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1.
N-(p-amylcinnamoyl)anthranilic acid (ACA), a phospholipase A2 (PLA2) inhibitor, is structurally-related to non-steroidal anti-inflammatory drugs (NSAIDs) of the fenamate group and may also modulate various ion channels. We used the whole-cell, patch-clamp technique at room temperature to investigate the effects of ACA on the Ca2+-activated chloride current (ICl(Ca)) and other chloride currents in isolated pig cardiac ventricular myocytes. ACA reversibly inhibited ICl(Ca) in a concentration-dependent manner (IC50 = 4.2 μM, nHill = 1.1), without affecting the L-type Ca2+ current. Unlike ACA, the non-selective PLA2 inhibitor bromophenacyl bromide (BPB; 50 μM) had no effect on ICl(Ca). In addition, the analgesic NSAID structurally-related to ACA, diclofenac (50 μM) also had no effect on ICl(Ca), whereas the current in the same cells could be suppressed by chloride channel blockers flufenamic acid (FFA; 100 μM) or 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid (DIDS;100 μM). Besides ICl(Ca), ACA (50 μM) also suppressed the cAMP-activated chloride current, but to a lesser extent. It is proposed that the inhibitory effects of ACA on ICl(Ca) are PLA2-independent and that the drug may serve as a useful tool in understanding the nature and function of cardiac anion channels.  相似文献   

2.
Human arylamine N-acetyltransferase 1 (NAT1) is a xenobiotic-metabolizing enzyme that biotransforms aromatic amine chemicals. We show here that biologically-relevant concentrations of inorganic (Hg2+) and organic (CH3Hg+) mercury inhibit the biotransformation functions of NAT1. Both compounds react irreversibly with the active-site cysteine of NAT1 (half-maximal inhibitory concentration (IC50) = 250 nM and kinact = 1.4 × 104 M−1 s−1 for Hg2+ and IC50 = 1.4 μM and kinact = 2 × 102 M−1 s−1 for CH3Hg+). Exposure of lung epithelial cells led to the inhibition of cellular NAT1 (IC50 = 3 and 20 μM for Hg2+ and CH3Hg+, respectively). Our data suggest that exposure to mercury may affect the biotransformation of aromatic amines by NAT1.  相似文献   

3.
We searched for novel agonists of TRP receptors especially for TRPA1 and TRPV1 in foods. We focused attention on garlic compounds, diallyl sulfide (DAS), diallyl disulfide (DADS), and diallyl trisulfide (DATS). In TRPA1 or TRPV1 heterogeneously expressed CHO cells, all of those compounds increased [Ca2+]i in concentration-dependent manner. The EC50 values of DADS and DATS were similar to that of allyl isothiocyanate (AITC) and that of DAS was 170-fold larger than that of AITC. Maximum responses of these sulfides were equal to that of AITC. The EC50 values of these compounds for TRPV1 were around 100 μM against that of capsaicin (CAP), 25.6 nM and maximum responses of garlic compounds were half to that of CAP. The Ca2+ responses were significantly suppressed by co-application of antagonist. We conclude that DAS, DADS, and DATS are agonist of both TRPA1 and TRPV1 but with high affinity for TRPA1.  相似文献   

4.
Prostaglandin (PG)E2 is a critical lipid mediator connecting chronic inflammation to cancer. The anti-carcinogenic epigallocatechin-3-gallate (EGCG) from green tea (Camellia sinensis) suppresses cellular PGE2 biosynthesis, but the underlying molecular mechanisms are unclear. Here, we investigated the interference of EGCG with enzymes involved in PGE2 biosynthesis, namely cytosolic phospholipase (cPL)A2, cyclooxygenase (COX)-1 and -2, and microsomal prostaglandin E2 synthase-1 (mPGES-1). EGCG failed to significantly inhibit isolated COX-2 and cPLA2 up to 30 μM and moderately blocked isolated COX-1 (IC50 > 30 μM). However, EGCG efficiently inhibited the transformation of PGH2 to PGE2 catalyzed by mPGES-1 (IC50 = 1.8 μM). In lipopolysaccharide-stimulated human whole blood, EGCG significantly inhibited PGE2 generation, whereas the concomitant synthesis of other prostanoids (i.e., 12(S)-hydroxy-5-cis-8,10-trans-heptadecatrienoic acid and 6-keto PGF) was not suppressed. Conclusively, mPGES-1 is a molecular target of EGCG, and inhibition of mPGES-1 is seemingly the predominant mechanism underlying suppression of cellular PGE2 biosynthesis by EGCG.  相似文献   

5.
Cytochrome P450 3A4 (CYP3A4) is the most abundant CYP enzyme in the liver and metabolizes approximately 50% of the drugs, including antiretrovirals. Although CYP3A4 induction by ethanol and impact of CYP3A4 on drug metabolism and toxicity is known, CYP3A4-ethanol physical interaction and its impact on drug binding, inhibition, or metabolism is not known. Therefore, we studied the effect of ethanol on binding and inhibition of CYP3A4 with a representative protease inhibitor, nelfinavir, followed by the effect of alcohol on nelfinavir metabolism. Our initial results showed that methanol, ethanol, isopropanol, isobutanol, and isoamyl alcohol bind in the active site of CYP3A4 and exhibit type I spectra. Among these alcohol compounds, ethanol showed the lowest KD (5.9 ± 0.34 mM), suggesting its strong binding affinity with CYP3A4. Ethanol (20 mM) decreased the KD of nelfinavir by >5-fold (0.041 ± 0.007 vs. 0.227 ± 0.038 μM). Similarly, 20 mM ethanol decreased the IC50 of nelfinavir by >3-fold (2.6 ± 0.5 vs. 8.3 ± 3.1 μM). These results suggest that ethanol facilitates binding of nelfinavir with CYP3A4. Furthermore, we performed nelfinavir metabolism using LCMS. Although ethanol did not alter kcat, it decreased the Km of nelfinavir, suggesting a decrease in catalytic efficiency (kcat/Km). This is an important finding because alcoholism is prevalent in HIV-1-infected persons and alcohol is shown to decrease the response to antiretroviral therapy.  相似文献   

6.
This research examines the in vitro interaction of phthalate diesters and monoesters with the G protein-coupled cannabinoid 1 (CB1) receptor, a presynaptic complex involved in the regulation of synaptic activity in mammalian brain. The diesters, n-butylbenzylphthalate (nBBP), di-n-hexylphthalate (DnHP), di-n-butylphthalate (DnBP), di-2-ethylhexylphthalate (DEHP), di-isooctylphthalate (DiOP) and di-n-octylphthalate (DnOP) inhibited the specific binding of the CB1 receptor agonist [3H]CP-55940 to mouse whole brain membranes at micromolar concentrations (IC50s: nBBP 27.4 μM; DnHP 33.9 μM; DnBP 45.9 μM; DEHP 47.4 μM; DiOP 55.4 μM; DnOP 75.2 μM). DnHP, DnBP and nBBP achieved full (or close to full) blockade of [3H]CP-55940 binding, whereas DEHP, DiOP and DnOP produced partial (55-70%) inhibition. Binding experiments with phenylmethane-sulfonylfluoride (PMSF) indicated that the ester linkages of nBBP and DnBP remain intact during assay. The monoesters mono-2-ethylhexylphthalate (M2EHP) and mono-isohexylphthalate (MiHP) failed to reach IC50 at 150 μM and mono-n-butylphthalate (MnBP) was inactive. Inhibitory potencies in the [3H]CP-55940 binding assay were positively correlated with inhibition of CB1 receptor agonist-stimulated binding of [35S]GTPγS to the G protein, demonstrating that phthalates cause functional impairment of this complex. DnBP, nBBP and DEHP also inhibited binding of [3H]SR141716A, whereas inhibition with MiHP was comparatively weak and MnBP had no effect. Equilibrium binding experiments with [3H]SR141716A showed that phthalates reduce the Bmax of radioligand without changing its Kd. DnBP and nBBP also rapidly enhanced the dissociation of [3H]SR141716A. Our data are consistent with an allosteric mechanism for inhibition, with phthalates acting as relatively low affinity antagonists of CB1 receptors and cannabinoid agonist-dependent activation of the G-protein. Further studies are warranted, since some phthalate esters may have potential to modify CB1 receptor-dependent behavioral and physiological outcomes in the whole animal.  相似文献   

7.
The current investigation demonstrates the antitumor effects of combined supplementations of vanadium (V) (4.27 µmol/L drinking water ad libitum) and1α, 25-dihydroxy vitamin D3 (Vitamin D3) (0.3 μg/100 μL propylene glycol per os twice a week) on 1, 2 dimethylhydrazine (DMH) (20 mg/kg body weight) induced rat colon carcinogenesis. There was a significant reduction in incidence (70%), multiplicity (P < 0.0001) and volume (P < 0.01) of colon tumors. HPLC-fluorescence assay detected the combinatorial actions of V and Vitamin D3 against DMH-induced colonic O6-methylguanine DNA adducts formation (at four sequential time points; ANOVA, F = 13.56, P < 0.01). Simultaneous inhibition of DNA single strand breaks (P < 0.001) indicates the potency of the combination regimen in limiting the initiation event of colon carcinogenesis. Immunohistochemical analysis revealed that the effect of V and vitamin D3 occurred through suppression of cell proliferation (BrdU-LI: P < 0.001) along with an induction of apoptosis (TUNEL-LI: P < 0.01). The immunoexpression of tumor suppressor p53 and downregulation of antiapoptotic protein BCl-2 in subsequent immunofluorescence assay further provide strong evidence for the combinatorial inhibitory actions of vanadium and vitamin D3 against DMH-induced rat colon carcinogenesis.  相似文献   

8.
Cytochrome bd is a terminal quinol:O2 oxidoreductase of respiratory chains of many bacteria. It contains three hemes, b558, b595, and d. The role of heme b595 remains obscure. A CO photolysis/recombination study of the membranes of Escherichia coli containing either wild type cytochrome bd or inactive E445A mutant was performed using nanosecond absorption spectroscopy. We compared photoinduced changes of heme d-CO complex in one-electron-reduced, two-electron-reduced, and fully reduced states of cytochromes bd. The line shape of spectra of photodissociation of one-electron-reduced and two-electron-reduced enzymes is strikingly different from that of the fully reduced enzyme. The difference demonstrates that in the fully reduced enzyme photolysis of CO from heme d perturbs ferrous heme b595 causing loss of an absorption band centered at 435 nm, thus supporting interactions between heme b595 and heme d in the di-heme oxygen-reducing site, in agreement with previous works. Photolyzed CO recombines with the fully reduced enzyme monoexponentially with τ ∼ 12 μs, whereas recombination of CO with one-electron-reduced cytochrome bd shows three kinetic phases, with τ ∼ 14 ns, 14 μs, and 280 μs. The spectra of the absorption changes associated with these components are different in line shape. The 14 ns phase, absent in the fully reduced enzyme, reflects geminate recombination of CO with part of heme d. The 14-μs component reflects bimolecular recombination of CO with heme d and electron backflow from heme d to hemes b in ∼ 4% of the enzyme population. The final, 280-μs component, reflects return of the electron from hemes b to heme d and bimolecular recombination of CO in that population. The fact that even in the two-electron-reduced enzyme, a nanosecond geminate recombination is observed, suggests that namely the redox state of heme b595, and not that of heme b558, controls the pathway(s) by which CO migrates between heme d and the medium.  相似文献   

9.
10.
Nature has provided inspiration for Drug Discovery studies and amphibian secretions have been used as a promising source of effective peptides which could be explored as novel drug prototypes for neglected parasitic diseases as Leishmaniasis and Chagas disease. In this study, we isolated four antimicrobial peptides (AMPs) from Phyllomedusa nordestina secretion, and studied their effectiveness against Leishmania (L.) infantum and Trypanosoma cruzi. The antiparasitic fractions were characterized by mass spectrometry and Edman degradation, leading to the identification of dermaseptins 1 and 4 and phylloseptins 7 and 8. T. cruzi trypomastigotes were susceptible to peptides, showing IC50 values in the range concentration of 0.25–0.68 μM. Leishmania (L.) infantum showed susceptibility to phylloseptin 7, presenting an IC50 value of 10 μM. Except for phylloseptin 7 which moderate showed cytotoxicity (IC50 = 34 μM), the peptides induced no cellular damage to mammalian cells. The lack of mitochondrial oxidative activity of parasites detected by the MTT assay, suggested that peptides were leishmanicidal and trypanocidal. By using the fluorescent probe SYTOX® Green, dermaseptins 1 and 4 and phylloseptins 7 and 8 showed time-dependent plasma membrane permeabilization of T. cruzi; phylloseptin 7 also showed a similar effect in Leishmania parasites. The present study demonstrates for the first time that AMPs target the plasma membrane of Leishmania and T. cruzi, leading to cellular death. Considering the potential of amphibian peptides against protozoan parasites and the reduced mammalian toxicity, they may contribute as scaffolds for drug design studies.  相似文献   

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