Pharmacokinetics of the glucuronide and sulfate conjugates of genistein and daidzein in men and women after consumption of a soy beverage.
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BACKGROUND: The soy isoflavones genistein and daidzein are found in blood and tissues as aglycones, glucuronides, and sulfates. Isoflavone conjugates may serve as sources of aglycones at specific target tissues and may have bioactivity. Yet, very little is known about the plasma pharmacokinetics of isoflavone conjugates after soy ingestion. OBJECTIVE: The objective of this study was to determine the plasma pharmacokinetics of glucuronide and sulfate conjugates of genistein and daidzein in humans after the consumption of a drink made with soy-protein isolate. DESIGN: Six men and 6 women ( +/- SD age: 40.8 +/- 3 y) consumed a soy-protein-isolate drink. The pharmacokinetics of isoflavone glucuronide and sulfate conjugates were studied with the use of beta-glucuronidase (EC 3.2.1.31) and sulfatase (EC 3.1.6.1) digestion and liquid chromatography-mass spectrometry. RESULTS: Glucuronides of genistein and daidzein made up a significantly lower percentage (P < 0.05) of the total isoflavone concentration in plasma (48% and 33%, respectively) than in urine. The percentages of sulfates of genistein and daidzein in plasma (8% and 26%, respectively) were 2- to 6-fold those in urine (P < 0.05). Approximately 30% of the total genistein or daidzein was comprised of mixed conjugates (one glucuronide and one sulfate). For daidzein sulfate, genistein sulfate, daidzein glucuronide, and genistein glucuronide, the time to peak (t(max)) was 4.5, 4.5, 4.5, and 6.0 h, respectively, and the apparent half-life (t(1/2 lambdaz)) was 3.1, 5.7, 3.2, and 8.4 h, respectively. CONCLUSIONS: These data suggest that there are significant differences in the pharmacokinetics of sulfate and glucuronide conjugates of isoflavones. This may have important implications for the meal frequency and maintenance of target tissue bioactivity required to elicit potential health benefits. (+info)
Rapid colorimetric quantification of lipo-chitooligosaccharides from Mesorhizobium loti and Sinorhizobium meliloti.
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Nod factors are lipids with a chitinlike headgroup produced by gram-negative Rhizobium bacteria. These lipo-chitooligosaccharides (LCOs) are essential signaling molecules for accomplishing symbiosis between the bacteria and roots of legume plants. Despite their important role in the Rhizobium-legume interaction, no fast and sensitive Nod factor quantification methods exist. Here, we report two different quantification methods. The first is based on the enzymatic hydrolysis of Nod factors to release N-acetylglucosamine (GlcNAc), which can subsequently be quantified. It is shown that the degrading enzyme, glusulase, releases exactly two GlcNAc units per pentameric nodulation factor from Mesorhizobium loti factor, allowing quantification of LCOs from Mesorhizobium loti. The second method is based on a specific type of Nod factors that are sulfated on the reducing GlcNAc, allowing quantification analogous to the quantification of sulfolipids. Here, a two-phase extraction method is used in the presence of methylene blue, which specifically forms an ion pair with sulfated lipids. The blue ion pair partitions into the organic phase, after which the methylene blue signal can be quantified. To enable Nod factor quantification with this method, the organic phase was modified and the partitioning was evaluated using fluorescent and radiolabeled sulfated Nod factors. It is shown that sulfated LCOs can be quantified with this method, using sodium dodecyl sulfate for calibration. Both methods allow Nod factor quantification in parallel enabling a fast and easy detection of nanomole quantities of Nod factors. Accurate Nod factor quantification will be crucial for characterization and cross-comparison of the affinity for Nod factors of newly identified Nod factor binding proteins or putative Nod factor receptors. (+info)
Cloning and characterization of two extracellular heparin-degrading endosulfatases in mice and humans.
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Here we report the cloning of a full-length cDNA encoding the human ortholog (HSulf-1) of the developmentally regulated putative sulfatases QSulf-1 (Dhoot, G. K., Gustafsson, M. K., Ai, X., Sun, W., Standiford, D. M., and Emerson, C. P., Jr. (2001) Science 293, 1663-1666) and RSulfFP1 (Ohto, T., Uchida, H., Yamazaki, H., Keino-Masu, K., Matsui, A., and Masu, M. (2002) Genes Cells 7, 173-185) as well as a cDNA encoding a closely related protein, designated HSulf-2. We have also obtained cDNAs for the mouse orthologs of both Sulfs. We demonstrate that the proteins encoded by both classes of cDNAs are endoproteolytically processed in the secretory pathway and are released into conditioned medium of transfected CHO cells. We demonstrate that the mammalian Sulfs exhibit arylsulfatase activity with a pH optimum in the neutral range; moreover, they can remove sulfate from the C-6 position of glucosamine within specific subregions of intact heparin. Taken together, our results establish that the mammalian Sulfs are extracellular endosulfatases with strong potential for modulating the interactions of heparan sulfate proteoglycans in the extracellular microenvironment. (+info)
Metabolism of bisphenol a in primary cultured hepatocytes from mice, rats, and humans.
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Studies have shown that in the rat, bisphenol A (BPA) is metabolized and eliminated primarily as a monoglucuronide, a metabolite without estrogenic activity. The purpose of this study was to determine the extent of monoglucuronide formation in monolayers of hepatocytes from rats, mice, and humans. Noncytotoxic concentrations of BPA (10, 20, and 35 microM; 1.0 microCi), as assessed by lactate dehydrogenase leakage, were incubated with isolated hepatocytes for 0-6 h. Media were collected and analyzed for metabolites by radiochemical high performance liquid chromatography and liquid chromatography-tandem mass spectrometry. The metabolites identified include a monoglucuronide (major metabolite), a sulfate conjugate, and a glucuronide/sulfate diconjugate (minor metabolites). In hepatocytes of male Fischer-344 rats, the predominate metabolite was the diconjugate (glucuronide/sulfate). Under these conditions, the extent of metabolism by 3 h was similar in all species tested because all BPA was converted to conjugates by 3 h. Initial rates of metabolism in hepatocytes followed the order of mice > rats > humans. However, when extrapolated to the whole liver (i.e., cells per liver), the hepatic capacity for BPA glucuronidation is predicted to be humans > rats > mice. This research was supported in part by The Society of Plastics Industry Inc., and Southwest Environmental Health Science Center (ES 06694). (+info)
Heparan sulfate fine structure and specificity of proteoglycan functions.
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Heparan sulfate chains have markedly heterogeneous structures in which distinct patterns of sulfation determine the binding specificity for ligand proteins. These "fine structures" of heparan sulfate are mainly produced by the regulated introduction of sulfate groups at the N-, 2-O-, 6-O-, and 3-O-positions of the sugar chain. Recent biochemical, histochemical, and genetic studies have demonstrated that different fine structures mediate distinct molecular recognition events to regulate a variety of cellular functions. In this review, we focus on the molecular basis of growth factor control by the sulfation status of heparan sulfate. (+info)
Posttranslational modification of serine to formylglycine in bacterial sulfatases. Recognition of the modification motif by the iron-sulfur protein AtsB.
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Calpha-formylglycine is the catalytic residue of sulfatases. Formylglycine is generated by posttranslational modification of a cysteine (pro- and eukaryotes) or serine (prokaryotes) located in a conserved (C/S)XPXR motif. The modifying enzymes are unknown. AtsB, an iron-sulfur protein, is strictly required for modification of Ser(72) in the periplasmic sulfatase AtsA of Klebsiella pneumoniae. Here we show (i) that AtsB is a cytosolic protein acting on newly synthesized serine-type sulfatases, (ii) that AtsB-mediated FGly formation is dependent on AtsA's signal peptide, and (iii) that the cytosolic cysteine-type sulfatase of Pseudomonas aeruginosa can be converted into a substrate of AtsB if the cysteine is substituted by serine and a signal peptide is added. Thus, formylglycine formation in serine-type sulfatases depends both on AtsB and on the presence of a signal peptide, and AtsB can act on sulfatases of other species. AtsB physically interacts with AtsA in a Ser(72)-dependent manner, as shown in yeast two-hybrid and GST pull-down experiments. This strongly suggests that AtsB is the serine-modifying enzyme and that AtsB relies on a cytosolic function of the sulfatase's signal peptide. (+info)
Mucopolysaccharides associated with nuclei of cultured mammalian cells.
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Mucopolysaccharides have been isolated, fractionated, and characterized from the nuclei of cultured B16 mouse melanoma cells grown in the presence of (3-H)-glucosamine and (35-S)sulfate. Digestion of the nuclei with DNase followed by Pronase gave a mixture of complex carbohydrates from which the mucopolysaccharides were isolated by precipitation with cetylpyridinium chloride. After fractionation by differential salt extraction and chromatography on controlled pore glass bead columns, the components were identified by chemical and enzymatic methods. The major polysaccharide components were a family of high-molecular-weight chondroitin sulfates with different degrees of sulfation; a minor component has been characterized as heparan sulfate. (+info)
Separation of a proteoglycan fraction from Kurloff cells stimulating protein synthesis in macrophages.
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Proteoglycan from Kurloff cells, when present in the medium in low concentrations, increased the incorporation of (3-H)leucine into trichloracetic acid-precipitable material by macrophages from peritoneal exudates, in addition to inhibiting their migration from capillary tubes, as observed previously. After treatment with 0.5 M-NaOH, followed by dialysis or ultrafiltration, material with the distinctive u.v. and i.r. spectra of the whole proteoglycan appeared in the diffusate, and biological activity was lost from the proteoglycan which remained in the dialysis residue. The diffusible material absorbed near 260 nm and had i.r. bands at 805 cm-minus-1 and 1260 cm-minus-1, but did not display the i.r. bands characteristic of chondrotin 4-sulphate. It contained little sulphate, no hexosamine and less than 1% of the uronic acid present in the whole proteoglycan, and there were only trace amounts of amino acids, xylose and galactose. However, significant amounts of ribose and organic phosphate were present, each representing about 1% of the whole proteoglycan. After proteolysis and chondroitanase digestion of the proteoglycan, a fraction with absorbance at 260 mn was eluted from Dowex 1 with water which stimulated the incorporation of (3H)leucine by macrophages and inhibited their migration from capillary tubes. (+info)