High-affinity inhibitors of human NAD-dependent 15-hydroxyprostaglandin dehydrogenase: mechanisms of inhibition and structure-activity relationships

Background

15-hydroxyprostaglandin dehydrogenase (15-PGDH, EC 1.1.1.141) is the key enzyme for the inactivation of prostaglandins, regulating processes such as inflammation or proliferation. The anabolic pathways of prostaglandins, especially with respect to regulation of the cyclooxygenase (COX) enzymes have been studied in detail; however, little is known about downstream events including functional interaction of prostaglandin-processing and -metabolizing enzymes. High-affinity probes for 15-PGDH will, therefore, represent important tools for further studies.

Principal Findings

To identify novel high-affinity inhibitors of 15-PGDH we performed a quantitative high-throughput screen (qHTS) by testing >160 thousand compounds in a concentration-response format and identified compounds that act as noncompetitive inhibitors as well as a competitive inhibitor, with nanomolar affinity. Both types of inhibitors caused strong thermal stabilization of the enzyme, with cofactor dependencies correlating with their mechanism of action. We solved the structure of human 15-PGDH and explored the binding modes of the inhibitors to the enzyme in silico. We found binding modes that are consistent with the observed mechanisms of action.

Conclusions

Low cross-reactivity in screens of over 320 targets, including three other human dehydrogenases/reductases, suggest selectivity of the present inhibitors for 15-PGDH. The high potencies and different mechanisms of action of these chemotypes make them a useful set of complementary chemical probes for functional studies of prostaglandin-signaling pathways.

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Distribution and breakdown of labeled coenzyme Q10 in rat

Radioactive coenzyme Q(10) ([(3)H]CoQ) was synthesized in a way that the metabolites produced retained the radioactivity. Administration of the lipid to rats intraperitoneally resulted in an efficient uptake into the circulation, with high concentrations found in spleen, liver, and white blood cells; lower concentrations in adrenals, ovaries, thymus, and heart; and practically no uptake in kidney, muscle, and brain. In liver homogenate most [(3)H]CoQ appeared in the organelles, but it was also present in the cytosol and transport vesicles. Mitochondria, purified on a metrizamide gradient, had a very low concentration of [(3)H]CoQ, which was mainly present in the lysosomes. All organs that took up the labeled lipid also contained water-soluble metabolites. The majority of metabolites excreted through the kidney and appeared in the urine. Some metabolites were also present in the feces, which further contained nonmetabolized [(3)H]CoQ, excreted through the bile. The major metabolites were purified from the urine, and the mass spectrometric fragmentation showed that these compounds, containing the ring with a short side chain, are phosphorylated. Thus, the results demonstrate that CoQ is metabolized in all tissues, the metabolites are phosphorylated in the cells, transported in the blood to the kidney, and excreted into the urine.     

Mechanisms ensuring optimal conditions of implantation and embryo development in the pig

The paper summarizes results of a series of studies concerning luteolysis and early pregnancy in pigs. The involvement of the oxytocin (OT)/OT receptor system in the mechanism of corpus luteum (CL) protection during early pregnancy as well as the implication of luteinizing hormone (LH) in the endometrial prostaglandin (PG) release and synthesis are described. In addition, the role of leptin in the regulation of ovarian steroidogenesis and the expression of leptin and its receptor (OB-Rb) genes in hypothalamus, pituitary and reproductive tissues are reported. Moreover, a strong emphasis was placed on the mechanism of PGE2 participation in the local endocrine regulations of reproductive processes occurring in the utero-ovarian area as well as on the vascular endothelial growth factor (VEGF) ligand-receptor system in the ovary and uterus.     

Serological and structural features of Hafnia alvei lipopolysaccharides containing d-3-hydroxybutyric acid

Abstract The serological heterogeneity of Hafnia alvei lipopolysaccharides from strains ATCC 13337, 1187, 1221, 114/60, 1211 and 1216, that contain d -3-hydroxybutyric acid, was analyzed by rocket immunoelectrophoresis, immunoblotting and passive hemagglutination. The significance of d -3-hydroxybutyric acid component for their cross-reactivity has been discussed. The results obtained allowed us to place four H. alvei strains (ATCC 13337, 1187, 1221 and 114/60) in one serotype (A) and to consider two other strains (1211 and 1216) as separate serotypes (B and C, respectively).     

Proinflammatory interferon-gamma--inducing monokines (interleukin-12, interleukin-18, interleukin-15)--serum profile in patients with systemic lupus erythematosus

Concentrations of three interferon-gamma - inducing monokines, IL-12, IL-18 and IL-15, were investigated in the serum of 60 patients with systemic lupus erythematosus (SLE) and 20 healthy subjects. IL-12 and IL-18 were detectable in the serum of all patients with SLE and in all healthy controls. The level of IL-12 was significantly higher in SLE patients (median 264.9 pg/ml) than in the control group (median 163.6 pg/ml, p < 0.02). Similar differences were observed in the level of IL-18 (median values 602.2 pg/ml and 252.7 pg/ml, respectively, p < 0.001). Correlations between serum levels of IL-12 and IL-18 and SLE activity were not statistically significant (p > 0.05). We found a significant, positive correlation between IL-12 and IL-18 (rh? = 0.419, p < 0.001) in SLE patients. The level of IL-18 was higher in the SLE patients with antinuclear antibodies (ANA) (median 660.0 pg/ml) than in ANA-negative patients (median 326.5 pg/ml, p < 0.03), as well as in patients with immunoglobulin deposits at the dermal-epidermal junction (median 746.0 pg/ml and 444.0 pg/ml respectively, p < 0.04). The level of IL-12 was also higher in patients with skin immunoglobulin deposits (328.9 pg/ml) than those without this phenomenon (257.0 pg/ml, p < 0.05). The levels of both cytokines in the patients treated with immunosuppressive drugs and those patients not treated were similar. The serum levels of IL-15 were low and not significant both in SLE patients (median 2.9 pg/ml), and in healthy controls (median 1.6 pg/ml). In conclusion, serum levels of IL-12 and IL-18 are higher in SLE patients than in healthy controls which may indicate a role in the disease pathogenesis. However, they do not correlate with disease activity and seem to be unresponsive to immunosuppressive treatment.     

The CysB motif of Rev3p involved in the formation of the four‐subunit DNA polymerase ζ is required for defective‐replisome‐induced mutagenesis

Eukaryotic DNA replication is performed by high‐fidelity multi‐subunit replicative B‐family DNA polymerases (Pols) α, δ and ?. Those complexes are composed of catalytic and accessory subunits and organized in multicomplex machinery: the replisome. The fourth B‐family member, DNA polymerase zeta (Pol ζ), is responsible for a large portion of mutagenesis in eukaryotic cells. Two forms of Pol ζ have been identified, a hetero‐dimeric (Pol ζ₂) and a hetero‐tetrameric (Pol ζ₄) ones and recent data have demonstrated that Pol ζ₄ is responsible for damage‐induced mutagenesis. Here, using yeast Pol ζ mutant defective in the assembly of the Pol ζ four‐subunit form, we show in vivo that [4Fe‐4S] cluster in Pol ζ catalytic subunit (Rev3p) is also required for spontaneous (wild‐type cells) and defective‐replisome‐induced mutagenesis – DRIM (pol3‐Y708A, pol2‐1 or psf1‐100 cells), when cells are not treated with any external damaging agents.     

Use of impedance methods for testing bactericidal activity of antiseptics

Standards PN-EN 1040 and EN 12054 describe test methods and minimum requirements for bactericidal activities of antiseptics. However, standard procedures are time consuming and require 48 hours of incubation. New alternative technique based on impedimetric procedure provides possibilities to reduce this time to several hours. The Bactometer (BioMerieux, Vitek System, USA) is a fully automated impedance technology system used to microbial quantitation of products. Impedance measures microbial activity by electrical methods. The aim of the study was to adaptate the impedimetric method utilising Bactometer--system to microbiological activity control of chemical antiseptics. Eight different products were utilised throughout the study. The samples for classical and alternative method were prepared in the same way as described in standards. The method of choice was dilution-neutralization method. All procedures conducted in Bactometer were verified by plate count method. The high correlation was observed between results obtained by normative methods and impedimetric measurement. All tested products meet requirements. The procedure utilising the Bactometer, provides a rapid and accurate system for the determination of bacterial content. The results of validation carried out during this study indicate, possibility to use impedimetric method alternatively to traditional methods.