Vascular sphingosine-1-phosphate S1P1 and S1P3 receptors

The sphingolipid sphingosine-1-phosphate (S1P) acts on five subtypes of G-protein- coupled receptors, termed S1P(1) (formerly endothelial differentiation gene-1 [Edg-1]), S1P(2) (Edg-5), S1P(3) (Edg-3), S1P(4) (Edg-6) and S1P(5) (Edg-8), and possibly several other "orphan" receptors, such as GPR3, GPR6 and GPR12. These receptors are coupled to different intracellular second messenger systems, including adenylate cyclase, phospholipase C, phosphatidylinositol 3-kinase/protein kinase Akt, mitogen-activated protein kinases, as well as Rho- and Ras-dependent pathways. Consistently with this receptor multiplicity and pleiotropic signaling mechanisms, S1P influences numerous cell functions. S1P(1)1, S1P(2) and S1P(3) receptors are the major S1P receptor subtypes in the cardiovascular system, where they mediate the effects of S1P released from platelets, and possibly other tissues (such as brain). Thus S1P(1) and S1P(3) receptors enhance endothelial and vascular smooth muscle cell proliferation and migration, playing a key role in developmental and pathological angiogenesis. In contrast, S1P(2) receptors inhibit migration of these cell types, probably because of their unique stimulatory effect on a GTPase-activating protein inhibiting the activity of Rac. S1P receptors can also cause relaxation and constriction of blood vessels. The former effect is mediated by pertussis toxin-sensitive receptors (possibly S1P(1)) located on the endothelium and stimulating phosphatidylinositol 3-kinase/Akt/endothelial nitric oxide synthase (eNOS). The vasoconstricting effect of S1P is likely to be mediated by S1P(2) and/or S1P(3) receptors, via Rho-Rho-kinase, and is more potent in coronary and cerebral blood vessels. Finally, S1P also protects endothelial cells from apoptosis through activation of phosphatidylinositol 3-kinase/Akt/eNOS via S1P(1) and S1P(3) receptors. The variety of these effects, taken together with the existence of multiple receptor subtypes, provides an abundance of therapeutic targets that currently still await the development of selective agents.     

Antimykobakterielle 1-Phenyl-1-alkylaminoalkane

Antimycobacterial 1-Phenyl-1-alkylaminoalkanes Synthesis and testing for antimycobacterial properties (M. tuberculosis H 37 Ra, Middlebrook-7H9-broth) of 1-phenyl-1-alkylaminoalkanes, which differ from antimycobacterial N-alkylbenzylamines by an additional alkyl chain in α-position, is described. By variation of both alkyl chains and introduction of one or two Cl-substituents in the aromatic ring the activity increases up to an optimum within the homologous series. Overstepping optimal lipophilicity or ramification of the alkyl chains decrease activity. Compounds 19, 20, 33-35, 51-53, 61-63, 65-67, 70-73, 96 and 102 - 104 inhibit the growth of M. tuberculosis in concentrations of 2 to 4 μg/ml.     

Sulfation of iodothyronines by human sulfotransferase 1C1 (SULT1C1)*

Sulfation is an important component of human thyroid hormone metabolism. The role of the human sulfotransferase 1C1 (SULT1C1) is not known. Because SULT1C1 is present in the adult thyroid, intra-thyroidal sulfation of thyroid hormones and their metabolites might occur. We tested this hypothesis by determining the ability of recombinant human SULT1C1 to catalyze iodothyronine sulfation. Apparent K(m) values for 3,3',5-triiodothyronine (T(3)), 3, 3'-diiodothyronine (3,3'-T(2)), 3',5',3-triiodothyronine (rT(3)), and 3,3',5,5'-tetraiodothyronine (T(4)) with SULT1C1 were 28.7, 10.3, 10.2, and 59.3 microM, respectively. Thermal stability and responses to inhibitors also were tested with T(3) as the substrate. Enzyme aliquots were measured simultaneously to determine SULT1C1 substrate preferences at optimal iodothyronine concentrations. SULT1C1 activity obtained with T(3) was used as 100%, and the activities with 3,3'-T(2), rT(3), T(4), and 3,5-diiodothyronine (3, 5-T(2)) were 614, 314, 25, and 4%, respectively. We report for the first time the characterization of human SULT1C1 with T(3) and the preferences of the enzyme for various iodothyronines. The presence of SULT1C1 in the adult thyroid gland raises the possibilities that the enzyme can contribute to intraglandular thyroid hormone processing and iodide reutilization.     

Phenotype of the Cyp1a1/1a2/1b1-/- triple-knockout mouse

Crossing the Cyp1a1/1a2(-/-) double-knockout mouse with the Cyp1b1(-/-) single-knockout mouse, we generated the Cyp1a1/1a2/1b1(-/-) triple-knockout mouse. In this triple-knockout mouse, statistically significant phenotypes (with incomplete penetrance) included slower weight gain and greater risk of embryolethality before gestational day 11, hydrocephalus, hermaphroditism, and cystic ovaries. Oral benzo[a]pyrene (BaP) daily for 18 days in the Cyp1a1/1a2(-/-) produced the same degree of marked immunosuppression as seen in the Cyp1a1(-/-) mouse; we believe this reflects the absence of intestinal CYP1A1. Oral BaP-treated Cyp1a1/1a2/1b1(-/-) mice showed the same "rescued" response as that seen in the Cyp1a1/1b1(-/-) mouse; we believe this reflects the absence of CYP1B1 in immune tissues. Urinary metabolite profiles were dramatically different between untreated triple-knockout and wild-type; principal components analysis showed that the shifts in urinary metabolite patterns in oral BaP-treated triple-knockout and wild-type mice were also strikingly different. Liver microarray cDNA differential expression (comparing triple-knockout with wild-type) revealed at least 89 genes up- and 62 genes down-regulated (P-value < or = 0.00086). Gene Ontology "classes of genes" most perturbed in the untreated triple-knockout (compared with wild-type) include lipid, steroid, and cholesterol biosynthesis and metabolism; nucleosome and chromatin assembly; carboxylic and organic acid metabolism; metal-ion binding; and ion homeostasis. In the triple-knockout compared with the wild-type mice, response to zymosan-induced peritonitis was strikingly exaggerated, which may well reflect down-regulation of Socs2 expression. If a single common molecular pathway is responsible for all of these phenotypes, we suggest that functional effects of the loss of all three Cyp1 genes could be explained by perturbations in CYP1-mediated eicosanoid production, catabolism and activities.     

间质作用因子1通过正调控窖蛋白1抑制FBJ-S1-H的迁移性

目的证明间质作用因子(stromal interaction molecule1,Stim1)在FBJ诱导的小鼠骨肉瘤细胞中的抑癌作用。方法在Stim1高表达的FBJ-S1-H细胞采用Stim1以siRNA干扰技术得到Stim1沉默的几株S1-H单克隆细胞株,通过细胞行为学方法和RT-PCR技术对其mRNA进行研究,通过明胶酶谱法对细胞基质金属酶活性进行研究。结果通过细胞行为学方法证明,Stim1的沉默提高了细胞的迁移性,通过对mRNA表达的研究发现,Stim1沉默引起了多种基因表达的变化,其中包括基质金属酶9(matrix mexalloprotelnase 9,MMP-9)的升高,窖蛋白(caveolinl,Cav1),甾醇调控因子Srebf1的降低等,提高单克隆细胞中的Cav1含量可以使细胞迁移性降低。结论实验结果证明在FBJ-S1-H细胞中,Stim1能够抑制细胞的移动性,沉默Stim1的表达能够提高细胞的迁移性。     

3-Sulfonyl-1-carba-1-dethiacephems

The stability of the 1-carba-1-dethiacephalosporin framework has allowed the synthesis of a range of 3-sulfonyl-1-carba-1-dethiacephems unavailable for a variety of reasons in the cephem arena. The known p-nitrobenzyl 7 beta-(phenoxyacetamido)-3-[[(trifluoromethyl)sulfonyl]oxy]-1-carba -1- dethia-3-cephem-4-carboxylate served as a precursor to this series of compounds. Displacement of the enol triflate with various sulfinates in acetonitrile or DMF and deprotection of the intermediates led to 7 beta-[(2-amino-4-thiazolyl)(methoxyimino)acetyl]amino]- 3-[alkyl(aryl)sulfonyl]-1-carba-1-dethia-3-cephem-4-carboxyl ic acids. The 3-sulfonyl-1-carba-1-dethiacephems display potent activity against both Gram-positive and Gram-negative bacteria. The following MIC's (microgram/mL) for the 3-cyclopropyl sulfone are representative: Staphylococcus aureus = 4, Streptococcus pyogenes = 1, Haemophilus influenzae = 0.25, Escherichia coli = 0.03, Enterobacter cloacae = 0.25, Proteus rettgeri = 0.25. The excellent in vitro antibacterial activity of this series indicates the potential of the carbacephalosporin framework for exploring substituents which are unknown or which produce unstable cephems.     

Substrate-dependent modulation of UDP-glucuronosyltransferase 1A1 (UGT1A1) by propofol in recombinant human UGT1A1 and human liver microsomes

Our previous study has shown that propofol, a probe substrate for human UDP-glucuronosyltransferase (UGT) 1A9, activated the glucuronidation of 4-methylumbelliferone (4-MU) by recombinant UGT1A1 in a concentration-dependent manner. In the present study, we investigated the mechanism of activation, and whether the stimulatory effect occurs when another substrate is used with human liver microsomes. The glucuronidation of 4-MU followed Michaelis-Menten kinetics with a K(m) value of 101 microM in the absence of propofol. In the presence of 200 microM propofol, a concentration that causes heterotopic activation of 4-MU glucuronidation (4-MUG), the V(max) value increased to 1.5-fold, while the K(m) value decreased to 0.53-fold. In order to assess whether propofol activates UGT1A1 activity for a substrate other than 4-MU, the effect of propofol on oestradiol 3beta-glucuronidation by recombinant UGT1A1 and in human liver microsomes was evaluated. In contrast to 4-MUG activity, propofol inhibited UGT1A1-catalysed oestradiol 3beta-glucuronidation in recombinant UGT1A1 as well as in human liver microsomes with IC(50) values of 59 and 228 microM, respectively. In addition, a known UGT1A1 modulator, 17alpha-ethynyloestradiol, stimulated oestradiol 3beta-glucuronidation slightly at a concentration of 5 microM, while it inhibited 4-MUG in recombinant UGT1A1 at all concentrations tested (5-100 microM). These findings indicate that the modulation of UGT1A1 by propofol is substrate-dependent, and thus care should be taken when extrapolating the stimulatory effects of drugs for one glucuronidation substrate.     

Preferential induction of CYP1A1 and CYP1B1 in CCSP-positive cells.

Both benzo[a]pyrene (BaP) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) are potent ligands of aryl hydrocarbon receptors (AhR). Although animal studies indicate that both compounds induce pathological changes in the peripheral lung, the specific cell type involved remains unclear. Clara cells, expressing Clara cell specific protein (CCSP) and abundant in cytochrome P450, are nonciliated bronchiolar epithelial cells in the peripheral lung. Here we explore the hypothesis that CCSP-positive Clara cells are highly responsive to AhR ligands and are the primary cell type involved in BaP- and TCDD-induced toxicities. The responsiveness to AhR ligands was evaluated by measuring the respective mRNA and protein levels of cytochrome P450 1A1 (CYP1A1) and 1B1 (CYP1B1) using real-time RT-PCR and immunocytochemistry assays. Two in vitro models were used: primary cultures of human small airway epithelial (SAE) cells and rat lung slice cultures. In the presence of calcium, human SAE cells differentiated into CCSP-positive cells. BaP- and TCDD-induced mRNA and protein levels of CYP1A1 and CYP1B1 levels were significantly elevated in CCSP-positive cell cultures. Similarly, AhR mRNA and protein levels were increased in CCSP-positive cell cultures, as determined by real-time RT-PCR and Western blot analysis. When rat lung slice cultures were treated with BaP or TCDD for 24 h, CYP1A1 and CYP1B1 proteins were strongly induced in Clara cells. These results indicate that, in the peripheral lung of both rats and humans, CCSP-positive cells (Clara cells) may be more sensitive to AhR ligands than other cell types.     

大豆异黄酮对YAC—1细胞和小鼠ESC艾氏肉瘤生长的抑制作用

目的 观察大豆异黄酮对YAC-1细胞及小鼠ESC艾氏肉瘤生长的抑制作用及对免疫功能的影响.方法用MTT法测定大豆异黄酮对YAC-1细胞生长的抑制率和小鼠淋巴细胞转化刺激指数;LDH释放法测定小鼠NK细胞活性;称重观察肿瘤生长和脾指数和胸腺指数.结果金雀异黄素和大豆甙元剂量依赖性抑制YAC-1细胞生长,并随时间延长作用增强;大豆异黄酮50、100、400 mg·kg     

2-ABT-S-oxide detoxification by glutathione S-transferases A1-1, M1-1 and P1-1: Implications for toxicity associated with zileuton

1. Zileuton, an agent which targets the leukotriene pathway through inhibition of 5-lipoxygenase (5-LO), was approved for the treatment of asthma in 1997. Shortly after its release, its use was restricted due to the observation of hepatotoxicity in patients.

2. Previous research from the authors’ laboratory demonstrated the formation of the reactive metabolite, 2-ABT-S-oxide (M1) from zileuton, and has identified a mercapturate of 2-ABT, C1, in the urine of rats dosed with zileuton. The reaction between M1 and glutathione (GSH) has been established in vitro; however, the potential for catalysis by glutathione transferases (GSTs) was not addressed.

3. The work presented here outlines a role for GSTs in the detoxification of M1. Non-enzymatic conjugation studies with M1 and GSH in control experiments led to a t_1/2 of 6.4 ± 0.4 h at pH 6.5. This rate was accelerated in the presence of GSTA1-1, GSTM1-1 and GSTP1-1 providing t_1/2 values of 2.6 ± 0.1, 0.53 ± 0.02, and 0.3 ± 0.04 h, respectively, at pH 6.5.

4. The inhibition of various GST enzymes was also studied. Results show that M1 inhibits GSTM1-1 and GSTP1-1 to a greater extent as compared with GSTA1-1. In the case of GSTA1-1, the inhibition was observed to be reversible, whereas M1 inhibition of GSTM1-1 and GSTP1-1 was found to be irreversible under identical conditions.

5. GSTM1-1 is present in liver and thus the finding of the alkylation and potential irreversible inactivation of this isoform in vivo could contribute to an understanding of the hepatotoxicity associated with zileuton.

     

Variation in induced CYP1A1 levels: relationship to CYP1A1, Ah receptor and GSTM1 polymorphisms

The genotypic basis of interindividual variation in levels of induced CYP1A1 activity has been investigated by screening both the CYP1A1 gene and the Ah receptor gene (AhR) for both previously described and novel polymorphisms. A 103-fold level of interindividual variation in induced CYPlA1 activity [ethoxyresorufin O-deethylase (EROD)] was observed in lymphocytes from a group of 30 Caucasian volunteers. High levels of induced EROD activity did not correlate with the presence of CYP1A1*2 or CYP1A1*4 alleles or with the GSTM1 null genotype. Novel CYP1A1 alleles with the base substitutions C4151T, G-469A and C-459T respectively, were detected by screening the coding exons and approximately 1 kb of upstream sequence in 20 individuals by single-strand conformational polymorphism (SSCP) analysis but none of the three novel alleles appeared to be associated with high induced CYP1A1 activity in the study group. Screening of the 11 exons of the AhR gene by SSCP analysis confirmed the existence of the previously described G1721A polymorphism in a Caucasian population and a novel allele (G1768A which results in the amino acid substitution V5701) was also detected. The novel allele was very rare in Caucasians though more common in African-Americans. Individuals with at least one copy of the G1721A AhR variant allele showed a significantly higher level of induced CYP1A1 activity compared with individuals negative for the polymorphism (P = 0.0001). A similar finding was obtained for induced CYP1A1 protein levels determined by immunoblotting. Levels of induced CYP1A1 activity were also found to show a sex difference with women showing a significantly lower induced activity compared with men. We conclude that genotypes for the G1721A AhR polymorphism and gender appear to be determinants of levels of induced CYP1A1 activity and that interindividual variation in levels of induced CYP1A1 activity appears to be associated more with regulatory factors than polymorphism in the CYP1A1 gene.