostilbene-2,2,-disulfonic acid (DIDS). Secretin increased bile flow and biliary excretion of HCO3, and Cl,. Interestingly, secretin effects were not observed in the absence of taurocholate. Whereas secretin effects were all blocked by intrabiliary NPPB, DIDS only inhibited secretin-induced increases in bile flow and HCO3, excretion but not the increased Cl, excretion, revealing a role of biliary Cl,/HCO3, exchange in secretin-induced, bicarbonate-rich choleresis in normal rats. Finally, small hairpin RNA adenoviral constructs were used to demonstrate the involvement of the Na+ -independent anion exchanger 2 (AE2) through gene silencing in normal rat cholangiocytes. AE2 gene silencing caused a marked inhibition of unstimulated and secretin-stimulated Cl,/HCO3, exchange. In conclusion, maintenance of the bile acid pool is crucial for secretin to induce bicarbonate-rich choleresis in the normal rat and that this occurs via a chloride,bicarbonate exchange process consistent with AE2 function. (HEPATOLOGY 2006;43:266,275.) [source] Cooperative antitumor effects of vitamin D3 derivatives and rosemary preparations in a mouse model of myeloid leukemiaINTERNATIONAL JOURNAL OF CANCER, Issue 12 2006Hagar Sharabani Abstract 1,,25-dihydroxyvitamin D3 (1,25D3) is a powerful differentiation agent, which has potential for treatment of myeloid leukemias and other types of cancer, but the calcemia produced by pharmacologically active doses precludes the use of this agent in the clinic. We have shown that carnosic acid, the major rosemary polyphenol, enhances the differentiating and antiproliferative effects of low concentrations of 1,25D3 in human myeloid leukemia cell lines (HL60, U937). Here we translated these findings to in vivo conditions using a syngeneic mouse leukemia tumor model. To this end, we first demonstrated that as in HL60 cells, differentiation of WEHI-3B D, murine myelomonocytic leukemia cells induced by 1 nM 1,25D3 or its low-calcemic analog, 1,25-dihydroxy-16-ene-5,6-trans-cholecalciferol (Ro25-4020), can be synergistically potentiated by carnosic acid (10 ,M) or the carnosic acid-rich ethanolic extract of rosemary leaves. This effect was accompanied by cell cycle arrest in G0+G1 phase and a marked inhibition of cell growth. In the in vivo studies, i.p. injections of 2 ,g Ro25-4020 in Balb/c mice bearing WEHI-3B D, tumors produced a significant delay in tumor appearance and reduction in tumor size, without significant toxicity. Another analog, 1,25-dihydroxy-16,23Z-diene-20-epi-26,27-hexafluoro-19-nor-cholecalciferol (Ro26-3884) administered at the same dose was less effective than Ro25-4020 and profoundly toxic. Importantly, combined treatment with 1% dry rosemary extract (mixed with food) and 1 ,g Ro25-4020 resulted in a strong cooperative antitumor effect, without inducing hypercalcemia. These results indicate for the first time that a plant polyphenolic preparation and a vitamin D derivative can cooperate not only in inducing leukemia cell differentiation in vitro, but also in the antileukemic activity in vivo. These data may suggest novel protocols for chemoprevention or differentiation therapy of myeloid leukemia. © 2006 Wiley-Liss, Inc. [source] Bis(4,7-dimethyl and 5-dinitro-1,10-phenanthroline) sulfato-oxovanadium(IV)-mediated in vivo male germ cell apoptosisJOURNAL OF APPLIED TOXICOLOGY, Issue 4 2001Osmond J. D'Cruz Abstract Oxovanadium(IV) [VO] complexes of 1,10-phenanthroline are a new class of potent apoptosis-inducing cytotoxic agents against human testicular cancer cells in vitro. The present study investigated the in vivo ability of four(bis)-chelated 1,10-phenanthroline [phen] complexes of sulfato-oxovanadium(IV),VO(phen)2, VO(Cl,phen)2, VO(Me2,phen)2 and VO(NO2,phen)2,with and without substitutions, to induce testicular germ cell apoptosis. Male germ cell loss in mice was measured by determining the epididymal sperm count, testicular weight and histological evaluation of the testes. Repetitive intratesticular injection (7.5 mg kg,1 testis,1) of bis-chelated 1,10-phenanthroline complexes of oxovanadium(IV) with 4,7-dimethyl [VO(Me2,phen)2] and 5-dinitro [VO(NO2,phen)2] substitution led to decreased sperm counts and reduced testicular weights. Histopathological examination of testicular sections from VO(Me2,phen)2 - and VO(NO2,phen)2 -treated mice revealed a marked inhibition of spermatogenesis and preferential loss of maturing, as well as elongated spermatids. In situ evaluation of seminiferous tubule cross-sections by terminal deoxynucleotidyl transferase-mediated FITC-deoxyuridine triphosphate nick end-labeling (TUNEL) and laser scanning confocal microscopy showed characteristic apoptotic germ cells delineating the periphery of the seminiferous tubules. The ability of bis-chelated 4,7-dimethyl- and 5-dinitro-substituted 1,10-phenanthroline complexes of oxovanadium(IV) to induce germ cell apoptosis in vivo may have potential utility in the treatment of human testicular germ cell tumors. Copyright © 2001 John Wiley & Sons, Ltd. [source] Protection against acetaminophen hepatotoxicity by clofibrate pretreatment: Role of catalase induction,JOURNAL OF BIOCHEMICAL AND MOLECULAR TOXICOLOGY, Issue 5 2002Chuan Chen Abstract Mice pretreated with the peroxisome proliferator clofibrate (CFB) are highly resistant to acetaminophen (APAP)-induced hepatotoxicity. The objective of the present study was to investigate whether the increase in hepatic catalase activity following CFB pretreatment plays a role in this hepatoprotection. An irreversible inhibitor, 3-amino-1,2,4-triazole (3-AT), was used to modulate catalase activity. Hepatic catalase activity in mice pretreated with CFB (500 mg/kg, i.p., for 10 days) was significantly inhibited by 3-AT (100 or 500 mg/kg, i.p.). In addition, the lower dose of 3-AT (100 mg/kg) had minimal effect on biliary and urinary excretion of APAP metabolites generated from a nontoxic dose, suggesting that APAP metabolism was not modulated by this dose of 3-AT. The mortality rate of corn-oil-pretreated mice challenged with APAP (800 mg/kg, p.o.) was significantly increased by 3-AT (100 mg/kg, i.p.) given 1 h before APAP. As expected, CFB pretreatment conferred full protection against APAP-induced hepatotoxicity. The same 3-AT treatment, however, did not abolish hepatoprotection in CFB-pretreated mice, despite the marked inhibition of hepatic catalase activity. In conclusion, these results indicate that elevated catalase activity in mice exposed to CFB does not appear to mediate the hepatoprotection, suggesting that other cellular defense mechanisms are involved. © 2002 Wiley Periodicals, Inc. J Biochem Mol Toxicol 16:227,234, 2002; Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/jbt.10043 [source] The regulation of osteogenesis by ECM rigidity in MC3T3-E1 cells requires MAPK activationJOURNAL OF CELLULAR PHYSIOLOGY, Issue 3 2007Chirag B. Khatiwala Once thought to provide only structural support to tissues by acting as a scaffold to which cells bind, it is now widely recognized that the extracellular matrix (ECM) provides instructive signals that dictate cell behavior. Recently we demonstrated that mechanical cues intrinsic to the ECM directly regulate the behavior of pre-osteoblastic MC3T3-E1 cells. We hypothesized that one possible mechanism by which ECM compliance exerts its influence on osteogenesis is by modulating the mitogen-activated protein kinase (MAPK) pathway. To address this hypothesis, the differentiation of MC3T3-E1 cells cultured on poly(ethylene glycol) (PEG)-based model substrates with tunable mechanical properties was assessed. Alkaline phosphatase (ALP) levels at days 7 and 14 were found to be significantly higher in cells grown on stiffer substrates (423.9 kPa hydrogels and rigid tissue culture polystyrene (TCPS) control) than on a soft hydrogel (13.7 kPa). Osteocalcin (OCN) and bone sialoprotein (BSP) gene expression levels followed a similar trend. In parallel, MAPK activity was significantly higher in cells cultured on stiffer substrates at both time points. Inhibiting this activation pharmacologically, using PD98059, resulted in significantly lower ALP levels, OCN, and BSP gene expression levels on the hydrogels. Interestingly, the effectiveness of PD98059 was itself dependent on substrate stiffness, with marked inhibition of MAPK phosphorylation in cells grown on compliant hydrogels but insignificant reduction in cells grown on TCPS. Together, these data confirm a role for MAPK in the regulation of osteogenic differentiation by ECM compliance. J. Cell. Physiol. 211: 661,672, 2007. © 2007 Wiley-Liss, Inc. [source] PGH2 -derived levuglandin adducts increase the neurotoxicity of amyloid ,1,42JOURNAL OF NEUROCHEMISTRY, Issue 4 2006Olivier Boutaud Abstract The body of evidence indicating that oligomers of amyloid ,1,42 (A,1,42) produce toxicity to neurons, together with our demonstration that prostaglandin H2 (PGH2) oligomerizes amyloid ,1,42, led to the examination of the neurotoxicity of amyloid ,1,42 treated with PGH2. The neurotoxic effects of A,1,42 incubated with PGH2 was examined in primary cultures of cerebral neurons of mice, monitoring the reduction of 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide (MTT) as an indicator of cell toxicity. Whereas A,1,42 itself, incubated for 24 h, has little or no effect on MTT reduction, A,1,42 24 h after exposure to PGH2 produced a marked inhibition of MTT reduction, comparable with the inhibition resulting from A,1,42 that has been oligomerized by incubation for 6 days. Similar results were obtained when A,1,42 was incubated with levuglandin E2 (LGE2), a reactive aldehyde formed by spontaneous rearrangement of PGH2. The oligomers formed from reaction of A,1,42 with LGE2 exhibit immunochemical similarity with amyloid-derived diffusible ligands (ADDLs), as determined by analysis of the products of reaction of A,1,42 with LGE2 using western blotting with an antibody that is selective for ADDLs. [source] Proteasomal inhibition causes the formation of protein aggregates containing a wide range of proteins, including nitrated proteinsJOURNAL OF NEUROCHEMISTRY, Issue 2 2003Dong-Hoon Hyun Abstract Mutations in Cu,Zn-superoxide dismutase (SOD-1) are associated with some familial cases of amyotrophic lateral sclerosis (ALS), but it is not known how they result in cell death. We examined effects of overexpression of wild-type SOD-1 or the G37R or G85R mutations on the accumulation of ubiquitinated and nitrated proteins, and on loss of cell viability induced by the proteasome inhibitor, lactacystin. Wild-type SOD-1 had no effect on proteasomal activity, but the mutants decreased it somewhat. Treatment with lactacystin (1 µm) caused only limited cell viability loss, even though it induced a marked inhibition of proteasomal activities. However, viability loss due to apoptosis was substantial in response to lactacystin when cells were overexpressing a mutant SOD-1. The frequency of cells showing immunoreactivity against ubiquitinated- or nitrated-proteins was enhanced when wild-type and mutant SOD-1 s were overexpressed. Ubiquitinated or nitrated ,-tubulin, SOD-1, ,-synuclein and 68K neurofilaments were observed in the aggregates. Similar aggregates were observed in cells overexpressing mutant parkin (Del3,5, T240R and Q311,X). The nitric oxide synthase inhibitor, l -NAME, decreased viability loss and aggregation, suggesting that nitration of proteins may play an important role in aggregation and in the cell death accompanying it. [source] Expression of CD73/ecto-5,-nucleotidase on human gingival fibroblasts and contribution to the inhibition of interleukin-1,-induced granulocyte-macrophage colony stimulating factor productionJOURNAL OF PERIODONTAL RESEARCH, Issue 1 2004Eiji Nemoto Background and objectives:, CD73/5,-nucleotidase (5,-NT) is an ectoenzyme that participates in immune/inflammatory reactions. We examined the possible expression of CD73/5,-NT on human gingival fibroblasts (hGF), which are important to the immune/inflammatory system in periodontal tissue. Methods and results:, We demonstrated that CD73/5,-NT was expressed on hGF by flow cytometry. We found that pre-treatment of hGF with 5,-AMP induced marked inhibition of granulocyte-macrophage colony-stimulating factor (GM-CSF) production from hGF upon stimulation with interleukin-1, (IL-1,) by enzyme-linked immunosorbent assay (ELISA). A specific inhibitor of 5,-NT, adenosine 5,-[,,,-methylene] diphosphate blocked the inhibition of GM-CSF production, suggesting that adenosine converted from 5,-AMP acts on the inhibitory effects. The GM-CSF inhibition suggested that A3 receptor might be involved. The rank order of agonists was found to be (N6 -benzyl-5,- N -ethylcarboxamidoadenosine) A3 receptor agonist , (2-chloroadenosine) non-selective agonist > (CGS-21680) A2A receptor agonist > adenosine , (N6 -cyclohexyladenosine) A1 agonist. Further support for the main role of A3 receptor was the binding A3 antagonist [9-chloro-2-(2-furanyl)-5-([phenylacetyl]amino)[1,2,4]-triazolo[1,5- c]quinazdine] reversed the effect of adenosine, but no significant reverse was observed by A1 (1,3-dipropyl-8-cyclopentylxanthine), A2 [3,7-dimethyl-1-(2-propargyl)xanthine], A2A[8-(3-chlorostyryl)caffeine], and A2B (alloxazine) antagonists. The CD73/5,-NT expression was increased upon stimulation with gamma-interferon, but not other stimulants such as tumor necrosis factor-alpha, IL-4, lipopolysaccharide from Porphyromonas gingivalis and Escherichia coli, and fimbriae from P. gingivalis, and this increase was correlated with the enhanced GM-CSF inhibition by 5,-AMP but not adenosine. Conclusions:, These findings suggested that CD73/5,-NT on hGF exerts an anti-inflammatory effects in periodontal disease by conversion from 5,-AMP to adenosine. [source] Antinociceptive action of the extract and the flavonoid quercitrin isolated from Bauhinia microstachya leavesJOURNAL OF PHARMACY AND PHARMACOLOGY: AN INTERNATI ONAL JOURNAL OF PHARMACEUTICAL SCIENCE, Issue 10 2005Vinícius M. Gadotti This study examined the antinociceptive effect of Bauhinia microstachya (Leguminosae), a native plant widely distributed in the South of Brazil, in several chemical and mechanical models of pain. The methanolic extract (ME) from B. microstachya (3,30 mg kg,1, i.p.) and the isolated compound quercitrin (1,10 mg kg,1, i.p.), given 30 min earlier, produced a dose-dependent inhibition of acetic-acid-induced visceral pain in mice, with a mean ID50 value (dose necessary to reduce the nociceptive response by 50% relative to the control value) of 7.9 and 2.4 mg kg,1, respectively. The ME of B. microstachya (3,100 mg kg,1, i.p., 30 min earlier) also caused a dose-dependent inhibition of capsaicin-induced pain, with a mean ID50 value of 18.8 mg kg,1. Moreover, the ME (3,100 mg kg,1, i.p., 30 min earlier) produced marked inhibition of both phases of formalin-induced pain, with mean ID50 values for the neurogenic and the inflammatory phases of 30.3 and 17.2 mg kg,1, respectively. In addition, the ME of B. microstachya (3,300 mg kg ,1, i.p., 30 min earlier) inhibited, in a graded manner, the hyperalgesia induced by bradykinin (3.2 ,g/paw), substance P (13.5 ,g/paw), carrageenan (300 ,g/paw), capsaicin (100 ,g/paw) and adrenaline (100ng/paw) in the rat paw, with mean ID50 values of 20.5, 17.9, 101.8, 54.2 and 99.7 mg kg,1, respectively. Taken together, these data demonstrate that ME of B. microstachya elicited a pronounced antinociceptive action against several chemical and mechanical models of pain in mice and rats. The precise mechanism responsible for the antinociceptive effect of the extract still remains unclear, but seems to be partly related to modulation of the release or action of pro-inflammatory mediators involved in the models of pain used. Finally, the flavonoid quercitrin isolated from this plant appears to contribute for the antinociceptive property of the methanolic extract. [source] Defective Translocation of PKC, in EtOH-Induced Inhibition of Mg2+ Accumulation in Rat HepatocytesALCOHOLISM, Issue 9 2010Lisa M. Torres Background:, Rats chronically fed ethanol for 3 weeks presented a marked decreased in total hepatic Mg2+ content and required approximately 12 days to restore Mg2+ homeostasis upon ethanol withdrawal. This study was aimed at investigating the mechanisms responsible for the EtOH-induced delay. Methods:, Hepatocytes from rats fed ethanol for 3 weeks (Lieber-De Carli diet,chronic model), rats re-fed a control diet for varying periods of time following ethanol withdrawal, and age-matched control rats fed a liquid or a pellet diet were used. As acute models, hepatocytes from control animals or HepG2 cells were exposed to varying doses of ethanol in vitro for 8 minutes. Results:, Hepatocytes from ethanol-fed rats presented a marked inhibition of Mg2+ accumulation and a defective translocation of PKC, to the cell membrane. Upon ethanol withdrawal, 12 days were necessary for PKC, translocation and Mg2+ accumulation to return to normal levels. Exposure of control hepatocytes or HepG2 cells to a dose of ethanol as low as 0.01% for 8 minutes was already sufficient to inhibit Mg2+ accumulation and PKC, translocation for more than 60 minutes. Also in this model, recovery of Mg2+ accumulation was associated with restoration of PKC, translocation. The use of specific antisense in HepG2 cells confirmed the involvement of PKC, in modulating Mg2+ accumulation. Conclusions:, Translocation of PKC, isoform to the hepatocyte membrane is essential for Mg2+ accumulation to occur. Both acute and chronic ethanol administrations inhibit Mg2+ accumulation by specifically altering PKC, translocation to the cell membrane. [source] Mechanisms involved in the relaxant action of the ethanolic extract of propolis in the guinea-pig trachea in-vitroJOURNAL OF PHARMACY AND PHARMACOLOGY: AN INTERNATI ONAL JOURNAL OF PHARMACEUTICAL SCIENCE, Issue 6 2002Niraldo Paulino This study examines the mechanisms by which the standardised ethanolic extract of propolis induces relaxation of the guinea-pig trachea in-vitro. In guinea-pig trachea with or without epithelium and contracted by histamine, the propolis extract caused reproducible and graded relaxation, with a mean EC50 value of 3.8 or 10.5 ,g mL,1 and Emax of 100%, respectively. The propolis extract-induced relaxation was markedly reduced (26 ± 9 and 96 ± 3%) when guinea-pig tracheas were exposed to Krebs solution containing elevated K+ in the medium (40 or 80 mM). Pre-incubation of guinea-pig tracheas with tetraethylamonium (100 mM) or with 4-aminopyridine (10 mM) reduced the propolis extract-induced relaxation by 31±10% and 28 ± 2%. Likewise, apamin (0.1 ,M), charybdotoxin (0.1 ,M) or iberiotoxin (0.1 ,M) caused marked inhibition of propolis extract-mediated relaxation in guinea-pig trachea (percentage of inhibition: 65 ± 3%, 60 ± 5% and 65 ± 9%, respectively). Also, glibenclamide (1 ,M) inhibited the relaxant response caused by the propolis extract by 57 ± 4%. ,-Conotoxin GIVA (0.1 ,M) or capsaicin (1 ,M) produced small but significant inhibition (30 ± 5% or 47 ± 7%, respectively) of the propolis extract-induced relaxation. The vasoactive intestinal peptide (VIP) antagonist D- P -CI-Phe6, Leu17[VIP] porcine (0.1 ,M) inhibited relaxation by 55 ± 5%, while propranolol (1 ,M) induced a parallel rightward displacement (about 20 fold) of the propolis extract concentration-response curve. Finally, the propolis extract-induced relaxation was inhibited by the nitric oxide synthase inhibitor L-NG -nitroarginine (L-NOArg, 100 ,M) (48 ± 6%), and by the soluble guanylate cyclase inhibitor methylene blue (10 ,M) (37 ± 6%), while the more selective soluble guanylate cyclase inhibitor 1H -[1,2,4]oxadiazolol[4,3-a]quinoxalin-1-one (ODQ, 1 ,M) produced only a parallel (about 3 fold) rightward displacement of the propolis extract concentration-response curve. Collectively, these results support the notion that the propolis extract-mediated relaxation in the guinea-pig trachea involves the release of nitric oxide, probably from sensory neurons, besides the activation of soluble guanylate cyclase and activation of Ca2+ - and ATP-sensitive K+channels. Furthermore, the stimulation of ,2 -adrenergic and VIP receptors also seems to account for its relaxant action. [source] Modulation of selenoprotein P expression by TGF-,1 is mediated by Smad proteinsBIOFACTORS, Issue 1-4 2001Volker Mostert Abstract Selenoprotein P (SeP) is a selenium-rich plasma protein which accounts for more than 50% this study, the effect of TGF-,1 on the expression of SeP in the human liver cell line HepG2 was investigated. Western analysis revealed a dose-dependent reduction of SeP content in cell supernatant. RT-PCR analysis of SeP-mRNA expression demonstrated a marked inhibition and a reporter gene under control of the SeP promoter was negatively regulated by TGF-,1. Smad proteins are the transcriptional mediators of TGF-, signaling. A putative Smad-binding element (SBE) is present in the SeP promoter. In electrophoretic-mobility-shift assays, TGF-,1 enhanced the binding of nuclear proteins to this SBE. Overexpression of Smad3 and 4 resulted in a downregulation of SeP-promoter activity whereas deletion of the SBE led to a loss of TGF-,1 responsiveness. We conclude that SeP expression is modulated by the binding of Smad3/4 complexes to a functional SBE in the SeP promoter. [source] Stereoselective modulatory actions of oleamide on GABAA receptors and voltage-gated Na+ channels in vitro: a putative endogenous ligand for depressant drug sites in CNSBRITISH JOURNAL OF PHARMACOLOGY, Issue 2 2000Bernard Verdon cis -9,10-octadecenoamide (,oleamide') accumulates in CSF on sleep deprivation. It induces sleep in animals (the trans form is inactive) but its cellular actions are poorly characterized. We have used electrophysiology in cultures from embryonic rat cortex and biochemical studies in mouse nerve preparations to address these issues. Twenty ,Mcis -oleamide (but not trans) reversibly enhanced GABAA currents and depressed the frequency of spontaneous excitatory and inhibitory synaptic activity in cultured networks. cis -oleamide stereoselectively blocked veratridine-induced (but not K+ -induced) depolarisation of mouse synaptoneurosomes (IC50, 13.9 ,M). The cis isomer stereoselectively blocked veratridine-induced (but not K+ -induced) [3H]-GABA release from mouse synaptosomes (IC50, 4.6 ,M). At 20 ,Mcis -oleamide, but not trans, produced a marked inhibition of Na+ channel-dependent rises in intrasynaptosomal Ca2+. The physiological significance of these observations was examined by isolating Na+ spikes in cultured pyramidal neurones. Sixty-four ,Mcis -oleamide did not significantly alter the amplitude, rate of rise or duration of unitary action potentials (1 Hz). cis -Oleamide stereoselectively suppressed sustained repetitive firing (SRF) in these cells with an EC50 of 4.1 ,M suggesting a frequency- or state-dependent block of voltage-gated Na+ channels. Oleamide is a stereoselective modulator of both postsynaptic GABAA receptors and presynaptic or somatic voltage-gated Na+ channels which are crucial for synaptic inhibition and conduction. The modulatory actions are strikingly similar to those displayed by sedative or anticonvulsant barbiturates and a variety of general anaesthetics. Oleamide may represent an endogenous modulator for drug receptors and an important regulator of arousal. British Journal of Pharmacology (2000) 129, 283,290; doi:10.1038/sj.bjp.0703051 [source] Tetramer-blocking assay for defining antigen-specific cytotoxic T lymphocytes using peptide-MHC tetramerCANCER SCIENCE, Issue 2 2006Hiroshi Yokouchi Peptide-MHC tetramers have been engineered to allow accurate detection of antigen-specific cytotoxic C lymphocytes (CTL) by flow cytometry. Here, we propose a novel use for peptide-MHC tetramers in the specific and sensitive analysis of the cytotoxic function of antigen-specific CTL by blocking MHC-restricted antigen-specific cytotoxicity. We found that pretreatment of ovalbumin (OVA)-specific CD8+ CTL (OT-1 CTL), derived from OT-1 T-cell receptor (TCR)-transgenic mice, with OVA257,264 peptide-H-2Kb tetramer caused a marked inhibition of the cytotoxicity against OVA-expressing EG-7 tumor cells. OVA257,264 peptide-H-2Kb tetramer did not block the cytotoxicity mediated by 2C mouse (H-2b)-derived CD8+ CTL, which recognize allo (H-2Ld) antigens. Moreover, OT-I CTL activity was not inhibited by an irrelevant HBV208,216 peptide-H-2Kb tetramer. These results indicate that the blocking of CTL activity with peptide-MHC tetramer was caused by interference with the interaction between the TCR and H-2Kb -OVA257,264 peptide complex, but not with the CD8-MHC class I interaction. The blocking activity of OVA257,264 peptide-H-2Kb tetramer was reversible because OT-I CTL pretreated with the tetramer recovered their cytotoxicity after culturing with interleukin-2 for 24 h. The same results were also demonstrated in freshly isolated, in vivo -primed OT-1 CTL sorted by the tetramer. These results demonstrate that peptide-MHC tetramer is a useful tool for defining MHC-restricted antigen-specific CTL function. Moreover, our finding implies that the measurement of CTL activity immediately after tetramer-guided sorting is not a suitable method for evaluating the function of in vivo -induced tetramer-positive CTL. We believe that the tetramer-blocking assay presented here will be useful for functionally monitor the induction of MHC-restricted antigen-specific CTL during vaccination therapy against tumor and infectious diseases. (Cancer Sci 2006; 97: 148 ,154) [source] A New Quinoline Derivative MS-209 Reverses Multidrug Resistance and Inhibits Multiorgan Metastases by P-glycoprotein-expressing Human Small Cell Lung Cancer CellsCANCER SCIENCE, Issue 7 2001Hiroshi Nokihara Development of distant metastases and acquired multidrug resistance (MDR) are major problems in therapy for human small cell lung cancer (SCLC). MS-209 is a novel quinoline compound, which reverses P-glycoprotein (P-gp)-mediated MDR. We previously reported that MS-209 reversed in vitro MDR of human SCLC (SBC-3/ADM and H69/VP) cells expressing P-gp. In the present study, we determined the therapeutic effect of MS-209 in combination with chemotherapy against multiorgan metastases of MDR SCLC cells. SBC-3/ADM cells expressing P-gp were highly resistant to etoposide (VP-16), adriamycin (ADM), and vincristine (VCR) in vitro, compared with parental SBC-3 cells lacking P-gp expression. MS-209 restored chemosensitivity of SBC-3/ADM cells to VP-16, ADM, and VCR in a dose-dependent manner in vitro. Intravenous injection with SBC-3 or SBC-3/ADM cells produced metastatic colonies in the liver, kidneys and lymph nodes in natural killer (NK) cell-depleted severe combined immunodeficiency (SCID) mice, though SBC-3/ ADM cells more rapidly produced metastases than did SBC-3 cells. Treatment with VP-16 and ADM reduced metastasis formation by SBC-3 cells, whereas the same treatment did not affect metastasis by SBC-3/ADM cells. Although MS-209 alone had no effect on metastasis by SBC-3 or SBC-3/ADM cells, combined use of MS-209 with VP-16 or ADM resulted in marked inhibition of metastasis formation by SBC-3/ADM cells to multiple organs. These findings suggest that MS-209 reversed the MDR of SBC-3/ADM cells, but not SBC-3 cells, growing in the various organs, and inhibited metastasis formation in vivo. Therefore, this chemosensitizing agent, MS-209, may be useful for treatment of refractory SCLC patients with multiorgan metastases. [source] | ||||||||||||||||