Segmented 3D Echo Planar Acquisition for Rapid Susceptibility-Weighted Imaging: Application to Microhemorrhage Detection in Traumatic Brain Injury.

BACKGROUND: Susceptibility-weighted imaging (SWI) provides superior image contrast of cerebral microhemorrhages (CMBs). It is based on a three-dimensional (3D) gradient echo (GRE) sequence with a relatively long imaging time. PURPOSE: To evaluate whether an accelerated 3D segmented echo planar imaging SWI is comparable to GRE SWI in detecting CMBs in traumatic brain injury (TBI). STUDY TYPE: Prospective. SUBJECTS: Four healthy volunteers and 46 consecutive subjects (38.0 ± 14.4 years, 16 females; 12 mild, 13 moderate, and 7 severe TBI). FIELD STRENGTH/SEQUENCE: A 3 T scanner/3D gradient echo and 3D segmented echo planar imaging (segEPI). ASSESSMENT: Brain images were acquired using GRE and segEPI in a single session (imaging time = 9 minutes 47 seconds and 1 minute 30 seconds, respectively). The signal-to-noise ratio (SNR) calculated from healthy volunteer thalamus and centrum semiovale were compared. CMBs were counted by three raters blinded to diagnostic information. STATISTICAL TESTS: A t-test was used to assess SNR difference. Pearson correlation and Wilcoxon signed-rank test were performed using CMB counts. The intermethod agreement was evaluated using Bland-Altman method. Intermethod and interrater reliabilities of image-based diffuse axonal injury (DAI) diagnoses were evaluated using Cohen's kappa and percent agreement. P ≤ 0.05 was considered statistically significant. RESULTS: Thalamus SNRs were 16.9 ± 2.2 and 16.5 ± 3 for GRE and segEPI (P = 0.84), respectively. Centrum semiovale SNRs were 25.8 ± 4.6 and 21.1 ± 2.7 (P = 0.13). The correlation coefficient of CMBs was 0.93, and differences were not significant (P = 0.56-0.85). For DAI diagnoses, Cohen's kappa was 0.62-0.84 and percent agreement was 85%-94%. DATA CONCLUSION: CMB counts on segEPI and GRE were highly correlated, and DAI diagnosis was made equally effectively. segEPI SWI can potentially replace GRE SWI in detecting TBI CMBs, especially when time constraints are critical. EVIDENCE LEVEL: 1 TECHNICAL EFFICACY: Stage 2.

Lung-Targeted Delivery of Dimethyl Fumarate Promotes the Reversal of Age-Dependent Established Lung Fibrosis.

Idiopathic pulmonary fibrosis (IPF), a severe and deadly form of lung fibrosis, is widely regarded as a disease of aging. We previously demonstrated that aged mice with persistent lung fibrosis and IPF lung myofibroblasts exhibit deficient Nrf2-mediated antioxidant responses. Tecfidera is an orally administered FDA-approved drug for the treatment of multiple sclerosis, where the active pharmaceutical ingredient is dimethyl fumarate (DMF), an active Nrf2 activator. However, no studies have evaluated the efficacy of DMF for age-associated persistent lung fibrosis. Here, we demonstrate that in IPF lung fibroblasts, DMF treatment inhibited both TGF-ß-mediated pro-fibrotic phenotypes and led to a reversal of established pro-fibrotic phenotypes. We also evaluated the pre-clinical efficacy of lung-targeted (inhaled) vs. systemic (oral) delivery of DMF in an aging murine model of bleomycin-induced persistent lung fibrosis. DMF or vehicle was administered daily to aged mice by oral gavage or intranasal delivery from 3-6 weeks post-injury when mice exhibited non-resolving lung fibrosis. In contrast to systemic (oral) delivery, only lung-targeted (inhaled) delivery of DMF restored lung Nrf2 expression levels, reduced lung oxidative stress, and promoted the resolution of age-dependent established fibrosis. This is the first study to demonstrate the efficacy of lung-targeted DMF delivery to promote the resolution of age-dependent established lung fibrosis.

Novel CD33 antibodies unravel localization, biology and therapeutic implications of CD33 isoforms.

The aim of this study was to establish the therapeutic relevance of the CD33D2 isoform by developing novel antibodies targeting the IgC domain of CD33. Two novel IgC-targeting antibodies, HL2541 and 5C11-2, were developed, and CD33 isoforms were assessed using multiple assays in cells overexpressing either CD33FL or CD33D2 isoforms, unmodified acute myeloid leukemia (AML) cell lines and primary AML specimens representing different genotypes for the CD33 splicing single nucleotide polymorphism. CD33D2 was recognized on cells overexpressing CD33D2 and unmodified AML cell lines; however, minimal/no cell surface detection of CD33D2 was observed in primary AML specimens. Both isoforms were detected intracellularly using novel antibodies. Minimal cell surface expression of CD33D2 on primary AML/progenitor cells warrants further studies on anti-CD33D2 immunotherapeutics.

Pharmacogenomics of poor drug metabolism in Greyhounds: Cytochrome P450 (CYP) 2B11 genetic variation, breed distribution, and functional characterization.

Greyhounds recover more slowly from certain injectable anesthetics than other dog breeds. Previous studies implicate cytochrome P450 (CYP) 2B11 as an important clearance mechanism for these drugs and suggest Greyhounds are deficient in CYP2B11. However, no CYP2B11 gene mutations have been identified that explain this deficiency in Greyhounds. The objectives of this study were to provide additional evidence for CYP2B11 deficiency in Greyhounds, determine the mechanisms underlying this deficiency, and identify CYP2B11 mutations that contribute to this phenotype in Greyhounds. Greyhound livers metabolized CYP2B11 substrates slower, possessed lower CYP2B11 protein abundance, but had similar or higher mRNA expression than other breeds. Gene resequencing identified three CYP2B11 haplotypes, H1 (reference), H2, and H3 that were differentiated by mutations in the gene 3'-untranslated region (3'-UTR). Compared with 63 other dog breeds, Greyhounds had the highest CYP2B11-H3 allele frequency, while CYP2B11-H2 was widely distributed across most breeds. Using 3'-UTR luciferase reporter constructs, CYP2B11-H3 showed markedly lower gene expression (over 70%) compared to CYP2B11-H1 while CYP2B11-H2 expression was intermediate. Truncated mRNA transcripts were observed in CYP2B11-H2 and CYP2B11-H3 but not CYP2B11-H1 transfected cells. Our results implicate CYP2B11 3'-UTR mutations as a cause of decreased CYP2B11 enzyme expression in Greyhounds through reduced translational efficiency.

Thoracic involvement in Erdheim-Chester disease: computed tomography imaging findings and their association with the BRAFV600E mutation.

OBJECTIVES: To investigate the computed tomography (CT) thoracic findings in Erdheim-Chester disease (ECD) and evaluate the association of these findings with the BRAFV600E mutation. METHODS: This was a prospective study of patients with ECD (n=61, men=46) who underwent thoracic CT imaging. CT examinations were independently interpreted by two experienced radiologists. Association of imaging findings with BRAFV600E was achieved via the Chi-square or Fisher's exact test and odds ratios (OR) with 95% confidence intervals (CI), as appropriate. RESULTS: Fifty-five ECD patients (90%) showed pulmonary findings, which included interlobular septal thickening (69%), pulmonary nodules (62%), airway thickening (13%) and ground glass opacities (36%). Pulmonary nodules were classified by the pattern of distribution: subpleural regions (36%), lung parenchyma (13%) and both regions (13%). Pleural and mediastinal involvement were present in 15% and 62% of cases, respectively. The most common mediastinal finding was sheathing of the right coronary artery (34%), followed by sheathing of the thoracic aorta (30%). The BRAFV600E mutation, positive in 31 patients, was associated with the frequency of sheathing of the coronary arteries (p = 0.01). CONCLUSIONS: Of the thoracic findings reported in this study, we found a statistically significant positive association between the BRAFV600E mutation and presence of coronary artery sheathing. KEY POINTS: • To assess the degree of thoracic involvement in ECD with CT. • BRAF V600E mutation has a high association with right coronary artery sheathing. • BRAF V600E genetic testing detects patients at high risk of developing RCA sheathing.

Abdominal involvement in Erdheim-Chester disease (ECD): MRI and CT imaging findings and their association with BRAFV600E mutation.

OBJECTIVES: To use magnetic resonance imaging (MRI) and computed tomography (CT) to define abdominal involvement in Erdheim-Chester disease (ECD), and to investigate the association between these findings and the BRAFV600E mutation. METHODS: This prospective study was performed on 61 ECD patients (46 men). The MRI and CT imaging studies were reviewed independently by two experienced radiologists. The association between BRAFV600E mutation and imaging findings was analysed using Fisher's exact test, and odds ratios with 95% confidence intervals. RESULTS: Perinephric infiltration was the most common finding (67%), followed by involvement of proximal ureters (61%). In 56% of cases, infiltration extended to the renal sinuses, and in 38% caused hydronephrosis. Adrenal gland infiltration was present in 48% of patients. Infiltration of renal artery (49%) and aorta (43%) were the most common vascular findings, followed by sheathing of celiac, superior mesenteric artery (SMA) or inferior mesenteric artery (IMA) (23%). The BRAFV600E mutation was positive in 53% of patients with interpretable BRAF sequencing. There was a statistically significant association between this mutation and perinephric infiltration (p = 0.003), renal sinus involvement (p < 0.001), infiltration of proximal ureters (p < 0.001), hydronephrosis (p < 0.001), adrenal gland involvement (p < 0.001), periaortic infiltration (p = 0.03), sheathing or stenosis of renal artery (p < 0.001) and sheathing of other aortic branches (p = 0.04). CONCLUSIONS: Renal and vascular structures are the most commonly affected abdominal organs in ECD patients. Some of these findings have significant positive association with the BRAFV600E mutation. KEY POINTS: • Abdominal imaging plays a crucial role in management of Erdheim-Chester disease. • Significant associations exist between BRAF V600E mutation and several abdominal imaging findings. • Considering several associations, evaluating BRAFV600E mutation status is recommended in ECD patients.

Identification and validation of the microRNA response elements in the 3'-untranslated region of the UDP glucuronosyltransferase (UGT) 2B7 and 2B15 genes by a functional genomics approach.

Posttranscriptional repression of UDP-glucuronosyltransferase (UGT) 2B7 and 2B15 expression by microRNAs (miRNAs) may be an important mechanism underlying inter-individual variability in drug glucuronidation. Furthermore, the UGT2B15 3'-UTR contains a common SNP (rs3100) that could influence miRNA binding. The aim of this study was to identify the complete complement of miRNAs that could regulate UGT2B7 and UGT2B15 expression through binding to the reference and/or variant 3'-UTRs. Luciferase reporter plasmids containing either the reference or variant 3'-UTRs were screened against a 2,048 human miRNA library to identify those miRNAs that decrease luciferase activity by at least 30% when co-transfected into HEK293 cells. Six novel miRNAs (miR-1293, miR-3664-3p, miR-4317, miR-513c-3p, miR-4483, and miR-142-3p) were identified that repressed the reference UGT2B7 3'-UTR, while twelve novel miRNAs (miR-770-5p, miR-103b, miR-3924, miR-376b-3p, miR-455-5p, miR-605, miR-624-3p, miR-4712-5p, miR-3675-3p, miR-6500-5p, miR-548as-3p, and miR-4292) repressed both the reference and rs3100 variant UGT2B15 3'-UTR. Deletion and mutagenesis studies confirmed the binding site location of each miRNA. Although the UGT2B15 rs3100 SNP was located within the miR-376c-3p response element, there was no effect on miRNA binding. miR-142-3p, miR-3664-3p, miR-4317, miR-455-5p, miR-376c-3p, miR-770-5p, miR-3675-3p, miR-331-5p, miR-605, and miR-376b-3p transcript levels were measured by quantitative PCR and correlated with UGT2B7 and UGT2B15 enzyme activities in 27 human liver samples. A significant negative correlation (Rs = -0.53; p = 0.005) was demonstrated between hepatic miR-455-5p transcript levels and UGT2B15-mediated S-oxazepam glucuronidation activities. Thus, the UGT2B7 and UGT2B15 3'-UTRs contain miRNA response elements for multiple miRNAs that may contribute to variable drug glucuronidation.

Marketed drugs used for the management of hypercholesterolemia as anticancer armament.

The design of novel pharmacologic agents as well as their approval for sale in markets all over the world is a tedious and pricey process. Inevitably, oncologic patients commonly experience unwanted effects of new anticancer drugs, while the acquisition of clinical experience for these drugs is largely based on doctor-patient partnership which is not always effective. The repositioning of marketed non-antineoplastic drugs that hopefully exhibit anticancer properties into the field of oncology is a challenging option that gains ground and attracts preclinical and clinical research in an effort to override all these hindrances and minimize the risk for reduced efficacy and/or personalized toxicity. This review aims to present the anticancer properties of drugs used for the management of hypercholesterolemia. A global view of the antitumorigenicity of all marketed antihypercholesterolemic drugs is of major importance, given that atherosclerosis, which is etiologically linked to hypercholesterolemia, is a leading worldwide cause of morbidity and mortality, while hypercholesterolemia and tumorigenesis are known to be interrelated. In vitro, in vivo and clinical literature data accumulated so far outline the mechanistic basis of the antitumor function of these agents and how they could find application at the clinical setting.

Identification and validation of microRNAs directly regulating the UDP-glucuronosyltransferase 1A subfamily enzymes by a functional genomics approach.

Posttranscriptional repression of UDP-glucuronosyltransferase (UGT) 1A expression by microRNAs (miRNAs) may be an important mechanism underlying interindividual variability in drug glucuronidation. Furthermore, the UGT1A 3'-UTR shared by all UGT1A enzymes is polymorphic, containing three linked SNPs (rs10929303, rs1042640, and rs8330) that could influence miRNA binding. The aim of this study was to identify the complete complement of miRNAs that could regulate UGT1A expression through binding to the reference and/or common variant UGT1A 3'-UTR. Luciferase reporter plasmids containing either the reference or variant UGT1A 3'-UTR were screened against a 2048 human miRNA library to identify those miRNAs that decrease luciferase activity by at least 30% when co-transfected into HEK293 cells. Four novel miRNAs (miR-103b, miR-141-3p, miR-200a-3p, and miR-376b-3p) were identified that repressed both reference and variant UGT1A 3'-UTR, while two other miRNAs selectively repressed the reference (miR-1286) or variant (miR-21-3p) 3'-UTR. Deletion and mutagenesis studies confirmed the binding site location for each miRNA. rs8330 disrupted miR-1286 binding to the reference UGT1A 3'-UTR, while rs10929303 enhanced miR-21-3p binding to the variant 3'-UTR. Transfection of miR-21-3p, miR-103b, miR-141-3p, miR-200a-3p, and miR-376b-3p mimics into LS180 human intestinal cells showed repression of UGT1A1 and UGT1A6 mediated glucuronidation and mRNA without affecting UGT2B7 activity or mRNA. Furthermore, transfection of miR-21-3p, miR-141-3p, and miR-200a-3p into primary human hepatocytes, repressed UGT1A1 activity and mRNA without affecting CYP3A activity. Finally, miR-21-3p and miR-200a-3p expression were negatively correlated with UGT1A6 activity and mRNA in human liver samples. Thus, UGT1A is regulated by multiple miRNAs with some showing allele-dependent effects.

Abnormalities of brain neural circuits related to obesity: A Diffusion Tensor Imaging study.

PURPOSE: Increased Body-Mass-Index (BMI) has been associated with brain atrophy in both gray and white matter structures. However, little is known concerning the integrity of white matter tracts in obesity. The purpose of the study was to evaluate the pattern of changes in white matter microstructure in human adiposity. MATERIAL AND METHODS: The study included 268 participants (52 obese, 96 overweight and 120 normal-weight) that were retrospectively evaluated by Diffusion Tensor Imaging. The fractional anisotropy, axial, radial and mean diffusivity values were compared between the above groups using Tract Based Spatial Statistics. RESULTS: The analysis revealed that the increased BMI was related with decreased fractional anisotropy in several white matter regions including the anterior and posterior thalamic radiation, the inferior fronto-occipital fasciculus, the inferior and superior longitudinal fasciculus, the corpus callosum (callosal body and forceps minor), the uncinate fasciculus, the internal capsule, the corticospinal tract and the cingulum (cingulate gyrus and hippocampus). CONCLUSIONS: Anisotropic diffusion of anatomic regions governing important brain circuits such as reward seeking inhibition, motivation/drive and learning/conditioning decreases with increasing BMI.

Transcriptome association analysis identifies miR-375 as a major determinant of variable acetaminophen glucuronidation by human liver.

Acetaminophen is the leading cause of acute liver failure (ALF) in many countries including the United States. Hepatic glucuronidation by UDP-glucuronosyltransferase (UGT) 1A subfamily enzymes is the major route of acetaminophen elimination. Reduced glucuronidation may predispose some individuals to acetaminophen-induced ALF, but mechanisms underlying reduced glucuronidation are poorly understood. We hypothesized that specific microRNAs (miRNAs) may reduce UGT1A activity by direct effects on the UGT1A 3'-UTR shared by all UGT1A enzyme transcripts, or by indirect effects on transcription factors regulating UGT1A expression. We performed an unbiased miRNA whole transcriptome association analysis using a bank of human livers with known acetaminophen glucuronidation activities. Of 754 miRNAs evaluated, 9 miRNAs were identified that were significantly overexpressed (p<0.05; >2-fold) in livers with low acetaminophen glucuronidation activities compared with those with high activities. miR-375 showed the highest difference (>10-fold), and was chosen for further mechanistic validation. We demonstrated using in silico analysis and luciferase reporter assays that miR-375 has a unique functional binding site in the 3'-UTR of the aryl hydrocarbon receptor (AhR) gene. Furthermore overexpression of miR-375 in LS180 cells demonstrated significant repression of endogenous AhR protein (by 40%) and mRNA (by 10%), as well as enzyme activity and/or mRNA of AhR regulated enzymes including UGT1A1, UGT1A6, and CYP1A2, without affecting UGT2B7, which is not regulated by AhR. Thus miR-375 is identified as a novel repressor of UGT1A-mediated hepatic acetaminophen glucuronidation through reduced AhR expression, which could predispose some individuals to increased risk for acetaminophen-induced ALF.

Secondary metabolism pathway polymorphisms and plasma efavirenz concentrations in HIV-infected adults with CYP2B6 slow metabolizer genotypes.

OBJECTIVES: Efavirenz is widely prescribed for HIV-1 infection, and CYP2B6 polymorphisms 516G→T and 983T→C define efavirenz slow metabolizer genotypes. To identify genetic predictors of higher plasma efavirenz concentrations beyond these two common functional alleles, we characterized associations with mid-dosing interval efavirenz concentrations in 84 HIV-infected adults, all carrying two copies of these major loss-of-function CYP2B6 alleles. METHODS: Study participants had been randomized to efavirenz-containing regimens in prospective clinical trials and had available plasma efavirenz assay data. Analyses focused on secondary metabolism pathway polymorphisms CYP2A6 -48T→G (rs28399433), UGT2B7 735A→G (rs28365062) and UGT2B7 802T→C (rs7439366). Exploratory analyses also considered 196 polymorphisms and 8 copy number variants in 41 drug metabolism/transport genes. Mid-dosing interval efavirenz concentrations at steady-state were obtained ≥8 h but <19 h post-dose. Linear regression was used to test for associations between polymorphisms and log-transformed efavirenz concentrations. RESULTS: Increased efavirenz concentrations were associated with CYP2A6 -48T→G in all subjects (P = 3.8 × 10(-4)) and in Black subjects (P = 0.027) and White subjects (P = 0.0011) analysed separately; and with UGT2B7 735 G/G homozygosity in all subjects (P = 0.006) and in Black subjects (P = 0.046) and White subjects (P = 0.062) analysed separately. In a multivariable model, CYP2A6 -48T→G and UGT2B7 735 G/G homozygosity remained significant (P < 0.05 for each). No additional polymorphisms or copy number variants were significantly associated with efavirenz concentrations. CONCLUSIONS: Among individuals with a CYP2B6 slow metabolizer genotype, CYP2A6 and possibly UGT2B7 polymorphisms contribute to even higher efavirenz concentrations.

Studies on the role of metabolic activation in tyrosine kinase inhibitor-dependent hepatotoxicity: induction of CYP3A4 enhances the cytotoxicity of lapatinib in HepaRG cells.

Idiosyncratic hepatotoxicity has been associated with the oral tyrosine kinase inhibitor lapatinib, which is used in metastatic breast cancer therapy. Lapatinib is extensively metabolized by cytochrome P450 3A4/5 to yield an O-debenzylated metabolite, which can undergo further oxidation to a reactive quinone imine. A recent clinical study reported that concomitant use of lapatinib with dexamethasone increased the incidence of hepatotoxicity in metastatic breast cancer patients treated with lapatinib, and so we hypothesized that induction of CYP3A enhances the bioactivation of lapatinib to reactive intermediates that contribute to hepatotoxicity. Therefore, we examined the effect of CYP3A4 induction on the cytotoxicity and metabolism of lapatinib in the HepaRG human hepatic cell line. Differentiated HepaRG cells were pretreated with dexamethasone (100 µM) or the prototypical CYP3A4 inducer rifampicin (4 µM) for 72 hours, followed by incubation with lapatinib (0-100 µM) for 24 hours. Cell viability was monitored using WST-1 assays, and metabolites were quantified by liquid chromatography coupled to tandem mass spectrometry. Induction of CYP3A4 by dexamethasone or rifampicin enhanced lapatinib-induced cytotoxicity, compared with treatment with lapatinib alone. A direct comparison of the cytotoxicity of lapatinib versus O-debenzylated lapatinib demonstrated that the O-debenzylated metabolite was significantly more cytotoxic than lapatinib itself. Furthermore, pretreatment with 25 µM l-buthionine sulfoximine to deplete intracellular glutathione markedly enhanced lapatinib cytotoxicity. Cytotoxicity was correlated with increased formation of O-debenzylated lapatinib and cysteine adducts of the putative quinone imine intermediate. Collectively, these data suggest that CYP3A4 induction potentiates lapatinib-induced hepatotoxicity via increased reactive metabolite formation.

Induction of hepatic CYP3A enzymes by pregnancy-related hormones: studies in human hepatocytes and hepatic cell lines.

CYP3A activity is induced by approximately 2-fold during the third trimester of human pregnancy. Placental growth hormone (PGH), estrogens (primarily 17ß-estradiol), cortisol, and progesterone have the potential to modulate CYP3A activity. Therefore, we determined whether the elevated plasma concentrations of these hormones during pregnancy induce hepatic CYP3A expression. We incubated sandwich-cultured human hepatocytes (SCHH) from premenopausal female donors (n = 2) with the physiologic (unbound, 1× total) and the 10× total third trimester hormone plasma concentrations (individually and in combination) and determined their effect on CYP3A activity and the transcripts of CYP3A4, CYP3A5, and the respective hormone receptors (growth hormone receptor, glucocorticoid receptor, and estrogen receptor alpha). Of all the hormones, cortisol was the most potent inducer of CYP3A activity and CYP3A4, CYP3A5 mRNA expression. The combination of PGH/growth hormone and cortisol induced CYP3A activity and expression significantly more than did cortisol alone. When incubated with the unbound or total plasma concentration of all the hormones, CYP3A activity in SCHH was induced to an extent comparable to that observed in vivo during the third trimester. These hormones had only a modest effect on the mRNA expression of the hormone receptors. The pattern of induction observed in SCHH was reproduced in HepaRG cells but not in HuH7/HepG2 cells. SCHH or HepaRG cells could be used to determine the mechanistic basis of CYP3A induction during pregnancy and to predict the magnitude of induction likely to be observed during the first and second trimesters, when phenotyping studies to measure in vivo CYP3A activity are logistically difficult to perform.

Placental Pathology and Blood Pressure's Level in Women with Hypertensive Disorders in Pregnancy.

Objective. The aim of this study was to investigate the extent of placental lesions associated with blood pressure (BP) levels in pregnancies complicated by hypertension. Methods. 55 singleton pregnancies complicated by mild hypertension were recruited and compared to 55 pregnancies complicated by severe hypertension. The histological assessment was carried out with regard to the following aspects: vessels number/field of vision, infarction, villous fibrinoid necrosis, villous hypermaturity, avascular villi, calcifications, lymphohistiocytic villitis, and thickened vessels. Statistical analysis was performed by SPSS. Results. All placental lesions were observed more often in the severe hypertension group. Vessels number was significantly decreased, and infarction and villous fibrinoid necrosis were significantly increased in the placentas of the severe hypertension group compared to the mild hypertension group (P < 0.001). Conclusion. This study supports that the extent of placental lesions in hypertensive pregnancies is correlated with hypertension level and so highlights blood pressure level as a mirror of placental function.

Dynamic elements at both cytoplasmically and extracellularly facing sides of the UapA transporter selectively control the accessibility of substrates to their translocation pathway.

In the UapA uric acid-xanthine permease of Aspergillus nidulans, subtle interactions between key residues of the putative substrate binding pocket, located in the TMS8-TMS9 loop (where TMS is transmembrane segment), and a specificity filter, implicating residues in TMS12 and the TMS1-TMS2 loop, are critical for function and specificity. By using a strain lacking all transporters involved in adenine uptake (DeltaazgA DeltafcyB DeltauapC) and carrying a mutation that partially inactivates the UapA specificity filter (F528S), we obtained 28 mutants capable of UapA-mediated growth on adenine. Seventy-two percent of mutants concern replacements of a single residue, R481, in the putative cytoplasmic loop TMS10-TMS11. Five missense mutations are located in TMS9, in TMS10 or in loops TMS1-TMS2 and TMS8-TMS9. Mutations in the latter loops concern residues previously shown to enlarge UapA specificity (Q113L) or to be part of a motif involved in substrate binding (F406Y). In all mutants, the ability of UapA to transport its physiological substrates remains intact, whereas the increased capacity for transport of adenine and other purines seems to be due to the elimination of elements that hinder the translocation of non-physiological substrates through UapA, rather than to an increase in relevant binding affinities. The additive effects of most novel mutations with F528S and allele-specific interactions of mutation R481G (TMS10-TMS11 loop) with Q113L (TMS1-TMS2 loop) or T526M (TMS12) establish specific interdomain synergy as a critical determinant for substrate selection. Our results strongly suggest that distinct domains at both sides of UapA act as selective dynamic gates controlling substrate access to their translocation pathway.