A case of multifocal Langerhans cell granulomatosis: a BAL follow up study

UDP-glucuronosyltransferase (UGT) 2B9, isolated from a cynomolgus monkey liver cDNA library, is 89% identical to human UGT2B7 in primary amino acid sequence, and the two expressed enzymes were previously shown to catalyze the glucuronidation of many common endogenous substrates. The purpose of the present study was to characterize the reactivity of expressed UGT2B9 with important therapeutic agents and other xenobiotics. UGT2B9, stably expressed in human embryonic kidney 293 cells, catalyzes the 3-O- and 6-O-glucuronidation of morphine and the 6-O-glucuronidation of codeine. A number of other morphinan (e.g. naloxone, naltrexone, and nalorphine) and oripavine (e.g. buprenorphine) derivatives are substrates for this enzyme. In general, morphinan derivatives are glucuronidated at higher rates, compared with oripavines; however, glucuronidation efficiency values (Vmax/KM) for the compounds are similar. Stably expressed UGT2B9 also catalyzes the glucuronidation of profen nonsteroidal anti-inflammatory drugs, fibrate hypolipidemic agents, and straight-chain fatty acids at the carboxylic acid moiety. Monoterpenoid alcohols and propanolol are glucuronidated at aliphatic hydroxyl positions. Expressed UGT2B9 exhibits enantioselective glucuronidation for (R/S)-ibuprofen, (R/S)-propanolol, and (+)/(-)-menthol. The data suggest that monkey UGT2B9 and human UGT2B7 are functionally similar.     

UDP-glucuronosyltransferases in human intestinal mucosa

While UDP-glucuronosyltransferases (UGTs) are known to be expressed at high levels in human liver, relatively little is known about extrahepatic expression. In the present study, UGT2B family isoforms involved in the glucuronidation of steroid hormones and bile acids have been characterized in microsomes prepared from jejunum, ileum and colon from six human subjects. Glucuronidation of androsterone and testosterone was highly significant and increased from proximal to distal intestine. In contrast, hyodeoxycholic acid was glucuronidated at a low level in jejunum and ileum and activity was barely detectable in colon. No significant glucuronidation of lithocholic acid was found. Small phenols were glucuronidated with much lower activity than found in liver. High levels of UGT protein were detected with polyclonal anti-rat androsterone- and testosterone-UGT antibodies, whereas UGT2B4, a major hepatic hyodeoxycholic acid-specific UGT, was undetectable using a highly specific anti-human UGT2B4 antibody. Screening for RNA expression by RT-PCR confirmed the absence of UGT2B4 and UGT1A6 and showed expression of UGT2B7, a hepatic isoform shown to glucuronidate androsterone, in all intestinal segments. To our knowledge, the presence of functional androsterone and testosterone directed isoforms in human intestine is a novel finding which supports the idea that the intestinal tract functions as a steroid-metabolizing organ and plays a significant role in steroid hormone biotransformation.     

Progesterone metabolites and bile acids in serum of patients with intrahepatic cholestasis of pregnancy: effect of ursodeoxycholic acid therapy

The concentrations in serum of sulfated metabolites of progesterone are known to be elevated in patients with intrahepatic cholestasis of pregnancy (ICP). The profiles of these metabolites and conjugated bile acids were analyzed in serum from 11 patients with ICP before and during administration of ursodeoxycholic acid (UDCA) (8 patients) or placebo (3 patients). The clinical condition of 7 of the patients given UDCA improved markedly, and 1 patient given placebo had a spontaneous remission of the disease. The total concentration of conjugated bile acids in the 11 patients was 25 +/- 6 micromol/L (mean +/- SEM) and decreased to 6.3 +/- 3.5 micromol/L in the 7 patients responding to treatment with UDCA. The level of 7alpha-hydroxy-4-cholesten-3-one was significantly lower (7.2 +/- 2.2 ng/mL) in patients with ICP than in healthy pregnancy (18 +/- 4.6 ng/mL) (P < .05). The concentrations of 5alpha-pregnane-3alpha,20alpha-diol mono- and disulfates decreased by 52% +/- 7.9% and 68% +/- 5.5%, respectively, in the patients responding to treatment. Similar decreases were observed for the mono- and disulfates of 5alpha-pregnane-3alpha,20alpha,21-triol and 5beta-pregnane-3alpha,20alpha-diol. The disulfate of 5alpha-pregnane-3beta,20alpha-diol showed a smaller decrease, while glucuronidated steroids were not affected. The 3alpha-/3beta-hydroxysteroid ratio and di-/monosulfate ratio decreased significantly during UDCA. The magnitudes of the changes of bile acid and steroid concentrations during UDCA were not correlated to each other. The results suggest that UDCA stimulates the biliary excretion of steroids with a 3alpha-sulfoxy group and disulfates. This effect seems to be independent of the effect on bile acid excretion, indicating the use of different transport proteins. The possibility of an effect of UDCA on the formation of the steroid sulfates cannot be excluded.     

Glucuronidation of retinoids by rat recombinant UDP: glucuronosyltransferase 1.1 (bilirubin UGT)

Rat liver recombinant BR1UGT1.1 was found to have significant activity toward retinoid substrates. UGT1.1 glucuronidation activity was 91 +/- 18 pmol/mg x min for atRA and 113 +/- 19 pmol/mg x min for 5,6-epoxy-atRA. The apparent K(M) and V(max) of atRA acid glucuronidation by UGT1.1 were 59.1 +/- 5.4 microM and 158 +/- 43 pmol/mg x min, respectively. SDS-PAGE and Western blot analysis of UGT1.1-transfected HK293 membrane proteins photolabeled with [11,12-3H]atRA revealed a protein of approximately 56 kDa that was labeled by [3H]atRA, detected by anti-pNP UGT antibody and not present in membranes from nontransfected HK293 cells. Liver microsomes from Gunn rats, which lack UGT1.1, had significant activity toward atRA (111 +/- 28 pmol/mg x min).     

Autotransplantation of the spleen in the rat: donor leukocytes of the splenic fragment survive implantation to migrate and proliferate in the host

A rabbit liver UDP-glucuronosyltransferase cDNA that is related to human and rat UGT1A7 has been identified. The predicted amino acid sequence of the UGT1A71 displays 80% similarity to that encoded by human HP4 (UGT1A9), but 81% to that predicted for human UGT1A7 and 77% to the rat UGT1A7 (UGTA2). The exons encoding human UGT1A7 and rat UGTA2 are the seventh of the series of cassette exons that flank the 3' common exon series of the UGT1A locus. Southern blot analysis demonstrates that the exon sequence encoding UGT1A71 is part of a larger cluster of highly related genes. The UGT1A71 RNA is expressed in both neonatal and adult liver, and unlike rat UGT1A2 which is inducible with Ah receptor ligands such as polycyclic aromatic hydrocarbons, rabbit UGT1A7 is not regulated when animals are exposed to these inducers. Following expression of UGT171 in COS-1 cells, glucuronidation activity was identified for small phenolic molecules like 4-nitrophenyl, bulky phenols as represented by 4-hydroxybiphenol and octylgallate, as well as 4-hydroxyestrone. In addition, UGT1A71 possesses catalytic activity toward tertiary amines like the tricyclic antidepressant imipramine. The pattern of UGT1A71 glucuronidation is similar to that observed for human UGT1A9, except tertiary amines are not subject to glucuronidation by human UGT1A9. Glucuronidation of tertiary amines is catalyzed principally by human UGT1A4 as well as rabbit UGT1A4. Although rabbit UGT1A7 catalyzes the formation of quarternary ammonium glucuronides, the Vmax is considerably less than that observed for rabbit UGT1A4. Overall, the characterization of rabbit UGT1A7 suggests that this protein represents the ortholog of the human UGT1A7, which to date has not been identified.     

IL-6 functions in cynomolgus monkeys blocked by a humanized antibody to human IL-6 receptor

A humanized antibody to the human interleukin-6 receptor (IL-6R), hPM-1, blocked the interleukin-6 (IL-6) functions in normal cynomolgus monkey lymphocytes in vitro. The binding activity of hPM-1 to non-human primate IL-6R was examined in peripheral blood lymphocytes by flow cytometry. PM-1 recognized the IL-6R on T lymphocytes of cynomolgus and rhesus monkeys, but did not on those of marmosets. The homology between human IL-6R and its cynomolgus monkey counterpart was 97.3% in the extracellular domain of the amino acid sequence, as determined by DNA sequencing of the PCR product from peripheral blood mononuclear cells. PM-1 inhibited two functional parameters in vitro in cynomolgus monkeys: (1), T-cell proliferation stimulated by phytohemaglutinin and human IL-6; (2), Immunoglobulin G-production evoked by Staphylococcus aureus Cowan-1- and human IL-6-stimulated B lymphocytes. These data show that hPM-1 binds to and functionally blocks the cynomolgus monkey IL-6 receptors.     

Excretion and metabolism of tipredane, a novel glucocorticoid, in the rat, mouse, monkey, and human

The excretion and metabolism of [3H]tipredane, a novel glucocorticoid, has been studied in mice, rats, marmosets, rhesus and cynomolgus monkeys, and humans. After oral administration, [3H]tipredane was rapidly absorbed, metabolized, and excreted into urine and feces. In mice and male rats, radioactivity was excreted primarily into feces or bile, whereas in female rats, monkeys, and humans, excretion was mainly via the renal route. Some sex differences in the proportions excreted into urine and feces were noted in rodents, with females eliminating relatively more radioactivity in urine. Tipredane was shown to be extensively metabolized, but the routes were highly species-dependent and, in the rat, they were sex-dependent. Unchanged tipredane was not detected in any urine, bile, or blood extracts. Urinary and blood extract profiles indicated that there were between 10 and 30 metabolites in rats and mice, the majority of which constituted < 2% of the dose. In these species, the major pathways involved loss of the thioethyl moiety, S-oxidation of the thiomethyl group, and saturation of the adjacent saturated C16-17 bond. Hydroxylation of the steroid B-ring was seen in the 7 alpha-position in mice and female rats, and in the 6 beta-position in male rats. Metabolism of tipredane in rhesus and cynomolgus monkeys and humans was similar, but less extensive and different to that seen in rodents. The major products, the 6 beta-hydroxylated sulfoxide and sulfone metabolites of tipredane, accounted for 21-36% of the dose in human and monkey urine, and were also major components in blood. In contrast to mice and rats, S-oxidation and an unsaturated C16-17 bond were evident in primates. Metabolism of tipredane was rapid and complex, with significant species differences, although the disposition in rhesus and cynomolgus monkeys seemed to be similar to humans.     

Discordant effects on eicosanoids and fibrin degradation products in two murine models of antiphospholipid antibody

Comparison of 7-hydroxylation of coumarin, a CYP2A6 substrate, in human and African green and cynomolgus monkey liver microsomes was made by means of an HPLC assay with UV detection. In human liver microsomes, the Km and Vmax values for the metabolic conversion were 2.1 microM and 0.79 nmol/mg/min, respectively. While African green monkey showed Km and Vmax values of 2.7 microM and 0.52 nmol/mg/min, which were similar to human, higher Km and Vmax values were found in cynomolgus monkey. Coumarin 7-hydroxylation in human and African green monkey was selectively inhibited by methoxsalen and pilocarpine (CYP2A6 inhibitors) but not by other inhibitors, i.e. alpha-naphthoflavone (CYP1A1), orphenadrine (CYP2B6), sulfaphenazole (CYP2C9), quinidine (CYP2D6) and ketoconazole (CYP3A4). Immunoinhibition results supported CYP2A6 involvement in human and its homolog in monkey in coumarin 7-hydroxylation, as only anti-CYP2A6, but not CYP2B1, CYP2C13, CYP2D6, CYP2E1 or CYP3A antibodies, inhibited this conversion. African green monkey was found to be similar to human in catalytic activity of coumarin 7-hydroxylation and response to CYP2A6 inhibitors or antibody inhibition. However, the monkey CYP2A6 is not identical to the human in that Ki values were different, and differences were observed with some CYP2A6 inhibitors, such as nicotine and methoxsalen, suggesting that, under some circumstances, studies of nicotine kinetics and drug taking behavior in monkey may not be comparable to human.     

Hepatic and extrahepatic dehydrogenation/isomerization of 5-cholestene-3 beta,7 alpha-diol: localization of 3 beta-hydroxy-delta 5-C27-steroid dehydrogenase in pig tissues and subcellular fractions

Conversion of 5-cholestene-3 beta,7 alpha-diol (7 alpha-hydroxycholesterol) into 7 alpha-hydroxy-4-cholesten-3-one was studied with microsomes from different pig tissues and with liver subcellular fractions. Dehydrogenase/isomerase activity was efficient in microsomes from liver, ovary and lung, but less efficient in microsomes from adrenal gland and kidney. Microsomes from these tissues, with the exception of lung, were also active in dehydrogenation/isomerization of dehydroepiandrosterone and pregnenolone. Inhibition studies were carried out with trilostane, a competitive inhibitor of 3 beta-hydroxysteroid dehydrogenases active in steroid hormone biosynthesis (C19/C21-dehydrogenases), and a monoclonal antibody raised against a purified hepatic 3 beta-hydroxy-delta 5-C27-steroid dehydrogenase. The results showed that the C27-dehydrogenase activity in the tissues was not dependent on the C19/C21 dehydrogenases, but was dependent on the 3 beta-hydroxy-delta 5-C27-steroid dehydrogenase. Liver mitochondria, cytosol and peroxisomes lacked dehydrogenase/isomerase activity towards 7 alpha-hydroxycholesterol when microsomal contamination was taken into account. Immunoblotting experiments with monoclonal antibodies raised against the 3 beta-hydroxy-delta 5-C27-steroid dehydrogenase showed immunoreactivity only with protein in liver microsomes. Immunohistochemical studies showed localization of the 3 beta-hydroxy-delta 5-C27-steroid dehydrogenase in the bile duct epithelium. It is concluded that 7 alpha-hydroxycholesterol is converted into 7 alpha-hydroxy-4-cholesten-3-one by the microsomal 3 beta-hydroxy-delta 5-C27-steroid dehydrogenase in liver and extrahepatic tissues.     

Sex hormones differentially regulate isoforms of UDP-glucuronosyltransferase

PURPOSE: To investigate the role of sex hormones in the regulation of UDP-glucuronosyltransferase (UGT). METHODS: We examined liver from adult, prepubertal, gonadectomised and gonadectomised plus hormone replaced rats of both sexes. Immunohistochemistry and immunoblots were performed using a polyclonal UGT antibody to a number of family 1 and family 2 UGT isoforms. Northern blot analysis was performed utilising cDNA probes to family 1 and family 2 isoforms. RESULTS: Immunohistochemistry demonstrated variations in intensity and distribution of staining in the hormonally manipulated rats. Immunoblots showed variations in individual band intensity between rat groups. Immunoblots using a more specific antibody (anti-17 beta-hydroxysteroid UGT, which recognises UGT2B3 and UGT2B2) demonstrated marked differences between male and female rats and significant alterations after gonadectomy and testosterone replacement in the male rats. In northern analysis, UGT2B3 and 2B1 mRNA were significantly higher in adult males than females, and in prepubertal males compared to prepubertal females. In male rats, gonadectomy resulted in a 45-53% reduction in UGT2B3 and 2B1 levels respectively, which increased significantly with testosterone treatment to greater than normal adult levels. No change in UGT2B3 or 2B1 occurred after gonadectomy in females. In contrast, UGT1*1 mRNA tended to be higher in adult female and prepubertal female rats than in their male counterparts. In females, gonadectomy resulted in significant up-regulation of UGT1*1, while gonadectomy plus oestradiol treatment resulted in markedly reduced levels. UGT1*1 mRNA was not significantly altered by gonadectomy in males. CONCLUSIONS: This study demonstrates the differential effects of sex hormones on the expression of isoforms from the two phylogenetically distinct UGT families.     

Hepatic metabolism and transport of bilirubin and other organic anions

Most of bilirubin, bile acids and other organic anions are preferentially taken up by the liver and excreted into bile. Recently many transporters on the sinusoidal and canalicular membranes of the hepatocytes have been reported for each ligand. complementary DNA was cloned for human Na+/taurocholate cotransporting polypeptide (NTCP) which mediates sodium dependent secondary active hepatic uptake of bile acids. For the hepatic uptake of non-bile acid-organic anions such as bilirubin, at least 4 transporters are postulated, i.e., bilirubin/BSP binding protein (BBBP), organic anion binding protein (OABP), bilitranslocase, and organic anion transporting polypeptide (OATP). In the hepatocytes, bilirubin is glucuronidated in the endoplasmic reticulum. The gene for UDP-glucuronosyltransferase (UGT) 1 family has been elucidated and differential splicing from several exons 1 (A to J) results in forming isozymes of UGT 1 including bilirubin UGT. At the canalicular membranes, two main ATP-dependent organic anion transporters have been reported, i.e., canalicular bile salt transporter (cBST) for bile acids and canalicular multispecific organic anion transporter (cMOAT) for non-bile acid organic anions. Recently multidrug resistance protein (MRP) is reported closely related to or identical to cMOAT. These canalicular ATP-dependent transporters are called ABC (ATP-binding cassette) transporters.     

Characterisation of a new human and murine member of the DnaJ family of proteins

We report the characterisation of a human gene, designated MCG18 (multiple endocrine neoplasia type 1 candidate gene 18), that encodes a new member of the DnaJ family of proteins. Database searches indicate that MCG18 also has the locus name HSPF2. MCG18 lies 250bp centromeric of the VRF/VEGFB gene on chromosome 11q13. The MCG18 cDNA is predicted to encode a 241 amino acid product that has partial homology to Escherichia coli dnaJ in that it contains the J domain. However, MCG18 has greatest similarity to a functionally undefined protein from Caenorhabditis elegans, both of which are predicted to have a membrane-spanning region adjacent to their J domains. The cDNA encoding the murine homolog (Mcg18) was also cloned and sequenced, and the encoded protein shares 81% similarity to MCG18. The coding region of MCG18 is interrupted by 4 introns and the mRNA is expressed as a 1.4kb message in all tissues examined, including those derived from the breast, ovary, bladder, lung and keratinocytes.     

Androgen deprivation impairs memory in older men.

Androgen deprivation leads to a profound loss of synaptic density in the hippocampus and changes in learning and memory in animal models. The authors examined group differences in verbal memory between men on androgen deprivation therapy (ADT), a commonly used treatment for prostate cancer, and healthy men. The authors found that men on ADT have a specific impairment of retention but normal encoding and retrieval processes on a word list-learning task. Speed and accuracy for both perceptual and semantic encoding, as well as retrieval at a very short retention interval, were not affected; however, recognition fell to chance after a 2-min retention interval in men on ADT. Healthy men showed only moderate forgetting, and performance was still above chance at 12 min. This pattern of preserved encoding and retrieval but impaired retention suggests that androgens play a role in hippocampally mediated memory processes, possibly having a specific affect on consolidation. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Zyme, a novel and potentially amyloidogenic enzyme cDNA isolated from Alzheimer's disease brain

The deposition of the beta amyloid peptide in neuritic plaques and cerebral blood vessels is a hallmark of Alzheimer's disease (AD) pathology. The major component of the amyloid deposit is a 4.2-kDa polypeptide termed amyloid beta-protein of 39-43 residues, which is derived from processing of a larger amyloid precursor protein (APP). It is hypothesized that a chymotrypsin-like enzyme is involved in the processing of APP. We have discovered a new serine protease from the AD brain by polymerase chain reaction amplification of DNA sequences representing active site homologous regions of chymotrypsin-like enzymes. A cDNA clone was identified as one out of one million that encodes Zyme, a serine protease. Messenger RNA encoding Zyme can be detected in some mammalian species but not in mice, rats, or hamster. Zyme is expressed predominantly in brain, kidney, and salivary gland. Zyme mRNA cannot be detected in fetal brain but is seen in adult brain. The Zyme gene maps to chromosome 19q13.3, a region which shows genetic linkage with late onset familial Alzheimer's disease. When Zyme cDNA is co-expressed with the APP cDNA in 293 (human embryonic kidney) cells, amyloidogenic fragments are detected using C-terminal antibody to APP. These co-transfected cells release an abundance of truncated amyloid beta-protein peptide and shows a reduction of residues 17-42 of Abeta (P3) peptide. Zyme is immunolocalized to perivascular cells in monkey cortex and the AD brain. In addition, Zyme is localized to microglial cells in our AD brain sample. The amyloidogenic potential and localization in brain may indicate a role for this protease in amyloid precursor processing and AD.     

Isolation of novel and known genes from a human fetal cochlear cDNA library using subtractive hybridization and differential screening

V79 (Chinese hamster lung fibroblast) cell lines expressing a functional recombinant phenobarbital-inducible rat liver UDP-glucuronosyltransferase (UGT), i.e., UGT2B1, were established. Western blot analysis of positive colonies, using anti-rat liver UGT antibodies, revealed the presence of an immunoreactive polypeptide of the expected molecular mass of 52 kDa. The substrate specificity of the recombinant enzyme toward > 100 compounds was determined. Phenolic and alcoholic substrates included 4-methylumbelliferone, 4-hydroxybiphenyl, chloramphenicol, and testosterone, but a range of carboxylic acids of both endogenous (medium-chain saturated fatty acids, long-chain polyunsaturated fatty acids, and bile acids) and exogenous (profen nonsteroidal anti-inflammatory drugs, fibrate hypolipidemic agents, and sodium valproate) origin were also accepted, indicating that the enzyme was capable of forming both ether- and ester-type glucuronides from various structurally unrelated compounds. Determination of apparent kinetic constants for the glucuronidation by UGT2B1 of selected aglycones revealed a high maximal velocity toward the 3-position of morphine (49.3 +/- 2.2 nmol/min/mg of protein), compared with other known substrates such as 4-methylumbelliferone (2.67 +/- 0.11 nmol/min/mg of protein) or clofibric acid (0.06 +/- 0.02 nmol/min/mg of protein). To gain a better insight into the mechanisms underlying the apparently wide substrate specificity of UGT2B1, series of structurally related compounds were tested as potential substrates. The rate of glucuronidation of unbranched saturated fatty acids and omega,omega,omega-triphenylalkanoic acids increased progressively with increasing alkyl chain length and then declined, with the best substrates in these two homologous series being decanoic acid and 4,4,4-triphenylbutanoic acid, respectively. Glucuronidation of para-substituted phenols always proceeded at a higher rate than that of the corresponding para-substituted benzoic acids. This could mean that the aglycon hydroxyl group was better positioned in the enzyme active site in the case of phenols. Alternatively, if the initial interaction with the enzyme required the aglycon to be in the protonated uncharged form, then the observation could be explained by the difference in ionization between phenols and benzoic acids at the incubation pH used. The introduction of a bulky alkyl group into the para-position led to increases of up to 300-fold in the rate of glucuronidation, probably as a result of the increased aglycon lipophilicity. Finally, the enzyme showed a degree of stereo- and regiospecificity, preferring (S)-ibuprofen to the R-enantiomer (Vmax/Km, 3.06 and 1.10 microliters/min/mg of protein, respectively) and glucuronidating lithocholic acid but not hyodeoxycholic acid, which differs by only a single hydroxyl group.(ABSTRACT TRUNCATED AT 400 WORDS)