A competitive enzyme linked immunosorbent assay for the determination of N-acetyltransferase (NAT2) phenotypes

The ratio of 5-acetylamino-6-amino-3-methyluracil (AAMU) to 1-methylxanthine (IX) in urine samples after caffeine ingestion can be used to indicate human N-acetyltransferase (NAT2) phenotypes. In previous studies, this ratio has been determined by LC or capillary electrophoresis. The possibility that this ratio could be determined by competitive antigen enzyme linked immunosorbent assays (ELISAs) has been investigated. Polyclonal antibodies were raised in rabbits against synthetic derivatives of AAMU and IX, and competitive antigen ELISAs were developed after isolation of the IgGs by ion-exchange chromatography. The competitive antigen ELISA correctly identified previously determined NAT2 phenotypes and gave the expected distribution of slow and fast N-acetylators within a group of 48 individuals.     

Paclitaxel (taxol) inhibits the arylamine N-acetyltransferase activity and gene expression (mRNA NAT1) and 2-aminofluorene-DNA adduct formation in human bladder carcinoma cells (T24 and TSGH 8301)

Acetylator polymorphism in man results from differential expression of human liver N-acetyltransferase. N-Acetyltransferase enzyme activity has been demonstrated to be involved in some types of chemical carcinogenesis. Paclitaxel (taxol) had been shown to affect N-acetyltransferase activity of human lung cancer cells. In this study, paclitaxel was chosen to investigate the effects of arylamine N-acetyltransferase activity (N-acetylation of substrate), gene expression and 2-aminofluorene-DNA adduct formation in human bladder carcinoma cell lines (T24 and TSGH 8301). The N-acetyltransferase activity (N-acetylation of substrates) was determined by high performance liquid chromatography assaying for the amounts of acetylated 2-aminofluorene and p-aminobenzoic acid and nonacetylated 2-aminofluorene and p-aminobenzoic acid. Intact human bladder carcinoma T24 and TSGH 8301 cells were used for examining N-acetyltransferase activity, gene expression and 2-aminofluorene-DNA adduct formation. The results demonstrated that the N-acetyltransferase activity, gene expression (NAT1 mRNA) and 2-aminofluorene-DNA adduct formation in intact human bladder carcinoma cells were inhibited and decreased by paclitaxel in a dose-dependent manner. The effects of paclitaxel on the apparent values of Km and Vmax of N-acetyltransferase enzyme from intact human bladder carcinoma cells were also determined in these cell lines. A marked influence of paclitaxel was observed on the decreasing apparent values of Km and Vmax from intact human bladder carcinoma cells (T24 and TSGH 8301). Thus, paclitaxel is an uncompetitive inhibitor to the NAT enzyme.     

The effect of paclitaxel on 2-aminofluorene-DNA adducts formation and arylamine N-acetyltransferase activity and gene expression in human lung tumor cells (A549).

In this study, paclitaxel was used to determine inhibition of arylamine N-acetyltransferase (NAT) activity, gene expression and 2-aminofluorene-DNA adduct formation in a human lung tumor cell line (A549). The activity of NAT was measured by HPLC assaying for the amounts of N-acetyl-2-aminofluorene (2-AAF) and remaining 2-aminofluorene (2-AF). Human lung tumor cell cytosols and intact cells were used for examining NAT activity and carcinogen-DNA adduct formation. The results demonstrated that NAT activity, gene expression (NAT1 mRNA) and 2-AF-DNA adduct formation in human lung tumor cells were inhibited and decreased by paclitaxel in a dose-dependent manner. The effects of paclitaxel on the values of the apparent Km and Vmax of NAT from human lung tumor cells were also determined in both examined systems. The result also indicated that paclitaxel decreased the apparent values of Km and Vmax from human lung tumor cells in both cytosol and intact cells. Thus, paclitaxel is an uncompetitive inhibitor to NAT enzyme.     

Nitric oxide protects murine embryonic liver cells (BNL CL.2) from cytotoxicity induced by glucose deprivation

We investigated the protective effects of nitric oxide on cell death of murine embryonic liver cells (BNL CL.2) after glucose deprivation. Endogenous nitric oxide production by BNL CL.2 cells was induced by 6 hr pretreatment with interferon-gamma and lipopolysaccharide. We used sodium nitroprusside and S-nitroso-L-glutathione as exogenous nitric oxide-generating compounds. All agents were used at doses that did not show direct cytotoxicity as measured by crystal violet staining assay. In the BNL CL.2 cells, the viability dropped very steeply after 24 hr incubation with glucose-free media. Endogenous nitric oxide produced by treatment of the cells with interferon-gamma and lipopolysaccharide protected the cells from glucose deprivation-induced cytotoxicity, but did not protect them in the presence of the nitric oxide synthesis inhibitor, N(G)-monomethyl-L-arginine. Exogenous nitric oxide protected the cells from glucose deprivation-induced cytotoxicity in a concentration-dependent manner. Cytoprotection by nitric oxide donors was abolished by the use of nitric oxide scavenger, 2-phenyl-4,4,5,5,-tetramethylimidazole, but not by the soluble guanosine cyclase inhibitor, 1H-[1,2,4]oxadiazole[4,3-a]quinoxalin-1-one. In addition, cytoprotective effects comparable to endogenous or exogenous nitric oxide were not observed when the cells were incubated with dibutyl guanosine 3',5'-cyclic monophosphate. Based upon these results, we suggest that nitric oxide may enhance the cell survival of BNL CL.2 cells after glucose deprivation via a guanosine 3',5'-cyclic monophosphate-independent pathway.     

Thymidylate synthase is the principal target enzyme for the cytostatic activity of (E)-5-(2-bromovinyl)-2'-deoxyuridine against murine mammary carcinoma (FM3A) cells transformed with the herpes simplex virus type 1 or type 2 thymidine kinase gene

Murine mammary carcinoma FM3A cells, deficient in cytosol thymidine (dThd) kinase (TK) activity and transformed by the herpes simplex virus type 1 (HSV-1) or type 2 (HSV-2) TK gene (designated FM3A TK-/HSV-1 TK+ and FM3A TK-/HSV-2 TK+, respectively) proved extremely sensitive to the cytostatic action of the potent antiherpetic drugs (E)-5-(2-bromovinyl)-2'-deoxyuridine (BVDU) and (E)-5-(2-iodovinyl)-2'-deoxyuridine (IVDU). The fact that FM3A TK-/HSV-2 TK+ cells were 5-fold more sensitive to the cytostatic action of BVDU and IVDU but incorporated [125I]IVDU to a 20-fold lower extent into their DNA than did FM3A TK-/HSV-1 TK+ cells led us to conclude that incorporation of these compounds into DNA of HSV TK gene-transformed cell lines is not directly related to their cytostatic action. In attempts to unravel the mechanism of the cytostatic effects of BVDU and IVDU on HSV TK gene-transformed FM3A cells, both compounds were submitted to an intensive biochemical study. Thymidylate synthase was identified as the principal target enzyme for the cytostatic action of BVDU and IVDU since (i) both compounds were far more inhibitory to 2(1)-deoxyuridine (dUrd) than to dThd incorporation into HSV TK gene-transformed FM3A cell DNA, (ii) the cytostatic action of BVDU and IVDU was more readily reversed by dThd than by dUrd, (iii) both compounds strongly inhibited the metabolic pathway leading to the incorporation of 2'-deoxycytidine (dCyd) into DNA thymidylate, (iv) BVDU and IVDU strongly inhibited tritium release from [5-3H]dCyd and [5-3H]dUrd in intact HSV TK gene-transformed FM3A cells, and (v) [125I]IVDU accumulated intracellularly as its 5'-monophosphate to concentration levels considerably higher than those required to inhibit partially purified thymidylate synthase. The inhibitory effects mentioned under (i) to (iv) were not observed with the parental FM3A/0 and FM3A/TK- cells; they were more pronounced for FM3A TK-/HSV-2 TK+ cells than for FM3A TK-/HSV-1 TK+ cells, which correlates with the differential cytostatic effects of BVDU and IVDU on these cells.     

Pharmacological characterization of MK-7246, a potent and selective CRTH2 (chemoattractant receptor-homologous molecule expressed on T-helper type 2 cells) antagonist

The chemoattractant receptor-homologous molecule expressed on T-helper type 2 cells (CRTH2) is a G protein-coupled receptor that has been reported to modulate inflammatory responses in various rodent models of asthma, allergic rhinitis and atopic dermatitis. In this study, we describe the biological and pharmacological properties of {(7R)-7-[[(4-fluorophenyl)sulfonyl](methyl)amino]-6,7,8,9-tetrahydropyrido[1,2-a]indol-10-yl}acetic acid (MK-7246), a novel synthetic CRTH2 antagonist. We show that MK-7246 1) has high affinity for the human, monkey, dog, rat, and mouse CRTH2, 2) interacts with CRTH2 in a reversible manner, 3) exhibits high selectivity over all prostanoid receptors as well as 157 other receptors and enzymes, 4) acts as a full antagonist on recombinant and endogenously expressed CRTH2, 5) demonstrates good oral bioavailability and metabolic stability in various animal species, 6) yields ex vivo blockade of CRTH2 on eosinophils in monkeys and sheep, and 7) significantly blocks antigen-induced late-phase bronchoconstriction and airway hyper-responsiveness in sheep. MK-7246 represents a potent and selective tool to further investigate the in vivo function of CRTH2.     

Highly selective cytostatic activity of (E)-5-(2-bromovinyl)-2'-deoxyuridine derivatives for murine mammary carcinoma (FM3A) cells transformed with the herpes simplex virus type 1 thymidine kinase gene

(E)-5-(2-Bromovinyl)-2'-deoxyuridine (BVDU) and various structurally related analogues thereof, i.e., (E)-5-(2-iodovinyl)-2'-deoxyuridine (IVDU) and (E)-5-(2-bromovinyl)-2'-deoxycytidine (BVDC), and the carbocyclic analogues of BVDU, IVDU, and BVDC, were evaluated for their inhibitory effects on the growth of murine mammary carcinoma FM3A cells, deficient in thymidine kinase (TK) activity but transformed with the herpes simplex virus type 1 (HSV-1) TK gene (designated FM3A/TK-/HSV-1 TK+). BVDU and its congeners were much more inhibitory to the growth of FM3A/TK-/HSV-1 TK+ than to the growth of the wild type (FM3A/0) cells. For BVDU, for example, the 50% inhibitory dose for the FM3A/TK-/HSV-1 TK+ cells was 0.5 ng/ml, as compared to 11 micrograms/ml for the FM3A/0 cells. Evidently, BVDU and its congeners required phosphorylation by the HSV-1 TK to exert their cytostatic action. In attempts to evaluate further the mechanism of this cytostatic action, BVDU, IVDU, and their carbocyclic analogues were evaluated for their inhibitory effects on thymidylate synthetase (TS) and their incorporation into DNA. TS was identified as one, but not the sole, target in the cytostatic activity of BVDU and its derivatives. With [125I]IVDU and its carbocyclic analogue C-[125I]IVDU, clear evidence was obtained for the incorporation of these radiolabeled analogues into DNA of the FM3A/TK-/HSV-1 TK+ cell line and a TS-deficient mutant thereof, FM3A/TK-/HSV-1 TK+/TS-. No incorporation was detected with [125I]IVDU or C-[125I]IVDU into DNA of FM3A/0 and FM3A/TS- cells. To what extent the incorporation of [125I]IVDU and C-[125I]IVDU contributed to their cytostatic action against FM3A/TK-/HSV-1 TK+ cells remains the subject of further study.     

Characterization of a human 5-hydroxytryptamine3 receptor type A (h5-HT3R-AS) subunit stably expressed in HEK 293 cells.

1. A cloned cDNA encoding a human 5-hydroxytryptamine3 receptor type A subunit (h5-HT3R-As) was transfected into human embryonic kidney (HEK 293) cells maintained in cell culture and a stable cell line expressing a high density of the recombinant receptor was selected. 2. Membrane homogenates prepared from transfected, but not untransfected, cells exhibited a homogeneous and saturable population (Bmax = 4.49 +/- 0.46 pmol mg-1 protein) of sites that bound the radiolabelled 5-HT3 receptor antagonist, [3H]-granisetron with high affinity (pKD = 8.87 +/- 0.08). Kinetic studies (at 37 degrees C) revealed rapid association (kappa +1 4.76 +/- 0.3 x 10(8) M-1 min-1) and dissociation (kappa -1 = 0.21 +/- 0.003 min-1) of the radioligand. 3. Selective and non-selective 5-HT3 receptor ligands competed for [3H]-granisetron binding with a rank order of potency (granisetron > ondansetron > meta-chlorophenylbiguanide > 5-HT > 2-methyl-5-HT > metoclopramide > > phenylbiguanide > cocaine > (+)-tubocurarine) identical to that established for 5-HT3 receptors endogenous to the human CNS. 4. In electrophysiological recordings performed on transfected cells, voltage-clamped at a holding potential of -60 mV, locally applied 5-HT (10 microM) evoked transient inward current responses that reversed in sign at a potential of -1.0 +/- 1.1 mV. Such responses were antagonized in a reversible manner by granisetron (1 nM). 5. The construction of a stable cell line expressing a high density of recombinant human 5-HT3 receptors which display appropriate pharmacology and function will assist in the further characterization of this receptor subtype and the exploration of species differences in 5-HT3 receptor pharmacology.     

Characterization of the human brain putative A2B adenosine receptor expressed in Chinese hamster ovary (CHO.A2B4) cells.

1. An [3H]-adenine pre-labelling methodology was employed to assay cyclic AMP generation by adenosine analogues in Chinese hamster ovary (CHO.A2B4) cells, transfected with cDNA which has been proposed to code for the human brain A2B adenosine receptor, and in guinea-pig cerebral cortical slices. 2. Adenosine analogues showing the following rank order of potency in the CHO.A2B4 cells (pD2 value): 5'-N-ethylcarboxamidoadenosine (NECA, 5.91) > adenosine (5.69) > 2-chloroadenosine (5.27) > N6-(2-(4-aminophenyl)-ethylamino)adenosine (APNEA, 4.06). The purportedly A2A-selective agonist, CGS 21680, failed to elicit a significant stimulation of cyclic AMP generation at concentrations up to 10 microM in CHO.A2B4 cells. In the guinea-pig cerebral cortex, NECA was more potent than APNEA with pD2 values of 5.91 and 4.60, respectively. 3. Of these agents, NECA was observed to exhibit the greatest intrinsic activity in CHO.A2B4 cells (ca. 10 fold stimulation of cyclic AMP), while, in comparison, maximal responses to adenosine (32% NECA response), 2-chloroadenosine (61%), and APNEA (73%) were reduced. 4. Antagonists of NECA-evoked cyclic AMP generation showed the rank order of apparent affinity (apparent pA2 value in CHO.A2B4 cells: guinea-pig cerebral cortex): XAC (7.89: 7.46) > CGS 15943 (7.75: 7.33) > DPCPX (7.16: 6.91) > PD 115,199 (6.95: 6.39) > 8FB-PTP (6.52: 6.55) > 3-propylxanthine (4.63: 4.59). 5. We conclude that, using the agents tested, the A2B adenosine receptor cloned from human brain expressed in Chinese hamster ovary cells exhibits an identical pharmacological profile to native A2B receptors in guinea-pig brain.     

Lack of association between the angiotensin-converting enzyme gene (I/D) polymorphism and diabetic nephropathy in Tunisian type 2 diabetic patients.

OBJECTIVE: The aim of the present study was to investigate whether the angiotensin-converting enzyme (ACE) insertion/deletion (I/D) polymorphism is associated with diabetic nephropathy and type 2 diabetes in the Tunisian population. DESIGN: A case-control study was conducted among 141 unrelated type 2 diabetic patients with (90 patients) or without nephropathy (51 patients) and 103 non-diabetic controls with normal fasting blood glucose. Genotyping was performed using a nested polymerase chain reaction amplification in order to identify correctly heterozygous individuals. RESULTS: The distribution of DD, ID and II genotypes did not significantly differ between type 2 diabetic patients with or without nephropathy (DD: 44%; ID: 46%; II: 10% vs. DD: 41%; ID: 47 %; II: 12%, respectively). There was also no significant statistical difference between the genotype distribution and allele frequencies of the (I/D) polymorphism in all type 2 diabetic subjects compared to non-diabetic controls with normal fasting blood glucose (DD: 43%; ID: 46%; II: 11% vs. DD: 37%; ID: 48%; II: 15%, respectively). CONCLUSIONS: In the present preliminary study, the (I/D) polymorphism within the ACE gene is likely not associated with diabetic nephropathy nor with type 2 diabetes in the Tunisian studied population.     

自体造血干细胞回输治疗Ⅰ型糖尿病2例

目的观察采用自体造血干细胞回输治疗Ⅰ型糖尿病的临床疗效。方法提取自体造血干细胞后,行T淋巴系统清除,再行干细胞回输,重建T淋巴系统,以清除原免疫系统对胰岛细胞的自身免疫反应,恢复胰岛细胞功能。结果2例患儿分别于干细胞回输后第14天、第20天停用胰岛素,安全出院。随访7个月,各项检查指标均正常。结论T淋巴系统清除和自体造血干细胞重建治疗为临床治疗Ⅰ型糖尿病提供了一种新方法,近期疗效好。     

A rapid and transient synthesis of nitric oxide (NO) by a constitutively expressed type II NO synthase in the guinea-pig suprachiasmatic nucleus.

1. We have measured extracellular NO/NO(2)(-) concentrations in guinea-pig suprachiasmatic nucleus (SCN) brain slices using fast cyclic voltammetry. A rapid and transient signal equivalent to 2.2+/-0.2 microM NO/NO(2)(-) (mean+/-s.e.mean, n=13) was detected at 1.26 V, the peak oxidation potential for NO, following local electrical stimulation (five pulses of 0.1 ms duration at 100 Hz, delivered every 5 min). 2. The NO/NO(2)(-) signal was inhibited by the non-selective nitric oxide synthase (NOS) inhibitors L-NAME, L-NMMA and the highly selective type II NOS (iNOS) inhibitor 1400 W (Garvey et al., 1997) in a concentration-dependent manner. IC(50) values were 229 microM (65 - 801, n=3, geomean and 95% confidence intervals (C.I.)), 452 nM (88 - 2310, n=5), and 14.2 microM (3.6 - 54.4, n=5), with maximum inhibitions of 82.8+/-6.7, 46.0+/-8.1, and 90.6+/-3.6%, respectively. 3. Exposure of the slices to the protein synthesis inhibitor cyclohexamide or the inhibitor of type II NOS induction dexamethasone immediately following slice cutting, and for a subsequent 4 - 5 h, did not inhibit the NO/NO(2)(-) signal. 4. The evoked NO/NO(2)(-) signal was not reduced following 6 h perfusion in Ca(2+)-free media, consistent with a Ca(2+)-independent type II NOS activity. 5. PCR for type II NOS revealed the presence of this isotype in the SCN, even immediately following removal of the brain. 6. These studies provide the first evidence to suggest a functional, constitutively-active type II NOS within the brain of normal, healthy adult animals, and add type II NOS to the multiple isotypes of NO synthase playing a role within the mammalian SCN.     

Characterization of human A(2B) adenosine receptors: radioligand binding, western blotting, and coupling to G(q) in human embryonic kidney 293 cells and HMC-1 mast cells.

Recombinant human A(2B) adenosine receptors (A(2B)ARs) and receptors extended on the amino terminus with hexahistidine and the FLAG epitope, DYKDDDDK (H/F-A(2B)) were stably overexpressed (to >20,000 fmol/mg protein) in human embryonic kidney 293 cells (HEK-A(2B)). By Western blotting, the H/F-A(2B) receptor runs as a 34.8-kDa glycoprotein. Pharmacological properties of A(2B)ARs were characterized with (125)I-3-aminobenzyl-8-phenyl-(4-oxyacetic acid)-1-propylxanthine (K(D), 36 nM). In competition binding assays, the affinity of agonists is reduced by substitution on either the N(6)- or the C-2 position of the adenine ring, whereas 5'-substitutions increase affinity, resulting in the potency order: 5'-N-ethylcarboxamidoadenosine (NECA) > N(6)-aminobenzyl-NECA approximately 2-chloroadenosine > 2-[4-(2-carboxyethyl)phenethylamino]-NECA (CGS21680) > N(6)-aminobenzyladenosine. The A(2B)AR is potently blocked by the A(2A)-selective antagonist 4-(2-[7-amino-2-[2-furyl][1,2, 4]triazolo-[2,3-a][1,3,5] triazin-5-yl-amino]ethyl)phenol (ZM241385; K(I), 32 nM for A(2B), 1.4 nM for A(2A)) and the A(1) selective antagonist 8-cyclopentyl-1,3-dipropylxanthine (K(I), 50.5 nM for A(2B); 2.5 nM for A(1)). The K(I) values for the antiasthmatic xanthines, theophylline (7.8 microM) and enprofylline (6.4 microM), are below their therapeutic plasma concentrations (20 to 50 microM), and agree with K(I) determinations for inhibition of NECA-stimulated cAMP accumulation in HEK-A(2B) cells. NECA or N(6)-(2-iodo)benzyl-5'-N-methylcarboxamidodoadenosine (IB-MECA) stimulate inositol trisphosphates and calcium accumulation in HEK-A(2B) or HEK-A(3) cells, respectively, but only the A(3) response is prevented by pertussis toxin. In human HMC-1 mast cells, A(2B)AR activation stimulates calcium mobilization and cAMP accumulation. We conclude that HEK-A(2B) cells and HMC-1 mast cells possess A(2B)AR glycoproteins that are coupled to both G(q/11) and G(s).     

Effects of YM471, a nonpeptide AVP V(1A) and V(2) receptor antagonist, on human AVP receptor subtypes expressed in CHO cells and oxytocin receptors in human uterine smooth muscle cells.

YM471, (Z)-4'-[4,4-difluoro-5-[2-(4-dimethylaminopiperidino)-2-oxoethylidene]-2,3,4,5-tetrahydro-1H-1-benzoazepine-1-carbonyl]-2-phenylbenzanilide monohydrochloride, is a newly synthesized potent vasopressin (AVP) receptor antagonist. Its effects on binding to and signal transduction by cloned human AVP receptors (V(1A), V(1B) and V(2)) stably expressed in Chinese hamster ovary (CHO) cells, and oxytocin receptors in human uterine smooth muscle cells (USMC) were studied. YM471 potently inhibited specific [(3)H]-AVP binding to V(1A) and V(2) receptors with K(i) values of 0.62 nM and 1.19 nM, respectively. In contrast, YM471 exhibited much lower affinity for V(1B) and oxytocin receptors with K(i) values of 16.4 microM and 31.6 nM, respectively. In CHO cells expressing V(1A) receptors, YM471 potently inhibited AVP-induced intracellular Ca(2+) concentration ([Ca(2+)](i)) increase, exhibiting an IC(50) value of 0.56 nM. However, in human USMC expressing oxytocin receptors, YM471 exhibited much lower potency in inhibiting oxytocin-induced [Ca(2+)](i) increase (IC(50)=193 nM), and did not affect AVP-induced [Ca(2+)](i) increase in CHO cells expressing V(1B) receptors. Furthermore, in CHO cells expressing V(2) receptors, YM471 potently inhibited the production of cyclic AMP stimulated by AVP with an IC(50) value of 1.88 nM. In all assays, YM471 showed no agonistic activity. These results demonstrate that YM471 is a potent, nonpeptide human V(1A) and V(2) receptor antagonist which will be a valuable tool in defining the physiologic and pharmacologic actions of AVP.     

Cell-type specific effects of endocytosis inhibitors on 5-hydroxytryptamine(2A) receptor desensitization and resensitization reveal an arrestin-, GRK2-, and GRK5-independent mode of regulation in human embryonic kidney 293 cells

The effect of endocytosis inhibitors on 5-hydroxytryptamine(2A) (5-HT(2A)) receptor desensitization and resensitization was examined in transiently transfected human embryonic kidney (HEK) 293 cells and in C6 glioma cells that endogenously express 5-HT(2A) receptors. In HEK-293 cells, 5-HT(2A) receptor desensitization was unaffected by cotransfection with a dominant-negative mutant of dynamin (DynK44A), a truncation mutant of arrestin-2 [Arr2(319-418)], or by two well-characterized chemical inhibitors of endocytosis: concanavalin A (conA) and phenylarsine oxide (PAO). In contrast, beta 2-adrenergic receptor desensitization was significantly potentiated by each of these treatments in HEK-293 cells. In C6 glioma cells, however, DynK44A, Arr2(319-418), conA, and PAO each resulted in the potentiation of 5-HT(2A) and beta-adrenergic receptor desensitization. The cell-type-specific effect of Arr2(319-418) on 5-HT(2A) receptor desensitization was not related to the level of GRK2 or GRK5 expression. Interestingly, although beta 2-adrenergic receptor resensitization was potently blocked by cotransfection with DynK44A, 5-HT(2A) receptor resensitization was enhanced, suggesting the existence of a novel cell-surface mechanism for 5-HT(2A) receptor resensitization in HEK-293 cells. In addition, Arr2(319-418) had no effect on 5-HT(2A) receptor resensitization in HEK-293 cells, although it attenuated the resensitization of the beta 2-adrenergic receptor. However, in C6 glioma cells, both DynK44A and Arr2(319-418) significantly reduced 5-HT(2A) receptor resensitization. Taken together, these results provide the first convincing evidence of cell-type-specific roles for endocytosis inhibitors in regulating GPCR activity. Additionally, these results imply that novel GRK and arrestin-independent mechanisms of 5-HT(2A) receptor desensitization and resensitization exist in HEK-293 cells.     

Inhibition of a type B monoamine oxidase inhibitor, (E)-2-(4-fluorophenethyl)-3-fluoroallylamine (MDL-72974A), on semicarbazide-sensitive amine oxidases isolated from vascular tissues and sera of different species.

(E)-2-(4-Fluorophenethyl)-3-fluoroallylamine hydrochloride (MDL-72974A) has been discovered recently to be a very potent and highly selective type B monoamine oxidase inhibitor. We have found that this inhibitor is also capable of inhibiting semicarbazide-sensitive amine oxidases (SSAOs) obtained from vascular tissues and sera of different species. The inhibition of SSAO by MDL-72974A was irreversible and time dependent. It was competitive without preincubation of the enzyme with the inhibitor and demonstrated a mixed-type of inhibition when the enzyme was preincubated with the inhibitor. The IC50 values were estimated to be 2 x 10(-9) M, 5 x 10(-9) M, 8 x 10(-8) M and 2 x 10(-8) M for SSAO from dog aorta, rat aorta, bovine aorta and human umbilical artery, respectively. SSAO obtained from bovine serum was relatively insensitive to MDL-72974A (IC50 = 3 x 10(-7) M. Following intraperitoneal administration of MDL-72974A, rat brain MAO-B was inhibited with the ED50 value being about 0.2 mg/kg. Rat aorta SSAO was also inhibited and to a similar extent by the same dose. MDL-72974A is the most potent SSAO inhibitor that has been described thus far.     

Suppressive effects of caraway (Carum carvi) extracts on 2, 3, 7, 8-tetrachloro-dibenzo-p-dioxin-dependent gene expression of cytochrome P450 1A1 in the rat H4IIE cells.

Cytochrome P450 1A1 (CYP1A1) is among the cytochrome P450 classes known to convert xenobiotics and endogenous compounds to toxic and/or carcinogenic metabolites. Suppression of CYP1A1 over expression by certain compounds is implicated in prevention of cancer caused by chemical carcinogens. Chemopreventive agents containing high levels of flavonoids and steroids-like compounds are known to suppress CYP1A1. This study was carried out for assessment of the genomic and proteomic effects of caraway (Carum carvi) extracts containing high levels of both flavonoids and steroid-like substances on ethoxy resorufin dealkylation (EROD) activity and CYP1A1 at mRNA levels. Rat hepatoma cells co-treated with a CYP1A1 inducer i.e. TCDD (2, 3, 7, 8-tetrachlorodibenzo-p-dioxin) and different preparations of caraway extracts at concentrations of 0, 0.13, 1.3, and 13 microM in culture medium. After incubation (37 degrees C and 7% CO2 for 20 h), changes in EROD specific activity recorded and compared in cells under different treatments. The results show that caraway seed extract prepared in three different organic solvents suppressed the enzyme activity in hepatoma cells in a dose-dependent manner. The extracts added above 0.13 microM could significantly inhibit EROD activity and higher levels of each extract (1.3 and 13 microM) caused approximately 10-fold suppression in the enzyme activity. Accordingly, data obtained from the RT-PCR (TaqMan) clearly showed the suppressive effects of plant extract on CYP1A1-related mRNA expression. These data clearly show that substances in caraway seeds extractable in organic solvents can potentially reverse the TCDD-dependent induction in cytochrome P450 1A1.     

The effect of vomitoxin (Deoxnivalenol) on testicular morphology, testicular spermatid counts and epididymal sperm counts in IL-6KO [B6129-IL6 [TmlKopf] (IL-6 gene deficient)] and WT [B6129F2 (wild type to B6129-IL6 with an intact IL-6 gene)] mice.

The potential of vomitoxin (VT) to affect testicular morphology and testicular and epididymal sperm counts was assessed in three strains of mice: IL-6KO [B6129-IL6 (tmlKopf) (IL-6 gene deficient)], WT [B6129F2 (wild type to B6129-IL6 with an intact IL-6 gene)] and B6C3F1 mice in a 90-day feeding study. The treated mice received VT at a concentration of 10 ppm in their diet. The body weight of VT-treated animals was significantly reduced compared with control animals. Slight changes, not statistically significant, were observed in relative testis weight and testicular spermatid counts. Histological changes were not apparent in the testes of VT-treated animals. The diameter of the seminiferous tubules, the height of the seminiferous epithelium and the number of Sertoli cell nucleoli per cross-sectioned seminiferous tubule in the VT-treated groups were not significantly different from their respective untreated controls. The IL-6KO and B6C3F1 VT-treated mice had significantly reduced cauda epididymal weights compared with their respective controls. These changes were not attributed to decreased sperm counts and this finding suggests that VT may exert an adverse affect on the epididymis.     

Elucidation of the effects of the CYP1A2 deficiency polymorphism in the metabolism of 4-cyclohexyl-1-ethyl-7-methylpyrido[2,3-d]pyrimidine-2-(1h)-one (YM-64227), a phosphodiesterase type 4 inhibitor, and its metabolites in dogs.

The canine CYP1A2 1117 C>T single nucleotide polymorphism is responsible for a substantial portion of the interindividual variability seen in the pharmacokinetics of 4-cyclohexyl-1-ethyl-7-methylpyrido[2,3-d]pyrimidine-2-(1H)-one (YM-64227). The purpose of this study is to investigate the contribution of CYP1A2 to the metabolism of YM-64227 and its five metabolites (MM-1 to MM-5), as well as to determine the interindividual variability between the pharmacokinetic profiles of the compounds with respect to the CYP1A2 deficiency polymorphism. alpha-Naphthoflavone and anti-CYP1A1/2 antibody inhibited the metabolic activities at which MM-2 and MM-3 were formed from YM-64227 in C/C- and C/T-type microsomes. In T/T type, the rate of MM-2 and MM-3 formation was lower, and alpha-naphthoflavone and anti-CYP1A1/2 antibody were shown to have no effect. A positive correlation between the overall metabolism of YM-64227 and phenacetin O-deethylation, a CYP1A2 activity marker, was observed in all the genotypes. The in vitro metabolic clearances in the T/T type of MM-1, MM-3, MM-4, and MM-5 were less than 50% lower than those in the C/C type. The anti-CYP1A1/2 antibody inhibited the metabolism of MM-1, MM-3, MM-4, and MM-5 in the C/C and C/T types. These results suggest that the formation of MM-2 and MM-3 from YM-64227 is catalyzed by CYP1A2, and that CYP1A2 contributes mainly to the subsequent metabolism of the primary metabolites of YM-64227, with the exception of MM-2. It is possible that the interindividual variability of YM-64227 with respect to the CYP1A2 deficiency polymorphism is caused by a decrease in the metabolic activities of both the unchanged drug and its metabolites.