Nonselective Type Research Articles

The Second Generation Type II JAK2 Inhibitor, AJ1-10502, Demonstrates Enhanced Selectivity, Improved Therapeutic Efficacy and Reduced Mutant Cell Fraction Compared to Type I JAK2 Inhibitors in Models of Myeloproliferative Neoplasms (MPNs)

Type I JAK inhibition (JAKi) represents a mainstay therapy for myelofibrosis and hydroxyurea-resistant high-risk polycythemia vera. Current type I JAKi, including ruxolitinib (RUX) and fedratinib, improve symptoms and outcomes in MPNs; however, JAK2VF allele burden remains essentially unchanged, and efficacy wanes over time. Sustained JAK/STAT signaling plays a critical role in MPN cell persistence in the setting of type I inhibition. CHZ868, a type II JAKi that binds the inactive conformation of the kinase domain, overcomes RUX persistence in vitro and reduces Jak2VF allele fraction in vivo suggesting improved JAK2 targeting might enhance clinical efficacy. However, current type II JAKi, including CHZ868, are limited by lack of kinome specificity and off-target toxicity. We therefore sought to develop novel type II JAK2i with improved potency and selectivity. Computational free energy perturbation and structure-activity relationship-based methods were used to identify lead type II inhibitor compounds. This work, followed by Absorption, Distribution, Metabolism and Excretion modeling, led to the development of AJ1-10502. Kinome selectivity profiling of AJ1-10502 revealed potent JAK2 selectivity with minimal kinase cross-reactivity compared to CHZ868, including among other JAK family tyrosine kinases (Figure 1A). Assessment of in vitro efficacy of AJ1-10502 in SET2 inhibitor naïve and SET2 RUX persistence (RUXper) cells revealed concentration-dependent inhibition of proliferation of RUXper cells comparable to that of CHZ868. The IC50 for AJ1-10502 in RUXper cells was similar to the IC50 observed in inhibitor naïve cells. We next evaluated AJ1-10502 in vivo using a novel dual Dre/Cre-recombinase Jak2VF knock-in/knock-out model allowing for the comparison of JAKi to Jak2VF genetic deletion. AJ1-10502 demonstrated reductions in leukocytosis comparable to that of RUX (K/uL: VEH 18.7 vs. RUX 10.1 vs. AJ1-10502 9.3) but dose-dependent improvements in hematocrit, platelet levels, and spleen weights superior to RUX, with reductions in spleen weights on par to that of Jak2VF deletion (mg: VEH 480 vs. RUX 255 vs. AJ1-10502 145 vs. del 107, p<0.05). A greater degree of restoration of splenic architecture was also observed with AJ1-10502 compared to type I JAKi. Critically, we observed reductions in peripheral blood (PB) and bone marrow (BM) mutant allele fraction with AJ1-10502 not observed with RUX, including within Mac1+Gr1+ myeloid cell fractions (VEH 94% vs. RUX 92% vs. AJ1-10502 78.5%, p<0.05), consistent with a mutant-biased reduction in myeloid output. In a separate series of competitive transplants using a Cre-inducible human JAK2VF transgenic mouse line, we validated the phenotypic changes in regard to leukocytosis, hematocrit, and spleen weight reduction seen with the DreCre model. Most importantly, we confirmed significant reductions in mutant cell fraction within the hematopoietic stem (HSC) compartment of BM and spleen, including among granulocytic-monocytic progenitor (GMP) and long-term HSC (LT-HSC) populations (Figure 1B). Finally, a separate study comparing AJ1-10502 to CHZ868 revealed no significant weight loss with AJ1-10502 (-0.5g) compared to CHZ868 (-2.2g, p<0.05) despite similar reductions in Jak2VF allele fraction suggesting comparable efficacy without systemic toxicity. In sum, AJ1-10502 is a potent, selective type II JAK2i with improved efficacy compared to RUX and an enhanced safety profile compared to previous non-selective type II JAKi. Most importantly, AJ1-10502 results in superior reductions in PB and BM mutant cell fraction in vivo not observed with type I JAKi. These data demonstrate the preclinical utility of type II JAKi with AJ1-10502 and inform a path to clinical development of type II JAKi for MPN patients. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Beta-blocker use and urothelial bladder cancer survival: a Swedish register-based cohort study

Background Recent observational studies linked β-adrenergic receptor blocker use with improved survival in patients with several cancer types, but there is no information on the potential effects of β-blockers in patients with bladder cancer. Literature from pre-clinical studies is also limited, but urothelial cancer can exhibit significant overexpression of β-adrenergic receptors relative to normal urothelial tissue, suggesting that urothelial cancer may benefit from β-blockade therapy. We thus aimed to explore the possible association between β-blocker use and bladder cancer-specific mortality (BCSM) among patients with urothelial bladder cancer. Material and methods Patients diagnosed during 2006–2014 and identified from the Swedish Cancer Register (n = 16,669) were followed until 31 December 2015. Cox regression was used to evaluate the association of β-blockers dispensed within 90 days prior to cancer diagnosis with BCSM (primary outcome) and all-cause mortality, while controlling for socio-demographic factors, tumor characteristics, comorbidity, other medications and surgical procedures. Hazard ratios (HR) with 95% confidence intervals (CI) were reported. Results Overall, β-blocker use was associated with lower BCSM [HR 0.88 (95%CI 0.81–0.96)]. Especially use of nonselective β-blockers showed a clear inverse association in comparison with both nonuse [0.66 (0.50–0.86)] and use of other antihypertensive medications [0.72 (0.54–0.95)]. The inverse association was most pronounced among patients with locally advanced/metastatic disease: [0.35 (0.18–0.68)]. A lower-magnitude inverse association was observed for selective β-blocker use [0.91 (0.83–0.99)]. Largely similar inverse associations were observed for hydrophilic [0.82 (0.70–0.95)] and lipophilic [0.91 (0.83–1.00)] β-blocker use. Conclusion β-blocker use, particularly of the nonselective type, was associated with lower BCSM, especially in patients with locally advanced/metastatic urothelial bladder cancer.

Identification of highly selective type II kinase inhibitors with chiral peptidomimetic tails

Identification of highly selective type II kinase inhibitors is described. Two different chiral peptidomimetic scaffolds were introduced on the tail region of non-selective type II kinase inhibitor GNF-7 to enhance the selectivity. Kinome-wide selectivity profiling analysis showed that type II kinase inhibitor 7a potently inhibited Lck kinase with great selectivity (IC50 of 23.0 nM). It was found that 7a and its derivatives possessed high selectivity for Lck over even structurally conserved all Src family kinases. We also observed that 7a inhibited Lck activation in Jurkat T cells. Moreover, 7a was found to alleviate clinical symptoms in DSS-induced colitis mice. This study provides a novel insight into the design of selective type II kinase inhibitors by adopting chiral peptidomimetic moieties on the tail region.

Endothelin receptor antagonists alleviate blood-brain barrier disruption and cerebral edema in a mouse model of traumatic brain injury: A comparison between bosentan and ambrisentan

Traumatic brain injury (TBI) is induced by the immediate physical disruption of brain tissue. TBI causes disruption of the blood–brain barrier (BBB) and brain edema. In the cerebrospinal fluid (CSF) of TBI patients, endothelin-1 (ET-1) is increased, suggesting that ET-1 aggravates TBI-induced brain damage. In this study, the effect of bosentan (ETA/ETB antagonist) and ambrisentan (ETA antagonist) on BBB dysfunction and brain edema were examined in a mouse model of TBI using lateral fluid percussion injury (FPI). FPI to the mouse cerebrum increased the expression levels of ET-1 and ETB receptors. Administration of bosentan (3 or 15 mg/kg/day) and ambrisentan (0.1 or 0.5 mg/kg/day) at 6 and 24 h after FPI ameliorated BBB disruption and cerebral brain edema. Delayed administration of bosentan from 2 days after FPI also reduced BBB disruption and brain edema, while ambrisentan had no significant effects. FPI-induced expression levels of ET-1 and ETB receptors were reduced by bosentan, but not by ambrisentan. In cultured mouse astrocytes and brain microvessel endothelial cells, ET-1 (100 nM) increased prepro--ET-1 mRNA, which was inhibited by bosentan, but not by ambrisentan. FPI-induced alterations of the expression levels of matrix metalloproteinase-9, vascular endothelial growth factor-A, and angiopoietin-1 in the mouse cerebrum were reduced by delayed administration of bosentan, while ambrisentan had no significant effects. These results suggest that ET antagonists are effective in improving BBB disruption and cerebral edema in TBI patients and that an ETA/ETB non-selective type of antagonists is more effective.

A transparent insulation façade enhanced with a selective absorber: A cooling energy load and validated building energy performance prediction model

Solar thermal façade concepts for building integration have a relevant contribution in the field of renewable solar energy exploitation. This study is focused on an experimental analysis of a transparent insulation façade (TIF) concept and the options for the prediction of its performance using building energy simulation (BES) modelling. A TIF prototype based on a transparent insulation material (TIM) and a selective absorber is tested and compared with a nonselective type of absorber. An experimental study was conducted involving full-scale climatic chamber and dynamic outdoor tests. The proposed TIF concept with a selective absorber and its solar effective layers provides the same thermal performance as conventional types of thermal insulation. As the TIM integration is potentially limited by overheating phenomena, a maximum cooling energy load obtained by dynamic outdoor conditions provides its real outdoor performance. The results show that a significant difference is measured when compared with the nonselective absorber type. Finally, a detailed comparative investigation of the thermal performance of both TIFs was conducted using the BES method. When applying the low-e functionality of the solar absorber, significant limitations were found specifically concerning the surface radiant heat exchange with the low emissivity surface affecting the air cavity enclosed zone.

Scintillation properties of phosphate-borate-fluoride glass doped with Tb3+/Pr3+

Scintillation glass doped with Tb3+ and Pr3+ ions with different concentrations were prepared by the melt-quenching method. Optical, photoluminescence and decay kinetic characteristics of the pulse cathodoluminescence (PCL) were investigated. It was shown that the absorption coefficient of the induced absorption in the visible range of the spectrum decreases significantly with the increase of the Pr2O3 content starting from 0.2 to 1 wt%. There was the difference in the luminescence spectra of the glass at a selective and non-selective type of excitation. The “green” emission (λem= 542 nm, 5D4→7F5 radiative transition of Tb3+ ions) was excited an electron beam. The “red” emission (λem= 600 nm, 3P0→3H6 radiative transition of Pr3+ ion) was observed under selective excitation action (λexc= 450 nm). It was demonstrated that decreasing of intensity the main bands of Tb3+ ions at 487, 544, 622 nm connected with increases of concentration Pr3+ ions. The luminescence decay time of terbium ions at 487, 544, 622 nm emission bands depend on Pr3+ concentration. The tendency of reducing the luminescence decay time in the main luminescence bands of Tb3+ ions at increasing the Pr3+ concentration was presented. The results showed that Tb3+/ Pr3+ co-doped phosphate-borate-fluoride glasses are promising non-crystalline scintillation materials.

A Non-Ventilated Solar Façade Concept Based on Selective and Transparent Insulation Material Integration: An Experimental Study

A new solar façade concept based on transparent insulation and a selective absorber is proposed, tested and compared with conventional insulation and a non-selective type of absorber, respectively. The presented study focuses on an experimental non-ventilated solar type of façade exposed to solar radiation both in the laboratory and in outdoor tests. Due to the high solar absorbance level of the façade, high- and low-emissivity contributions were primarily analysed. All of the implemented materials were contrasted from the thermal and optical point of view. An analysis was made of both thermodynamic and steady state procedures affecting the proposed solar façade concept. Experimental full scale tests on real building components were additionally involved during summer monitoring. An indicator of the temperature response generated by solar radiation exposure demonstrates the outdoor performance of the façade is closely related to overheating phenomena. From the thermal point of view, the proposed transparent insulation and selective absorber concept corresponds to the performance of conventional thermal insulation of identical material thickness; however, the non-selective prototype only provides 50% thermal performance. The results of the solar-based experiments show that with a small-scale experimental prototype, approximately no significant difference is measured when compared with a non-selective absorber type. The only difference was achieved at the maximum of 2.5 K, when the lower temperature was obtained in the solar selective concept. At the full-scale outdoor mode, the results indicate a maximum of 3.0 K difference, however the lower temperature achieves a non-selective approach. This solar façade can actively contribute to the thermal performance of building components during periods of heating.

ATP-Competitive MLKL Binders Have No Functional Impact on Necroptosis.

MLKL is a pore forming pseudokinase involved in the final stage of necroptosis, a form of programmed cell death. Its phosphorylation by RIPK3 is necessary for triggering necroptosis but not for triggering apoptosis, which makes it a unique target for pharmacological inhibition to block necroptotic cell death. This mechanism has been described as playing a role in disease progression in neurodegenerative and inflammatory diseases. A type II kinase inhibitor (cpd 1) has been described that reportedly binds to the MLKL pseudokinase domain and prevents necroptosis. Here we describe five compounds that bind to the MLKL ATP-binding site, however the four MLKL-selective compounds have no activity in rescuing cells from necroptosis. We use kinase selectivity panels, crystallography and a new conformationally sensitive method of measuring protein conformational changes (SHG) to confirm that the one previously reported compound that can rescue cells (cpd 1) is a non-selective type II inhibitor that also inhibits the upstream kinase RIPK1. Although this compound can shift the GFE motif of the activation loop to an “out” position, the accessibility of the key residue Ser358 in the MLKL activation loop is not affected by compound binding to the MLKL active site. Our studies indicate that an ATP-pocket inhibitor of the MLKL pseudokinase domain does not have any impact on the necroptosis pathway, which is contrary to a previously reported study.

Post-diagnostic use of beta-blockers and the risk of death in patients with prostate cancer

BackgroundRecent observational studies have produced conflicting results with respect to beta-blocker use after prostate cancer diagnosis and mortality outcomes. ObjectiveTo determine whether post-diagnostic use of beta-blockers is associated with prostate cancer mortality and all-cause mortality. Patients and methodsA cohort of 6270 men newly-diagnosed with non-metastatic prostate cancer between 1st April 1998, and 31st December 2009, followed until 1st October 2012, was identified using large population-based electronic databases from the United Kingdom. Time-dependent Cox proportional hazards models were used to estimate adjusted hazard ratios (HRs) with 95% confidence intervals (CIs) of mortality outcomes associated with post-diagnostic use of beta-blockers. Secondary analyses were performed to examine the independent effects of non-selective beta-blockers, as well as cumulative duration of use. ResultsDuring a mean follow-up time of 3.8years (standard deviation: 2.7years), 1761 deaths occurred, including 715 from prostate cancer. Post-diagnostic use of beta-blockers was not associated with a decreased risk of prostate cancer mortality (HR: 0.97, 95% CI: 0.72–1.31) and all-cause mortality (HR: 0.97, 95% CI: 0.81–1.16). There was no statistically significant association for non-selective beta-blockers (prostate cancer mortality, HR: 1.05, 95% CI: 0.72–1.53 and all-cause mortality, HR: 0.94, 95% CI: 0.74–1.18), and no statistically significant trends of cumulative duration of use for both mortality outcomes. ConclusionThe use of beta blockers, including those of the non-selective type, was not associated with a decreased risk of prostate cancer and all-cause mortality.

Performance and Economic Analysis of Hybrid PVT Collectors in Solar DHW System

Performance of different concepts of solar hybrid PVT liquid collectors has been analyzed for domestic hot water application in a block of flats. A comparison with conventional installation of photovoltaic (PV) and photothermal (PT) solar collectors with the same total collector area 100 m2 has been done for identical load conditions. Conventional solar system has been considered with different fraction of PV collector to PT collector area for comparison with the PVT system. Economic analysis based on the performance results, energy prices and conventional PV and PT collector prices has revealed several important facts. Unglazed PVT collector cannot be competitive with conventional PV-PT collectors in given solar DHW system. Competitive price of unglazed PVT collector is negative in most of variants. Competitive price of glazed PVT collector available at the market is about less than half of its real today market price. Competitive price range has been set in advance for solar PVT collectors being under development with novel siloxane lamination: 290-410 €/m2 for nonselective type and 370-500 €/m2 for selective type.

Vasodilatory activity and antihypertensive profile mediated by inhibition of phosphodiesterase type 1 induced by a novel sulfonamide compound

LASSBio-985 is a sulfonamide compound designed as a simplified structure of a nonselective phosphodiesterase type 4 (PDE-4) inhibitor that promotes vasodilatory activity in vitro. PDE are enzymes responsible for the hydrolysis of cyclic adenosine 3',5'- monophosphate and cyclic guanosine 3',5'-monophosphate. Five different isozymes of PDE are found in vascular smooth muscle (PDE1-PDE5). Aortic rings, with or without endothelium, from male normotensive and spontaneously hypertensive rats (SHR) were prepared for isometric tension recording. Blood pressure was measured in Wistar Kyoto (WKY) rats and SHR during intravenous infusion of LASSBio-985 (10 mg/kg/min) during 15 min. LASSBio-985 induced a concentration-dependent vasodilation in aortic rings from normotensive and SHR, which was almost completely inhibited in endothelium-denuded vessels. Vasodilatory activity was also reduced in endothelium-intact aortic rings that had been pretreated with N(ω)-nitro-L-arginine methyl ester hydrochloride (L-NAME), a nitric oxide synthase inhibitor and 1H-[1,2,4]oxadiazolod[4,3-a]quinoxalin-1-one (ODQ), a guanylate cyclase inhibitor. LASSBio-985-induced vasodilation was also inhibited by sildenafil (100 μm) and SQ 22536, a PDE5 inhibitor and adenylate cyclase inhibitor, respectively. To evaluate the involvement of some endothelial receptors, atropine, diphenhydramine, HOE 140, naloxone, propranolol, indomethacin, and wortmannin were tested, but none inhibited the effects of LASSBio-985. The residual effect observed on endothelium-denuded aortic rings was abolished by nicardipine, a voltage-sensitive-Ca(2+)-channel blocker. Intravenous infusion of LASSBio-985 (10 mg/kg/min) significantly reduced systolic and diastolic pressures in both WKY and SHR. LASSBio-985 is a compound with vasodilatory activity, which could be consequent to PDE1 inhibition and voltage-sensitive-Ca(2+)-channel blockade.

The adsorption of Naproxen enantiomers on the chiral stationary phase ( [formula omitted])-Whelk-O1 under reversed-phase conditions: The effect of mobile phase composition

The adsorption of the Naproxen enantiomers on the Whelk-O1 chiral stationary phase from acidic (0.01 M CH 3COOH) methanol–water solutions was studied using the frontal analysis method and band profile calculations. The adsorption model that best accounted for these data is one assuming the coexistence of three types of adsorption sites, two of them being enantioselective, associated with the chiral selectors, the third type being the nonselective type of sites associated with residual surface silanol groups. The adsorption equilibrium is influenced by secondary processes that result in variations of the model parameters with the mobile phase composition. Several qualitative correlations between the model parameters and the methanol–water ratio were observed and are discussed.

Muscle pathology in 57 patients with myotonic dystrophy type 2.

We evaluated muscle biopsies from 57 patients with genetically confirmed myotonic dystrophy type 2/proximal myotonic myopathy (DM2/PROMM). Light microscopy showed myopathic together with "denervation-like" changes in almost all biopsies obtained from four different muscles: increased fiber size variation, internal nuclei, small angulated fibers, pyknotic nuclear clumps, and predominant type 2 fiber atrophy. Quantitative morphometry in 18 biopsies that were immunostained for myosin heavy chain confirmed a predominance of nonselective type 2 fiber atrophy. These histological changes were similar in all patients regardless of the site of biopsy, the predominant clinical symptoms and signs, and the clinical course. It is likely that, in a number of undiagnosed patients, DM2 is the underlying disorder. With a better understanding of the histopathological pattern in DM2, biopsies from patients with undiagnosed neuromuscular disorders can now be reevaluated.

Formation of Conjugated Linoleic Acids in Soybean Oil during Hydrogenation with a Nickel Catalyst As Affected by Sulfur Addition

The effects of sulfur addition on the formation of conjugated linoleic acid (CLA) isomers were studied during the hydrogenation of soybean oil with a nonselective type nickel catalyst. Sulfur addition greatly promoted CLA formation in soybean oil during hydrogenation. As the amount of sulfur increased to a certain level, the maximal quantity of CLA in soybean oil during hydrogenation increased greatly. However, further increase in sulfur addition above the certain level decreased CLA formation. The optimal sulfur level for the promotion of CLA formation differed greatly with the amount of nickel used. It was of great interest to find that the optimal ratio of sulfur to nickel for the promotion of CLA formation was always 0.06:1, regardless of the nickel amount used. At the same ratio of sulfur to nickel, higher nickel content induced significantly higher production of CLA (p < 0.05). At the optimal sulfur to nickel ratio, an increase in the nickel amount from 0.05 to 0.15% produced approximately 1.5 times higher levels of CLA during hydrogenation under the tested conditions. The CLA isomer compositions were greatly affected by both sulfur addition and amounts of nickel used for treatment. This is the first report of the possibility that the total quantity of CLA and their isomer composition could be manipulated during hydrogenation by controlling the amounts of sulfur and nickel.

Task integration as a factor in secondary-task effects on sequence learning

In several studies it has been found that implicit sequence learning is impaired by the concurrent performance of a secondary task. In most studies the task was to count high-pitched tones when high-pitched and low-pitched tones were presented in a random sequence. In this paper the hypothesis is examined that dual-task interference in the particular combination of tasks results from task integration, in particular from the learning of an integrated visual-auditory sequence in which every second (auditory) element is random. Instead of the tone-counting task a similar go/no-go task was used in which foot responses to high-pitched tones were performed. In Exp. 1 the sequence of tones was random in one condition, but in two other conditions repeated tone sequences of 5 and 6 elements were combined with visual sequences of 6 elements. Under dual-task test conditions, implicit learning of the visual as well as the auditory sequences was better with the auditory sequence of 6 elements than of 5 elements, while under single-task test conditions the nature of the tone sequence had no effect. In Exp. 2 the superior implicit learning with the 6-element sequence was replicated with different test procedures in which either the visual or auditory sequence was changed to random or in which the two sequences remained intact but were shifted by one element relative to each other. Randomization of the visual or auditory sequences not only impaired visual or auditory RT, respectively, but also impaired RT to stimuli in the other modality, and this cross-modal effect was almost as strong as the intra-modal effect of randomization. Finally, in Exp. 3 it was shown that integrated visual-auditory sequences are learned only when responses to both of them are required, but not when the tones can be neglected. These results are consistent with a conception of implicit learning as (at least partly) a basic and nonselective type of learning of all potentially behaviorally relevant relations between stimuli in the environment and one's own actions.

P-16-2 - Effects of metyrapone treatment upon hormonal secretion and sleep

The enzyme 11β-hydroxysteroid dehydrogenase (11β-HSD) is considered to confer mineralocorticoid specificity on the non-selective Type I adrenocorticoid receptor by converting active 11-hydroxyglucocorticoids to receptor-inactive 11-oxo metabolites, in mineralocorticoid target tissues like the kidney. However, 11β-HSD is also present in the liver, where it may regulate steroid exposure to the glucocorticoid Type II receptor. Because of the much higher activities compared to that in kidney, liver 11β-HSD possibly has additional functions besides the metabolism of glucocorticoids. In the present investigation we have isolated 11β-HSD from mouse liver microsomes and demonstrate that the homogeneously purified enzyme is also capable of catalyzing the reductive metabolism of xenobiotic carbonyl compounds such as metyrapone, p-nitroacetophenone and p-nitrobenzaldehyde. Enzyme kinetic studies revealed that, in addition to NADP+, mouse liver 11β-HSD also accepts NAD+ as cosubstrate for glucocorticoid 11β-dehydrogenation. NADH as cosubstrate for 11-oxoreduction plays only a minor role compared to that with NADPH, a fact which is also true for xenobiotic carbonyl reduction. Inhibition experiments revealed strong sensitivity of xenobiotic carbonyl reduction to glucocorticoids. The competitive nature of this inhibition suggests that both glucocorticoids and xenobiotic carbonyl substances bind to the same catalytically active site of 11β-HSD. High enzyme activities were also found in microsomal fractions of the ovary and adrenal gland but, although expressing considerable glucocorticoid 11-dehydrogenation activity (one third that of liver), almost no carbonyl reduction was detectable in kidney microsomes. Immunoblot analysis with polyclonal antibodies directed against the liver 11β-HSD did not yield an immunological crossreaction in the same tissues. In conclusion, corresponding to the cytosolic aldo-keto reductases, microsomal 11β-HSD of liver may be considered to play a role in the phase I biotransformation of pharmacologically relevant carbonyl substances as well as protecting organisms against toxic carbonyl compounds by converting them to less lipophilic and more soluble and conjugatable metabolites. Discrepancies in bioactivity together with the lack of response to anti-liver 11β-HSD antibodies strongly indicate the existence of distinct forms of 11β-HSD to be present in kidney, adrenal gland and ovary. The ability of xenobiotic carbonyl reduction might be another distinguishing feature among the various 11β-HSD isozymes.

11β-hydroxysteroid dehydrogenase mediates reductive metabolism of xenobiotic carbonyl compounds

The enzyme 11β-hydroxysteroid dehydrogenase (11β-HSD) is considered to confer mineralocorticoid specificity on the non-selective Type I adrenocorticoid receptor by converting active 11-hydroxyglucocorticoids to receptor-inactive 11-oxo metabolites, in mineralocorticoid target tissues like the kidney. However, 11β-HSD is also present in the liver, where it may regulate steroid exposure to the glucocorticoid Type II receptor. Because of the much higher activities compared to that in kidney, liver 11β-HSD possibly has additional functions besides the metabolism of glucocorticoids. In the present investigation we have isolated 11β-HSD from mouse liver microsomes and demonstrate that the homogeneously purified enzyme is also capable of catalyzing the reductive metabolism of xenobiotic carbonyl compounds such as metyrapone, p-nitroacetophenone and p-nitrobenzaldehyde. Enzyme kinetic studies revealed that, in addition to NADP +, mouse liver 11β-HSD also accepts NAD + as cosubstrate for glucocorticoid 11β-dehydrogenation. NADH as cosubstrate for 11-oxoreduction plays only a minor role compared to that with NADPH, a fact which is also true for xenobiotic carbonyl reduction. Inhibition experiments revealed strong sensitivity of xenobiotic carbonyl reduction to glucocorticoids. The competitive nature of this inhibition suggests that both glucocorticoids and xenobiotic carbonyl substances bind to the same catalytically active site of 11β-HSD. High enzyme activities were also found in microsomal fractions of the ovary and adrenal gland but, although expressing considerable glucocorticoid 11-dehydrogenation activity (one third that of liver), almost no carbonyl reduction was detectable in kidney microsomes. Immunoblot analysis with polyclonal antibodies directed against the liver 11β-HSD did not yield an immunological crossreaction in the same tissues. In conclusion, corresponding to the cytosolic aldo-keto reductases, microsomal 11β-HSD of liver may be considered to play a role in the phase I biotransformation of pharmacologically relevant carbonyl substances as well as protecting organisms against toxic carbonyl compounds by converting them to less lipophilic and more soluble and conjugatable metabolites. Discrepancies in bioactivity together with the lack of response to anti-liver 11β-HSD antibodies strongly indicate the existence of distinct forms of 11β-HSD to be present in kidney, adrenal gland and ovary. The ability of xenobiotic carbonyl reduction might be another distinguishing feature among the various 11β-HSD isozymes.

Localization of endothelin receptor messenger ribonucleic acid in the rat ovary and fallopian tube by in situ hybridization.

Rat mRNAs encoding two subtypes of the endothelin (ET) receptor (ET(A) and ETB) were studied in the rat ovary and fallopian tube by means of Northern blotting and in situ hybridization. The mRNA transcripts for the endothelin-1-specific type receptor (ET(A)) in pooled RNA from the ovary and fallopian tube were 4.2 and 5.2 kilonucleotides, and that for the nonselective type receptor (ETB) was 4.7 kilonucleotides; these were similar to transcripts for endothelin receptors from other tissues. ET(A) mRNA expression was abundant in the muscle cell layer of the fallopian tube, but low in the ovary. On the other hand, ETB mRNA was abundant in the granulosa cells in the developing follicles, but low in atretic follicles and absent in the fallopian tube. These results demonstrated that the mRNAs for the two subtypes of the rat endothelin receptor have different expression profiles in the ovary and fallopian tube. ETs may mainly affect the granulosa cells in the dominant follicles as well as the muscle cells of the fallopian tube through ETB and ET(A), respectively.

Alternative transcript of the nonselective-type endothelin receptor from rat brain.

A novel cDNA encoding the nonselective type of endothelin (ET) receptor was isolated from a rat brain cDNA library. The cloned cDNA encoded a 442-amino acid protein with seven putative transmembrane domains. Nucleotide sequence analysis showed that the rat brain cDNA differed from the cloned rat lung nonselective ET receptor (ETB) cDNA by three extra nucleotides in its coding regions, which produced an encoded protein with four amino acid substitutions. In addition, both the 5' and 3' noncoding sequences of the rat brain cDNA were divergent from those of rat lung cDNA. Expression of the rat brain cDNA in COS-1 cells demonstrated that the encoded receptor displayed equal affinity toward the three ET isopeptides. However, Southern blot analysis indicated a single-copy gene for the rat ETB receptor. Further genomic cloning and sequence analysis demonstrated that rat brain cDNA encoded the authentic protein sequences of the rat ETB receptor. Moreover, the 5' noncoding sequences in rat brain cDNA that were divergent from those in rat lung cDNA were encoded by a distinct region, an upstream exon, in the rat ETB genome. All the findings suggest that rat brain cDNA represents an alternative transcript of the rat ETB gene. Preliminary Northern blot analysis indicated that the expression of this ETB cDNA sequence might be not only in the brain but also in other tissues, whereas its expression might be somehow tissue-specifically regulated.

Tissue-specific expression of an 11 beta-hydroxysteroid dehydrogenase with a truncated N-terminal domain. A potential mechanism for differential intracellular localization within mineralocorticoid target cells.

The enzyme 11 beta-hydroxysteroid dehydrogenase (11-HSD) is thought to confer specificity on the nonselective Type I adrenocorticoid receptor by converting glucocorticoids to receptor-inactive metabolites in mineralocorticoid target tissues. S1 nuclease analyses using a rat liver 11-HSD probe demonstrated tissue-specific expression of the 5' region of the 11-HSD gene in the liver, lung, and kidney not evident in previous studies. Renal tissue contained a unique protected species which mapped to a position within the coding region, consistent with a divergence in liver and kidney protein sequences. Screening of a rat kidney cDNA library resulted in the isolation of several clones (11-HSD1B) noncolinear in their 5' regions with the liver sequence (11-HSD1A). Nucleic acid sequence analysis showed that the divergent clones code for a protein lacking a 26-amino acid NH2-terminal putative membrane-spanning signal peptide. The deletion of the leader sequence from the microsomal 11-HSD1A protein may result in a nuclear localization of the 11-HSD1B isoform. The renal 11-HSD1A and 11-HSD1B species increased coordinately during ontogeny and in parallel with the developmental surge in glucocorticoids. At least three alternate sites of polyadenylation were found to be utilized by the 11-HSD gene. Southern blot analysis showed the presence of a single gene in the rat. This study shows the expression of a kidney-specific 11-HSD isoform which may protect the Type I adrenocorticoid receptor from occupation by glucocorticoids in the nucleus of a mineralocorticoid target cell.