Potent and selective inhibition of squalene epoxidase by synthetic galloyl esters

n-Alkyl esters (ethyl, octyl, dodecyl, and cetyl) of gallic acid were evaluated as enzyme inhibitors of recombinant rat squalene epoxidase (SE), a rate-limiting enzyme of cholesterol biogenesis. Dodecyl (6) (IC(50) = 0.061 microM) showed the most potent inhibition, which was far more potent than those of previously reported naturally occurring gallocatechins. Octyl gallate (5) (IC(50) = 0.83 microM) and cetyl gallate (7) (IC(50) = 0.59 microM) also showed good inhibition, while gallic acid (IC(50) = 73 microM) itself was not so active. In addition, chemically synthesized galloyl ester of cholesterol (9) (IC(50) = 3.9 microM), farnesol derivative (10) (IC(50) = 0.57 microM), and dodecyl galloyl amide (8) (IC(50) = 3.0 microM) were also potent inhibitors of SE. Inhibition kinetics revealed that dodecyl gallate inhibited SE in competitive (K(I) = 0.033 microM) and no-time-dependent manner. The potent inhibition of the flavin monooxygenase would be caused by specific binding to the enzyme, and by scavenging reactive oxygen species required for the epoxidation reaction.     

Antioxidative galloyl esters as enzyme inhibitors of p-hydroxybenzoate hydroxylase

Gallic acid and its esters were evaluated as enzyme inhibitors of recombinant p-hydroxybenzoate hydroxylase (PHBH), a NADPH-dependent flavin monooxygenase from Pseudomonas aeruginosa. n-Dodecyl gallate (DG) (IC(50)=16 microM) and (-)-epigallocatechin-3-O-gallate (EGCG) (IC(50)=16 microM), a major component of green tea polyphenols, showed the most potent inhibition, while product-like gallic acid did not inhibit the enzyme significantly (IC(50)>250 microM). Inhibition kinetics revealed that both DG and EGCG inhibited PHBH in a non-competitive manner (K(I)=18.1 and 14.0 microM, respectively). The enzyme inhibition was caused by specific binding of the antioxidative gallate to the enzyme, and by scavenging reactive oxygen species required for the monooxygenase reaction. Molecular modeling predicted that EGCG binds to the enzyme in the proximity of the FAD binding site via formation of three hydrogen bonds.     

Insulin-mediated inhibition of the induction of tyrosine aminotransferase by dexamethasone.

Insulin-mediated regulation of glucocorticoid-induced expression of the liver-specific gene tyrosine aminotransferase was studied in a clone of the Reuber rat hepatoma cells. Insulin inhibited dexamethasone-induced chloramphenicol acetyltransferase expression from approximately 4 kb of TAT 5' flanking sequence. The degree of this inhibition was comparable to the response of the endogenous gene. A construct of approximately 3 kbp of 5' flanking sequence exhibited no significant basal expression but retained sensitivity to glucocorticoids and to insulin inhibition of the glucocorticoid response. Results of further analysis of the insulin response in deletion constructs and constructs containing glucocorticoid responsive elements ligated to a heterologous promoter suggest that in addition to the glucocorticoid response elements a region close to the start site in the TAT promoter is necessary for insulin to inhibit glucocorticoid-mediated induction of expression.     

Imidazo[1,2-a]pyridine derivatives as inhibitors of TNF-alpha expression in T cells

Novel hexahydroimidazo[1,2-a]pyridines prepared by the addition of ethyl (1-benzylimidazolidin-2-ylidene)acetate (2) to the fungal metabolite podoscyphic acid (1a) and esters of 1a have been evaluated for their ability to inhibit the inducible TNF-alpha promoter activity in T cells. The methyl ester 3b is the most potent, inhibiting the TNF-alpha driven reporter gene expression in Jurkat T cells with an IC(50)-value of 2.0 microg/ml (3.6 microM). In addition, compound 3b inhibited the inducible TNF-alpha production in the myelomonocytic U937 cells with an IC(50)-value of 4.6 microM.     

Effect of n-3 fatty acids on serum lipid levels and hepatic fatty acid metabolism in BALB/c.KOR-Apoeshl mice deficient in apolipoprotein E expression

N-3 fatty acids exert a potent serum lipid-lowering effect in rodents mainly by affecting hepatic fatty acid oxidation and synthesis. However, it has been observed that fish oil and docosahexaenoic acid ethyl ester do not lower serum lipid levels in apolipoprotein E (apoE)-knockout (Apoetm1Unc) mice generated by gene targeting. To test the hypothesis that apoE expression is required for n-3 fatty acid-dependent regulation of serum lipid levels and hepatic fatty acid metabolism, we examined the effect of fish oil and n-3 fatty acid ethyl esters on the activity and gene expression of hepatic enzymes involved in fatty acid oxidation and synthesis using an alternative apoE-deficient mouse model with the BALB/c genetic background (BALB/c.KOR-Apoeshl). ApoE-deficient mice were fed diets containing 9.4% palm oil, fish oil, or 5.4% palm oil and 1% EPA plus 3% DHA ethyl esters for 15 days. In contrast to the reported data on apoE-knockout mice, fish oil and n-3 fatty acid ethyl esters greatly decreased serum triacylglycerol, cholesterol, and phospholipid levels in the Apoeshl mice. The decreases were greater with fish oil than with ethyl esters. The alterations by dietary n-3 fatty acids of serum lipid levels were accompanied by parallel changes in the activity and mRNA levels of enzymes involved in hepatic fatty acid oxidation and synthesis. The reason for the discrepancy between the results of the current study and previous studies is unknown. However, our study at least indicates that a lack of apoE expression does not necessarily accompany deficits in the n-3 fatty acid-dependent regulation of serum lipid levels and hepatic fatty acid metabolism.     

Inhibition of eukaryotic dna polymerase alpha by persimmon (Diospyros kaki) extract and related polyphenols.

The effects of persimmon extract (Diospyros kaki) and related polyphenols on eukaryotic DNA polymerase alpha were examined. It was found that persimmon extract, epigallocatechin gallate, and epicatechin gallate strongly inhibited the activity of DNA polymerase alpha purified from calf thymus. Among these polyphenols, persimmon extract had the most potent effect on DNA polymerase alpha activity and the concentration of persimmon extract producing 50% inhibition of the activity was 0.191 microM. Persimmon extract showed a weaker effect on DNA polymerase beta and slightly inhibited primase and DNA polymerase I. The inhibition of DNA polymerase alpha by persimmon extract was competitive with the template-primer and noncompetitive with dTTP substrate. The Ki value of DNA polymerase alpha for persimmon extract was estimated to be 70 nM. Moreover, persimmon extract inhibited [3H]thymidine incorporation of human peripheral lymphocyte cells stimulated by PHA.     

Synthetic tyrosyl gallate derivatives as potent melanin formation inhibitors

Three tyrosyl gallate derivatives (1-3) with variable hydroxyl substituent at the aromatic ring of tyrosol were synthesized and evaluated as potent inhibitors on tyrosinase activity and melanin formation in melan-a cells. Among three tyrosyl gallate derivatives, 4-hydroxyphenethyl 3,4,5-trihydroxybenote (1) (IC(50)=4.93 microM), 3-hydroxyphenethyl 3,4,5-trihydroxybenote (2) (IC(50)=15.21 microM), and 2-hydroxyphenethyl 3,4,5-trihydroxybenote (3) (IC(50)=14.50 microM) exhibited significant inhibitory effect on tyrosinase activity. Compound 1 was the most active compound, though it did not show the inhibitory effect on melanin formation in melan-a cells. However, compounds 2 (IC(50)=8.94 microM) and 3 (IC(50)=13.67 microM) significantly suppressed the cellular melanin formation without cytotoxicity. This study shows that the position of hydroxyl substituent at the aromatic ring of tyrosol plays an important role in the intracellular regulation of melanin formation in cell-based assay system.     

Homologous regulation of the gonadotropin-releasing hormone receptor gene is partially mediated by protein kinase C activation of an activator protein-1 element

Homologous regulation of GnRH receptor (GnRHR) gene expression is an established mechanism for controlling the sensitivity of gonadotropes to GnRH. We have found that expression of the GnRHR gene in the gonadotrope-derived alpha T3-1 cell line is mediated by a tripartite enhancer that includes a consensus activator protein-1 (AP-1) element, a binding site for SF-1 (steroidogenic factor-1), and an element we have termed GRAS (GnRHR-activating sequence). Further, in transgenic mice, approximately 1900 b.p. of the murine GnRHR gene promoter are sufficient for tissue-specific expression and GnRH responsiveness. The present studies were designed to further delineate the molecular mechanisms underlying GnRH regulation of GnRHR gene expression. Vectors containing 600 bp of the murine GnRHR gene promoter linked to luciferase (LUC) were transiently transfected into alpha T3-1 cells and exposed to treatments for 4 or 6 h. A GnRH-induced, dose-dependent increase in LUC expression of the -600 promoter was observed with maximal induction of LUC noted at 100 nM GnRH. We next tested the ability of GnRH to stimulate expression of vectors containing mutations in each of the components of the tripartite enhancer. GnRH responsiveness was lost in vectors containing mutations in AP-1. Gel mobility shift data revealed binding of fos/jun family members to the AP-1 element of the murine GnRHR promoter. Treatment with GnRH or phorbol-12-myristate-13-acetate (PMA) (100 nM), but not forskolin (10 microM), increased LUC expression, which was blocked by the protein kinase C (PKC) inhibitor, GF109203X (100 nM), and PKC down-regulation (10 nM PMA for 20 h). In addition, a specific MEK1/MEK2 inhibitor, PD98059 (60 microM), reduced the GnRH and PMA responses whereas the L-type voltage-gated calcium channel agonist, +/- BayK 8644 (5 microM), and antagonist, nimodipine (250 nM), had no effect on GnRH responsiveness. Furthermore, treatment of alpha T3-1 cells with 100 nM GnRH stimulated phosphorylation of both p42 and p44 forms of extracellular signal-regulated kinase (ERK), which was completely blocked with 60 microM PD98059. We suggest that GnRH regulation of the GnRHR gene is partially mediated by an ERK-dependent activation of a canonical AP-1 site located in the proximal promoter of the GnRHR gene.