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Biomedical subjects

M Beaulieu

Publications and source records attributed to M Beaulieu.

At least 19 recordsLinked to original sources
PubMed2001

Isolation and characterization of the UGT2B28 cDNA encoding a novel human steroid conjugating UDP-glucuronosyltransferase.

UDP-glucuronosyltransferase (UGT) enzymes belonging to the UGT2B subfamily catalyze the transfer of glucuronic acid to a large number of endogenous compounds, particularly steroids, to facilitate their elimination from target cells. A novel human UGT2B cDNA of 1666 bp was isolated and encodes a 529-amino acid protein named UGT2B28 type I. Glucuronidation assays demonstrated that UGT2B28 type I catalyzes the conjugation of endogenous and exogenous compounds. The tissue distribution of UGT2B28 revealed the expression of the type I transcript in the liver, breast, and LNCaP cells. Two other UGT2B cDNAs were isolated, and sequence analysis led to the identification of two truncated UGT2B28 species. UGT2B28 type II differs from type I by a deletion of 308 bp in the cofactor binding domain, whereas UGT2B28 type III lacks 351 bp in the putative substrate binding domain. All UGT2B28 isoforms are encoded by a single UGT2B28 gene which has a genomic organization similar to that of the other UGT2B genes characterized thus far. Although no substrates could be identified for the shorter isoforms, the three subtypes were shown to be located in the endoplasmic reticulum and the perinuclear membrane, demonstrating that the missing domains are not required for the subcellular localization of these UGT2B proteins. However, all the domains remain necessary for observing glucuronidation activity. The expression of UGT2B28 type I in the breast and liver suggests a role of this enzyme in the androgen and estrogen metabolism in these tissues.

Alternative Splicing↗
PubMed2001

PCR candidate region mismatch scanning: adaptation to quantitative, high-throughput genotyping.

Linkage and association analyses were performed to identify loci affecting disease susceptibility by scoring previously characterized sequence variations such as microsatellites and single nucleotide polymorphisms. Lack of markers in regions of interest, as well as difficulty in adapting various methods to high-throughput settings, often limits the effectiveness of the analyses. We have adapted the Escherichia coli mismatch detection system, employing the factors MutS, MutL and MutH, for use in PCR-based, automated, high-throughput genotyping and mutation detection of genomic DNA. Optimal sensitivity and signal-to-noise ratios were obtained in a straightforward fashion because the detection reaction proved to be principally dependent upon monovalent cation concentration and MutL concentration. Quantitative relationships of the optimal values of these parameters with length of the DNA test fragment were demonstrated, in support of the translocation model for the mechanism of action of these enzymes, rather than the molecular switch model. Thus, rapid, sequence-independent optimization was possible for each new genomic target region. Other factors potentially limiting the flexibility of mismatch scanning, such as positioning of dam recognition sites within the target fragment, have also been investigated. We developed several strategies, which can be easily adapted to automation, for limiting the analysis to intersample heteroduplexes. Thus, the principal barriers to the use of this methodology, which we have designated PCR candidate region mismatch scanning, in cost-effective, high-throughput settings have been removed.

Adenosine Triphosphatases↗
PubMed2001

Fast and robust optic disc detection using pyramidal decomposition and Hausdorff-based template matching.

We report about the design and test of an image processing algorithm for the localization of the optic disk (OD) in low-resolution (about 20 micro/pixel) color fundus images. The design relies on the combination of two procedures: 1) a Hausdorff-based template matching technique on edge map, guided by 2) a pyramidal decomposition for large scale object tracking. The two approaches are tested against a database of 40 images of various visual quality and retinal pigmentation, as well as of normal and small pupils. An average error of 7% on OD center positioning is reached with no false detection. In addition, a confidence level is associated to the final detection that indicates the "level of difficulty" the detector has to identify the OD position and shape.

Algorithms↗
PubMed1999

Alteration of human UDP-glucuronosyltransferase UGT2B17 regio-specificity by a single amino acid substitution.

The glucuronidation of steroid hormones is catalyzed by a family of UDP-glucuronosyltransferase (UGT) enzymes. Previously, two cDNA clones, UGT2B15 and UGT2B17, which encode UGT enzymes capable of glucuronidating C19steroids, were isolated and characterized. These proteins are 95% identical in primary structure; however, UGT2B17 is capable of conjugating C19steroid molecules at both the 3alpha and 17beta-OH positions, whereas UGT2B15 is only active at the 17beta-OH position. To identify the amino acid residue(s) which may account for this difference in substrate specificity, a comprehensive study on the role of 15 residues which differ between UGT2B15 and UGT2B17 was performed by site-directed mutagenesis. The stable expression of UGT2B17 mutant proteins into HK293 cells demonstrated that the mutation of isoleucine 125, valine 181 and valine 455 to the residues found in UGT2B15 did not alter enzyme activity nor substrate specificity. Furthermore, mutation of the variant residues in UGT2B15 (serine 124, asparagine 125, phenylalanine 165) to the amino acid residues found in UGT2B17 did not alter enzyme activity nor substrate specificity. However, mutation of the serine residue at position 121 of UGT2B17 to a tyrosine, as found in UGT2B15, abolished the ability of UGT2B17 to conjugate androsterone at the 3alpha position, but still retained activity for dihydrotestosterone and 5alpha-androstane-3alpha, 17beta-diol, which have an OH-group at the 17beta position. Interestingly, mutation of tyrosine 121 in UGT2B15 to a serine abolished activity for C19steroids. It is suggested that the serine residue at position 121 in UGT2B17 is required for activity towards the 3alpha and not for the 17beta position of C19steroids, whereas the tyrosine 121 in UGT2B15 is necessary for UGT activity. Despite the high homology between UGT2B15 and UGT2B17, it is apparent that different amino acid residues in the two proteins are required to confer conjugation of C19steroid molecules.

Amino Acid Sequence↗
PubMed1999

Stability of PO2, PCO2, and pH in fresh blood samples stored in a plastic syringe with low heparin in relation to various blood-gas and hematological parameters.

OBJECTIVE: To assess the stability of PO2, PCO2 and pH in fresh individual blood samples drawn in a plastic syringe with low heparin and stored at 22 degrees C or on ice in relation to various biochemical and hematological parameters of the samples. METHODS: PO2, PCO2, and pH were measured at determined times in samples kept at 22 degrees C or on ice. The magnitude of change in blood-gas parameters over time for each group of samples was determined. The change in PO2 was examined according to its initial value, the oxygen content (CaO2), the hemoglobin concentration, the leukocyte and platelet counts of each sample. RESULTS: The changes in PO2, PCO2, and pH over time were about three times lower in samples stored on ice than in the ones at 22 degrees C. Nevertheless, rapid increases in PO2 were observed for samples stored on ice with an initial PO2 between 50 and 250 mm Hg. Samples with PO2 over 250 mmHg exhibited a decrease in PO2 over time. No correlation was observed between the change in PO2 and the oxygen content, the hemoglobin concentration or the leukocyte and platelet counts for both groups of samples stored at 22 degrees C or on ice. CONCLUSION: PO2 in samples with an initial PO2 between 50 and 250 mm Hg stored on ice should be analyzed within 30 min. However, PCO2 and pH didn't exhibit clinically significant changes within 60 min under similar conditions. The magnitude of change in PO2 for samples kept on ice was dependent on their hemoglobin capacity for buffering oxygen, which is in converse relation with the oxygen saturation level of hemoglobin (SatHbO2). It is believed that the alteration in PO2 for samples stored on ice is produced predominantly by diffusion of oxygen through the plastic wall of the syringe, whereas it would be mainly of metabolic origin in samples at room temperature.

Blood Gas Analysis↗
PubMed1999

Dose rate effect of gamma irradiation on phenolic compounds, polyphenol oxidase, and browning of mushrooms (Agaricus bisporus).

To enhance the shelf life of edible mature mushrooms, Agaricus bisporus, 2 kGy ionizing treatments were applied at two different dose rates: 4.5 kGy/h (I(-)) and 32 kGy/h (I(+)). Both I(+) and I(-) showed a 2 and 4 day shelf-life enhancement compared to the control (C). Before day 9, no significant difference (p>0.05) in L value was detected in irradiated mushrooms. However, after day 9, the highest observed L value (whiteness) was obtained for the mushrooms irradiated in I(-). Analyses of phenolic compounds revealed that mushrooms in I(-) contained more phenols than I(+) and C, the latter containing the lower level of phenols. The fluctuation of the precursors of glutaminyl-4-hydroxyaniline (GHB) was less in I(-) than in I(+). The polyphenol oxidase (PPO) activities of irradiated mushrooms, analyzed via catechol oxidase, dopa oxidase, and tyrosine hydroxylase substrates, were found to be significantly lowered (p = 0.05) compared to C, with a further decrease in I(+). Analyses of the enzymes indicated that PPO activity was lower in I(+), contrasting with its lower phenols concentration. The observation of mushrooms' cellular membranes, by electronic microscopy, revealed a better preserved integrity in I(-) than in I(+). It is thus assumed that the browning effect observed in I(+) was caused by both the decompartmentation of vacuolar phenol and the entry of molecular oxygen into the cell cytoplasm. The synergetic effect of the residual active PPO and the molecular oxygen, in contact with the phenols, allowed an increased oxidation rate and, therefore, a more pronounced browning I(+) than in I(-).

Agaricales↗
PubMed1999

Expression of the androgen metabolizing enzyme UGT2B15 in adipose tissue and relative expression measurement using a competitive RT-PCR method.

OBJECTIVES: We have demonstrated previously that obesity in men was significantly associated with low plasma testosterone levels and higher concentrations of the androgen metabolite androstane-3 alpha, 17 beta-diol glucuronide, suggesting that androgen metabolism and elimination is increased in this condition. The objective of the present study was to investigate whether adipose tissue was a site of expression of the androgen metabolizing enzymes UDP-glucuronosyltransferases (UGT) 2B15 and 2B17. DESIGN AND PATIENTS: Subcutaneous and visceral adipose tissue was obtained from male patients subjected to various abdominal surgeries. MEASUREMENTS AND RESULTS: By performing reverse transcriptase-PCR (RT-PCR) amplification of mRNA extracted from adipose tissue samples, UGT2B15 transcript was detected in both subcutaneous and omental adipose tissue while UGT2B17 transcript expression was very low, or undetectable. A quantitative, competitive RT-PCR method was established and used to quantify UGT2B15 messenger RNA. The level of UGT2B15 expression was also measured in other human tissues. Although the major sites of expression were the liver and the lung, expression in adipose tissue was similar to levels found in the prostate, testis and mammary gland. CONCLUSIONS: These results demonstrate for the first time that both subcutaneous and visceral adipose tissue express androgen metabolizing enzyme UGT2B15 mRNA and further support the role of adipose tissue as a site of steroid metabolism.

Adipose Tissue↗
PubMed1999

Characterization and substrate specificity of UGT2B4 (E458): a UDP-glucuronosyltransferase encoded by a polymorphic gene.

Variations in glucuronidation activities among different individuals have been reported; however, genetic polymorphisms in the genes encoding phase II drug metabolizing UDP-glucuronosyltransferases have not been studied extensively. A novel UGT2B cDNA clone UGT2B4(E458) was isolated from human prostate and LNCaP cell cDNA libraries. The cDNA encoding UGT2B4(E458) is 2097 bp in length and has an open reading frame of 1584 nucleotides encoding a protein of 528 amino acids. Characterization of the UGT2B4(E458) cDNA revealed nucleotide differences with the previously published UGT2B4 and UGT2B11 cDNAs. These variations in the UGT2B4 sequence lead to an amino acid change from aspartic acid to glutamic acid at position 458. In the previous UGT2B11 cDNA (which has subsequently been renamed UGT2B4 (L109,396, D458)), leucine residues are found at positions 109 and 396, whereas phenylalanines are present at these positions in the UGT2B4(D458) and UGT2B4(E458) enzymes. Analysing the genomic DNA of 26 unrelated Caucasian individuals demonstrated the presence of variant alleles encoding UGT2B4(D458) and UGT2B4(E458). Stable expression of UGT2B4(E458) cDNA in HK293 cells demonstrates the presence of a 52 kDa protein, which is in agreement with other characterized (UGT2B proteins. UGT2B4(E458) conjugates hyodeoxycholic acid (HDCA) as well as 4-hydroxyestrone (4-OH-E1), androstane-3alpha,17beta-diol (3alpha-diol) and androsterone (ADT). Specific reverse transcriptase-polymerase chain reaction analysis revealed expression of UGT2B4(D458) and UGT2B4(E458) transcripts in a wide range of extrahepatic tissues, including the liver, kidney, testis, mammary gland, prostate, placenta, adipose, adrenal, skin and lung. Our results suggest that UGT2B4(E458) and UGT2B(E458) are two widely expressed isoenzymes, and that polymorphism in the UGT2B4 gene might be responsible for differences in UGT2B4 enzymatic properties.

Base Sequence↗
PubMed1998

Isolation and characterization of a human orphan UDP-glucuronosyltransferase, UGT2B11.

Glucuronidation is an important metabolic pathway for both endogenous and exogenous compounds. To isolate novel UGT2B cDNA clones, human prostate and LNCaP cell cDNA libraries were screened using a pool of steroid-specific UGT2B cDNA as probes. We have isolated a novel human cDNA of 1.7 kb in length containing an open reading frame of 1587 pb which encodes a deduced protein of 529 residues named UGT2B11. UGT2B11 share 91% identity in amino acids with UGT2B10, a UDP-glucuronosyltransferase (UGT) protein with unknown function. In agreement with other characterized UGT2B proteins, a Western blot analysis showed high levels of a 52-kDa protein present in a microsome preparation from HK293 cells stably transfected with the UGT2B11 cDNA. Despite the screening of 100 potential substrates, glucuronidation activity was not detected for the stably expressed UGT2B11 protein. However, UGT2B11 specific RT-PCR analysis revealed expression of the transcripts in a wide range of human tissues including the liver, kidney, mammary gland, prostate, skin, adipose, adrenal, and lung. The biological function of the UGT2B11 protein is unknown but its wide expression in human tissues raises the possibility that UGT2B11 may constitute an orphan UGT enzyme whose substrates specificity remain to be identified.

Amino Acid Sequence↗
PubMed1998

Isolation and characterization of a simian UDP-glucuronosyltransferase UGT2B18 active on 3-hydroxyandrogens.

A monkey cDNA, UGT2B18, encoding a UDP-glucuronosyltransferase (UGT) active on 3-hydroxyandrogens, has been isolated and characterized. Previous results suggested that the monkey represents the most appropriate animal model for studying the physiologic relevance of steroid UGTs. UGT2B18 was isolated from a cynomolgus monkey prostate cDNA library using human UGT2B7, UGT2B10 and UGT2B15 cDNA as probes. The cDNA is 1748 bp in length and contains an open reading frame of 1587 bp encoding a protein of 529 residues. The UGT2B18 cDNA clone was transfected into HK293 cells and a stable cell line expressing UGT2B18 protein was established. Western blot analysis of the UGT2B18-HK293 cell line using a human UGT2B17 polyclonal antibody (EL-93) revealed high expression of a 53 kDa UGT2B protein. The transferase activity of UGT2B18 was tested with over 60 compounds and was demonstrated to be principally active on C19 steroids having an hydroxyl group at position 3alpha of the steroid molecule. UGT2B18 was also active on planar phenols and bile acids. Kinetic analysis revealed that UGT2B18 glucuronidates 3-hydroxyandrogens with high velocity and affinity. Using cell homogenates, Km values of 5.1, 7.8 and 23 microM for androsterone (ADT), etiocholanolone and androstane-3alpha, 17beta diol (3alpha-diol) were obtained, respectively. Specific RT-PCR analysis demonstrated the expression of UGT2B18 transcripts in several tissues including liver, prostate, kidney, testis, adrenal, bile duct, bladder, colon, small intestine, cerebellum and pancreas suggesting a contribution of this isoenzyme to the high plasma levels of glucuronidated ADT and 3alpha-diol found in the cynomolgus monkey.

Amino Acid Sequence↗
PubMed1998

Effect of fibroblastic growth factors (FGF) on steroid UDP-glucuronosyltransferase expression and activity in the LNCaP cell line.

It is now widely accepted that factors other than androgens are crucial in the normal and abnormal growth of the prostate. In addition to hormones, many polypeptide growth factors, including the fibroblast growth factor family (FGF), can act as potent mitogens on cell proliferation. The FGF family of growth factors are essential factors for both normal and abnormal proliferation of prostate cells. To study the effect of FGFs on steroid glucuronidation, we used the human prostate cancer LNCaP cell line which is known to be stimulated by FGF resulting in increased cell proliferation. LNCaP cells express steroid metabolizing enzymes including uridine diphosphoglucuronosyltransferases (UGTs). In addition, LNCaP cells treated with dihydrotestosterone (DHT) and epidermal growth factor (EGF) express differential levels of the human UGT2B15 and UGT2B17 transcripts. In the present study, we examined the possible interaction between FGF and steroid UGT enzymes. Results show a dose dependent inhibition of DHT glucuronide (DHT-G) formation following treatment (6 days) with acidic FGF (aFGF) and basic FGF (bFGF). When cells were treated with 10 ng/ ml of FGFs, we observed 33 and 51% inhibition of glucuronidation activity using aFGF and bFGF respectively. Ribonuclease protection analyses revealed a 2 and 3 fold increase of UGT2B15 mRNA expression following treatment with aFGF (50 ng/ml) and bFGF (10 ng/ml) respectively. However, a slight decrease in UGT2B17 transcripts was observed, demonstrating a differential regulation. Since a reduction in the glucuronidation of DHT or its 5alpha-reduced metabolites may contribute to an increase in intraprostatic androgen levels, down-regulation of UGTs by growth factors such as FGFs may increase the proliferation of androgen-dependent tumors.

Androgens↗
PubMed1998

Characterization and regulation of UDP-glucuronosyltransferases in steroid target tissues.

Conjugation of compounds by glucuronidation is a pathway found in all vertebrates studied to date. Although, it is widely recognized that the liver is a major site of glucuronidation, it is now clear that extrahepatic tissues are also involved in the conjugation of compounds to which these tissues are exposed. High levels of androsterone glucuronide and androstane-3alpha,17beta-diol glucuronide found in the human prostate, breast cyst fluid and ovary follicular fluid suggest that glucuronidation of 5alpha-reduced C19 steroids occurs in these tissues. Recently, we have reported the tissue distribution of UGT2B15, which can conjugate steroids in several human extrahepatic steroid target tissues including the skin, breast and prostate. We have also isolated a new UGT2B cDNA encoding UGT2B17, that conjugates ADT which is the major 5alpha-reduced C19 steroid glucuronide in the circulation of humans. UGT2B17 is also widely distributed in several human steroid target tissues. This gene was mapped to human chromosome 4q13 and has an exon/intron structure similar to that of rat UGT2B1 and UGT2B2. Both UGT2B15 and UGT2B17, which are able to catalyze the glucuronidation of DHT, are expressed in LNCaP cells. Interestingly, glucuronidation of steroids is markedly regulated by several factors including androgens and growth factors. Treatment of LNCaP cells with dihydrotestosterone (DHT) and epidermal growth factor (EGF) caused a decrease of DHT glucuronidation and UGT2B mRNA levels. RNase protection assays showed a specific decrease of UGT2B17 transcript in LNCaP cells treated with DHT and EGF however, the level of UGT2B15 mRNA was not affected. As well, Western blot analysis demonstrated a diminution of UGT2B17 protein level in response to DHT and EGF. These results demonstrate a differential regulation of different isoforms of steroid conjugating UGTs present in human prostate LNCaP cells. In addition, UGT2B17 was shown to be more labile than UGT2B15 indicating that regulation of UGT2B17 expression would lead to a more rapid change in the level of glucuronidated steroids. Expression of exogenous UGT2B17 in LNCaP cells by gene transfer led to a significant decrease in the androgen response. This result indicates the ability of UGT enzymes to regulate the androgen response by conjugating androgens which abolishes their interaction with their receptor and facilitates their clearance from the cell. The glucuronidation of steroids by UGT enzymes is an important mechanism by which the levels of steroids is regulated in steroid target tissues.

Amino Acid Sequence↗
PubMed1998

Effect of interleukins on UGT2B15 and UGT2B17 steroid uridine diphosphate-glucuronosyltransferase expression and activity in the LNCaP cell line.

Cytokines are known to modulate the level of both phase 1 and phase 2 drug-metabolizing enzymes in hepatocytes. Although the effects of cytokines on cytochrome P450 (CYP450) enzymes are well understood, there is limited knowledge on how cytokines may affect steroid UDP-glucuronosyltransferase (UGT) phase 2 enzyme activity and expression in different cell types, including hepatocytes and steroid target cells. LNCaP cells, which is a human prostate cancer cell line, is a good model to study the effect of cytokines in steroid target cells because it is known to express steroidogenic enzymes, including UGT2B15 and UGT2B17, which are widely expressed steroid UGT enzymes known to conjugate androgens. In this study, we examined the possible interaction among interleukin-1alpha (IL-1alpha), IL-4, IL-6, and steroid UGT enzymes (UGT2B15 and UGT2B17). Treatment of LNCaP cells with IL-1alpha led to a dose-dependent inhibition of dihydrotestosterone (DHT) glucuronidation. IL-1alpha decreased both UGT activity and LNCaP cell proliferation in the absence and presence of DHT (0.5 nM); a maximal inhibition of 70% was observed. IL-6 inhibited LNCaP cell proliferation as well as the DHT-induced proliferation of these cells. However, neither IL-4 nor IL-6 significantly affected the formation of DHT glucuronide. Ribonuclease protection and Western blot analyses demonstrated a specific reduction of UGT2B17 transcript and protein levels in IL-1alpha-treated LNCaP cells. The level of UGT2B15 was not affected by cytokine treatments, indicating a differential regulation between these two UGT enzymes. Transfection experiments performed with the UGT2B17 gene promoter region indicates that the regulation occurs at the transcription level via putative cis-acting elements. This study indicates that cell proliferation and UGT expression in steroid-responsive cancer cells are differentially regulated depending on the cytokines present in the cell microenvironment.

Cell Division↗
PubMed1997

Upregulation of alpha1A- and alpha1B-adrenergic receptor mRNAs in the heart of cardiomyopathic hamsters.

An increased density of alpha1-adrenergic receptors (AR) has been linked to the development of necrotic lesions in the heart of hamsters with hereditary cardiomyopathy. To determine whether this increase results from an upregulation of the receptor mRNA(s), Northern blot analyses were carried out in the heart of 60-day-old control and cardiomyopathic hamsters using selective DNA probes for the three subtypes of alpha1-ARs (alpha1A, alpha1B and alpha1D). Transcripts for the three alpha1-ARs were detected in both control and cardiomyopathic hamsters. A two-fold increase in the alpha1A- and alpha1B-AR mRNA levels was observed in the cardiomyopathic hearts when compared to controls. In contrast, no change in the alpha1D-AR mRNA level could be detected. The enhancement in alpha1A- and alpha1B-AR mRNA levels was paralleled by a 20% increase in the total number of alpha1-ARs, as assessed by [3H]prazosin radioligand binding assays. Competition binding assays using subtype selective ligands indicated that the increased density of both alpha1A and alpha1B receptors contributes to the total alpha1-AR upregulation. Taken together, these data suggest that the early development of hereditary cardiomyopathy in hamsters is accompanied by a specific overexpression of the alpha1A- and alpha1B-ARs. A discrete increase of the alpha1-AR density could contribute to eliciting coronary microspasms, therefore participating in the development of focal necrotic lesions that are characteristic of the hamster cardiomyopathic model.

Animals↗
PubMed1997

Chromosomal localization, structure, and regulation of the UGT2B17 gene, encoding a C19 steroid metabolizing enzyme.

UGT2B17 is a UDP-glucuronosyltransferase enzyme expressed in several extrahepatic steroid target tissues, including the human prostate, where it glucuronidates C19 steroids such as dihydrotestosterone (DHT), androsterone (ADT), and androstane-3alpha, 17beta-diol (3alpha-diol). To determine if UGT2B17 is regulated by physiological effectors of the human prostate, DHT and epidermal growth factor (EGF) were demonstrated to specifically down-regulate the steady-state levels of UGT2B17 transcript and protein in LNCaP cells (Guillemette et al., 1997). These results implicate regulation of UGT2B17 at the level of gene transcription, therefore, a P-1-derived artificial chromosome (PAC) clone of 120 kb containing the entire UGT2B17 gene was isolated. The gene is comprised of six exons spanning approximately 30 kb, and fluorescence in situ hybridization of the UGT2B17 PAC clone to normal human lymphocyte chromosomes, mapped the gene to chromosome 4q13. To determine if the 5'-flanking DNA of the UGT2B17 gene is sufficient to confer gene expression, a 2,942-bp fragment was subcloned into a luciferase reporter plasmid and yielded an activity of 25-fold over background when transfected in LNCaP cells. However, transfection of the construct into HK-293, MCF-7, JEG-3, and HepG2 cells yielded only a moderate activity of two- to five-fold over background. Treatment of transfected LNCaP cells with 10 nM R1881, a nonmetabolizable analog of DHT, and 10 ng/ml EGF decreased the luciferase activity by 60%. This suggests that at least part, if not all, of the inhibitory effect of EGF and DHT on UGT2B17 is at the level of transcription. Progressive 5' deletions of the UGT2B17 5'-flanking region in the luciferase constructs alleviated the inhibition by R1881 and EGF, and revealed several potential responsive elements that may confer the observed regulation of the UGT2B17 gene. This study demonstrates regulation of the UGT2B17 gene by physiological effectors of the human prostate and supports the hypothesis that UGT enzymes are involved in steroid metabolism in extrahepatic tissues.

Amino Acid Sequence↗
PubMed1997

Expression and characterization of a novel UDP-glucuronosyltransferase, UGT2B9, from cynomolgus monkey.

Uridine diphosphate glucuronosyltransferases (UGTs) are important phase II detoxification enzymes. Despite the expression of UGT proteins in many species, previous results have suggested that simians represent the most appropriate animal model to study the glucuronidation of steroids in extrahepatic steroid target tissues. Northern blot analysis using a pool of human UGT2B cDNA probes demonstrated the expression of homologous UGT2B transcripts in several tissues including the liver, kidney, adrenal, breast, testis, and prostate of the cynomolgus monkey (Macacafascicularis). Western blot analyses using a polyclonal antibody raised against human UGT2B17 protein also demonstrated expression of homologous UGT2B proteins in monkey tissues. cDNA libraries were constructed from monkey liver and prostate mRNA and a novel UGT2B cDNA, UGT2B9, was isolated from both libraries. The UGT2B cDNA from the prostate library is 2,648 bp in length and contains an open reading frame of 1,587 bp encoding a protein of 529 residues. In vitro transcription/translation of the cDNA clone produced a protein of 52 kD. The UGT2B9 cDNA clone was transfected into HK293 cells and a stable cell line expressing UGT2B9 protein was established. The activity of UGT2B9 was tested with over 60 compounds and was demonstrated to be active on C18, C19, and C21 steroids, bile acids, and several xenobiotics including eugenol, 1-naphthol, and p-nitrophenol. Kinetic analysis revealed that UGT2B9 glucuronidates steroids with high affinity and efficiency with Km values of 0.2, 3.2, 0.2, and 1.8 microM for dihydrotestosterone, testosterone, androsterone, and 1,3,5,10-estratrien-3,4-diol-17-one, respectively. It is apparent that this simian UGT2B enzyme is specific for more different classes of steroids than any other UGT enzyme characterized to date, and may be related to the high plasma levels of glucuronidated C19 steroids found in the cynomolgus monkey.

Amino Acid Sequence↗
PubMed1997

Triplex-forming oligonucleotides with unexpected affinity for a nontargeted GA repeat sequence.

We examined the affinity and the specificity of triplex formation for different purine ODNs directed against two portions of a purine sequence derived from the mouse fli-1 gene. As expected, the ODNs antiparallel to the purine strand of their target can form triplex DNA. One parallel ODN showed binding to its target sequence. We explain this unusual binding by an interaction of the ODN with a GA repetition present in the sequence. We further examined the interaction of this ODN with a target composed of 14 GA repetitions. Unexpectedly, one ODN shows higher affinity for a partially complementary GA target relative to its completely complementary target. For another ODN, the binding to the GA target is weaker and might involve skipping of bases in a way that resembles alternate strand triplex formation.

Animals↗