Reaction of a redox-active ligand complex of nickel with dioxygen probes ligand-radical character.

Bis(imino)pyridine complex [Ni{2,6-(ArN=CMe)(2)C(5)H(3)N}Cl] (where Ar = 2,6-(i)Pr(2)C(6)H(3)) was synthesized by reduction of the corresponding dichloride complex and characterized as a ligand-radical complex of Ni(II). Reaction of this complex with O(2) caused intraligand C-C bond cleavage to afford the Ni complex of the new iminoethylpyridylcarboxamidato ligand, which also was isolated as the corresponding carboxamide, 6-(ArN=CMe)C(5)H(3)N-2-C(O)NHAr. This reaction serves as an example of small-molecule activation effected directly at the redox-active bis(imino)pyridine ligand without an overall oxidation state change at the Ni center.

Oxidase activity of a flavin-dependent thymidylate synthase.

Flavin-dependent thymidylate synthases (FDTS) catalyze the production of dTMP from dUMP and N(5),N(10)-methylene-5,6,7,8-tetrahydrofolate (CH(2)H(4)folate). In contrast to human and other classical thymidylate synthases, the activity of FDTS depends on a FAD coenzyme, and its catalytic mechanism is very different. Several human pathogens rely on this recently discovered enzyme, making it an attractive target for novel antibiotics. Like many other flavoenzymes, FDTS can function as an oxidase, which catalyzes the reduction of O(2) to H(2)O(2), using reduced NADPH or other reducing agents. In this study, we exploit the oxidase activity of FDTS from Thermatoga maritima to probe the binding and release features of the substrates and products during its synthase activity. Results from steady-state and single-turnover experiments suggest a sequential kinetic mechanism of substrate binding during FDTS oxidase activity. CH(2)H(4)folate competitively inhibits the oxidase activity, which indicates that CH(2)H(4)folate and O(2) compete for the same reduced and dUMP-activated enzymatic complex (FDTS-FADH(2)-NADP(+)-dUMP). These studies imply that the binding of CH(2)H(4)folate precedes NADP(+) release during FDTS activity. The inhibition constant of CH(2)H(4)folate towards the oxidase activity was determined to be rather small (2 microm), which indicates a tight binding of CH(2)H(4)folate to the FDTS-FADH(2)-NADP(+)-dUMP complex.

As epidermal stem cells age they do not substantially change their characteristics.

In this study, we ask the basic question: do stem cells age? We demonstrated that epidermal stem cells isolated from neonatal mice had the capacity to form multiple cell lineages during development. Here we demonstrate the cell lineages are clonal, and that epidermal stem cells isolated from the footpad epithelium of old mice have similar capabilities. Using Hoechst dye exclusion and cell size, we isolated viable homogenous populations of epidermal stem and transit-amplifying (TA) cells from GFP-transgenic mice, and injected these cells into 3.5-d blastocysts. Only the stem-injected blastocysts produced mice with GFP+ cells in their tissues. Furthermore, aged and young stem cells showed similar gene and protein expression profiles that showed some differences from the TA cell profiles. These data suggest that there may be a fundamental difference between somatic stem and TA cells, and that an epidermal stem cell placed in a developmental environment can alter its fate determination no matter what its age.

Effect of the delta6-desaturase inhibitor SC-26196 on PUFA metabolism in human cells.

The objective of this study was to determine the effect of 2,2-diphenyl-5-(4-[[(1 E)-pyridin-3-yl-methylidene]amino]piperazin-1-yl)pentanenitrile (SC-26196), a delta6-desaturase inhibitor, on PUFA metabolism in human cells. SC-26196 inhibited the desaturation of 2 microM [1-14C] 18:2n-6 by 87-95% in cultured human skin fibroblasts, coronary artery smooth muscle cells, and astrocytes. By contrast, SC-26196 did not affect the conversion of [1-14C]20:3n-6 to 20:4 in the fibroblasts, demonstrating that it is selective for delta6-desaturase. The IC50 values for inhibition of the desaturation of 2 microM [1-14C] 18:3n-3 and [3-14C]24:5n-3 in the fibroblasts, 0.2-0.4 microM, were similar to those for the inhibition of [1-14C 18:2n-6 desaturation, and the rates of recovery of [1-14C]18:2n-6 and [3-14C]24:5n-3 desaturation after removal of SC-26196 from the culture medium also were similar. SC-26196 reduced the conversion of [3-14C]22:5n-3 and [3-14C]24:5n-3 to DHA by 75 and 84%, respectively, but it had no effect on the retroconversion of [3-14C]24:6n-3 to DHA. These results demonstrate that SC-26196 effectively inhibits the desaturation of 18- and 24-carbon PUFA and, therefore, decreases the synthesis of arachidonic acid, EPA, and DHA in human cells. Furthermore, they provide additional evidence that the conversion of 22:5n-3 to DHA involves delta6-desaturation.