Traditional plant functional groups explain variation in economic but not size-related traits across the tundra biome.

AIM: Plant functional groups are widely used in community ecology and earth system modelling to describe trait variation within and across plant communities. However, this approach rests on the assumption that functional groups explain a large proportion of trait variation among species. We test whether four commonly used plant functional groups represent variation in six ecologically important plant traits. LOCATION: Tundra biome. TIME PERIOD: Data collected between 1964 and 2016. MAJOR TAXA STUDIED: 295 tundra vascular plant species. METHODS: We compiled a database of six plant traits (plant height, leaf area, specific leaf area, leaf dry matter content, leaf nitrogen, seed mass) for tundra species. We examined the variation in species-level trait expression explained by four traditional functional groups (evergreen shrubs, deciduous shrubs, graminoids, forbs), and whether variation explained was dependent upon the traits included in analysis. We further compared the explanatory power and species composition of functional groups to alternative classifications generated using post hoc clustering of species-level traits. RESULTS: Traditional functional groups explained significant differences in trait expression, particularly amongst traits associated with resource economics, which were consistent across sites and at the biome scale. However, functional groups explained 19% of overall trait variation and poorly represented differences in traits associated with plant size. Post hoc classification of species did not correspond well with traditional functional groups, and explained twice as much variation in species-level trait expression. MAIN CONCLUSIONS: Traditional functional groups only coarsely represent variation in well-measured traits within tundra plant communities, and better explain resource economic traits than size-related traits. We recommend caution when using functional group approaches to predict tundra vegetation change, or ecosystem functions relating to plant size, such as albedo or carbon storage. We argue that alternative classifications or direct use of specific plant traits could provide new insights for ecological prediction and modelling.

Unravelling the transcriptomic landscape of the major phase II UDP-glucuronosyltransferase drug metabolizing pathway using targeted RNA sequencing.

A comprehensive view of the human UDP-glucuronosyltransferase (UGT) transcriptome is a prerequisite to the establishment of an individual's UGT metabolic glucuronidation signature. Here, we uncover the transcriptome landscape of the 10 human UGT gene loci in normal and tumoral metabolic tissues by targeted RNA next-generation sequencing. Alignment on the human hg19 reference genome identifies 234 novel exon-exon junctions. We recover all previously known UGT1 and UGT2 enzyme-coding transcripts and identify over 130 structurally and functionally diverse novel UGT variants. We further expose a revised genomic structure of UGT loci and provide a comprehensive repertoire of transcripts for each UGT gene. Data also uncover a remodelling of the UGT transcriptome occurring in a tissue- and tumor-specific manner. The complex alternative splicing program regulating UGT expression and protein functions is likely critical in determining detoxification capacity of an organ and stress-related responses, with significant impact on drug responses and diseases.

A relativistic type Ibc supernova without a detected gamma-ray burst.

Long duration gamma-ray bursts (GRBs) mark the explosive death of some massive stars and are a rare sub-class of type Ibc supernovae. They are distinguished by the production of an energetic and collimated relativistic outflow powered by a central engine (an accreting black hole or neutron star). Observationally, this outflow is manifested in the pulse of gamma-rays and a long-lived radio afterglow. Until now, central-engine-driven supernovae have been discovered exclusively through their gamma-ray emission, yet it is expected that a larger population goes undetected because of limited satellite sensitivity or beaming of the collimated emission away from our line of sight. In this framework, the recovery of undetected GRBs may be possible through radio searches for type Ibc supernovae with relativistic outflows. Here we report the discovery of luminous radio emission from the seemingly ordinary type Ibc SN 2009bb, which requires a substantial relativistic outflow powered by a central engine. A comparison with our radio survey of type Ibc supernovae reveals that the fraction harbouring central engines is low, about one per cent, measured independently from, but consistent with, the inferred rate of nearby GRBs. Independently, a second mildly relativistic supernova has been reported.

Recommendations on breast cancer screening and prevention in the context of implementing risk stratification: impending changes to current policies.

In recent years, risk stratification has sparked interest as an innovative approach to disease screening and prevention. The approach effectively personalizes individual risk, opening the way to screening and prevention interventions that are adapted to subpopulations. The international perspective project, which is developing risk stratification for breast cancer, aims to support the integration of its screening approach into clinical practice through comprehensive tool-building. Policies and guidelines for risk stratification-unlike those for population screening programs, which are currently well regulated-are still under development. Indeed, the development of guidelines for risk stratification reflects the translational aspects of perspective. Here, we describe the risk stratification process that was devised in the context of perspective, and we then explain the consensus-based method used to develop recommendations for breast cancer screening and prevention in a risk-stratification approach. Lastly, we discuss how the recommendations might affect current screening policies.

Contribution of (1,3)-beta-D-glucan to diagnosis of invasive candidiasis after liver transplantation.

Invasive candidiasis (IC) causes high morbidity and mortality rates after liver transplantation, in part due to delayed diagnosis. The fungal cell wall component (1,3)-beta-d-glucan (BG) could be an early biomarker of IC. This preliminary prospective study was designed to evaluate the contribution of BG measurements to the diagnosis of IC after liver transplantation. All consecutive patients who underwent liver transplantation at Henri Mondor Hospital in France between January and June 2013 were enrolled prospectively in the study. They were monitored weekly for colonization by Candida, and colonization index values were calculated. Serum samples were tested for BG (Fungitell; Cape Cod Inc.) at least weekly between liver transplantation and discharge from the hospital. A total of 52 patients (including 39 male patients) were enrolled, with a median age of 55 years (range, 31 to 69 years). The median Model for End-Stage Liver Disease (MELD) score was 27 (range, 6 to 40). Cultures from 42 patients (81%) yielded Candida spp., with the most common Candida species isolated being Candida glabrata (47%). Six cases of documented IC were found for four of the 52 patients. On the day the clinical diagnosis of IC was made, analysis based on combining two sequential BG-positive samples (>146 pg/ml) and a colonization index of ≥0.5 revealed sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) results of 83%, 89%, 50%, and 97.6%, respectively. The detection of BG associated with Candida colonization may be a promising tool based on a high NPV that can rule out IC among high-risk patients.

A novel UGT1 marker associated with better tolerance against irinotecan-induced severe neutropenia in metastatic colorectal cancer patients.

The risk of severe irinotecan-induced neutropenia has been shown to be related to the UGT1 variant UGT1A1*28, which increases exposure to the potent metabolite SN-38. Our goal was to identify a novel UGT1 marker(s) using 28 haplotype-tagged single nucleotide polymorphisms genotyped by mass spectrometry. By characterizing the UGT1 sequence from a cohort of 167 Canadian metastatic colorectal cancer (mCRC) patients and a validation cohort of 250 Italian mCRC patients, we found rs11563250G, located in the intergenic region downstream of UGT1, to be significantly associated with reduced risk of severe neutropenia (odds ratio (OR)=0.21; P=0.043 and OR=0.27; P=0.036, respectively, and OR=0.31 when combined; P=0.001), which remained significant upon correction for multiple testing in the combined cohort (P=0.041). For the two-marker haplotype rs11563250G and UGT1A1*1 (rs8175347 TA6), the OR was of 0.17 (P=0.0004). Genetic testing of this marker may identify patients who might benefit from increased irinotecan dosing.

Candida vascular complication in a liver transplant recipient due to yeast contamination of preservation solution.

Infections remain a major cause of morbidity and mortality after liver transplantation. One possible cause of infection is preservation fluid contamination. Donor-derived pathogens, such as Candida albicans, have occasionally produced life-threatening complications in organ recipients, already described in renal transplantation. In the present case, we report the loss of a liver graft secondary to vascular complications because of C. albicans found in the preservation fluid. Our case report raises the question of implementing procedures, similar to those in renal transplantation, including early antifungal treatment and repeated radiological monitoring for the prevention and detection of vascular complications.

Prehabilitation in hepato-pancreato-biliary surgery: A systematic review and meta-analysis. A necessary step forward evidence-based sample size calculation for future trials.

INTRODUCTION: Prehabilitation is defined as preoperative conditioning of patients in order to improve post-operative outcomes. Some studies showed an increase in functional recovery following colorectal surgery, but its effect in hepato-pancreato-biliary (HPB) surgery is unclear. The aim of this study was to realize a systematic literature review and meta-analysis on the current available evidence on prehabilitation in HPB surgery. MATERIALS AND METHODS: A systematic review and a metanalysis were carried out on prehabilitation (physical, nutritional and psychological interventions) in HPB surgery (2009-2019). Assessed outcomes were postoperative complications, length of stay (LOS), 30-day readmission, and mortality. MAIN RESULTS: Four studies among the 191 screened were included in this systematic review (3 randomized controlled trials, 1 case-control propensity score study), involving 419 patients (prehabilitation group, n=139; control group, n=280). After pooling, no difference was observed on LOS ((-4.37 days [95% CI: -8.86; 0.13]) or postoperative complications (RR 0.83 [95%CI: 0.62; 1.10]), reported by all the included studies. Two trials reported on readmission rate, but given the high heterogeneity, a meta-analysis was not realized. No deaths were reported among the included studies. CONCLUSION: No effect of prehabilitation programs in HPB surgery was observed on LOS or postoperative complications rate. Future trials with standardized outcomes of measure, and adequately powered samples calculations are thus required. PROSPERO REGISTRATION: CRD42020165218.

Non-invasive ICG-clearance: a useful tool for the management of hepatic artery thrombosis following liver transplantation.

BACKGROUND: The clinical presentation of hepatic artery thrombosis (HAT) post-liver transplantation (LT) varies considerably. Doppler ultrasonography (Doppler US) is the first line investigation, with a diagnostic sensitivity for HAT as high as 92%. Because indocyanine green (ICG) elimination from the blood depends among other factors on the hepatic blood flow, we hypothesized that plasma disappearance rate of indocyanine green (PDR-ICG) can be influenced by the flow in the hepatic artery. Thus, we evaluated the role of PDR-ICG measurement in HAT diagnosis in post-LT patients. PATIENTS AND METHODS: Fourteen liver transplant patients with no visible flow in the hepatic artery (Doppler US) were identified. Of the 14, seven patients had HAT confirmed by CT-angiography. The PDR-ICG measurement, an investigation routinely used in our center, was performed in all 14 patients. RESULTS: The PDR-ICG in patients with HAT was significantly lower than in patients without HAT (5.8 ± 4.3 vs. 23.8 ± 7.4%/min, p= 0.0009). In patients with HAT, after the revascularization, the PDR-ICG value increased (5.8 ± 4.3 vs. 15.6 ± 3.5%/min, p = 0.006). CONCLUSION: The ICG elimination may be an adjunct diagnostic tool in the management of patients with suspected HAT following LT.

A case of recurrent vulvar carcinoma treated with erlotinib, an EGFR inhibitor.

BACKGROUND: There is no effective therapy for patients with regional and/or distant recurrence of vulvar carcinoma. Recently two case reports about the use of erlotinib, an EGFR (epithelial growth factor receptor) inhibitor, in the context of recurrent vulvar cancer were published with a good clinical response reported. CASE: We report a case where erlotinib was used in a 67-year-old patient with recurrent and multi-treated vulvar carcinoma. Utilization of erlotinib was started with rapid clinical improvement. The treatment was well tolerated with palliation of symptoms. A CT scan also showed cutoff "net" improvement, with regression of size and number of hilar and pulmonary metastases. After one month of improvement, despite continuous treatment with erlotinib, dyspnea returned. A new CT scan showed an increased number of hilar nodes, a new hepatic lesion and increase in the size of the known pelvic lesion. CONCLUSION: EGFR inhibitors appear to be promising agents for this devastating and fatal disease. As with other studies with these agents, our patient showed a rapid response with important palliation of symptoms, however of short duration.

Expression of stromelysin-3 in atherosclerotic lesions: regulation via CD40-CD40 ligand signaling in vitro and in vivo.

Stromelysin-3 is an unusual matrix metalloproteinase, being released in the active rather than zymogen form and having a distinct substrate specificity, targeting serine proteinase inhibitors (serpins), which regulate cellular functions involved in atherosclerosis. We report here that human atherosclerotic plaques (n = 7) express stromelysin-3 in situ, whereas fatty streaks (n = 5) and normal arterial specimens (n = 5) contain little or no stromelysin-3. Stromelysin-3 mRNA and protein colocalized with endothelial cells, smooth muscle cells, and macrophages within the lesion. In vitro, usual inducers of matrix metalloproteinases such as interleukin-1, interferon-gamma, or tumor necrosis factor alpha did not augment stromelysin-3 in vascular wall cells. However, T cell-derived as well as recombinant CD40 ligand (CD40L, CD154), an inflammatory mediator recently localized in atheroma, induced de novo synthesis of stromelysin-3. In addition, stromelysin-3 mRNA and protein colocalized with CD40L and CD40 within atheroma. In accordance with the in situ and in vitro data obtained with human material, interruption of the CD40-CD40L signaling pathway in low density lipoprotein receptor-deficient hyperlipidemic mice substantially decreased expression of the enzyme within atherosclerotic plaques. These observations establish the expression of the unusual matrix metalloproteinase stromelysin-3 in human atherosclerotic lesions and implicate CD40-CD40L signaling in its regulation, thus providing a possible new pathway that triggers complications within atherosclerotic lesions.

Management of liver failure in general intensive care unit.

OBJECTIVE: To produce French guidelines on Management of Liver failure in general Intensive Care Unit (ICU). DESIGN: A consensus committee of 23 experts from the French Society of Anesthesiology and Critical Care Medicine (Société française d'anesthésie et de réanimation, SFAR) and the French Association for the Study of the Liver (Association française pour l'étude du foie, AFEF) was convened. A formal conflict-of-interest (COI) policy was developed at the start of the process and enforced throughout. The entire guideline process was conducted independently of any industrial funding. The authors were advised to follow the principles of the Grading of Recommendations Assessment, Development and Evaluation (GRADE) system to guide their assessment of the quality of evidence. The potential drawbacks of making strong recommendations in the presence of low-quality evidence were emphasised. Some recommendations were ungraded. METHODS: Two fields were defined: acute liver failure (ALF) and cirrhotic patients in general ICU. The panel focused on three questions with respect to ALF: (1) Which etiological examinations should be performed to reduce morbidity and mortality? (2) Which specific treatments should be initiated rapidly to reduce morbidity and mortality? (3) Which symptomatic treatment should be initiated rapidly to reduce morbidity and mortality? Seven questions concerning cirrhotic patients were addressed: (1) Which criteria should be used to guide ICU admission of cirrhotic patients in order to improve their prognosis? (2) Which specific management of kidney injury should be implemented to reduce morbidity and mortality in cirrhotic ICU patients? (3) Which specific measures to manage sepsis in order to reduce morbidity and mortality in cirrhotic ICU patients? (4) In which circumstances, human serum albumin should be administered to reduce morbidity and mortality in cirrhotic ICU patients? (5) How should digestive haemorrhage be treated in order to reduce morbidity and mortality in cirrhotic ICU patients? (6) How should haemostasis be managed in order to reduce morbidity and mortality in cirrhotic ICU patients? And (7) When should advice be obtained from an expert centre in order to reduce morbidity and mortality in cirrhotic ICU patients? Population, intervention, comparison and outcome (PICO) issues were reviewed and updated as required, and evidence profiles were generated. An analysis of the literature and recommendations was then performed in accordance with the GRADE® methodology. RESULTS: The SFAR/AFEF Guidelines panel produced 18 statements on liver failure in general ICU. After two rounds of debate and various amendments, a strong agreement was reached on 100% of the recommendations: six had a high level of evidence (Grade 1 ±), seven had a low level of evidence (Grade 2 ±) and six were expert judgments. Finally, no recommendation was provided with respect to one question. CONCLUSIONS: Substantial agreement exists among experts regarding numerous strong recommendations on the optimum care of patients with liver failure in general ICU.

Use of targeted therapy in patients with metastatic renal cell carcinoma: clinical and economic impact in a Canadian real-life setting.

Introduction: Outside of randomized controlled clinical trials, the understanding of the effectiveness and costs associated with targeted therapies for metastatic renal cell carcinoma (mrcc) is limited in Canada. The purpose of the present study was to use real-world prospective data to assess the effectiveness and cost of targeted therapies for patients with mrcc. Methods: The Canadian Kidney Cancer Information System, a pan-Canadian database, was used to identify prospectively collected data relating to patients with mrcc. First- and subsequent-line time to treatment termination (ttt) was determined from therapy initiation time (sunitinib or pazopanib) to discontinuation of therapy. Kaplan-Meier survival curves were used to estimate the unadjusted and adjusted overall survival (os) by treatment. Unit treatment cost was used to estimate the cost by line of treatment and the total cost of therapy for the management of patients with mrcc. Results: The study included 475 patients receiving sunitinib or pazopanib in the first-line setting. Patients were treated mostly with sunitinib (81%); 19% of patients were treated with pazopanib. The median ttt in the first line was 7.7 months for patients receiving sunitinib and 4.6 months for those receiving pazopanib (p < 0.001). The adjusted os was 32 months with sunitinib and 21 months with pazopanib (hazard ratio: 1.61; p < 0.01). The total median cost of first- and second-line treatments was $56,476 (interquartile range: $23,738-$130,447) for patients in the sunitinib group and $46,251 (interquartile range: $28,167-$91,394) for those in the pazopanib group. Conclusions: For the two therapies, os differed significantly, with a higher median os being observed in the sunitinib group. The cost of treatment was higher in the sunitinib group, which is to be expected with longer survival.

The impact of UGT1A8, UGT1A9, and UGT2B7 genetic polymorphisms on the pharmacokinetic profile of mycophenolic acid after a single oral dose in healthy volunteers.

We studied whether polymorphisms in the UGT1A8, UGT1A9, and UGT2B7 genes, the enzymes producing the phenolic (MPAG) and acyl (AcMPAG) glucuronides of mycophenolic acid (MPA), could contribute to the interindividual variation observed in mycophenolate mofetil (MMF) pharmacokinetics (PKs). This study enrolled 17 healthy volunteers with no polymorphisms (controls) and 17 carriers of UGT1A9 -275/-2152 selected among 305 individuals genetically screened for UDP-glucuronosyltransferase (UGT) polymorphisms. Additional investigative groups included carriers of UGT1A8*2 (A173G) (n=9), UGT1A8*3 (C277Y) (n=4), and UGT1A9*3 (M33T) (n=5). Genetic analysis also included UGT2B7 to detect UGT2B7*2 (His268Tyr) and the promoter haplotype -1248A>G, -1241T>C, -1054T>C, -842G>A, -268A>G, -102T>C. Kinetics were measured in plasma and urine after a single 1.5 g oral dose of MMF, by high-performance liquid chromatography coupled with tandem mass spectrometry, over 12 h after drug intake. Compared to controls, MPA exposure was significantly lower for UGT1A9 -275/-2152 carriers, with no significant changes in MPAG. The estimates of enterohepatic (re)cycling (area under the concentration-time curve (AUC6-12 h/AUC0-12 h)) were significantly lower for MPA, MPAG, and AcMPAG in UGT1A9 -275/-2152 subjects. Compared with controls, UGT1A9*3 carriers had higher MPA and AcMPAG exposure, whereas homozygosity for the UGT1A8*2 allele and heterozygosity for UGT1A8*3 allele had no impact on MPA PKs. Compared with UGT2B7*1/*1 individuals (n=10), UGT2B7*2/*2 subjects (n=17) presented significantly higher free MPA C(max) values and elevated free and total MPA. Results indicate that after a single oral dose of MMF in healthy volunteers, specific UGT genotypes significantly alter MPA PKs and this clearly warrants additional studies with complete and detailed genetic profiling of UGT1A8, UGT1A9, and UGT2B7 genes.

Effects of alloantibodies to human leukocyte antigen on endothelial expression and serum levels of thrombomodulin.

Essentials The effect of alloantibodies on the endothelial expression of thrombomodulin is unknown. Thrombomodulin was quantified in stimulated endothelial cells and measured in serum samples. Anti-human leukocyte antigen (HLA) I vs. II antibodies have different effects on thrombomodulin. Anti-HLA II antibodies may promote a prothrombotic state and contribute to microangiopathy. SUMMARY: Rationale Thrombomodulin (TBM) is an anticoagulant and anti-inflammatory transmembrane protein expressed on endothelial cells. Donor-specific alloantibodies, particularly those against human leukocyte antigen (HLA) class II, are associated with microvascular endothelial damage in solid allografts. Objective Our aim was to characterize the effects of anti-HLA antibodies on endothelial expression of TBM, and in particular, the differential effects of anti-HLA class I compared with those of anti-HLA class II. Methods We used human glomerular microvascular endothelial cells to examine TBM expression on anti-HLA-treated cells, and we tested sera from transplant recipients for soluble TBM. Results We found that whereas membrane TBM expression increased in a dose-dependent manner in the presence of anti-HLA class I antibodies, treatment with anti-HLA class II led to minimal TBM expression on the endothelial surface but to a cytosolic accumulation. Platelet adhesion studies confirmed the functional impact of anti-HLA class II. Quantitative densitometry of the membrane lysates further suggested that anti-HLA class II impairs TBM glycosylation. Furthermore, we found a significant association between the presence of circulating anti-HLA class II antibodies in transplant recipients and low serum levels of TBM. Conclusion These results indicate that ligation of anti-HLA class I and II antibodies produces different effects on the endothelial expression of TBM and on serum levels of TBM in transplant recipients. Anti-HLA class II antibodies may be associated with a prothrombotic state, which could explain the higher occurrence of microangiopathic lesions in the allograft and the poor outcomes observed in patients with these alloantibodies.

Isolation and characterization of the human UGT2B15 gene, localized within a cluster of UGT2B genes and pseudogenes on chromosome 4.

Glucuronidation is a major pathway of androgen metabolism and is catalyzed by UDP-glucuronosyltransferase (UGT) enzymes. UGT2B15 and UGT2B17 are 95% identical in primary structure, and are expressed in steroid target tissues where they conjugate C19 steroids. Despite the similarities, their regulation of expression are different; however, the promoter region and genomic structure of only the UGT2B17 gene have been characterizedX to date. To isolate the UGT2B15 gene and other novel steroid-conjugating UGT2B genes, eight P-1-derived artificial chromosomes (PAC) clones varying in length from 30 kb to 165 kb were isolated. The entire UGT2B15 gene was isolated and characterized from the PAC clone 21598 of 165 kb. The UGT2B15 and UGT2B17 genes are highly conserved, are both composed of six exons spanning approximately 25 kb, have identical exon sizes and have identical exon-intron boundaries. The homology between the two genes extend into the 5'-flanking region, and contain several conserved putative cis-acting elements including Pbx-1, C/EBP, AP-1, Oct-1 and NF/kappaB. However, transfection studies revealed differences in basal promoter activity between the two genes, which correspond to regions containing non-conserved potential elements. The high degree of homology in the 5'-flanking region between the two genes is lost upstream of -1662 in UGT2B15, and suggests a site of genetic recombination involved in duplication of UGT2B genes. Fluorescence in situ hybridization mapped the UGT2B15 gene to chromosome 4q13.3-21.1. The other PAC clones isolated contain exons from the UGT2B4, UGT2B11 and UGT2B17 genes. Five novel exons, which are highly homologous to the exon 1 of known UGT2B genes, were also identified; however, these exons contain premature stop codons and represent the first recognized pseudogenes of the UGT2B family. The localization of highly homologous UGT2B genes and pseudogenes as a cluster on chromosome 4q13 reveals the complex nature of this gene locus, and other novel homologous UGT2B genes encoding steroid conjugating enzymes are likely to be found in this region of the genome.

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.

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.

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.