Modulation of the chemokines KC and MCP-1 by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in mice

Activation of the aryl hydrocarbon receptor (AhR) by TCDD may lead to the induction of proinflammatory cytokines in various cell types and organs such as liver leading to active chronic inflammation. Here we studied the expression of the chemokines keratinocyte chemoattractant (KC) and monocyte chemoattractant protein 1 (MCP-1) in different organs of mice after exposure to TCDD. TCDD exposure led to an early and clear induction of KC in liver and spleen on day 1 which was sustained over a period of 10 days. The level of MCP-1 mRNA was induced by TCDD on day 1 in spleen, lung, kidney, and liver, which was further increased at day 7. Increase of KC and MCP-1 at day 7 in liver, thymus, kidney, adipose, and heart was associated with elevated levels of the macrophage marker F4/80, indicating the infiltration of macrophages in these organs. Induction of KC requires a functional AhR since mice with a mutation in the AhR nuclear localization domain (AhR(nls)) were found to be resistant to TCDD-induced expression of KC. These results are the first showing the induction of the chemokines KC and MCP-1 in multiple organs of mice associated with an increase of the macrophage marker F4/80 indicating the involvement in TCDD's inflammatory response like infiltration of macrophages.     

Evolution of CHR-2 SINEs in cetartiodactyl genomes: possible evidence for the monophyletic origin of toothed whales

Short interspersed repetitive elements (SINEs) are a kind of retroposons dispersed among the eukaryotic genomes. Previously, we isolated and characterized a new SINE family, named CHR-2, members of which are distributed in the genomes of cetaceans, hippopotamuses, and ruminants. We analyzed systematically more than a hundred members of the CHR-2 SINEs, which were isolated from the genomes of cetaceans and cow, together with the additional data available in the DNA databases, and showed that these SINEs are divided into at least five distinct subfamilies that share diagnostic nucleotides and/or deletions. A hybridization analysis clearly demonstrated that, among these five subfamilies, two subfamilies, named CD and CDO, are specific to cetaceans and toothed whales, respectively. We reconstruct the evolutionary history of the CHR-2 SINEs during evolution of cetartiodactyl genomes. Received: 13 June 2001 / Accepted: 12 July 2001     

Metabolic flux analysis of the phenylpropanoid pathway in wound-healing potato tuber tissue using stable isotope-labeled tracer and LC-MS spectroscopy

The metabolic flux of two phenylpropanoid metabolites, N-p-coumaroyloctopamine (p-CO) and chlorogenic acid (CGA), in the wound-healing potato tuber tissue was quantitatively analyzed by a newly developed method based upon the tracer experiment using stable isotope-labeled compounds and LC-MS. Tuber disks were treated with aqueous solution of L-phenyl-d(5)-alanine, and the change in the ratio of stable isotope-labeled compound to non-labeled (isotope abundance) was monitored for p-CO and CGA in the tissue extract by LC-MS. The time-dependent change in the isotope abundance of each metabolite was fitted to an equation that was derived from the formation and conversion kinetics of each compound. Good correlations were obtained between the observed and calculated isotope abundances for both p-CO and CGA. The rates of p-CO formation and conversion (i.e. fluxes) were 1.15 and 0.96 nmol (g FW)(-1) h(-1), respectively, and for CGA, the rates 4.63 and 0.42 nmol (g FW)(-1) h(-1), respectively. This analysis enabled a direct comparison of the biosynthetic activity between these two compounds.     

C17,20-lyase inhibitors I. Structure-based de novo design and SAR study of C17,20-lyase inhibitors

Novel nonsteroidal C(17,20)-lyase inhibitors were synthesized using de novo design based on its substrate, 17 alpha-hydroxypregnenolone, and several compounds exhibited potent C(17,20)-lyase inhibition. However, in vivo activities were found to be short-lasting, and in order to improve the duration of action, a series of benzothiophene derivatives were evaluated. As a result, compounds 9h, (S)-9i, and 9k with nanomolar enzyme inhibition (IC(50)=4-9 nM) and 9e (IC(50)=27 nM) were identified to have powerful in vivo efficacy with extended duration of action. The key structural determinants for the in vivo efficacy were demonstrated to be the 5-fluoro group on the benzothiophene ring and the 4-imidazolyl moiety. Superimposition of 9k and 17 alpha-hydroxypregnenolone demonstrated their structural similarity and enabled rationalization of the pharmacological results. In addition, selected compounds were also identified to be potent inhibitors of human enzyme with IC(50) values of 20-30 nM.     

Substrate Recognition by the Multifunctional Cytochrome P450 MycG in Mycinamicin Hydroxylation and Epoxidation Reactions

The majority of characterized cytochrome P450 enzymes in actinomycete secondary metabolic pathways are strictly substrate-, regio-, and stereo-specific. Examples of multifunctional biosynthetic cytochromes P450 with broader substrate and regio-specificity are growing in number and are of particular interest for biosynthetic and chemoenzymatic applications. MycG is among the first P450 monooxygenases characterized that catalyzes both hydroxylation and epoxidation reactions in the final biosynthetic steps, leading to oxidative tailoring of the 16-membered ring macrolide antibiotic mycinamicin II in the actinomycete Micromonospora griseorubida. The ordering of steps to complete the biosynthetic process involves a complex substrate recognition pattern by the enzyme and interplay between three tailoring modifications as follows: glycosylation, methylation, and oxidation. To understand the catalytic properties of MycG, we structurally characterized the ligand-free enzyme and its complexes with three native metabolites. These include substrates mycinamicin IV and V and their biosynthetic precursor mycinamicin III, which carries the monomethoxy sugar javose instead of the dimethoxylated sugar mycinose. The two methoxy groups of mycinose serve as sensors that mediate initial recognition to discriminate between closely related substrates in the post-polyketide oxidative tailoring of mycinamicin metabolites. Because x-ray structures alone did not explain the mechanisms of macrolide hydroxylation and epoxidation, paramagnetic NMR relaxation measurements were conducted. Molecular modeling based on these data indicates that in solution substrate may penetrate the active site sufficiently to place the abstracted hydrogen atom of mycinamicin IV within 6 Å of the heme iron and ∼4 Å of the oxygen of iron-ligated water.     

Chloroplast outer envelope protein CHUP1 is essential for chloroplast anchorage to the plasma membrane and chloroplast movement

Chloroplasts change their intracellular distribution in response to light intensity. Previously, we isolated the chloroplast unusual positioning1 (chup1) mutant of Arabidopsis (Arabidopsis thaliana). This mutant is defective in normal chloroplast relocation movement and shows aggregation of chloroplasts at the bottom of palisade mesophyll cells. The isolated gene encodes a protein with an actin-binding motif. Here, we used biochemical analyses to determine the subcellular localization of full-length CHUP1 on the chloroplast outer envelope. A CHUP1-green fluorescent protein (GFP) fusion, which was detected at the outermost part of mesophyll cell chloroplasts, complemented the chup1 phenotype, but GFP-CHUP1, which was localized mainly in the cytosol, did not. Overexpression of the N-terminal hydrophobic region (NtHR) of CHUP1 fused with GFP (NtHR-GFP) induced a chup1-like phenotype, indicating a dominant-negative effect on chloroplast relocation movement. A similar pattern was found in chloroplast OUTER ENVELOPE PROTEIN7 (OEP7)-GFP transformants, and a protein containing OEP7 in place of NtHR complemented the mutant phenotype. Physiological analyses of transgenic Arabidopsis plants expressing truncated CHUP1 in a chup1 mutant background and cytoskeletal inhibitor experiments showed that the coiled-coil region of CHUP1 anchors chloroplasts firmly on the plasma membrane, consistent with the localization of coiled-coil GFP on the plasma membrane. Thus, CHUP1 localization on chloroplasts, with the N terminus inserted into the chloroplast outer envelope and the C terminus facing the cytosol, is essential for CHUP1 function, and the coiled-coil region of CHUP1 prevents chloroplast aggregation and participates in chloroplast relocation movement.     

The levels of main urinary metabolite of prostaglandin F1α and F2α in human subjects measured by radioimmunoassay

Radioimmunoassay technique for measuring 5α,7α-dihydroxy-11-keto-tetranorprosta-1,16-dioic acid, the main urinary metabolite of PGF₁α and PGF₂α (PGF₂α-MUM), was further improved.It was postulated based on some experimental data that the PGF₂α-MUM exists in the urine mostly as dioic acid form, not as δ-lactone formThe daily excretion of PGF₂α-MUM in men ranged from 14.43 μg to 36.14 μg and in women from 5.21 μg to 14.25 μg.