Experimentally induced mastitis and metritis modulate soy bean derived isoflavone biotransformation in diary cows

The present study compared the changes in isoflavone (daidzein and genistein) and their metabolite (equol and para-ethyl-phenol) concentrations in the blood plasma of cows with induced mastitis and metritis after feeding with soy bean. Sixteen cows were divided into four groups: control for mastitis group, cows with induced mastitis group, control for metritis group, and cows with induced metritis group. All cows were fed a single dose of 2.5 kg of soy bean and then blood samples were taken from the jugular vein for 8 h at predetermined intervals. The concentrations of soy bean-derived isoflavones and their active metabolites were measured in the blood plasma on HPLC system. β-Glucuronidase activity in the blood plasma of cows was measured by fluorometric method. In the blood plasma of cows with induced mastitis and metritis, we found considerably higher concentrations and time-dependent increase in isoflavone metabolites (equol and para-ethyl-phenol) with reference to cyclic cows (P < 0.05). Moreover, we noticed significant decrease of genistein in the blood plasma of the cows with induced metritis compared with control cows (P < 0.05). In addition, in the blood plasma of the cows with induced metritis, we found an increase in β-glucuronidase activity compared with control cows (P < 0.05). In conclusion, health status of the females influenced the concentrations of isoflavone metabolites in the blood plasma of the cows. Experimentally induced mastitis and metritis increased isoflavone absorption, biotransformation and metabolism. Therefore, we suggest that cows with induced mastitis and metritis are more exposed to active isoflavone metabolite actions than healthy cows.     

Experimentally induced mastitis and metritis modulate soy bean derived isoflavone biotransformation in diary cows

The present study compared the changes in isoflavone (daidzein and genistein) and their metabolite (equol and para-ethyl-phenol) concentrations in the blood plasma of cows with induced mastitis and metritis after feeding with soy bean. Sixteen cows were divided into four groups: control for mastitis group, cows with induced mastitis group, control for metritis group, and cows with induced metritis group. All cows were fed a single dose of 2.5 kg of soy bean and then blood samples were taken from the jugular vein for 8 h at predetermined intervals. The concentrations of soy bean-derived isoflavones and their active metabolites were measured in the blood plasma on HPLC system. β-Glucuronidase activity in the blood plasma of cows was measured by fluorometric method. In the blood plasma of cows with induced mastitis and metritis, we found considerably higher concentrations and time-dependent increase in isoflavone metabolites (equol and para-ethyl-phenol) with reference to cyclic cows (P < 0.05). Moreover, we noticed significant decrease of genistein in the blood plasma of the cows with induced metritis compared with control cows (P < 0.05). In addition, in the blood plasma of the cows with induced metritis, we found an increase in β-glucuronidase activity compared with control cows (P < 0.05). In conclusion, health status of the females influenced the concentrations of isoflavone metabolites in the blood plasma of the cows. Experimentally induced mastitis and metritis increased isoflavone absorption, biotransformation and metabolism. Therefore, we suggest that cows with induced mastitis and metritis are more exposed to active isoflavone metabolite actions than healthy cows.     

Clearance of lysosomal hydrolases following intravenous infusion. The role of liver in the clearance of beta-glucuronidase and N-acetyl-beta-D-glucosaminidase.

Certain highly purified forms of rat lysosomal glycosidases, β-glucuronidase and N-acetyl-β-d-glucosaminidase, are rapidly cleared from the circulation following intravenous infusion. Several lines of evidence are presented which indicate that the primary site of enzyme uptake is the liver. Clearance of the two enzymes was unaffected by nephrectomy, whereas it was abolished by evisceration. Tissue distribution experiments with native and [¹²⁵I]β-glucuronidase indicate the liver as the major, if not exclusive, site of enzyme uptake. Experiments with the isolated perfused liver showed clearance of certain enzyme preparations but not others. Those enzymes cleared by the isolated perfused liver were likewise cleared in vivo. Liver fractionation studies following infusion of large doses of β-glucuronidase revealed a rapid, short-lived increase in microsomal β-glucuronidase and a slower but larger increase in lysosomal β-glucuronidase. The results indicate that β-glucuronidase, N-acetyl-β-d-glucosaminidase, and probably other glycosidases are rapidly incorporated into the lysosomal compartment of liver.     

New oxandrolone derivatives by biotransformation using Rhizopus stolonifer

Structural transformation of the steroidal lactone, oxandrolone (1), by suspended-cell cultures of the plant pathogen fungus Rhizopus stolonifer, resulted in the production of three new metabolites. These metabolites were identified as 11α-hydroxyoxandrolone (2), 6α-hydroxyoxandrolone (3) and 9α-hydroxyoxandrolone (4), by different spectroscopic methods and single-crystal X-ray diffraction analysis for metabolite 2. Compounds 1 and 3 showed a significant β-glucuronidase inhibitory activity.     

Beta-glucuronidase in the serum and hemolymph cells of Mercenaria mercenaria and Crassostrea virginica (Mollusca: Pelecypoda).

The optimal pH of β-glucuronidase in the serum and cells of the hemolymph of the quahaug clam, Mercenaria mercenaria, and the American oyster, Crassostrea virginica, has been determined to be 4.5 by employing 0.2 M phosphate, 0.2 M glycylglycine, and 0.2 M acetate as the buffers.By comparing the optimal pH of lysozyme from C. virginica (which is either 5.0 or 5.5, depending on the buffer employed) with that of β-glucuronidase, it is concluded that if both of these lysosomal hydrolytic enzymes are to be concurrently operative at maximal efficiency, the hemolymph pH should be maintained between 4.5 and 5.5.The exact physiologic role of β-glucuronidase in M. mercenaria and C. virginica is undetermined; however, since this enzyme can hydrolyze acid mucopolysaccharides, which are constituents of bacterial walls, it may play a role in internal defense by acting on susceptible invading bacteria. Furthermore, the β-glucuronidase in cells may be correlated with cell proliferation.     

Analytical limits of four β-glucuronidase and β-galactosidase-based commercial culture methods used to detect Escherichia coli and total coliforms

Colilert^® (Colilert), Readycult^® Coliforms 100 (Readycult), Chromocult^® Coliform agar ES (Chromocult), and MI agar (MI) are β-galactosidase and β-glucuronidase-based commercial culture methods used to assess water quality. Their analytical performance, in terms of their respective ability to detect different strains of Escherichia coli and total coliforms, had never been systematically compared with pure cultures. Here, their ability to detect β-glucuronidase production from E. coli isolates was evaluated by using 74 E. coli strains of different geographic origins and serotypes encountered in fecal and environmental settings. Their ability to detect β-galactosidase production was studied by testing the 74 E. coli strains as well as 33 reference and environmental non-E. coli total coliform strains. Chromocult, MI, Readycult, and Colilert detected β-glucuronidase production from respectively 79.9, 79.9, 81.1, and 51.4% of the 74 E. coli strains tested. These 4 methods detected β-galactosidase production from respectively 85.1, 73.8, 84.1, and 84.1% of the total coliform strains tested. The results of the present study suggest that Colilert is the weakest method tested to detect β-glucuronidase production and MI the weakest to detect β-galactosidase production. Furthermore, the high level of false-negative results for E. coli recognition obtained by all four methods suggests that they may not be appropriate for identification of presumptive E. coli strains.     

Alterations in rat liver microsomal and lysosomal β-glucuronidase by compounds which induce hepatic drug-metabolizing enzymes

Chlordane, dieldrin, piperonyl butoxide, and benzpyrene, which induce the hepatic microsomal mixed function oxidases and UDP glucuronyltransferases, decreased activity of smooth and rough endoplasmic reticulum β-glucuronidase. The reduction occurred when either p-nitrophenyl β-D-glucuronide or phenolphthalein mono-β-glucuronide was used as the substrate. Chlordane or dieldrin pretreatment of rats for 3 days resulted in a 2.5-fold reduction in endoplasmic reticulum activity while the reduction was less for piperonyl butoxide or benzpyrene. On the other hand, aminopyrine demethylase and UDP glucuronyltransferase were increased 2-fold by chlordane or dieldrin pretreatments. Decreases in microsomal β-glucuronidase activity might be directly or indirectly involved in the induction process since decreases in β-glucuronidase activity are quantitatively similar to increases in activity of the drug-metabolizing enzymes. Lysosomal β-glucuronidase also decreased following pretreatment of rats with inducing agents, but the reduction was less than that observed in the endoplasmic reticulum fractions. Analysis of pH optima, temperature optima, K_m values, heat denaturation data, and effects of Triton X-100 on activities of various liver fractions suggests that β-glucuronidase from the endoplasmic reticulum and lysosomes have similar properties.     

Human placental N-acetyl-beta-D-hexosaminidase isozymes. Activity toward native hyaluronic acid.

The activity of purified human hexosaminidases A and B toward hyaluronic acid (HA) isolated from cultured human skin fibroblasts was investigated. The cleavage of N-acetylglucosaminyl residues to monosaccharide N-acetylglucosamines by hexosaminidase isozymes was determined in the presence and absence of purified human β-glucuronidase. The pH optima of this reaction, with and without β-glucuronidase, were 4.5 for hexosaminidase A and 4.0 for hexosaminidase B. The hydrolysis of HA by both hexosaminidase isozymes proceeds linearily for at least 18 h in the presence of β-glucuronidase. Concentrations of 0.5–5 units of either isozyme showed a linear relationship with rate of hydrolysis. Without β-glucuronidase, hexosaminidase only cleaved the terminal N-acetylglucosamine residue. However, under optimal conditions, with β-glucuronidase, the hydrolytic activity of hexosaminidase B was about 30% as efficient as that of hexosaminidase A. Approximately 70% of the HA could be degraded by 5 units of hexosaminidase A in the presence of 0.5 unit of β-glucuronidase, as opposed to 25% degraded by hexosaminidase B. These results probably reflect intrinsic differences in the activities of the two isozymes. Since the substrate (HA) did not inhibit the hydrolysis of a synthetic substrate (4-methylumbelliferyl-β-glucosaminide) by hexosaminidase B, the linear kinetics of HA hydrolysis implies no product inhibition. These data indicate that native HA can be hydrolyzed by the combined activities of β-glucuronidase with hexosaminidase A or hexoaminidase B.     

Progressive induction of β-glucuronidase in individual kidney epithelial cells

The magnitude and kinetics of β-glucuronidase induction in mouse kidney are determined by a cis-acting regulatory gene, Gus-r, that is closely linked to the enzyme structural gene. The accumulation of β-glucuronidase mRNA during induction is much slower than the turnover time of the mRNA, suggesting progressive acquisition of mRNA synthesizing capacity during induction. Counts of the numbers of induced cells present at various times of induction in strains carrying three different alleles of Gus-r show that all potentially responsive cells respond immediately. The level of induction is progressive in individual cells and does not involve continued recruitment of new cells into the induced population. It appears that during induction each chromosome becomes progressively more active in directing the synthesis of β-glucuronidase.     

Clearance of lysosomal hydrolases following intravenous infusion. Kinetic and competition experiments with beta-glucuronidase and N-acetyl-beta-D-glucosaminidase.

Clearance experiments with highly purified lysosomal glycosidases, β-glucuronidase and N-acetyl-β-d-glucosaminidase, following intravenous infusion revealed widely varying clearance profiles which depended on the tissue source of the enzyme. Normal rat serum β-glucuronidase and epididymal N-acetyl-β-d-glucosaminidase were cleared slowly from the circulation when compared with rat preputial gland β-glucuronidase, liver lysosomal β-glucuronidase, and liver lysosomal N-acetyl-β-d-glucosaminidase, respectively, which were cleared rapidly. Experiments comparing the catalytic properties and molecular dimensions of the enzymes revealed no differences between rapid and slow clearance forms. Kinetic analysis using the rapid clearance forms of β-glucuronidase has allowed the resolution of at least two components, rapid and slow. Clearance of the rapid component is saturable and appears to reflect binding or uptake by a limited number of sites. By contrast, the clearance rate of the slow component increased linearly with respect to dose and may be due to nonspecific or low-affinity binding. Competition experiments with β-glucuronidase-free lysosomal extract and highly purified lysosomal enzymes, but not serum glycoproteins or colloidal silver, suggest that one lysosomal enzyme inhibits clearance of others and that a common mechanism may be involved in their binding.     

Aggregation of sponge cells: a novel mechanism of controlled intercerllular adhesion

From the marine sponge Geodia cydonium a series of macromolecules have been isolated and characterized which are involved in the control of aggregation and separation of sponge cells; these include aggregation factor, aggregation receptor, anti-aggregation receptor, β-glucuronidase, β-glucuronosyltransferase, β-galactosyltransferase, β-galactosidase and a lectin. These components might be linked in the following sequence: (a) activation of the aggregation receptor by its enzymic glucuronylaion; (b) adhesive recognition of the cells, mediated by the aggregation factor and the glucuronylated aggregation receptor; (c) inactivation of the aggregation receptor by its deglucuronylation with the membrane-associated β-glucuronidase; (d) cell separation due either to the loss of the recognition site (glucuronic acid) of the aggregation receptor for the aggregation factor or to an inactivation of the aggregation factor by the anti-aggregation receptor. The activity of the anti-aggregation receptor is probably controlled by the Geodia lectin.     

Measuring Faecal Epi-Androsterone as an Indicator of Gonadal Activity in Spotted Hyenas (Crocuta crocuta)

Enzyme immunoassays (EIA) that measure faecal testosterone metabolites (fTM) are useful tools to monitor gonadal activity. The aim of this study was to validate an “in-house” epiandrosterone EIA to monitor fTM in spotted hyenas. FTM were characterised in a male and a female hyena that each received an injection of ³H-testosterone. High-performance liquid chromatography (HPLC) analyses revealed a cluster of highly polar enzyme-hydrolysable hormone metabolite conjugates. We performed hydrolysis using β-glucuronidase to deconjugate metabolites and improve sensitivity of the assay. Because β-glucuronidase from Helix pomatia has been reported to bias testosterone measurements in some species, we compared the enzymatic activity of the commonly used β-glucuronidase extracted from H. pomatia with the same enzyme from Escherichia coli. Our results showed that β-glucuronidases from both sources produced similar results from spotted hyena faeces. We therefore hydrolysed samples with H. pomatia enzymes. HPLC analyses also demonstrated that following hydrolysis the epiandrosterone EIA measured significant amounts of immunoreactive metabolites corresponding to radiolabelled metabolites in both sexes. Additionally, HPLC and GC-MS analyses confirmed the presence of epiandrosterone in faeces of spotted hyenas. The biological relevance of the epiandrosterone EIA was validated by demonstrating (1) a significant increase in fTM levels in response to a testosterone injection within 16 h, (2) no biological responsiveness to an adrenocorticotropic hormone (ACTH) injection and (3) significant differences in fTM levels between juvenile males and adult immigrant males in a free-ranging wild population. Our results clearly demonstrate that the epiandrosterone EIA is a reliable non-invasive method to monitor gonadal activity in spotted hyenas.     

Lipoate metabolism in Pseudomonas putida LP: thiolsulfinates of lipoate and bisnorlipoate.

Lipoate thiolsulfinate and two bisnorlipoate thiolsulfinates, as well as the previously identified products of β-oxidation (bisnorlipoate, tetranorlipoate, and β-hydroxybisnorlipoate), were isolated and identified as catabolites of [¹⁴C]lipoate from cultures of Pseudomonas putida LP, an organism capable of growth on lipoic acid as a sole source of carbon and sulfur. The newly identified metabolites were characterized by ion-exchange and paper chromatography and infrared, ultraviolet, and mass spectroscopies. Comparison of the isolated catabolites with synthetic standards implies that the lipoic thiolsulfinate isolated is the S-1 monoxide of 1,2-dithiolane-3-valeric acid; one bisnorlipoic thiolsulfinate isolated is the S-1 monoxide, the other apparently the S-2 monoxide. Metabolic studies with P. putida show that lipoate thiolsulfinate is taken up by this microorganism in an energy-dependent process, but less readily than lipoate; lipoate thiolsulfinate supports oxygen consumption in short-term experiments but does not support growth. These results are interpreted as meaning that the thiolsulfinates are “dead-end” metabolites, not intermediates in the sulfur metabolism of this organism. Lipoate thiolsulfinate is not detectably β-oxidized to bisnorlipoate thiolsulfinate under the usual culture conditions.     

Identification of new urinary metabolites of trimeprazine in rats by gas chromatography—mass spectrometry

The metabolites of trimeprazine were identified in urine of rats by gas chromatography—mass spectrometry. After the oral administration of trimeprazine, the urinary metabolites were extracted with diethyl ether before or after hydrolysis with β-glucuronidase. The identified metabolites were N-demethyltrimeprazine, 3-hydroxytrimeprazine, N-demethyl-3-hydroxytrimeprazine and trimeprazine sulphoxide.     

Isolation and preliminary characterization of new cytochrome c from autotrophic haloalkaliphilic sulfur-oxidizing bacterium Thioalkalivibrio nitratireducens

New small cytochrome c (TniCYT) was purified from haloalkaliphilic sulfur-oxidizing bacterium Thioalkalivibrio nitratireducens. The protein was analyzed by mass spectrometry as well as using visible, CD and EPR spectroscopy. It was found that TniCYT is a monomer with a molecular mass of 9461 Da which contains two hemes per molecule. The data of CD and EPR spectroscopy showed that two hemes possess different optical activity and are in distinct, high and low spin states. TniCYT was also demonstrated to have unusual characteristics in the visible spectrum, namely, the splitting of characteristic peaks was observed in α- and β-bands of the heme spectrum when the reduced form of cytochrome was analyzed. The α-band has two peaks with maximum at 548 and 556 nm whereas the β-band showes ones at 520 and 528 nm. According to the MALDI finger-print analysis, the new cytochrome has a unique amino acid sequence.     

Metabolism of 13-cis-retinoic acid: Identification of 13-cis-retinoyl-and 13-cis-4-oxoretinoyl-β-glucuronides in the bile of vitamin A-normal rats

The metabolism of 13-cis-[11-³H]retinoic acid has been examined in vitamin A-normal rats. Within 24 h after intravenous administration of the parent retinoid (15 μg/kg) to animals with biliary fistulas, 69 ± 9% of the dose was detected in the bile with 9 ± 6% being found in the urine. Analysis of the bile by reverse-phase high-pressure liquid chromatography demonstrated that the retinoic acid was being metabolized to several more polar compounds. A number of these compounds were sensitive to incubation with β-glucuronidase as evidenced by a change in their chromatographic behavior after treatment with the enzyme. Two of the metabolites have been identified as 13-cis-4-oxoretinoyl-β-glucuronide (8.1 ± 1.0% of the dose during the first 4 h after administration of the parent compound) and 13-cis-retinoyl-β-glucuronide (7.0 ± 4.4% of the dose). A comparison of the chromatographic profiles of bile from 13-cis- versus all-trans-retinoic acid-treated rats indicated a difference in their metabolism, with a greater proportion of the all-trans-retinoic acid being converted to compounds that eluted in the more polar regions of the column effluent.     

Chondroitin sulfate-degrading enzymes in human polymorphonuclear leukocytes: characteristics and evidence for concerted mechanism.

Human polymorphonuclear leukocyte-derived enzymes, β-glucuronidase, β-N-acetylgalactosaminidase, and aryl sulfatase were studied for their ability to degrade chondroitin sulfate. Evidence is presented which implies a concerted, synergistic mechanism of action for these enzymes in glycosaminoglycan degradation excluding the possibility of endoglycosidase activity. Conclusions are based upon results derived from pH optimum, inhibitor studies, kinetics, and partial purification of these enzymes. The data presented here demonstrate that the polymorphonuclear leukocytes contain all of the enzymes necessary for the solubilization and complete degradation of the chondroitin-4-sulfate of cartilage matrix.     

Gene sequencing and characterization of the light-harvesting complex 2 from thermophilic purple sulfur bacterium Thermochromatium tepidum

In this study, gene sequences coding for the light-harvesting (LH) 2 polypeptides from a thermophilic purple sulfur bacterium Thermochromatium tepidum are reported and characterization of the LH2 complex is described. Three sets of pucBA genes have been identified, and the gene products have been analyzed by electrophoresis and reversed-phase chromatography. The result shows that all of the genes are expressed but the distribution of the expression is not uniform. The gene products undergo post-translational modification, where two of the β-polypeptides appear to be N-terminally methylated. Absorption spectrum of the purified LH2 complex exhibits Q _y transitions at 800 and 854?nm in dodecyl β-maltopyranoside solution, and the circular dichroism spectrum shows a “molischianum”-like characteristic. No spectral change was observed for the LH2 when the bacterium was cultured under different conditions of light intensity. In lauryl dimethylamine N-oxide (LDAO) solution, significant changes in the absorption spectrum were observed. The B850 peak decreased and blue-shifted with increasing the LDAO concentration, whereas the B800 intensity increased without change in the peak position. The spectral changes can be partially or almost completely reversed by addition of metal ions, and the divalent cations seem to be more effective. The results indicate that ionic interactions may exist between LH2, detergent molecules and metal ions. Possible mechanisms involved in the detergent- and cation-induced spectral changes are discussed.     

Complete 1H, 15N and 13C resonance assignments of Bacillus cereus metallo-β-lactamase and its complex with the inhibitor R-thiomandelic acid

β-Lactamases inactivate β-lactam antibiotics by hydrolysis of their endocyclic β-lactam bond and are a major cause of antibiotic resistance in pathogenic bacteria. The zinc dependent metallo-β-lactamase enzymes are of particular concern since they are located on highly transmissible plasmids and have a broad spectrum of activity against almost all β-lactam antibiotics. We present here essentially complete (>96 %) backbone and sidechain sequence-specific NMR resonance assignments for the Bacillus cereus subclass B1 metallo-β-lactamase, BcII, and for its complex with R-thiomandelic acid, a broad spectrum inhibitor of metallo-β-lactamases. These assignments have been used as the basis for determination of the solution structures of the enzyme and its inhibitor complex and can also be used in a rapid screen for other metallo-β-lactamase inhibitors.