The Effects of Magnesium Sulphate on Integrated Pulmonary Index Scores and Propofol Consumption During Endobronchial Ultrasonography: A Retrospective Study.

Aim Our aim in this study was to investigate the effect of inhaled and intravenous (iv) magnesium (Mg) use on Integrated Pulmonary Index (IPI) score and propofol consumption in patients undergoing endobronchial ultrasonography (EBUS) procedure under sedoanalgesia. Materials and methods After obtaining the approval of the local ethics committee, the files of 96 patients aged 18-75 who underwent EBUS were reviewed retrospectively. Patients using Mg were classified as the M group, and patients not using Mg were classified as the control (C) group. IPI values, amount of propofol consumed, and intubation scores of group M and group C were evaluated. Results When the intubation score values ​​at the time of the bronchoscope passing through the vocal cords (assessment of vocal cord movement, cough reflex, and leg movement) during the EBUS procedure were compared, the intubation conditions were found to be significantly better in the M group than in the C group (p<0.05). Group M had less cough reflex than group C (p<0.05). IPI scores were significantly higher in the M group than in the C group at the 10th and 15th minutes (p<0.05). Total propofol consumption was found to be significantly lower in the M group (254.61±82.80 mg) than in the C group (321.25±90.04 mg) (p<0.05). Conclusion According to our results, the use of intravenous and inhaler Mg in addition to propofol sedation during the EBUS procedure may improve the respiratory parameters and can also significantly reduce the propofol dose.

Determination of wild animal sources of fecal indicator bacteria by microbial source tracking (MST) influences regulatory decisions.

Fecal indicator bacteria (FIB) are used to assess fecal pollution levels in surface water and are among the criteria used by regulatory agencies to determine water body impairment status. While FIB provide no information about pollution source, microbial source tracking (MST) does, which contributes to more direct and cost effective remediation efforts. We studied a watershed in Florida managed for wildlife conservation that historically exceeded the state regulatory guideline for fecal coliforms. We measured fecal coliforms, enterococci, a marker gene for avian feces (GFD), and a marker gene for human-associated Bacteroides (HF183) in sediment, vegetation, and water samples collected monthly from six sites over two years to: 1) assess the influence of site, temporal factors, and habitat (sediment, vegetation, and water) on FIB and MST marker concentrations, 2) test for correlations among FIB and MST markers, and 3) determine if avian feces and/or human sewage contributed to FIB levels. Sediment and vegetation had significantly higher concentrations of FIB and GFD compared to water and thus may serve as microbial reservoirs, providing unreliable indications of recent contamination. HF183 concentrations were greatest in water samples but were generally near the assay limit of detection. HF183-positive results were attributed to white-tailed deer (Odocoileus virginianus) feces, which provided a false indication of human sewage in this water body. FIB and GFD were positively correlated while FIB and HF183 were negatively correlated. We demonstrated that birds, not sewage, were the main source of FIB, thus avoiding implementation of a total maximum daily load program (TMDL). Our results demonstrate that the concomitant use of FIB and MST can improve decision-making and provide direction when water bodies are impaired, and provides a strategy for natural source exclusion in water bodies impacted by wild animal feces.

Photoaffinity labeling probe for the substrate binding site of human phenol sulfotransferase (SULT1A1): 7-azido-4-methylcoumarin.

A novel fluorescent photoactive probe 7-azido-4-methylcoumarin (AzMC) has been characterized for use in photoaffinity labeling of the substrate binding site of human phenol sulfotransferase (SULT1A1 or P-PST-1). For the photoaffinity labeling experiments, SULT1A1 cDNA was expressed in Escherichia coli as a fusion protein to maltose binding protein (MBP) and purified to apparent homogeneity over an amylose column. The maltose moiety was removed by Factor Xa cleavage. Both MBSULT1A1 and SULT1A1 were efficiently photolabeled with AzMC. This labeling was concentration dependent. In the absence of light, AzMC competitively inhibited the sulfation of 4MU catalyzed by SULT1A1 (Ki = 0.47 +/- 0.05 mM). Moreover, enzyme activity toward 2-naphthol was inactivated in a time- and concentration-dependent manner. SULT1A1 inactivation by AzMC was protected by substrate but was not protected by cosubstrate. These results indicate that photoaffinity labeling with AzMC is highly suitable for the identification of the substrate binding site of SULT1A1. Further studies are aimed at identifying which amino acids modified by AzMC are localized in the binding site.

The chemical modification of human liver UDP-glucuronosyltransferase UGT1*6 reveals the involvement of a carboxyl group in catalysis.

The treatment of UDP-glucuronosyltransferase UGT1*6 stably expressed in V79 cells with three carboxyl-specific reagents, dicyclohexylcarbodiimide, 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide and N-ethyl-5-phenylisoxazolium-3'-sulfonate (Woodward's reagent K), resulted in a fast, dose-dependent decrease of the 4-methylumbelliferone glucuronidation. The inactivation reactions followed pseudo-first order kinetics. The pKa of the modified residue was close to 5.0. A partial protection against inactivation by Woodward's reagent was observed at pH 7.4 in the presence of UDP-glucuronic acid, UDP, and, to a lesser extent, in the presence of 4-methylumbelliferone. Dicyclohexylcarbodiimide significantly decreased the Vmax, without affecting the apparent Km towards UDP-glucuronic acid and 4-methylumbelliferone. The results support the involvement of a carboxyl group in the catalytic process.

Photoaffinity labeling of the aglycon binding site of the recombinant human liver UDP-glucuronosyltransferase UGT1A6 with 7-azido-4-methylcoumarin.

7-Azido-4-methylcoumarin (AzMC) is a fluorescent photoactive compound structurally related to 4-methylumbelliferone (4-MU), a marker substrate of the human liver recombinant UDP-glucuronosyltransferase (UGT) 1A6. AzMC was synthesized and utilized to label the substrate binding site of UGT1A6. AzMC exhibits a fluorescence spectrum with maximum excitation and emission wavelengths of 380 and 442 nm, respectively. Upon irradiation, the probe irreversibly inhibited glucuronidation activity measured with para-nitrophenol (pNP) as substrate and interacted with UGT1A6 according to a saturable process indicative of reversible binding before covalent incorporation of the photoaffinity label. This inhibition was both time and concentration dependent and led to the calculation of an inhibition constant, k(2) = 0.113 mM min(-1), and dissociation constant, K(d) = 2.89 mM, for the reaction. Partial photoinactivation of UGT1A6 with AzMC revealed that the probe decreased the apparent V(max) of the pNP glucuronidation reaction, but not the K(m). Moreover, inhibition was partially prevented by 1-naphthol, a surrogate substrate for the enzyme, or by preincubation with an active-site directed inhibitor, 5'-O-[[(2-decanoylamino-3-phenyl-propyloxycarbonyl)amino]-su lfonyl]-2 ',3'-O-isopropylideneuridine. In contrast, UDP-glucuronic acid (UDP-GlcUA) did not have any protective effect against photoinactivation and AzMC did not affect the photoaffinity labeling of UGT1A6 by 5-[beta-(32)P]N(3)UDP-GlcUA, a photoaffinity analog of UDP-GlcUA. Additionally, in the absence of irradiation, AzMC was found to be a competitive inhibitor of 4MU glucuronidation. Collectively, these results strongly indicate that AzMC specifically binds to the UGT1A6 aglycon binding site. Amino acid alignment of phenol-binding proteins revealed a conserved motif, YXXXKXXPXP. It is possible that this motif is involved in phenol binding to UGT1A6 and other phenol-accepting proteins.

Characterization of the UDP-glucuronosyltransferases involved in the glucuronidation of an antithrombotic thioxyloside in rat and humans.

To investigate the glucuronidation on the hydroxyl group of carbohydrate-containing drugs, the in vitro formation of glucuronides on the thioxyloside ring of the antithrombotic drug, LF 4.0212, was followed in rat and human liver microsomes and with recombinant UDP-glucuronosyltransferases (UGT). The reaction revealed a marked regioselectivity in rat and humans. Human liver microsomes glucuronidated the compound mainly on the 2-hydroxyl position of the thioxyloside ring, whereas rat was able to form glucuronide on either the 2-, 3-, or 4- hydroxyl group of the molecule, although to a lower extent. LF 4.0212 was a much better substrate of human UGT than the rat enzyme (Vmax/Km 30.0 and 0.06 microl/min/mg, respectively). Phenobarbital, 3-methylcholanthrene, and clofibrate enhanced the glucuronidation of LF 4.0212 on positions 2, 3, and 4 of the thioxyloside ring, thus indicating that several UGT isoforms were involved in this process. The biosynthesis of the 2-O-glucuronide isomer was catalyzed by the human UGT1A9 and 2B4, but not by UGT1A6 and 2B11. By contrast, the rat liver recombinant UGT1A6 and 2B1 failed to form the 2-O-glucuronide isomers. From all the recombinant UGTs tested, none catalyzed the formation of the 3-O-glucuronide isomer. Interestingly, glucuronidation on the 4-position was found in all the metabolic competent V79 cell lines considered, including the nontransfected V79 cells, suggesting the presence of an endogenous UGT in fibroblasts able to actively glucuronidate the drug. This activity, which was nonsensitive to the inhibitory effect of 7,7,7-triphenylheptanoic acid, a potent UGT inhibitor, could reflect the existence of a different enzyme.

Human and rat liver UDP-glucuronosyltransferases are targets of ketoprofen acylglucuronide.

Acylglucuronides formed from carboxylic acids by UDP-glucuronosyltransferases (UGTs) are electrophilic metabolites able to covalently bind proteins. In this study, we demonstrate the reactivity of the acylglucuronide from the nonsteroidal anti-inflammatory drug, ketoprofen, toward human and rat liver UGTs. Ketoprofen acylglucuronide irreversibly inhibited the glucuronidation of 1-naphthol and 2-naphthol catalyzed by human liver microsomes or by the recombinant rat liver isoform, UGT2B1, which is the main isoform involved in the glucuronidation of the drug. A decrease of about 35% in the glucuronidation of 2-naphthol was observed when ketoprofen acylglucuronide was produced in situ in cultured V79 cells expressing UGT2B1. Inhibition was always associated with the formation of microsomal protein-ketoprofen adducts. The presence of these covalent adducts within the endoplasmic reticulum of cells expressing UGT2B1 was demonstrated following addition of ketoprofen to culture medium by immunofluorescence microscopy with antiketoprofen antibodies. Immunoblots of liver microsomes incubated with ketoprofen acylglucuronide and probed with antiketoprofen antibodies revealed the presence of several protein adducts; among those was a major immunoreactive protein at 56 kDa, in the range of the apparent molecular mass of UGTs. The adduct formation partially prevented the photoincorporation of the UDP-glucuronic acid (UDP-GlcUA) analog, [beta-32P]5N3UDP-GlcUA, on the UGTs, suggesting that ketoprofen glucuronide covalently reacted with the UDP-GlcUA binding domain. Finally, UGT purification from rat liver microsomes incubated with ketoprofen glucuronide led to the isolation of UGT adducts recognized by both anti-UGT and antiketoprofen antibodies, providing strong evidence that UGTs are targets of this metabolite.

Photoaffinity labeling studies of the human recombinant UDP-glucuronosyltransferase, UGT1*6, with 5-azido-UDP-glucuronic acid.

Recombinant human liver UDP-glucuronosyltransferase (UGT), UGT1*6, which catalyzes the glucuronidation of small phenols, previously expressed in a V79 cell line (1) was photolabeled with [beta-32P]5N3UDP-glucuronic acid ([beta-32P]5N3UDP-GlcUA). Two polypeptides with an approximate molecular weight of 54 kDa were extensively photolabeled in the recombinant cell line while the nontransfected cell line showed no photoincorporation in this area. The identity of the two polypeptides as UGTs, which correspond to two different glycosylation forms of the same enzyme, was confirmed by Western blot using a polyclonal monospecific antibody directed against the 120 amino acids of the N-terminal end of UGT1*6. Preincubation with UDP-glucuronic acid (UDP-GlcUA) inhibited the photoincorporation of the probe into the polypeptides indicating competition of both the photoprobe and the nucleotide-sugar for the same binding site. It was further shown that photoincorporation of [beta-32P]5N3UDP-GlcUA into the UDP-GlcUA-binding site was saturable. The lack of photoincorporation of a related photoprobe, [beta-32P]5N3UDP-glucose ([beta-32P]5N3UDP-Glc), into UGT1*6 demonstrated specificity of this enzyme for UDP-GlcUA. In enzymatic assays, unlabeled 5N3UDP-GlcUA was shown to be an effective cosubstrate of the glucuronidation of 4-nitrophenol catalyzed by UGT1*6. The studies were further extended by demonstrating that photolabeling of UGT1*6 was inhibited by several active site-directed inhibitors. Finally, photoaffinity labelling was used in the purification of the labeled UGT1*6 using preparative gel electrophoresis. In conclusion, we have demonstrated that photoaffinity labeling with [beta-32P]5N3UDP-GlcUA is an effective tool for the characterization of enzymes such as recombinant UGTs that use UDP-GlcUA.

Interaction of periodate-oxidized UDP-glucuronic acid with recombinant human liver UDP-glucuronosyltransferase 1A6.

Sodium periodate reacts with UDP-glucuronic acid (UDP-GlcUA) to generate a reactive derivative [periodate-oxidized UDP-GlcUA (o-UDP-GlcUA)]. The ability of this analog of UDP-GlcUA to inactivate and label the human recombinant UDP-glucuronosyltransferase (UGT) UGT1A6 via the UDP-GlcUA binding site was investigated. At an o-UDP-GlcUA concentration of 20 mM, the enzymatic activity of UGT1A6 was totally inactivated after 30 min of incubation at pH 7.4. Inhibition was irreversible, time-dependent, and concentration-dependent and exhibited pseudo-first order kinetics (kinact = 4.0 M-1.min-1). Cosubstrate protection with UDP-GlcUA was biphasic, with no protection in the first phase and almost total protection in the second phase, suggesting that at least 65% of the cross-linking occurs at the cosubstrate binding site. Partial inactivation by o-UDP-GlcUA led to a decrease in Vmax, suggesting that o-UDP-GlcUA can act as an active site-directed inhibitor. Furthermore, proteins, including the UGTs, from membrane fractions of a recombinant V79 cell line expressing the UGT1A6 enzyme and from rat liver microsomes were cross-linked by in situ periodate oxidation of [beta-32P]UDP-GlcUA. The present results suggest that periodate-oxidized UDP-GlcUA, which inactivates UGT1A6 by the possible formation of a Schiff base adduct with active site lysyl residues, can be used as a new affinity label for the UDP-GlcUA binding site.

The EXT1/EXT2 tumor suppressors: catalytic activities and role in heparan sulfate biosynthesis.

The D-glucuronyltransferase and N-acetyl-D-glucosaminyltransferase reactions in heparan sulfate biosynthesis have been associated with two genes, EXT1 and EXT2, which are also implicated in the inherited bone disorder, multiple exostoses. Since the cell systems used to express recombinant EXT proteins synthesize endogenous heparan sulfate, and the EXT proteins tend to associate, it has not been possible to define the functional roles of the individual protein species. We therefore expressed EXT1 and EXT2 in yeast, which does not synthesize heparan sulfate. The recombinant EXT1 and EXT2 were both found to catalyze both glycosyltransferase reactions in vitro. Coexpression of the two proteins, but not mixing of separately expressed recombinant EXT1 and EXT2, yields hetero-oligomeric complexes in yeast and mammalian cells, with augmented glycosyltransferase activities. This stimulation does not depend on the membrane-bound state of the proteins.

Arginine 52 and histidine 54 located in a conserved amino-terminal hydrophobic region (LX2-R52-G-H54-X3-V-L) are important amino acids for the functional and structural integrity of the human liver UDP-glucuronosyltransferase UGT1*6.

The hepatic UDP-glucuronosyltransferase UGT1*6 is actively involved in the glucuronidation of short and planar phenols in humans. Based on the irreversible inhibition of the enzyme on chemical modification by 2,3-butanedione and diethyl pyrocarbonate, the roles of His54 and Arg52 were investigated by oligonucleotide site-directed mutagenesis. These amino acids belong to a consensus sequence LX2-R52-G-H54-X3-V-L located in a conserved hydrophobic region of the variable amino-terminal domain of UGT. Arg52 was replaced by alanine (mutant R52A), and His54 was replaced by alanine or glutamine (mutants H54A and H54Q). The immunological and catalytic properties of UGT1*6 and mutants were examined after stable expression in V79 cell lines. Immunoblots and immunoprecipitation studies revealed that the mutant and UGT1*6 proteins were expressed in the microsomal membranes in similar amounts. However, replacement of His54 by glutamine led to a complete loss of activity toward 4-methylumbelliferone, and the Vmax value was decreased 4-5-fold in the mutants R52A and H54A compared with the wild-type enzyme. The dissociation constants that characterize the binding of 4-methylumbelliferone and UDP-glucuronic acid to UGT1*6 were not greatly affected by the mutations. Interestingly, H54Q was not recognized by specific antibodies to the amino-terminal portion of UGT1*6, thereby indicating that this amino acid was critical to antibody recognition. In contrast, the mutants R52A and H54A could not be differentiated from the wild-type protein by pH optimum or thermal denaturation. Furthermore, these mutants were still sensitive to irreversible inhibition by diethyl pyrocarbonate and 2,3-butanedione, with second-order inactivation constant values similar to those obtained for UGT1*6. Altogether, the strict conservation of His54 and Arg52 and the mutational analysis of these residues suggest that these amino acids in the hydrophobic amino-terminal consensus sequence LX2-R52-G-H54-X3-V-L are important for the function and the structure required for optimal catalytic efficiency of UGT1*6.