Inhibitory Effects of Quercetin and Its Human and Microbial Metabolites on Xanthine Oxidase Enzyme.

Quercetin is an abundant flavonoid in nature and is used in several dietary supplements. Although quercetin is extensively metabolized by human enzymes and the colonic microflora, we have only few data regarding the pharmacokinetic interactions of its metabolites. Therefore, we investigated the interaction of human and microbial metabolites of quercetin with the xanthine oxidase enzyme. Inhibitory effects of five conjugates and 23 microbial metabolites were examined with 6-mercaptopurine and xanthine substrates (both at 5 µM), employing allopurinol as a positive control. Quercetin-3'-sulfate, isorhamnetin, tamarixetin, and pyrogallol proved to be strong inhibitors of xanthine oxidase. Sulfate and methyl conjugates were similarly strong inhibitors of both 6-mercaptopurine and xanthine oxidations (IC50 = 0.2-0.7 µM); however, pyrogallol inhibited xanthine oxidation (IC50 = 1.8 µM) with higher potency vs. 6-MP oxidation (IC50 = 10.1 µM). Sulfate and methyl conjugates were approximately ten-fold stronger inhibitors (IC50 = 0.2-0.6 µM) of 6-mercaptopurine oxidation than allopurinol (IC50 = 7.0 µM), and induced more potent inhibition compared to quercetin (IC50 = 1.4 µM). These observations highlight that some quercetin metabolites can exert similar or even a stronger inhibitory effect on xanthine oxidase than the parent compound, which may lead to the development of quercetin-drug interactions (e.g., with 6-mercaptopurin or azathioprine).

Interaction of Chrysin and Its Main Conjugated Metabolites Chrysin-7-Sulfate and Chrysin-7-Glucuronide with Serum Albumin.

Chrysin (5,7-dihydroxyflavone) is a flavonoid aglycone, which is found in nature and in several dietary supplements. During the biotransformation of chrysin, its conjugated metabolites chrysin-7-sulfate (C7S) and chrysin-7-glucuronide (C7G) are formed. Despite the fact that these conjugates appear in the circulation at much higher concentrations than chrysin, their interactions with serum albumin have not been reported. In this study, the complex formation of chrysin, C7S, and C7G with human (HSA) and bovine (BSA) serum albumins was investigated employing fluorescence spectroscopic, ultrafiltration, and modeling studies. Our major observations/conclusions are as follows: (1) Compared to chrysin, C7S binds with a threefold higher affinity to HSA, while C7G binds with a threefold lower affinity; (2) the albumin-binding of chrysin, C7S, and C7G did not show any large species differences regarding HSA and BSA; (3) tested flavonoids likely occupy Sudlow's Site I in HSA; (4) C7S causes significant displacement of Sudlow's Site I ligands, exerting an even stronger displacing ability than the parent compound chrysin. Considering the above-listed observations, the high intake of chrysin (e.g., through the consumption of dietary supplements with high chrysin contents) may interfere with the albumin-binding of several drugs, mainly due to the strong interaction of C7S with HSA.

Design of a colicin E7 based chimeric zinc-finger nuclease.

Colicin E7 is a natural bacterial toxin. Its nuclease domain (NColE7) enters the target cell and kills it by digesting the nucleic acids. The HNH-motif as the catalytic centre of NColE7 at the C-terminus requires the positively charged N-terminal loop for the nuclease activity-offering opportunities for allosteric control in a NColE7-based artificial nuclease. Accordingly, four novel zinc finger nucleases were designed by computational methods exploiting the special structural features of NColE7. The constructed models were subjected to MD simulations. The comparison of structural stability and functional aspects showed that these models may function as safely controlled artificial nucleases. This study was complemented by random mutagenesis experiments identifying potentially important residues for NColE7 function outside the catalytic region.

Analysis of the influence of simulation parameters on biomolecule-linked water networks.

Advancement of computational molecular dynamics allows rapid calculation of large biomolecular systems in their water surroundings. New approaches of prediction of hydration networks of biomolecular surfaces and complex interfaces are also based on molecular dynamics (MD). Calculations with explicit solvent models can trace thousands of water molecules individually on a real time scale, yielding information on their mobility, and predicting their networking with biomolecular solutes and other water partners. Here, we investigate the effect of key parameters of molecular dynamics simulations on the quality of such predictions. Accordingly, systematic scans on temperature, pressure, force field, explicit water model and thermodynamic ensemble are performed. Explanations of optimal parameter values are provided using structural examples and analyses of the corresponding hydration networks.

Serum galectin-9 as a noninvasive biomarker for the detection of endometriosis and pelvic pain or infertility-related gynecologic disorders.

OBJECTIVE: To investigate the usefulness of soluble galectin-9 (Gal-9) in the noninvasive laboratory diagnosis of endometriosis and various gynecologic disorders. DESIGN: Prospective case-control study. SETTING: University medical centers. PATIENT(S): A total of 135 women of reproductive age were involved in the study, 77 endometriosis patients, 28 gynecologic controls, and 30 healthy women. INTERVENTION(S): Diagnostic laparoscopy and collection of tissue biopsies, peritoneal cells, and native peripheral blood from different case groups of gynecology patients and healthy women. MAIN OUTCOME MEASURE(S): The expression of mRNA and serum concentration of Gal-9. RESULT(S): Semiquantitative reverse transcription-polymerase chain reaction analysis and serum soluble Gal-9 ELISA were performed on three different cohorts of patients: those with endometriosis, those with benign gynecologic disorders, and healthy controls. Differences in the Gal-9 concentrations between the investigated groups and the stability of Gal-9 in the serum and diagnostic characteristics of Gal-9 ELISA were determined by statistical evaluation and receiver operating characteristic (ROC) curve analysis. Significantly elevated Gal-9 levels were found in both minimal-mild (I-II) and moderate-severe (III-IV) stages of endometriosis in comparison with healthy controls. At a cutoff of 132 pg/mL, ROC analysis revealed an excellent diagnostic value of Gal-9 ELISA in endometriosis (area under the curve = 0.973) with a sensitivity of 94% and specificity of 93.75%, indicating better diagnostic potential than that of other endometriosis biomarkers. Furthermore, various pelvic pain or infertility-associated benign gynecologic conditions were also associated with increased serum Gal-9 levels. CONCLUSION(S): Our results suggest that Gal-9 could be a promising noninvasive biomarker of endometriosis and a predictor of various infertility or pelvic pain-related gynecologic disorders.

Preorganization of the catalytic Zn(2+)-binding site in the HNH nuclease motif--A solution study.

The structure of the active site in a metalloenzyme can be a key determinant of its metal ion binding affinity and catalytic activity. In this study, the conformational features of the Zn(2+)-binding HNH motif were investigated by CD-spectroscopy in combination with isothermal microcalorimetric titrations. Various point mutations, including T454A, K458A and W464A, were introduced into the N-terminal loop of the nuclease domain of colicin E7 (NColE7). We show that the folding of the proteins was severely disturbed by the mutation of the tryptophan residue. This points to the importance of W464, being a part of the hydrophobic core located close to the HNH-motif. ITC demonstrated that the Zn(2+)-binding of the mutants including the W464 site became weak, and according to CD-spectroscopic measurements the addition of the metal ion itself cannot fully recover the functional structure. Titrations with Zn(2+)-ion in the presence and absence of the Im7 protein proved that the structural changes in the unfolded mutant included the HNH-motif itself. The metal-binding of the NColE7 mutants could be, however, fully rescued by the complexation of Im7. This suggests that the formation of a preorganized metal-binding site--existing in the wild-type enzyme but not in the W464 mutants--was induced by Im7. The low nuclease activity of all W464A mutants, however, implies that the interactions of this tryptophan residue are required for precise location of the catalytic residues, i.e. for stabilization of the fine-structure and of the tertiary structure. Our results contribute to the understanding of the metal binding site preorganization.