Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “TMB”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 991 records · Page 55Linked to original sources
PubMed2000

Mechanisms involved in UTP-induced contraction in isolated rat aorta.

The mechanisms of UTP-induced contractions in the rat aorta strips were studied. These were only partially inhibited in a Ca(2+)-free medium or by incubation with verapamil or nifedipine. Successive challenges did not decrease the magnitude of the contraction in the absence of external Ca(2+). Quin 2(acetoxymethyl) ester (Quin 2AM), 8-(N,N-diethylamino)octyl 3,4,5-trimetoxybenzoate (TMB-8), thapsigargin and ryanodine inhibited these contractions. The participation of protein kinase C is also very likely, since downregulation by the phorbol 12,13 dibutyrate (PDB) decreased UTP-induced contraction, and staurosporine and 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H-7) antagonized UTP-induced contractions and relaxed UTP-induced tonic contractions. Therefore, different pools of intracellular Ca(2+) and protein kinase C seem to participate in UTP-induced contraction and in the mechanisms of maintenance in a Ca(2+)-free medium.

Animals↗
PubMed1997

Regulation of protein kinases in steady-state contraction of cat gastric smooth muscle.

Cat gastric smooth muscle strips were used to investigate the involvement of protein kinases in the steady-state contraction induced by 1 microM acetylcholine or 20 mM KCI. The steady-state contraction induced by acetylcholine or KCl was inhibited by EGTA dose dependently. Voltage-dependent Ca2+ channel antagonists dose dependently inhibited the contractions induced by KCI as well as by acetylcholine. Inhibitory effects of voltage-dependent Ca2+ channel antagonists were significantly more prominent on KCI-induced contractions than on acetylcholine-induced contractions. The acetylcholine-induced contraction was dose dependently inhibited by 8-(N,N-diethylamino)octyl-3,4,5-trimethoxybenzoate (TMB-8, a blocker of intracellular Ca2+ release), but the KCl-induced contraction was not inhibited at all. Therefore both intracellular Ca2+ release and extracellular Ca2+ influx seem to be necessary for the acetylcholine-induced contraction, but intracellular Ca2+ release is not necessary for the KCl-induced contraction. Protein kinase C inhibitors, 10 microM 1-(5-isoquinolinylsulfonyl)-2-methyl-piperazine 2HCl (H-7) and 1 microM staurosporine, significantly inhibited the contraction induced by acetylcholine or KCl. Calmodulin antagonists, 30 microM trifluoperazine and 50 microM N-(6-aminohexyl)-5-chloro-2-naphthalenesulfonamide HCI (W-7), however, significantly inhibited the contraction induced by acetylcholine but not by KCl. A tyrosine kinase inhibitor, 50 microM genistein, did not affect the acetylcholine-induced contraction but significantly inhibited the KCl-induced contraction. These results strongly suggest that the involvement of protein kinases in regulation of the steady-state contraction may be agonist-dependent.

Acetylcholine↗
PubMed1998

Bradykinin-evoked Ca2+ mobilization in Madin Darby canine kidney cells.

We studied the mechanisms underlying the bradykinin-evoked changes in intracellular calcium concentration ([Ca2+]i) in Madin Darby canine kidney (MDCK) cells. Bradykinin evoked a [Ca2+]i transient in a dose-dependent manner, measured by fura-2 fluorimetry and digital video imaging. The transient consisted of a rise and a decay and [Ca2+]i returned to baseline without oscillations. External Ca2+ influx occurred, as demonstrated by Mn2+ quench and external Ca2+ removal measurements. Bradykinin acted by stimulating bradykinin B2 receptors as evidenced by blockade by D-arginyl-L-arginlyl-L-prolyl-trans-4-hydroxy-L-prolylglycyl -3-(2-thienyl)-L-alanyl-L-seryl-D-1,2,3,4-tetrahydro-3-isoquinolineca rbonyl-L-(2alpha,3beta,7alphabeta)-octahydro-1 H-indole-2-carbonyl-L-arginine (HOE 140) but not by D-arginyl-L-arginlyl-L-prolyl-trans-4-hydroxy-L-proylglycyl- 3-(2-thienyl)-L-alanyl-L-seryl-D-1,2,3,4-tetrahydro-3-isoquinolinecar bonyl-L-(2alpha,3beta,7alphabeta)-octahydro-1 H-indole-2-carbonyl ([Des-Arg]HOE 140). The [Ca2+]i signal was abolished by 1-(6-((17beta-3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1 H-pyrrole-2,5-dione (U73122) and partially inhibited by neomycin, implying mediation by phospholipase C. The transient was initiated by a release of Ca2+ from internal stores since it was abolished by pretreatment with thapsigargin or cyclopiazonic acid. The mobilization of the internal Ca2+ store subsequently triggered a 1-[beta-[3-(4-methoxyphenyl)propoxy]-4-methoxyphenethyl]-1 H-imidazole hydrochloride (SKF 96365)-insensitive Ca2+ entry. Pretreatment with carbonylcyanide m-chlorophynylhydrozone and gly-phe-beta-naphthylamide did not alter the transient, thus excluding the participation of mitochondria and lysosomes. Efflux via Ca2+ pumps contributed to the decay of the transient. Efflux via Na+/Ca2+ exchange or sequestration by mitochondria and lysosomes was insignificant. The transient was blunted by the protein kinase C activator phorbol 12-myristate 13-acetate, and was enhanced by the protein kinase C inhibitors sphingosine and chelerythrine, the protein kinase A inhibitor 2,5-di-(t-butyl)-1,4-hydroquinone, N-[2-(p-bromocinnamylamino)ethyl]5-isoquinolinesulfonamide (H-89), the agent 8-(diethylamino)octyl 3,4,5-trimethoxybenzoate (TMB-8), and agents that elevated levels of 3',5'-cyclic guanosine monophosphate. The transient did not heterologously desensitize with that evoked by ATP, ADP or UTP.

Animals↗
PubMed1992

Regulatory volume decrease by cultured non-pigmented ciliary epithelial cells.

Cells (ODM C1-2/SV40) derived from human non-pigmented ciliary epithelial cells were studied by electronic cell sizing. The time course of the cell volume (vc) was monitored after suspending cells in paired experimental and control, isosmotic and hyposmotic solutions of identical ionic composition. Following anisosmotic cell swelling, the cells displayed the regulatory volume decrease (RVD) previously described. The RVD primarily reflects loss of cell KCl since: (1) the K(+)-channel blockers quinidine and Ba2+ both inhibit the RVD; and (2) replacement of external Cl- with gluconate or addition of the Cl- channel blocker NPPB also inhibits the RVD. Bicarbonate has previously been reported to speed the RVD. This action likely reflects pH dependence of the channels since: (1) increasing the external pH speeds the RVD, whether or not HCO3- is present; and (2) DIDS (a blocker of Cl- channels and of Cl-/HCO3- exchange) is an effective inhibitor of the RVD, even after blocking Cl-/HCO3- exchange by removing external HCO3-. The RVD could also be inhibited by reducing the availability of Ca2+, either by omitting Ca2+ from the external medium or by blocking mobilization of intracellular Ca2+ with TMB-8. Furthermore, the RVD was slowed and incomplete in the presence of the calcium/calmodulin blocker trifluoperazine. We conclude that anisosmotic swelling triggers a series of events, mediated at least in part by calcium/calmodulin, leading to the extrusion of KCl through parallel K+ and Cl- channels.

Bicarbonates↗
PubMed2000

Persistent increase in the amount of aquaporin-5 in the apical plasma membrane of rat parotid acinar cells induced by a muscarinic agonist SNI-2011.

SNI-2011 induces the long-lasting increase in the amount of aquaporin-5 (AQP5) in apical plasma membranes (APMs) of rat parotid acini in a concentration-dependent manner. This induction was inhibited by p-F-HHSiD, U73122, TMB-8, or dantrolene but not by bisindolmaleimide or H-7, indicating that SNI-2011 acting at M(3) muscarinic receptors induced translocation of AQP5 via [Ca(2+)](i) elevation but not via the activation of protein kinase C. In contrast, acetylcholine induced a transient translocation of AQP5 to APMs. SNI-2011 induces long-lasting oscillations of [Ca(2+)](i) in the presence of extracellular Ca(2+). Thus, SNI-2011 induces a long-lasting translocation of AQP5 to APMs coupled with persistent [Ca(2+)](i) oscillations.

Animals↗
PubMed2002

Increased intracellular calcium is required for neurite outgrowth induced by a synthetic peptide ligand of NCAM.

We have recently identified a synthetic peptide, termed C3, capable of binding the first immunoglobulin-like module of neural cell adhesion molecule (NCAM) by means of combinatorial chemistry and shown that this NCAM ligand promotes neurite outgrowth. By means of single cell calcium imaging using the calcium-sensitive probe fura-2-acetomethyl ester, we here show that the C3-peptide induced an increase in intracellular calcium in primary hippocampal neurons and PC12-E2 cells, presumably requiring mobilization of calcium from both extracellular and intracellular stores. We further observed that C3-induced neurite outgrowth was inhibited by antagonists of voltage-dependent calcium channels as well as by an inhibitor of intracellular calcium mobilization, TMB-8. These findings demonstrate at the single cell level that a synthetic NCAM ligand directly can induce an increase in intracellular calcium and suggest that NCAM-dependent neurite outgrowth requires calcium mobilization from both extracellular and intracellular calcium stores. Thus, the C3-peptide may be regarded as a useful tool for the study of NCAM-dependent signal transduction. Furthermore, the peptide may be of considerable therapeutical interest for the treatment of neurodegenerative disorders.

Animals↗
PubMed1997

Intracellular Ca2+ dependence of nitric oxide mediated enhancement of interleukin-8 secretion in human endothelial cells.

Nitric oxide (NO.) can induce transient [Ca2+] changes in endothelial cells not different from receptor mediated signalling. Whether this Ca2+ signal may provide a link with IL-8 secretion induced by NO. donors was investigated in human endothelial cells. Sodium nitroprusside (SNP) and S-nitroso-N-acetyl-DL-penicillamine (SNAP) dose dependently increased IL-8 production in this cell type. Additive IL-8 secretion was found with TNFalpha. Buffering intracellular Ca2+ with MAPT/AM suppressed NO. induced [Ca2+]i changes and reduced subsequent IL-8 secretion. The additive effect of both NO. donors on TNFalpha induced IL-8 secretion was completely blocked in the presence of MAPT/AM. SKF 96365, which has been shown to block receptor mediated Ca2+ entry, and TMB-8, which blocks intracellular Ca2+ release, both inhibited IL-8 secretion, particularly when TNFalpha was used as a costimulator, indicating that [Ca2+]i changes are important components of IL-8 induction by NO..

Calcium↗
PubMed1997

Activation of mitogen-activated protein kinase is involved in sphingosine 1-phosphate-stimulated interleukin-6 synthesis in osteoblasts.

We previously showed that sphingosine 1-phosphate (SPP) acts as a second messenger for tumor necrosis factor alpha-induced interleukin-6 (IL-6) synthesis in osteoblast-like MC3T3-E1 cells. In the present study, we further investigated the mechanism of IL-6 synthesis induced by SPP in MC3T3-E1 cells. SPP significantly induced p42/p44 mitogen-activated protein (MAP) kinase activity. PD98059, an inhibitor of MAP kinase kinase, suppressed SPP-induced IL-6 synthesis as well as SPP-induced MAP kinase activation. The patterns of both inhibitions were similar. TMB-8, an inhibitor of Ca2+ mobilization from intracellular Ca2+ stores, significantly suppressed the SPP-induced IL-6 synthesis. These results strongly suggest that SPP-induced IL-6 synthesis is mediated via p42/p44 MAP kinase activation in osteoblast-like cells and that the SPP-induced IL-6 synthesis is dependent on intracellular Ca2+ mobilization.

3T3 Cells↗
PubMed2001

Separation of ternary mixtures by pseudo-simulated moving bed chromatography.

Modeling and simulation are presented of the separation of a ternary mixture by pseudo-simulated moving bed chromatography, according to the JO process of Japan Organo Co. The process cycle is divided into two steps. In step 1, feed and eluent streams are introduced into the system, equivalent to a series of preparative chromatographic columns, and the intermediate component is produced. In step 2, similar to a simulated moving bed (SMB), there is no feed and the less adsorbed species is collected in the raffinate while the more retained species is collected in the extract; this step is described by an equivalent TMB model. A parametric study is presented in order to analyze the sensitivity of the model to variations on the duration of step 1 and step 2 and on the intraparticle mass transfer coefficient.

Chromatography↗
PubMed1999

Enantiomers separation by simulated moving bed chromatography. Non-instantaneous equilibrium at the solid-fluid interface.

The simulated moving bed (SMB) technology, first conceived for large bulk-scale separations in the petrochemical industry, has found increasingly new applications in the pharmaceutical industry. Among these, the separation of fine chemicals has been the subject of considerable study and research. This work presents the modeling, simulation and design of the operation of a SMB plant in order to separate a binary chiral mixture. The usual assumption of instantaneous equilibrium at the solid-fluid interface is questioned and a first-order kinetics of adsorption is taken into account. The cases of linear, Langmuir and modified Langmuir equilibria are studied. The equivalent true moving bed (TMB) model was used assuming axial dispersion for the fluid flow and plug flow for the solid-phase flow. Intraparticle diffusion was described by a linear driving force (LDF) approximation. Simulation results indicate that, under certain conditions, equilibrium is not actually reached at the adsorbent surface. This leads to different unit performances, in terms of product purities and recoveries, as compared to those predicted assuming instantaneous equilibrium. Moreover, SMB units may be improperly designed by the usual methods (flow-rate ratio separation regions) if non-equilibrium effects are overlooked.

Adsorption↗
PubMed2000

Optimization of simulated moving bed plants with low efficient stationary phases: separation of fructose and glucose.

An optimization procedure for simulated moving bed (SMB) plants with low efficient stationary phases is presented. The new aspect is that the desorbent consumption can be cut by 70% by running the plant with lower internal liquid flows and a corresponding larger switch time while the productivity is kept constant. This concept was validated by the separation of fructose and glucose in water on a calcium resin with an eight-column SMB plant. The separation can be predicted well by a true moving bed (TMB) and a simulated moving bed simulation. Adsorption isotherms were determined up to 300 kg/m3 for glucose and 500 kg/m3 for fructose from 25 to 80 degrees C. Experimental SMB runs were performed over a wide range of feed concentrations (10-350 kg/m3) and temperatures (25-80 degrees C). The strong influence of the delay volume is pointed out. For an experimental run with high feed concentration a complete set of data is presented. To reduce biological growth separation at 80 degrees C is recommended.

Fructose↗
PubMed2001

Optimisation of an ELISA for the serodiagnosis of visceral leishmaniasis using in vitro derived promastigote antigens.

An antibody detection ELISA was developed for diagnosis of visceral leishmaniasis. Antigens released by Leishmania donovani promastigotes into a protein-free medium were used. SDS-PAGE analysis has indicated that Ld-ESM contain several protein antigens. Titration and chequer-board analyses were performed to optimise the assay protocol. Optimal results were obtained when antigen (50 microg/ml) was coated with PBS-methyl glyoxal buffer, and wells blocked with 0.5% casein. A serum dilution of 1:500 in antigen-coated wells, blocked with 0.5% casein, generated lowest absorbance with Ref-ve sera and higher absorbance with Ref+ve sera. All steps of the ELISA were performed at room temperature. The S/N ratio, the differential absorbance between the negative sample vs. the test or Ref+ve sample, was used to quantify the specific antigen and antibody reactions. An anti-human monoclonal antibody conjugated with HRP (MAb-conjugate) outperformed a commercially available anti-human polyclonal antibody conjugate (PAb-conjugate). The MAb-conjugate gave minimal background reactions with endemic sera. Optimised final assay steps mentioned below were used to evaluate sera samples from field trials. ELISA wells were coated with 50 microg/ml Ld-ESM mixed in PBS-methyl glyoxal overnight, and after removing the antigen, blocked with 0.5% casein for 1 h at RT. Patient sera along with control sera, diluted to 1:500 in PBS/T, were reacted for 1 h at RT. After washing the plate with PBS/T, wells were reacted with MAb-conjugate for 40 min at RT, and after washing, binding of antibodies was visualized by using TMB as a chromogen substrate. The relative specific binding was quantified by the S/N ratio. A batch of n=22 endemic sera from North Africa were evaluated and resulted with 100% specificity and sensitivity, 99.99% PPV and 95.45% NPV. The specificity and sensitivity of this assay will be further evaluated in planned retrospective and prospective multi-site trials.

Animals↗
PubMed1998

Intracellular calcium in prothoracic glands of Manduca sexta.

Cytosolic free calcium was measured in individual prothoracic gland cells of Manduca larvae with Fura-2. During the last larval instar there was no correlation between intracellular calcium concentration and ecdysteroid secretion by the glands. The addition of prothoracicotropic hormone (PTTH) from brains of Manduca larvae to prothoracic glands in vitro resulted in a significant increase in the calcium concentration of the gland cells. The effect of PTTH was inhibited by the inorganic calcium channel antagonists, cadmium, lanthanum and nickel, and by the antagonist of T-type calcium channels, amiloride, whereas all the other antagonists tested failed to block the action of PTTH. TMB-8, an inhibitor of intracellular calcium mobilization, did not reduce the PTTH-induced rise in calcium, which suggests that IP(3)-dependent intracellular calcium stores are not involved in the calcium-mediated stimulation of ecdysteroid synthesis. Moreover, PTTH is thought to increase intracellular calcium in prothoracic glands of Manduca by influencing calcium channels in the plasma membrane.

Journal Article↗
PubMed1993

The action of diazepam in the isolated rat detrusor muscle.

Diazepam is one of the benzodiazepines, a group of drugs that depresses the central nervous system. It also inhibits the contractility of smooth muscles in the periphery, but the mechanism of this inhibitory action has not been clarified. Our study was undertaken to investigate the effect of diazepam on the contractility of the detrusor muscle. Detrusor muscle strips isolated from rat urinary bladder were examined by isometric myography. Diazepam, as well as baclofen, a gamma-aminobutyric acid (GABA)B receptor agonist, reduced the electric field stimulation-induced contractions; delta-aminovaleric acid, a GABAB receptor antagonist, completely antagonized the inhibitory effect of baclofen, but not the inhibitory action of diazepam. Diazepam reduced the basal tone of detrusor muscle concentration dependently, and this inhibitory action was not affected by tetrodotoxin. Diazepam suppressed the contractile responses to bethanechol, adenosine triphosphate and potassium chloride. Diazepam diminished the calcium-induced recovery of tension in calcium-free PSS. A23187, a calcium ionophore, partially recovered the basal tone which had been reduced by diazepam in normal physiologic salt solution (PSS). The loss of tension in calcium free PSS containing diazepam could not be recovered by addition of A23187. On the other hand, the loss of tension in calcium-free PSS containing 3,4,5-trimethoxybenzoic acid 8(diethylamino)octyl ester (TMB-8), an inhibitor of intracellular calcium release, was considerably recovered by addition of A23187. Based on these results, it is suggested that diazepam inhibits the contractility of detrusor muscle acting directly on the smooth muscle cell, which is unrelated to the activation of GABA receptors. Its inhibitory action appears to be mediated through interference with the influx of extracellular calcium.

Adenosine Triphosphate↗
PubMed2003

Magnesium-deficient medium enhances NO production in alveolar macrophages isolated from rats.

Magnesium deficiency has been shown to increase nitric oxide (NO) levels in plasma and to aggravate endotoxin lethality. The present study was performed to examine the effects of magnesium (Mg(2+))-deficient culture medium, with and without endotoxin (LPS), on NO release and inducible NOS (iNOS) mRNA levels in alveolar macrophages isolated from rats. Decreasing the Mg(2+) concentration in the culture medium from 0.39 mM (normal-Mg(2+) medium) to 0.021 mM (Mg(2+)-deficient medium) increased NO release from alveolar macrophages for 2 h. However, LPS stimulation in Mg(2+)-deficient medium had little effect on NO release. The increased NO release in Mg(2+)-deficient medium was suppressed completely by L-NAME and aminoguanidine. Dexamethasone, pyrrolidine dithiocarbamate and curcumin strongly inhibited NO release. Verapamil, U73122, TMB-8 and W-7 had no significant effect on NO release induced by Mg(2+) deficiency. Preculture of macrophages with Mg(2+)-deficient medium for 22 h markedly increased NO release and iNOS mRNA levels for a further 2 h; these increments were suppressed completely by curcumin. These results suggest that Mg(2+) deficiency enhances NO production via iNOS by alveolar macrophages. In this experimental condition, we can not suggest that NO production from alveolar macrophage plays an essential role in the pathogenesis of enhanced endotoxin lethality in Mg-deficient rats.

Animals↗
PubMed1997

P2X-purinoceptors in the heart: actions of ATP and UTP.

Positive inotropic effects of ATP and UTP (1 microM - 1mM) were studied in isolated rat and guinea pig cardiac tissues. The potency order obtained was ATP>UTP in both species, suggesting possible interaction with P2X-purinoceptors. Binding studies using [(3)H]alpha,beta-methylene ATP as marker of P2X-purinoceptors revealed two receptor sites: one high-, the other low-affinity, in atria and ventricles from rat and guinea pig. Both ATP and UTP were found to bind high-affinity sites of [(3)H]alpha,beta-methylene ATP. The effects of various calcium inhibitors such as nifedipine, dantrolene, ryanodine and TMB-8 on positive inotropic effects induced by ATP and UTP were also studied. The results suggest that ATP and UTP may increase inotropism by interaction with P2X-purinoceptors by means of a calcium-dependent mechanism.

Adenosine Triphosphate↗
PubMed1999

Pharmacomechanical coupling in rat vas deferens: effects of agents that modulate intracellular release of calcium and protein kinase C activation.

The effects of agents that modulate intracellular release of calcium and protein kinase C (PKC) activation on noradrenaline (NA)-induced contractions of epididymal vas deferens in calcium-free/EGTA (1 mM) medium were investigated. NA (100 microM) or methoxamine (100 microM) evoked repeatable contractions. Clonidine (100-300 microM) was ineffective. The contractions to NA were reduced by procaine (1-10 mM) but not by thapsigargin (0.1-30 microM), ryanodine (1-30 microM) or TMB-8 (1-30 microM). Contractions to cumulative additions of NA (1-100 microM) were enhanced in the presence of cyclopiazonic acid (10 & 30 microM) but not ryanodine (10 & 30 microM). Sequential contractions to NA were not blocked by PKC inhibitors, calphostin C (1 microM) or Ro 31-8220 (1-30 microM) but were reduced by H-7 (1-30 microM), a broad spectrum protein kinase inhibitor. Although RT-PCR experiments detected mRNA for some Ca2+-dependent/DAG-activated and Ca2+-independent/DAG-activated PKC isoforms in epididymal vas deferens, the PKC activators, phorbol 12, 13-dibutyrate (100 microM) or phorbol 12-myristate 13-acetate (100 microM) failed to activate the tissues in calcium-free medium but enhanced subsequent contractions to NA. These results indicate a limited role for intracellular calcium stores and phorbol ester/DAG-sensitive PKC isoforms in NA-induced contraction of epididymal rat vas deferens in calcium-free medium. The results suggest that pharmacomechanical coupling triggered by NA may involve the sensitization of contractile myofilaments to Ca2+ or a Ca2+-independent mechanism. The possible involvement of Ca2+-independent/DAG-insensitive PKC isoforms and agonist-dependent but PKC-independent sensitization pathway is discussed.

Animals↗
PubMed1999

Involvement of calcium in ACC-oxidase activity from Cicer arietinum seed embryonic axes.

Both in vivo and in vitro ACC-oxidase activities as well as ethylene production from embryonic axes of chickpea seeds were strongly inhibited by EGTA, a selective extracellular Ca2+ ion chelator, indicating that the influx of Ca2+ is important for enzymatic activity. EGTA inhibition was restored by exogenous Ca2+. Treatments of embryonic axes with either Verapamil and LaCl3 (both Ca2+ channel blockers) or TMB-8 (an intracellular Ca2+ antagonist) provoked an inhibition of both ACC-oxidase activity and ethylene production. These results suggest an involvement of calcium fluxes and intracellular calcium levels in the activity of the last step of the ethylene biosynthetic pathway, which is, in turn, intimately correlated with germination of Cicer arietinum seeds.

Amino Acid Oxidoreductases↗