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Stabilization of TMB reaction product for electron microscopic retrograde and anterograde fiber tracing.

Use of the highly sensitive tetramethylbenzidine (TMB) method of horseradish peroxidase histochemistry for electron microscopy has been limited by the solubility of the reaction product in aqueous and alcoholic solutions. We have found that following the TMB reaction with a diaminobenzidine-cobalt (DAB-Co) step causes the TMB crystals to become coated with DAB-Co. The resultant reaction complex is insoluble, and easily localized using electron microscopy. By systematically varying the pH at which the TMB reaction is run, the size and shape of the reaction complex can be controlled. The pH 4.0 reaction complex was the most suitable for electron microscopic identification of labeled structures less than 1.0 micron in diameter (e.g., axon terminals).

Animals↗

Abrogation of DTH response and mitogenic lectin- and alloantigen-induced activation of lymphocytes by calcium inhibitors TMB-8 and BAPTA-AM.

In vitro treatment of mouse lymphocytes with an intracellular calcium chelator BAPTA-AM significantly decreased lectin Concanavalin-A (Con-A)-induced and mixed lymphocyte reaction (MLR) mediated, alloantigen-induced lymphocyte activation as indicated by decreased percentage of lymphoblasts among the BAPTA-treated lymphocytes. In vivo treatment of mice with intracellular Ca(2+) antagonist TMB-8 was found to substantially impair delayed type hypersensitivity (DTH; cell mediated immune) response, as indicated by decreased footpad swelling on tuberculin challenge of mice sensitized with BCG, after a single treatment with a low dose of 0.01mg of TMB-8 per mouse. Interestingly, a second injection of a higher dose of TMB-8 (0.1mg per mouse) resulted in very significant (p=0.001) abrogation of DTH as indicated by complete absence of swelling of foot pad after PPD challenge in BCG-primed treated mice. All mice in this group showed fully impaired DTH response. Lymphocytes of allosensitized mice gave a significantly higher (p<0.05) MLR response than the naïve mice. However, a single treatment of allosensitized mice with 0.1mg TMB-8 resulted into a lower MLR response, comparable in magnitude to that of untreated naïve mice.

Animals↗

TMB-Hunt: a web server to screen sequence sets for transmembrane beta-barrel proteins.

TMB-Hunt is a program that uses a modified k-nearest neighbour (k-NN) algorithm to classify protein sequences as transmembrane beta-barrel (TMB) or non-TMB on the basis of whole sequence amino acid composition. By including differentially weighted amino acids, evolutionary information and by calibrating the scoring, a discrimination accuracy of 92.5% was achieved, as tested using a rigorous cross-validation procedure. The TMB-Hunt web server, available at www.bioinformatics.leeds.ac.uk/betaBarrel, allows screening of up to 10,000 sequences in a single query and provides results and key statistics in a simple colour coded format.

Algorithms↗

Effect of TMB-8 on the pulmonary vasoconstrictor action of prostaglandin F2 alpha and the thromboxane mimic, U 46619.

1 TMB-8 (8-(N,N-diethylamino)octyl-3,4,5 trimethoxybenzoate HCl), an intracellular calcium antagonist, had no direct action on the pulmonary vasculature of the perfused canine lung lobe preparation. 2 The pulmonary pressor response to the thromboxane mimic, U46619, was not affected by TMB-8. 3 The vasopressor response to prostaglandin F2 alpha (PGF 2 alpha) was significantly attenuated but not completely blocked by TMB-8. 4 We conclude that the pulmonary pressor response to PGF 2 alpha is dependent on both intracellular and extracellular calcium pools for contraction and that U46619 facilitates either solely extracellular calcium influx or mobilizes an intracellular calcium pool not inhibited by TMB-8.

Animals↗

Genetic analysis of chromosomal operons involved in degradation of aromatic hydrocarbons in Pseudomonas putida TMB.

The catabolic pathway for the degradation of aromatic hydrocarbons encoded by Pseudomonas putida TMB differs from the TOL plasmid-encoded pathway as far as regulation of the upper pathway is concerned. We found, by analyzing Tn5-induced mutants and by Southern blot hybridization with appropriate probes derived from the TOL plasmid pWW0, that the catabolic genes of strain TMB were located on the bacterial chromosome and not on the 84-kb plasmid harbored by this strain. The catabolic genes of TMB and pWW0 had sequence homology, as shown by Southern blot hybridization, but differed significantly in their restriction patterns. The analysis of the mutants suggests that a regulatory mechanism similar to that present in pWW0 coexists in TMB with a second mode of regulation which is epistatic on the former and that the chromosomal region carrying the catabolic genes is prone to rearrangements and deletions.

Chromosome Mapping↗

Unexpected potentiation of insulin release by the calcium store blocker TMB-8.

Insulin release from rat pancreatic islets, stimulated by cAMP, 3-isobutyl-1-methylxanthine, or forskolin, is potentiated by TMB-8 [8-(N,N-diethylamino)octyl 3,4,5-trimethoxybenzoate], a drug that blocks the efflux of calcium from intracellular calcium stores without affecting influx. There is a short latent period before the potentiation occurs which can be reduced by preincubation with the drug. TMB-8 alone neither stimulates nor inhibits insulin release. The results suggest that an intracellular store fills with calcium because of the blocked efflux and unchanged influx, and loses its ability to regulate the cytosol Ca++ concentration. Under basal conditions, in the presence or absence of TMB-8, the plasma membrane is able to regulate the cytosol Ca++ concentration at low levels so that no change in insulin release occurs. However, when 3-isobutyl-1-methylxanthine, cAMP, or forskolin add an additional Ca++ load to the cytosol, the regulator capacity of the plasma membrane is overwhelmed, and cytosol Ca++ rises to higher levels than in the absence of TMB-8 because the filled store is no longer regulating. Thus, insulin release is potentiated.

1-Methyl-3-isobutylxanthine↗

[The effects of TMB-8, an intracellular Ca2+ antagonist, on impaired left ventricular relaxation by global ischemia in dog].

The effects of TMB-8, an inhibitor of Ca2+ release from the intracellular store site, on impaired left ventricular relaxation caused by global ischemia were examined in 45 anesthetized dogs. In the non-ischemic group, the intracoronary infusion of 100 micrograms/min TMB-8 suppressed the decrease in T, an index of left ventricular relaxation, by increasing the atrial pacing rate. In the ischemic group, the intracoronary infusion of 30 and 100 micrograms/min TMB-8 suppressed the increases of T and left ventricular end-diastolic pressure caused by ischemia and increase of pacing rate. The results indicate that TMB-8 suppresses the ability of relaxation in non-ischemic myocardium and prevents the impairment of left ventricular relaxation induced by left ventricular global ischemia.

Animals↗

Inhibition of interleukin 1-induced biosynthesis of stromelysin by the calcium antagonist TMB-8 (8-(N, N-diethylamino)octyl-3,4,5-trimethoxybenzoate HCl).

This study was done to identify agents that can inhibit interleukin 1 (IL1)-induced stromelysin biosynthesis and to gain insight into the mechanism of IL1 action. For this purpose, various agents known to modulate calcium-dependent signal transduction pathway were evaluated in rabbit synovial fibroblast (RSF) cultures. Only the conditioned medium from RSF treated with the intracellular calcium antagonist TMB-8 (8-(N,N-diethylamino)-octyl 3,4,5-trimethoxybenzoate hydrochloride) had significantly lower proteoglycan-degrading metalloproteinase activity than controls. Biosynthetic labeling, immunoprecipitation and immunohistochemical studies, using a polyclonal antibody against rabbit stromelysin, demonstrated that TMB-8 inhibited synthesis stromelysin, the proteoglycan-degrading matrix metalloproteinase. Further evaluation of the TMB-8 effect revealed that the compound had no effect on secretion and that it was not acting by preventing activation of the proenzyme or by inhibiting the enzyme activity. These results suggest that TMB-8 may be inhibiting stromelysin synthesis by limiting intracellular calcium levels.

Animals↗

Effect of TMB-8 on histamine release from isolated rat mast cells.

TMB-8 was capable of complete inhibition of the histamine release induced by antigen, compound 48/80, and the ionophore A23187 (2 microM). The effective concentrations for 50% inhibition (IC50) were 1.2 X 10(-4) M for antigen, 1.6 X 10(-4) M for compound 48/80 both in the absence and in the presence of calcium, and 0.7 X 10(-4) M for the ionophore. The inhibitory action was not affected by increased calcium concentration in the medium from 1 to 2m M. The presence of glucose in the medium counteracted the inhibition by TMB-8 and, for low concentrations of the ionophore (0.25 microM), TMB-8 caused a pronounced enhancement of the histamine release. Our results indicate that TMB-8 primarily exerts its effects by interference with oxidative metabolism rather than by affecting the intracellular calcium availability.

Animals↗

Effects of nifedipine and TMB-8 on angiotensin II-induced mesenteric vasoconstriction in dogs.

The effects of a Ca2+ entry blocker, nifedipine, and a putative intracellular Ca2+ release inhibitor, 8-(N, N-diethylamino) octyl-3,4,5-trimethoxybenzoate (TMB-8), on mesenteric vasoconstriction, induced by angiotensin II, were examined in anesthetized dogs. Injection of angiotensin II (5 and 10 ng/kg) into the mesenteric artery decreased the mesenteric blood flow, which was suppressed during intramesenteric arterial infusion of TMB-8 (30 and 100 micrograms/kg/min) but not of nifedipine (0.03 and 0.1 microgram/kg/min). A higher dose of nifedipine (0.3 microgram/kg/min) only slightly attenuated the mesenteric blood flow response. Intravenous injection of angiotensin II (100 ng/kg) decreased the mesenteric and renal blood flow. Both blood flow responses were suppressed during intravenous infusion of TMB-8 (1 and 2 mg/kg/min). Intravenous infusion of nifedipine (0.1-1.0 microgram/kg/min) suppressed the renal blood flow response, whereas the mesenteric blood flow response was relatively resistant to nifedipine. The present results suggest that a TMB-8-sensitive Ca2+ movement pathway participates in the angiotensin II-induced contraction of the dog mesenteric vasculature in vivo. The Ca2+ influx through dihydropyridine-sensitive Ca2+ channels may not play a significant role in the angiotensin II-induced mesenteric vasoconstriction.

Angiotensin II↗

Effects of nifedipine and TMB-8 on angiotensin II-induced antinatriuresis in anesthetized dogs.

A calcium entry blocker, nifedipine, or an intracellular calcium release inhibitor TMB-8, was infused into the renal artery before and during intravenous infusion of angiotensin II in anesthetized dogs. In the control period, nifedipine (0.1 microgram/kg/min) or TMB-8 (75 micrograms/kg/min) increased urine flow rate, urinary sodium excretion and fractional sodium excretion, with little change in renal blood flow or glomerular filtration rate. Angiotensin II (10 ng/kg/min) elevated blood pressure and reduced urine flow rate, urinary sodium excretion and fractional sodium excretion. In the angiotensin II infusion period, nifedipine increased urine flow rate, urinary sodium excretion and fractional sodium excretion to levels higher than those observed in the control period. TMB-8 also caused augmented urinary responses. The results suggest that the angiotensin II-induced antinatriuresis depends both on the calcium influx through dihydropyridine-sensitive calcium channels, and on the calcium release from TMB-8-sensitive calcium stores at the renal tubular sites.

Angiotensin II↗

Adsorption Behavior of N(2), Water, C(6) Hydrocarbons, and Bulkier Benzene Derivative (TMB) on Na-X Zeolite and Its K(+)-, Rb(+)-, and Cs(+)-Exchanged Analogues.

The hydrothermal crystallization of X-type zeolite with a Si/Al ratio of 1.15 was achieved from the Na(2)O-Al(2)O(3)-SiO(2)-H(2)O system at 368 K under static conditions. The post-synthesis modification was carried out by a conventional ion-exchange technique to obtain K(+)-, Rb(+)-, and Cs(+)-exchanged samples with different degrees of exchange. All the samples were characterized using chemical analysis, IR, SEM, powder XRD, low-temperature nitrogen adsorption, and equilibrium sorption uptakes of different probe molecules. The relative intensities of the XRD peaks of cation-exchanged zeolite were found to be affected to different extents, depending on the nature and the concentration of nonframework cationic size, without any shift in the positions of reflection. The sorptive properties of the K-, Rb-, and Cs-exchanged samples were studied using nitrogen, water, and different C(6) hydrocarbons including bulkier benzene derivative 1,3,5-trimethylbenzene (TMB) as probe molecules. The trend observed in chemical potential estimated as a function of nitrogen coverage indicates different sorption selectivity because of differences in the cationic size and population. Sorption uptake kinetics for probe molecules such as water, n-hexane, cyclohexane, benzene, and TMB were also studied. The samples with higher degrees of exchange and/or cationic size have shown a decrease in hydrophilic character due to the formation of irregular networks of water molecules connected with preadsorbed water molecules, framework oxygen ions, and nonframework cations. Among C(6) hydrocarbons including TMB, the benzene molecule is found to be the most promising probe for the estimation of openness of structure and surface heterogeneity as well. Copyright 2001 Academic Press.

Journal Article↗

Effect of trifluoperazine, compound 48/80, TMB-8 and verapamil on the rate of calmodulin binding to erythrocyte Ca2+-ATPase.

The erythrocyte Ca2+-ATPase shifts reversibly between two states, the calmodulin-deficient A-state and the calmodulin-saturated B-state, dependent on calcium and calmodulin. The effects on this system of the four drugs, trifluoperazine, compound 48/80, TMB-8 and verapamil were studied. All four drugs inhibited the maximum activity of the B -state Ca2+-ATPase and, in addition, trifluoperazine and compound 48/80 in higher doses inhibited the A-state. Furthermore, the four drugs decreased the calmodulin sensitivity of the Ca2+-ATPase in the order of decreasing effect: trifluoperazine greater than compound 48/80 greater than TMB-8 greater than verapamil. In the same order of decreasing effect the drugs increased the time required for full calmodulin activation of the A-state of Ca2+-ATPase, whereas the drugs had only small effects on the rate of deactivation of the B-state, caused by dissociation of calmodulin from the enzyme. It is discussed whether the effects on calmodulin activation were caused by a reduction of free calmodulin due to the formation of drug-calmodulin complexes or whether the drugs, especially trifluoperazine, compound 48/80 and TMB-8, by binding to the Ca2+-ATPase, decreased the rate constants for association of calmodulin and enzyme.

Calcium Channel Blockers↗

Stimulation of renin secretion by 8-(N,N-diethylamino)octyl-3,4,5-trimethoxybenzoate (TMB-8).

The intracellular calcium antagonist 8-(N,N-diethylamino)octyl-3,4,5-trimethoxybenzoate (TMB-8) prevents the release of Ca2+ from cell storage sites. The effect of this compound on renin secretion from rat renal cortical slices in vitro was investigated. TMB-8 was a potent stimulant of renin secretion within the concentration range 10(-5)M to 5 X 10(-4)M with an optimum concentration of 2 X 10(-4)M. TMB-8 overcame the inhibition of renin secretion by angiotensin II, ouabain, 60 mM KCl and A23187. The results add to the existing evidence that Ca2+ is a common inhibitory messenger for a number of compounds which affect renin release and suggest a role for intracellular calcium stores in the regulation of juxtaglomerular cell Ca2+ levels.

Animals↗

The effects of TMB-8 on acetylcholine release from frog motor nerve: interactions with adenosine.

The putative intracellular calcium (Ca) antagonist TMB-8 was shown to reduce postjunctional sensitivity and quantal acetylcholine (ACh) release at low micromolar concentrations. At 10-fold higher concentrations, TMB-8 also blocked caffeine-induced Ca release (as monitored electrophysiologically by changes in ACh release) but did not impair the ability of adenosine to inhibit quantal ACh release. This last result implies that TMB-8 and adenosine exert their inhibitory actions at different steps in the depolarization-secretion coupling sequence.

Acetylcholine↗

Differential allosteric effects of 8-(N,N-diethylamino)octyl-3,4,5-trimethoxybenzoate-HCl (TMB-8) on muscarinic receptor subtypes.

TMB-8, a putative inhibitor of intracellular calcium mobilization, prevents the binding of the muscarinic ligand N-[3H]methylscopolamine. The inhibition was observed in four tissues from guinea pig; cortex, heart, pancreas, and ileum, representing M1, cardiac M2, glandular M2, and heterogeneous M2 subtypes of muscarinic receptors, respectively. The Ki values for all four tissues were approx. 4 microM. However, dissociation kinetics revealed that TMB-8 interacted with an allosteric site of three muscarinic receptor subtypes but not the subtype from pancreas. These results indicate that TMB-8 interacts with muscarinic receptors, and therefore would disrupt calcium mobilization or any second messenger system coupled to these receptors.

Allosteric Regulation↗

Effect of an intracellular calcium antagonist (TMB-8) on carbamylcholine-induced amylase release from dispersed rat pancreatic acini.

During 10-min incubation with increasing concentrations of carbamylcholine (carbachol), amylase release from dispersed rat pancreatic acini increased, became maximal at 2 X 10(-6)M and then decreased. In the concentration range of 10(-7)M to 10(-4)M, 8-(N,N-diethylamino)-octyl 3,4,5-trimethoxybenzoate hydrochloride (TMB-8) caused a dose-dependent inhibition of amylase release induced by a submaximal concentration of carbachol. No inhibitory effect was observed on basal and secretin-stimulated amylase release. TMB-8 showed a significantly greater ability of blocking the action of carbachol than verapamil and diltiazem. TMB-8 could reverse the submaximal stimulation of amylase release caused by supramaximal concentrations of carbachol to a maximal stimulation, while verapamil and diltiazem could not. These results confirm the hypothesis that mobilization of intracellular calcium is the primary step in the action of carbachol on pancreatin acinar cells and contributes to the submaximal secretory response of acinar cells induced by high concentrations of carbachol.

Amylases↗

Effects of TMB-8 and dantrolene on ACTH- and angiotensin-induced steroidogenesis by frog interrenal gland: evidence for a role of intracellular calcium in angiotensin action.

The influence of intracellular calcium on the steroidogenic response of adrenocortical tissue to ACTH and angiotensin has been studied in the frog, using a perifusion system technique. The release of corticosterone, aldosterone and prostaglandins in the effluent medium was monitored by specific radioimmunoassays. TMB-8 and dantrolene, two potential blockers of calcium mobilization from intracellular pool(s), were tested. Dantrolene (5 X 10(-5) M) significantly reduced basal and angiotensin-induced corticosterone and aldosterone production but had little effect on ACTH-evoked steroid release. Conversely TMB-8 (10(-4) M) profoundly depressed spontaneous as well as ACTH- and angiotensin II-induced corticosteroid secretion, suggesting that this compound may affect not only calcium mobilization from the endoplasmic reticulum pool but also calcium influx. Adrenal glands perifused with both dantrolene and calcium-free medium showed no response to angiotensin II. Conversely, in calcium-free conditions and in the presence of dantrolene, angiotensin II still caused an increase in prostaglandin synthesis. Taken together, these results indicate that 1) dantrolene is a more specific agent than TMB-8 in inhibiting calcium mobilization from intracellular pool(s); 2) ACTH increases corticosteroidogenesis without inducing mobilization of intracellular calcium; 3) angiotensin II stimulates both the efflux of calcium from the endoplasmic reticulum and the influx of calcium through the plasma membrane; 4) calcium is required after prostaglandin production in the steroidogenic response of frog interrenal gland to angiotensin II.

Adrenal Cortex Hormones↗