Yuri Belosludtsev

Post-receptor signal transduction and regulation of 14(R),15(S)-epoxyeicosatrienoic acid (14,15-EET) binding in U-937 cells

14(R),15(S)-epoxyeicosatrienoic acid (14,15-EET), a cytochrome P-450 monooxygenase (epoxygenase) metabolite of arachidonic acid has been reported to induce adhesion of a monocyte cell line (U-937) to cultured endothelial cells. In this study, we identified a population of specific, high affinity binding sites for 14(R),15(S)-EET in U-937 cell surface with Kd of 13.84 +/- 2.58 nM and Bmax of 3.54 +/- 0.28 pmol/10(6) cells. The specific binding of [3H]-14,15-EET on U-937 cells is more effectively displaced by 14(R),15(S)-EET than the 14(S),15(R)-isomer thus indicating stereospecificity. The binding was sensitive to various protease treatments suggesting the binding site is protein in nature. 14,15-EET binding in U937 cells is attenuated by cholera toxin (CT) and dibutyryl cAMP. Mean binding site density (Bmax) decreased 31.61% and 34.8% by the pretreatment with cholera toxin (200 micrograms/ml) and dibutyryl cAMP (300 nM), respectively, without affecting the dissociation constant. Under similar conditions, pertussis toxin (20-200 ng/ml) was less effective as compared to CT and dibutyryl cAMP. The down regulation of 14,15-EET binding caused by dibutyryl cAMP in U-937 cell was reversed by a specific protein kinase A (PKA) inhibitor, H-89, but not by the PKC inhibitor K252a. Thus, the results suggest that the specific binding site of 14,15-EET in U-937 cells is associated with a receptor that could be down regulated through an increase in intracellular cAMP and activation of a PKA signal transduction mechanism. We propose that the signal transduction mechanism of 14,15-EET begins with the binding of the receptor, which leads to the increase of intracellular cAMP levels and the activation of PKA, and finally with the down regulation of 14,15-EET receptor binding.

16(R)-hydroxy-5,8,11,14-eicosatetraenoic acid, a new arachidonate metabolite in human polymorphonuclear leukocytes

Intact human polymorphonuclear leukocytes (PMNL) incubated with substimulatory amounts of arachidonic acid in the absence of a calcium ionophore formed four metabolites that were isolated by reverse-phase HPLC and characterized structurally by GC/MS. A major metabolite eluting as the most abundant peak of radioactivity lacked UV chromophores above 215 nm, and its formation was sensitive to 2-diethylaminoethyl-2,2-diphenylvalerate hydrochloride (SKF525A) but not 3-amino-1-[m(trifluoromethyl)phenyl]-2-pyrazoline (BW755C), suggesting that it was likely to be a product of cytochrome P450. The GC/MS analysis revealed the presence of two components: 20-hydroxy-5,8,11,14-eicosatetraenoic acid (20-HETE) and 16-hydroxy-5,8,11,14-eicosatetraenoic acid (16-HETE) in an approximate ratio of 4:1. The minor metabolites were identified as 15-HETE and 5-HETE. Although 20-HETE has been observed previously as a product of arachidonic acid metabolism in PMNL, the occurrence of 16-HETE was a novel finding. The stereochemistry of the hydroxyl group in PMNL-derived 16-HETE was established by analysis of 1-pentafluorobenzyl-16-naphthoyl derivatives on a chiral-phase chromatographic column and comparison with authentic synthetic stereoisomers. The PMNL-derived radioactive metabolite co-eluted with the synthetic 16(R)-HETE stereoisomer. Analysis of the total lipid extracts from intact PMNL followed by mild alkaline hydrolysis resulted in detectable amounts of 16-HETE (108+/-26 pg/10(8) cells) and 20-HETE (341+/-69 pg/10(8) cells), which suggested that these HETEs were formed from endogenous arachidonic acid and esterified within PMNL lipids. Thus, in contrast to calcium ionophore-stimulated neutrophils that generate large amounts of 5-lipoxygenase products, the intact PMNL generate 20-HETE and 16(R)-HETE via a cytochrome P450 omega- and omega-4 oxygenase(s).

C-Glycosides: Pd/Cu co-catalyzed thiocarboxylation of stannyl glucopyranosides

Abstract Stereospecific Pd/Cu co-catalyzed cross-couplings of tributylstannyl glucopyranosides with thiono- and thiolchloroformates afford good to excellent yields of C-glucosyl thiocarboxylates, versatile precursors for more complex C-glucoside homologs.

Eicosanoid biosynthesis: Differential inhibition of cytochrome P450 epoxygenase and ω-hydroxylase

Abstract Biphenyl 4 and vinyldibromide 8 were prepared on a multigram scale and shown to be comparatively specific arachidonic acid epoxygenase and ω-hydroxylase inhibitors, respectively, in rat kidney microsomal fractions.

ACCELERATION OF NUCLEIC ACID HYBRIDIZATION ON DNA MICROARRAYS DRIVEN BY pH TUNABLE MODIFICATIONS

A series of peptides containing histidine residues were designed as potential hybridization rate enhancers within a polymeric matrix of DNA microarrays. The polymeric matrix modified with these peptides showed strong attraction to DNA molecules under conditions of induction. DNA probes on the peptide-modified sites rapidly hybridized to their complementary targets with single base pair mismatch discrimination.

Hepoxilins B3: Synthesis of all four stereoisomers and a glutathione adduct

Utilizing (−)-quinic acid as a differentiated bis-aldehyde chiron, both pairs of hepoxilin B3 enantiomers and a glutathione adduct were synthesized by regiospecific functionalization of an acyclic vic-diol.

Transparent Electronically Controlled DNA Chips1

A new electronically controlled DNA chip that consists of a glass substrate and indium tin oxide electrodes (ITO) has been developed. The glass substrate with ITO electrodes makes the chip transparent and enables detection of hybridization from below the surface of the chip via a CCD array which allows simplified detection optics and, possibly, improved sensitivity. ITO, however, is not inert and is susceptible to degradation or modification due to electrolysis, at the electrodes. A chip design that incorporates donut-shaped platinum electrodes with ITO in the center hole was made to demonstrate functionality of the new transparent chips hybridization and single base discrimination was demonstrated using a 20 nucleotide, perfectly matched duplex and single base mismatched duplexes with the mismatch located at different positions along the duplex. Single base discrimination was confirmed between the perfect match and all single base mismatched duplexes with the exception of that at the 3′ and 5′ ends. No noticeable degradation of the reactive ITO surface was seen during the tests indicating that the use of the transparent chips with ITO electrodes may be a feasible alternative to present silicon based chips with the advantage of simplified detection optics and possible improved sensitivity.