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PubMed1995

Role of Ca2+ and calmodulin in ehrlichial infection in macrophages.

Replication of Ehrlichia risticii was inhibited in P388D1 cells and murine peritoneal macrophages when a calmodulin antagonist (W-7, chlorpromazine, or trifluoperazine); a Ca2+ channel blocker (verapamil, diltiazem, nifedipine, or flunarizine); an extracellular Ca2+ chelator, EGTA [ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid]; an inhibitor of intracellular Ca2+ mobilization, TMB-8; or Ca2+ ionophore A23187 was added after internalization of the organism at 3 h postincubation. When intracellular ehrlichiae at their logarithmic stage of growth were treated with these reagents, not only was further proliferation prevented but also there was significant reduction in numbers of intracellular ehrlichiae. These reagents prevented spreading of E. risticii from P388D1 cells to THP-1 cells. None of these reagents prevented binding of [35S]methionine-labeled E. risticii to P388D1 cells, but all of these reagents prevented internalization of [35S]methionine-labeled E. risticii. Protein kinase C inhibitors, H-7 and staurosporin, had no effect. 14CO2 production from L-[14C]glutamine in Percoll-density-gradient-purified E. risticii was inhibited by A23187 but not by W-7 or verapamil, suggesting that Ca2+ but not calmodulin directly regulates ehrlichials glutamine oxidation. Pretreatment of E. risticii with W-7 or verapamil did not reduce its infectivity. These results indicate that calmodulin and Ca2+ are essential for ehrlichial internalization, replication, and spreading in macrophages but are not essential for binding.

Animals↗
PubMed1999

Hypoxia/aglycemia increases endothelial permeability: role of second messengers and cytoskeleton.

The effects of hypoxia/aglycemia on microvascular endothelial permeability were evaluated, and the second messenger systems and the cytoskeletal-junctional protein alterations in this response were also examined. Monolayers of human dermal microvascular endothelial cells on microcarrier beads were exposed to either thioglycolic acid (5 mM, an O(2) chelator), glucose-free medium, or both stresses together. Permeability measurements were performed over a 90-min time course. Although neither hypoxia alone nor aglycemia alone increased endothelial permeability significantly, the combination of both increased significantly as early as 15 min. Intracellular Ca(2+) measurements with fura 2-AM showed that hypoxia/aglycemia treatment increased Ca(2+) influx. To determine the second messengers involved in increased permeability, monolayers were incubated for 30 min with the cytosolic Ca(2+) scavenger 3,4, 5-trimethoxybenzoic acid 8-(diethylamino)octyl ester (TMB-8, 0.1 mM), a classical protein kinase C (PKC) blocker, Gö-6976 (10 nM), a cGMP-dependent protein kinase (PKG) antagonist, KT-5823 (0.5 microM), or the mitogen-activated protein (MAP) kinase inhibitor PD-98059 (20 microM). Hypoxia/aglycemia-mediated permeability changes were blocked by chelating cell Ca(2+), PKC blockade, PKG blockade, and by inhibiting p38 MAP kinase-1. Finally, changes in the binding of junctional proteins to the cytoskeleton under the same conditions were assessed. The concentrations of occludin and pan-reactive cadherin binding to the cytoskeleton were significantly decreased by only both stresses together. However, these effects were also blocked by pretreatment with TMB-8, Gö-6976, KT-5823 (not in occludin), and PD-98059. These data suggest that hypoxia/aglycemia-mediated endothelial permeability may occur through PKC, PKG, MAP kinase, and Ca(2+) related to dissociation of cadherin-actin and occludin-actin junctional bonds.

Alkaloids↗
PubMed1986

Influence of calcium and other divalent cations on protein turnover in rat skeletal muscle.

When rat muscles were incubated in Ca2+-free media, their rates of protein break-down were significantly lower than in complete medium (2.58 mM Ca2+). Dantrolene and 3,4,5-trimethoxybenzoic acid 8-(diethylamino)octyl ester, inhibitors of Ca2+ release from the sarcoplasmic reticulum, also reduced muscle proteolysis. When Ca2+ was added (up to 5.16 mM), proteolysis increased progressively up to 70% in the intact soleus and extensor digitorum longus muscles and up to 300% in the cut diaphragm preparation. Addition of Ca2+ did not affect the muscles' ATP or phosphocreatine content and increased protein synthesis slightly or not at all. Sr2+, Ba2+, and Mn2+ also increased proteolysis, but were less effective than Ca2+. Mg2+ inhibited the enhancement of proteolysis by Ca2+. This stimulation by Ca2+ was not affected by inhibitors of voltage-dependent Ca2+ channels, calmodulin, metalloendoproteases, microfilament or microtubule formation, or mersalyl. High Ca2+ levels also increased prostaglandin (PG) E2 production, although a rise in PGE2 did not appear essential for the increased proteolysis. The proteolysis induced by Ca2+ was prevented in muscles treated with Ep-475 or leupeptin. By contrast, these inhibitors of thiol proteases did not affect protein breakdown in Ca2+-free medium. Thus extracellular Ca2+ activates and Mg2+ inhibits a proteolytic pathway involving thiol proteases.

Animals↗
PubMed2006

Protection from experimental colitis by theaflavin-3,3'-digallate correlates with inhibition of IKK and NF-kappaB activation.

BACKGROUND AND PURPOSE: Inflammatory bowel disease (IBD) is associated with activation of nuclear factor kappa B (NF-kappaB) involved in regulating the expression of inducible nitric oxide synthase (iNOS) and proinflammatory cytokine genes. As theaflavin-3,3'-digallate (TFDG), the most potent anti-oxidant polyphenol of black tea, down-regulates NF-kappaB activation, we investigated if TFDG is beneficial in colonic inflammation by suppressing iNOS and proinflammatory cytokines. EXPERIMENTAL APPROACH: The in vivo efficacy of TFDG was assessed in mice with trinitrobenzene sulfonic acid (TNBS)-induced colitis. Both mRNA and protein levels of proinflammatory cytokines and iNOS were analyzed in colon tissue treated with or without TFDG. NF-kappaB activation was determined by electrophoretic mobility shift assay and levels of NF-kappaB inhibitory protein (IkappaBalpha) were analyzed by Western blotting. KEY RESULTS: Oral administration of TFDG (5 mg kg(-1) daily i.g.) significantly improved TNBS-induced colitis associated with decreased mRNA and protein levels of TNF-alpha, IL-12, IFN-gamma and iNOS in colonic mucosa. DNA binding and Western blotting revealed increase in NF-kappaB activation and IkappaBalpha depletion in TNBS-treated mice from Day 2 through Day 8 with a maximum at Day 4, which resulted from increased phosphorylation of IkappaBalpha and higher activity of IkappaB kinase (IKK). Pretreatment with TFDG markedly inhibited TNBS-induced increases in nuclear localization of NF-kappaB, cytosolic IKK activity and preserved IkappaBalpha in colon tissue. CONCLUSIONS AND IMPLICATIONS: TFDG exerts protective effects in experimental colitis and inhibits production of inflammatory mediators through a mechanism that, at least in part, involves inhibition of NF-kappaB activation.

Animals↗
PubMed2001

Effects of regular ingestion of black tea on haemostasis and cell adhesion molecules in humans.

OBJECTIVE: To assess the effects in humans of regular ingestion of black tea on haemostasis-related variables and cell adhesion molecules. DESIGN: Twenty-two subjects were recruited from the general population to a randomised-controlled crossover study. Subjects stopped drinking tea, apart from that provided, for the duration of the study. During a 4-week baseline period all subjects drank 5 cups/day (250 ml) of hot water. The effects of 5 cups/day of black tea for 4 weeks were then compared with hot water. Platelet aggregation in response to three doses of collagen and ADP, plasma concentrations of coagulation and fibrinolytic factors (fibrinogen, factor VII, tPA, PAI-1) and plasma concentrations of cell adhesion molecules (soluble P-selectin, E-selectin, ICAM-1, VCAM-1) were assessed twice, one week apart, at the end of each period. Twenty-four hour urinary concentration of 4-O-methylgallic acid (4OMGA), assessed once at the end of each period, was used as a marker of black tea polyphenol intake. RESULTS: The 24 h urinary excretion of 4OMGA was increased during regular ingestion of black tea in comparison to hot water (P<0.0001). Black tea resulted in lower soluble P-selectin (P=0.01) in comparison to hot water, but did not influence other adhesion molecules. Soluble P-selectin was significantly correlated with mean collagen-stimulated platelet aggregation at baseline (r=0.61, P=0.003), and during regular ingestion of hot water (r=0.70, P<0.0001) and black tea (r=0.51, P=0.01). However, platelet aggregation was not different between the black tea and hot water periods for collagen- or ADP-stimulated aggregation at any dose. Coagulation and fibrinolytic factors were also not different between periods. CONCLUSIONS: The effect of black tea on soluble P-selectin provides a potential mechanism for cardiovascular benefits of regular ingestion of tea. SPONSORSHIP: This study was supported by grants from the Tea Trade Health Research Association and the National Heart Foundation of Australia.

Adult↗
PubMed1993

Effects of modulators of cytosolic Ca2+ on phytohemagglutin-dependent Ca2+ response and interleukin-2 production in Jurkat cells.

We have previously reported the presence, in Jurkat T cells, of outward K+ currents and inward currents that have been attributed to Ca2+ channels. Here, we have studied the effects of dimethyl 1,4-dihydro-2,6-dimethyl-4-(2-nitrophenyl)-3,5-pyridine-dicarboxylate (nifedipine) and 4-(2,1,3-benzoxadiazol-4-yl)-1,4-dihydro-2,6-dimethyl-5- methoxy-carbonylpyridine-3-carboxylate (PN200-110), two dihydropyridines (DHPs) known to inhibit voltage-dependent Ca2+ channel activity in different types of cells, and two inhibitors of internal Ca2+ release (muscle cells), ryanodine and 3,4,5-trimethoxybenzoic acid 8-(diethylamino)octyl ester (TMB-8), on the Phaseolus vulgaris phytohemagglutinin (PHA)-dependent responses in Jurkat T lymphocytes. Our results show that nifedipine and PN200-110 inhibit the PHA-dependent production of interleukin-2 except when 12-O-tetradecanoyl-13-O-acetyl phorbol is added to the cultures. Ryanodine and TMB-8 are not inhibitors. The PHA-dependent Ca2+ response is significantly reduced when the cells are preincubated in the presence of the DHPs. Under these conditions, ryanodine has only a small inhibitory effect and TMB-8 has no effect. In contrast, only ryanodine (50 microM) decreases the PHA-dependent cytosolic release of Ca2+i when the cells are bathed in a medium containing a low concentration of Ca2+ (60 nM). The inhibitory effects of nifedipine and PN200-110 may result from the binding of these DHPs to specific receptor sites as revealed by studies using [3H]PN200-110 (KD = 8.5 +/- 3.1 nM; 2300 +/- 500 apparent binding sites/cell). Photoaffinity labeling studies using [3H]azidopine as a probe showed specific incorporation of label into three glycoproteins of molecular mass (+/- SD) 170 +/- 13, 110 +/- 25, and 60 +/- 17 kd as analyzed by electrophoresis under reducing conditions.

Affinity Labels↗
PubMed1985

Effect of retinoic acid, butylated hydroxytoluene, propylgallate and selenium on 2-acetylaminofluorene induced hepatotoxicity.

Rats of the Sprague-Dawley strain were administered 2-acetylaminofluorene (2-AAF) in different diet regimens to ascertain the effect of retinoic acid(RA), butylated hydroxytoluene (BHT), propylgallate (PG), and selenium on induced hepatotoxicity. In the first study, groups of young male albino rats of the Sprague-Dawley strain were fed a diet containing 0.05% 2-AAF. Carcinogen was added to the diet for 3 weeks, omitted 1 week, added 2 weeks, omitted 2 weeks, added 3 weeks, and omitted for the final 4 weeks. Animals were terminated at the end of 15 weeks. Supplementation of the diet by 0.5% BHT, PG or 4 ppm selenium as sodium selenite in the drinking water either throughout the entire feeding period or only during the administration of the carcinogen-free diet greatly inhibited the hepatotoxicity of 2-AAF. In contrast, addition of 0.02% RA to the diet in the above feeding regimens enhanced the hepatotoxicity of 2-AAF. In the second study, male and female Sprague-Dawley rats were administered a diet containing 0.03% 2-AAF for 16 weeks or a diet containing 0.03% 2-AAF supplemented with 0.02% RA for the first 4 weeks and the last 6 weeks of the 16-week feeding period. Addition of the 0.02% RA to the diet enhanced the hepatotoxicity of 2-AAF, especially in the female rats.

2-Acetylaminofluorene↗
PubMed1992

Thrombin-stimulated phosphorylation of myosin light chain and its possible involvement in endothelin-1 secretion from porcine aortic endothelial cells.

Thrombin-stimulated secretion of endothelin-1 (ET-1) from porcine aortic endothelial cells was inhibited in the presence of 3,4,5-trimethoxybenzoic acid 8-(diethylamino)octyl ester (TMB-8), trifluoperazine and N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7). 1-(5-Chloronaphthalene-1-sulfonyl)-1H-hexahydro-1,4-diazepine (ML-9) also prevented the thrombin-stimulated secretion of ET-1 but it enhanced the accumulation of ET-1 in the endothelial cells. When the endothelial cells were treated with thrombin, the phosphorylation of a 20-kDa protein which was identified as myosin light chain (MLC) was detected. Phosphorylation was augmented in a time-dependent manner. As in the case of ET-1 secretion, MLC phosphorylation was prevented by TMB-8, trifluoperazine, W-7 and ML-9 at the same concentrations which were effective in inhibiting the ET-1 secretion. The site of phosphorylation of MLC was identified as a serine residue. Parallel to the phosphorylation of MLC, thrombin increased the amounts of the 43- and 200-kDa proteins in the Triton-insoluble fraction; these proteins were identified as actin and myosin heavy chain, respectively. These results suggest that the MLC phosphorylation elicited by MLC kinase may facilitate the formation of filamentous myosin and actin which are probably involved in ET-1 secretion, possibly in the transport of ET-1-containing vesicles in thrombin-stimulated endothelial cells.

Animals↗
PubMed1986

A characterization of the antinociception produced by intracerebroventricular injection of 8-(N,N-diethylamino)octyl-3,4,5-trimethoxybenzoate in mice.

There is evidence that 8-(N,N-diethylamino)octyl-3,4,5-trimethoxybenzoate (TMB-8) acts as an intracellular Ca++ antagonist producing decreases in free intracellular Ca++ and inhibiting many cellular processes dependent upon intracellular Ca++. Intracerebroventricularly administered TMB-8 was active in the mouse tail-flick test (ED50 = 50 micrograms), and this antinociceptive response was antagonized by naloxone (AD50 = 0.28 mg/kg s.c.), Ca++ (0.2-0.4 mumol i.c.v.) and, to a lesser degree, by ethylene glycol bis (beta-aminoethyl ether)N,N1-tetraacetic acid (0.02 and 0.06 mumol i.c.v.). TMB-8 (i.c.v.) was only marginally active in the p-phenylquinone test. The potency of TMB-8 (i.c.v.) was potentiated 10-fold in morphine-tolerant mice in the tail-flick test (ED50 = 2.5 micrograms). TMB-8 inhibited contraction of stimulated ilea (IC50 = 2.2 microM), an effect which was neither antagonized nor reversed by naloxone (1 microM). TMB-8 did not potentiate morphine, or was it potentiated by morphine, in the stimulated guinea pig ileum. Procaine, but not lidocaine showed dose-dependent activity in the tail-flick and p-phenylquinone tests (ED50 values, 136 and 83 micrograms, respectively, i.c.v.). The antinociception produced by procaine (i.c.v.) in the tail-flick test was antagonized by naloxone (AD50 = 0.4 mg/kg s.c.) Lidocaine (100 micrograms i.c.v.) produced only 30% maximum possible effect in the tail-flick tests, and was inactive in p-phenylquinone tests. Doses of lidocaine greater than 100 micrograms i.c.v. resulted in lethality of greater than 50% of the animals tested. Thus, the activity of TMB-8 resembles that of opiates in that both are antagonized by Ca++ or naloxone in vivo.(ABSTRACT TRUNCATED AT 250 WORDS)

Analgesia↗
PubMed1986

Identification of the cellular mechanisms responsible for platelet-derived growth factor induced alterations in cytoplasmic vinculin distribution.

Exposure of quiescent density arrested BALB/c-3T3 cells (clone A31) to platelet-derived growth factor (PDGF; 6-12 ng/ml) results in a rapid, reversible, time- and dose-dependent removal of vinculin from adhesion plaques (Herman and Pledger, 1985). Potential cellular mechanisms involved in PDGF-induced removal of vinculin from adhesion plaques were examined. Removal of vinculin from adhesion plaques following exposure of cells to PDGF was temperature dependent, occurred in many fibroblast cell lines, and could be mimicked by 12-tetradecanoyl phorbol-13-acetate (TPA; 5-125 nM) or melittin (0.35 microM). Unlike the effect of PDGF, TPA- or melittin-induced vinculin disruption was not reversible. The removal of vinculin from adhesion plaques was inhibited by trifluoroperazine (TFP; 2.5 microM). 8-(N,N-diethylamino) octyl-3,4,5-trimethoxy benzoate (TMB-8; 1.0 microM), mepacrine (220 microM), n-alpha-p-tosyl-L-lysine chloromethylketone (TLCK; 100 microM), phenylmethoxysulphonylfluoride (PMSF; 500 microM), and epsilon-aminocaproic acid (epsilon-ACA; 100 microM); however, amiloride (100 microM), A23187 (20 microM), and chloroquine (1 mM) were unable to inhibit this effect. Melittin disruption of vinculin was inhibited by (in order of decreasing effectiveness) mepacrine greater than TMB-8 greater than TFP greater than leupeptin greater than PMSF, whereas A23187 and amiloride had no effect. The return of vinculin to adhesion plaques following PDGF treatment required de novo mRNA transcription and protein synthesis and was associated with PDGF-stimulated synthesis of vinculin. The observation that both PDGF- and melittin-induced removal of vinculin from adhesion plaques is inhibited by mepacrine suggests that phospholipase activation may be an early and important step in PDGF-induced disruption of vinculin from adhesion plaques. In addition, TFP, TMB-8 and protease inhibitor inhibition of both the PDGF and melittin effects on vinculin distribution, coupled with the finding that TPA can mimic the PDGF or melittin response, suggests that Ca2+, calmodulin, protein kinase C, and/or proteolysis may play an important role(s) in the removal of vinculin from adhesion plaques following PDGF addition. The lack of effect of A23187 addition on vinculin distribution suggests that alterations in cellular Ca2+ is necessary but not sufficient for vinculin removal from adhesion plaques.

Amiloride↗
PubMed1994

Characterization of PD 121981- and CGP 42112-induced unmasking of low concentration effects of angiotensin II in rabbit abdominal aorta.

The unmasking of the low concentration effect of angiotensin II (AII) was identified within the concentration ranges of 10(-13) to 10(-11) M of AII by PD 121981 (5-diphenylacetyl-1-(4-methoxy-3-methylbenzyl)- 4,5,6,7-tetrahydro-1H-imidazo[4,5-c]-pyridine-6-carboxylic acid) and 10(-12) to 3 x 10(-10) M of AII by CGP 42112 (nicotinic acid-Tyr-(N alpha-benzyl-oxycarbonyl-Arg)Lys-His-Pro-IIe-OH), AT2 antagonists, in association with the ordinary contraction curve, i.e., high concentration effect (at 3 x 10(-10)-10(-6) M of AII), in the rabbit abdominal aorta. Thus, they showed clear biphasic features of AII-induced contraction curves. However, this was not the case for angiotensin I and angiotensin III. This PD 121981-evoked low concentration effect of AII was selectively inhibited by DuP 753 (0.01-1 nM), dithiothreitol (10 and 100 microM), pertussis toxin (50 and 300 ng/ml, for 2 hr), nifedipine (1 and 10 microM) and 8-(diethylamino)octyl 3,4,5-trimethoxybenzoate hydrochloride (1 and 3 microM), which suggests the receptors were the AT1 subtype. However, the high concentration effect of AII was not affected by these drugs within the concentration ranges used in the present studies. These myographic results were almost consistent with the features of the intracellular Ca++ changes. Thus, it was concluded that the receptors that mediate the low concentration effect of AII belong to the AT1 subtype. However, the current study did not determine the mechanism by which PD 121981 and CGP 42112 evoked the up-regulation of the AT1 receptors.

Angiotensin II↗
PubMed2004

A novel tetrahydrofolate-dependent O-demethylase gene is essential for growth of Sphingomonas paucimobilis SYK-6 with syringate.

Sphingomonas paucimobilis SYK-6 degrades syringate to 3-O-methylgallate (3MGA), which is finally converted to pyruvate and oxaloacetate via multiple pathways in which protocatechuate 4,5-dioxygenase, 3MGA dioxygenase, and gallate dioxygenase are involved. Here we isolated the syringate O-demethylase gene (desA), which complemented the growth deficiency on syringate of a Tn5 mutant of the SYK-6 derivative strain. The desA gene is located 929 bp downstream of ferA, encoding feruloyl-coenzyme A synthetase, and consists of a 1,386-bp open reading frame encoding a polypeptide with a molecular mass of 50,721 Da. The deduced amino acid sequence of desA showed 26% identity in a 325-amino-acid overlap with that of gcvT of Escherichia coli, which encodes the tetrahydrofolate (H(4)folate)-dependent aminomethyltransferase involved in glycine cleavage. The cell extract of E. coli carrying desA converted syringate to 3MGA only when H(4)folate was added to the reaction mixture. DesA catalyzes the transfer of the methyl moiety of syringate to H(4)folate, forming 5-methyl-H(4)folate. Vanillate and 3MGA were also used as substrates for DesA; however, the relative activities toward them were 3 and 0.4% of that toward syringate, respectively. Disruption of desA in SYK-6 resulted in a growth defect on syringate but did not affect growth on vanillate, indicating that desA is essential to syringate degradation. In a previous study the ligH gene, which complements the growth deficiency on vanillate and syringate of a chemical-induced mutant of SYK-6, DC-49, was isolated (S. Nishikawa, T. Sonoki, T. Kasahara, T. Obi, S. Kubota, S. Kawai, N. Morohoshi, and Y. Katayama, Appl. Environ. Microbiol. 64:836-842, 1998). Disruption of ligH resulted in the same phenotype as DC-49; its cell extract, however, was found to be able to convert vanillate and syringate in the presence of H(4)folate. The possible role of ligH is discussed.

Base Sequence↗
PubMed1984

Cements containing syringic acid esters -- o-ethoxybenzoic acid and zinc oxide.

Fissure caries is reduced when syringic acid is incorporated into a cariogenic diet of rats. It was therefore of interest to synthesize n-hexyl and 2-ethylhexyl syringate and to evaluate the properties of cements with these compounds as ingredients. Liquids containing the esters dissolved in o-ethoxybenzoic acid (EBA) - when mixed with powders made up from zinc oxide, aluminum oxide, and hydrogenated rosin - hardened in from four to nine min. Properties of the cements were determined, when possible, according to ANSI/ADA specification tests. Depending on the powder-liquid ratio employed, we obtained compositions with varying physical properties desirable for different dental applications. The syringate cements, compared with the commonly used ZOE materials, have improved compressive and tensile strength, lower water solubility, do not inhibit polymerization, and are compatible with acrylic monomers. These cements pass, and mostly greatly exceed, the requirements for ZOE-type restorative materials. They also bond significantly to resins, composites, and non-precious metals. The bond strength is somewhat less than that of n-hexyl vanillate-EBA cement, but greatly exceeds the adhesion to various substrates of ZOE luting agents. Cements containing n-hexyl syringate were somewhat brittle. Best results were obtained with liquid compositions containing 5% 2-ethylhexyl syringate, 7% n-hexyl vanillate, and 88% EBA, which yielded non-brittle materials. These cements, because of the syringate ingredient, may possess caries-reducing properties. Thus, perhaps in conjunction with fluoride additives, they would be useful as insulating bases, pulp capping agents, root canal sealers, soft tissue packs, or intermediate restoratives.

Chemical Phenomena↗
PubMed1985

Effect of calcium antagonists on the biosynthesis and contractile effects of peptidoleukotrienes in rhesus monkey lung.

Anti-human Ig (immunoglobulin) E induced the release of 2.84 +/- 0.33 ng/ml of immunoreactive leukotrienes (iLTs) from passively sensitized, fragmented rhesus monkey lung, whereas tissue not challenged with anti-IgE released 0.21 +/- 0.08 ng/ml of iLTs spontaneously. Whereas the preferential lipoxygenase inhibitor, nordihydroguaiaretic acid (100 microM), inhibited completely anti-IgE-induced release of iLTs, the calcium channel entry blockers, nifedipine and verapamil (1 and 10 microM), and the purported intracellular calcium antagonist, TMB-8 (100 microM), were without affect on iLT release. The cyclooxygenase inhibitor, indomethacin (5 microM), potentiated iLT release by an average 27.7%. Anti-IgE also induced a contraction of the sensitized monkey lung parenchyma, which was partially suppressed by the antihistamine, mepyramine (10 microM). Against the residual contraction elicited by anti-IgE in the presence of mepyramine, neither FPL 55712 (10 microM) nor verapamil (10 microM) significantly suppressed the contractile activity of anti-IgE. On the monkey lung parenchyma, LTD4 elicited a concentration-dependent contraction, which was antagonized by FPL 55712 (KB = 1 microM) and suppressed by 40 to 50% by verapamil (10 microM). On monkey tracheal rings, the contraction elicited by LTD4 (30 nM) was suppressed by an average 83% by FPL 55712 (10 microM), 47% by verapamil (1 microM) and 45% by TMB-8 (100 microM). In contrast, the KCI-induced contraction was suppressed completely by verapamil and suppressed 79% by TMB-8, suggesting that LTD4 does not elicit contraction of the monkey trachea simply via voltage sensitive calcium entry.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed2004

Identification of the rabbit liver UDP-glucuronosyltransferase catalyzing the glucuronidation of 4-ethoxyphenylurea (dulcin).

Dulcin (DL), 4-ethoxyphenylurea, a synthetic chemical about 200 times as sweet as sucrose, has been proposed for use as an artificial sweetener. DL is excreted as a urinary ureido-N-glucuronide after oral administration to rabbits. The phenylurea N-glucuronide is the only ureido conjugate with glucuronic acid known at present; therefore, DL is interesting as a probe to search for new functions of UDP-glucuronosyltransferases (UGTs). Seven UGT isoforms (UGT1A3, UGT1A4, UGT1A6, UGT1A7, UGT2B13, UGT2B14, and UGT2B16) have been identified from rabbit liver, but these UGTs have not been investigated using DL as a substrate. In this work, the identities of UGT isoforms catalyzing the formation of DL glucuronide were investigated using rabbit liver microsomes (RabLM) and cloned/expressed as rabbit UGT isoforms. DL-N-glucuronide (DNG) production was determined quantitatively in RabLM and homogenates of COS-7 cells expressing each UGT isoform by using electrospray liquid chromatography-tandem mass spectrometry. Analysis of DNG formation using RabLM, by Eadie-Hofstee plot, gave a Vmax of 0.911 nmol/min/mg protein and the Km of 1.66 mM. DNG formation was catalyzed only by cloned expressed rabbit UGT1A7 and UGT2B16 (Vmax of 3.98 and 1.16 pmol/min/mg protein and a Km of 1.23 and 1.69 mM, respectively). Substrate inhibition of UGT1A7 by octylgallate confirmed the significant contribution of UGT1A7 to the formation of DNG. Octylgallate was further shown to competitively inhibit DNG production by RabLM (Ki = 0.149 mM). These results demonstrate that UGT1A7 is the major isoform catalyzing the N-glucuronidation of DL in RabLM.

Animals↗
PubMed1990

Structure of 3,4,5-trihydroxybenzoic acid octyl ester (octyl gallate) dihydrate at 123 K. A non-mesogenic amphiphilic molecule.

The crystal structure of 3,4,5-trihydroxybenzoic acid octyl ester (octyl gallate) dihydrate, C15H22O5.2H2O, is triclinic, P1, with a = 6.617 (1) [6.648 (1)], b = 9.956 (2) [9.995 (3)], c = 14.088 (4) [14.327 (7)] A, alpha = 79.08 (2) [79.78 (3)], beta = 85.58 (3) [86.59 (4)], gamma = 70.80 (2) [70.99 (3)] degrees, V = 860.5 A3, Z = 2, Dx = 1.229 1.229 [1.183] g cm-3, lambda(Mo K alpha) = 0.7093 A, mu = 0.58 cm-1, F(000) = 344 at 123 K [290 K]. The structure was solved by direct methods and refined to R(F2) = 0.059 for 302 parameters and 3655 observations. The alkyl chain of the molecule is in the fully extended conformation. The molecular packing is head-to-head bilayer with interdigitizing alkyl chains. The gallate head groups are hydrogen bonded with the water molecules to form a strong system of intra- and intermolecular hydrogen bonds consisting of finite and infinite chains. The crystals undergo crystal-to-crystal phase transitions at 365 K on heating and at 333 K on cooling, but despite the molecular packing which is analogous to that of the mesogenic alkyl glycosides, there is no thermotropic liquid crystal phase prior to the formation of the isotropic liquid phases at 376 K. Similary, no lyotropic liquid crystal phases are observed at room temperature in contact with a mixture of water and propanediol in which the crystals are soluble.

Calorimetry, Differential Scanning↗
PubMed1998

Effects of recycled paper dust extracts on isolated guinea pig trachea.

The effect of paper dust collected at two different locations in a paper recycling plant (PD1 and PD2) on isolated nonsensitized guinea pig tracheal smooth muscle was studied in vitro. Dust extracts were prepared as a 1:10 w/v aqueous solution. Dose-related contractions of guinea pig tracheal rings were elicited with both PD1 and PD2. Pharmacologic studies were performed with atropine (10(-6) M), indometacin (10(-6) M), pyrilamine (10(-6) M), LY171883 (10(-5) M), nordihydroguaiaretic acid (10(-5) M), and TMB8 (10(-5) M). The possible role of endogenous neuropeptides in this constrictor process was studied by depleting neural mediators with capsaicin (5 x 10(-6) M) before challenge with dust extracts. Constrictor effects were partially inhibited by a wide variety of the mediator blocking agents. The effects of both extracts were almost totally inhibited by the anticholinergic agent atropine, suggesting that a principal pathway mediating this response may involve the parasympathetic nervous system. The intracellular calcium-blocking agent TMB8 also induced a reduction of the contractile responses to PD1 and PD2 consistent with the well established role of intracellular calcium in smooth muscle constriction. Pretreatment with capsaicin significantly increased the contractile activity of paper dust extracts but only at the higher doses of these extracts. This suggests that the effect of paper dust is not initiated by the release of mediators stored in sensory nerves but that the prerelease of these mediators may enhance the constrictor effects of these dusts. We suggest that paper dust extracts cause dose-related airway smooth muscle constriction possibly associated with the release of cholinergic as well as other mediators. The constrictor effect does not require tissue presensitization or the release of neuropeptides from sensory nerves.

Animals↗
PubMed2001

Biological characterization of eugeniin as an anti-herpes simplex virus type 1 compound in vitro and in vivo.

Eugeniin exhibits antiviral activity against acyclovir and phosphonoacetic acid (PAA)-resistant herpes simplex virus type 1 (HSV-1) as well as the wild-type HSV-1 in vitro. In this study, we characterized the biological activity of eugeniin in cutaneously HSV-1-infected mice and its interaction with HSV-1 DNA polymerase. The oral and intraperitoneal administrations of eugeniin at 0.3 mg/kg showed similar therapeutic efficacy in retarding the development of skin lesions of HSV-1-infected mice. The two routes of administration at 6 or 50 mg/kg significantly prolonged the mean survival times and/or reduced mortality without toxicity. The oral administration of eugeniin at 50 mg/kg reduced virus yields in the skin and brain of infected mice. Thus, the therapeutic efficacy of oral administration at the various doses of eugeniin was similar to that of intraperitoneal administration, suggesting that the oral bioavailability of eugeniin was high with respect to absorption. Furthermore, the anti-HSV-1 activity of eugeniin was characterized by isobolograms analyzing its combined effects with acyclovir or PAA in HSV-1-infected Vero cells. Eugeniin enhanced the anti-HSV-1 activity of acyclovir but was suggested to be antagonistic with PAA. The interaction of eugeniin and PAA on the activity of partially purified HSV-1 DNA polymerase suggested that eugeniin interacted with the polymerase in the vicinity of PAA-binding site. Thus, eugeniin showed different anti-HSV-1 action from acyclovir and PAA and therapeutic anti-HSV-1 activity in mice.

Acyclovir↗