RBE of thermal neutrons for induction of chromosome aberrations in human lymphocytes

The induction of chromosome aberrations in human lymphocytes irradiated in vitro with slow neutrons was examined to assess the maximum low-dose RBE (RBE_M) relative to ⁶⁰Co γ-rays. For the blood irradiations, cold neutron beam available at the prompt gamma activation analysis facility at the Munich research reactor FRM II was used. The given flux of cold neutrons can be converted into a thermally equivalent one. Since blood was taken from the same donor whose blood had been used for previous irradiation experiments using widely varying neutron energies, the greatest possible accuracy was available for such an estimation of the RBE_M avoiding the inter-individual variations or differences in methodology usually associated with inter-laboratory comparisons. The magnitude of the coefficient α of the linear dose–response relationship (α = 0.400 ± 0.018 Gy^?1) and the derived RBE_M of 36.4 ± 13.3 obtained for the production of dicentrics by thermal neutrons confirm our earlier observations of a strong decrease in α and RBE_M with decreasing neutron energy lower than 0.385 MeV (RBE_M = 94.4 ± 38.9). The magnitude of the presently estimated RBE_M of thermal neutrons is—with some restrictions—not significantly different to previously reported RBE_M values of two laboratories.     

Interlaced proton grid therapy – Linear energy transfer and relative biological effectiveness distributions

PurposeInterlaced beams have previously been proposed for delivering proton grid therapy. This study aims to assess dose-averaged LET (LET_d) and RBE-weighted dose (D_RBE) distributions of such beam geometries, and compare them with conventional intensity modulated proton therapy (IMPT).MethodsIMPT plans and four different interlaced proton grid therapy plans were generated for five patient cases (esophagus, lung, liver, prostate, anus). The constant RBE = 1.1 was assumed for optimization. The LET_d was subsequently Monte Carlo calculated for each plan and used as input for two LET-dependent variable RBE models. The fulfilment of clinical goals, along with DVH and spatial distribution evaluations, were then assessed and compared.ResultsAll plans fulfilled the clinical target goals assuming RBE = 1.1. The target coverage was slightly compromised for some grid plans when assuming the variable RBE models. All IMPT plans, and 18 of 20 grid plans, fulfilled all clinical goals for the organs at risk when assuming RBE = 1.1, whereas most plans failed at least one goal when assuming the variable RBE models. Compared with the IMPT plans, the grid plans demonstrated substantially different LET_d distributions due to the fundamentally different beam geometries. However, D_RBE distributions in the target were similar.ConclusionsDespite the unconventional beam geometries of interlaced proton grid plans, with resulting alternating dose and LET_d patterns, the fulfillment of realistic clinical goals seems to be comparable to regular IMPT plans, both assuming RBE = 1.1 and variable RBE models. In addition, the alternating grid patterns do not seem to give rise to unexpected D_RBE hot-spots.     

Synthesis of blockwise alkylated (1→4) linked trisaccharides as surfactants: influence of configuration of anomeric position on their surface activities

New carbohydrate-based surfactants consisting of hydrophilic cellobiosyl and hydrophobic glucosyl residues, methyl β-d-glucopyranosyl-(1→4)-α-d-glucopyranosyl-(1→4)-2,3,6-tri-O-methyl-α-d-glucopyranoside 1 (GβGαMα, G: glucopyranosyl residue, α and β: α-(1→4)- and β-(1→4) glycosidic bonds, M: methyl group), 2 (G_βG_βM_α), 3 (G_βG_αM_β), 4 (G_βG_βM_β), 5 (G_βG_αE_α, E: ethyl group), 6 (G_βG_βE_α), 7 (G_βG_αE_β), 8 (G_βG_βE_β) and eight α-and β-glycoside mixtures (a mixture of 1 and 2: 1/2 = 62/38 (9), 32/68 (10); a mixture of 3 and 4: 3/4 = 69/31 (11), 32/68 (12); a mixture of 5 and 6: 5/6 = 62/38 (13), 33/67 (14); a mixture of 7 and 8: 7/8 = 59/41 (15), 29/71 (16)) were synthesized via combined methods consisting of acid-catalyzed alcoholysis of cellulose ethers and glycosylation of phenyl thio-cellobioside derivatives. Their surface activities in aqueous solution depended on their chemical structures: α- or β-(1→4) linkage between hydrophilic cellobiosyl and hydrophobic glucosyl blocks, methyl or ethyl groups of hydrophobic glucosyl block, and α- or β-linked ether group at the C-1 of hydrophobic glucosyl block. The mixing effect of α- and β-glycosides on surface activities was also investigated. As a result, ethyl β-d-glucopyranosyl-(1→4)-α-d-glucopyranosyl-(1→4)-2,3,6-tri-O-ethyl-β-d-glucopyranoside 7 (G_βG_αE_β) had the highest surface activity, and its critical micellar concentration (CMC) and γ_CMC (surface tension at CMC) values of compound 7 were 0.5 mM (ca. 0.03 wt %) and 34.5 mN/m, respectively. The surface tensions of α- and β-glycoside mixtures except for compounds 9 and 10 were almost equal to those of pure compounds. The syntheses of the mixtures of α- and β-glycosides without purification process are easier than those of pure compounds. Thus, the mixtures should be more practical compounds for industrial use as a surfactant.     

Synthesis of Novel Heterobranched β-Cyclodextrins from α-D-Mannosyl- Maltotriose and β-Cyclodextrin by the Reverse Action of Pullulanase,and Isolation and Characterization of the Products

α-D-Mannosyl-maltotriose (Man-G3) were synthesized from methyl α-mannoside and maltotriose by the transfer action of α-mannosidase. (Man-G3)-βCD and (Man-G3)₂-βCD were produced in about 20% and 4% yield, respectively when Aerobacter aerogenes pullulanase (160 units per 1 g of Man-G3) was incubated with the mixture of 1.6 M Man-G3 and 0.16 M βCD at 50°C for 4 days. The reaction products, (Man-G3)-βCD were separated to three peaks by HPLC analysis on a YMC-PACK A-323-3 column and (Man-G3)₂-βCD were separated to several peaks by HPLC analysis on a Daisopak ODS column. The major product of (Man-G3)-βCDs was identified as 6-O-α-(6³-O-α-D-mannosyl-maltotriosyl)-βCD by FAB-MS and NMR spectroscopies. The structures of (Man-G3)₂-βCDs were analyzed by TOF-MS and NMR spectroscopies, and confirmed by comparison of elution profiles of their hydrolyzates by α-mannosidase and glucoamylase on a graphitized carbon column with those of the authentic di-glucosyl-βCDs. The structures of three main components of (Man-G3)₂-βCDs were identified as 6¹,6²-, 6¹,6³- and 6¹,6⁴-di-O-(6³-O-α-D-mannosyl-maltotriosyl)-βCD.     

Increased PKC activity in cultured human keratinocytes and fibroblasts after treatment with 1α,25-dihydroxyvitamin D3

1α,25-Dihydroxyvitamin D₃ (10^?12 M to 10^?8 M) caused a dose dependent increase in PKC activity in the solubilized membrane fractions of cultured human keratinocytes and in the cytosolic fractions of cultured human fibroblasts. Maximum activity was induced by 1α,25-dihydroxyvitamin D₃ at 24 h. Sphingosine, which is believed to inhibit PKC mediated biological responses, blunted 1α,25(OH)₂D₃′s inducement of PKC activity in both keratinocytes and fibroblasts. Identical hormone treatment of vitamin D receptor deficient fibroblasts did not increase PKC activity. Treatment of keratinocytes and fibroblasts with 1β,25-dihydroxyvitamin D₃, which is believed to be ineffective in inducing genomic responses, did not induce PKC activity.     

A 13c-n.m.r. study of a di- and a tri-saccharide containing the α-d-galactopyranosyl group

The ¹³C-n.m.r. spectra of methyl 4-O-α-d-galactopyranosyl-α-d-galactopyranoside (1) and methyl 4-O-[4-O-(α-d-galactopyranosyl)-β-d-galactopyranosyl]-β-d-glucopyranoside (2) in D₂O were recorded. Comparison of these spectra with the spectra of methyl α-d-galactopyranoside (4) and methyl β-lactoside (5) provided substantial confirmation of the structures of 1 and 2.     

Chemical synthesis of [1β-3H]1α,25-dihydroxyvitamin D3 and [1α-3H]1β,25-dihydroxyvitamin D3: Biological activity of 1β,25-dihydroxyvitamin D3

The simple three-step preparation of [1β-³H]1α,25-dihydroxyvitamin D₃ and [1α-³H]1β,25-dihydroxyvitamin D₃ from 1α,25-dihydroxyvitamin D₃ is described. In the rat, 1β,25-dihydroxyvitamin D₃, when compared with its α-epimer, did not stimulate intestinal calcium transport or bone calcium mobilization at doses 1000-fold higher than the doses of the natural hormone, 1α,25-dihydroxyvitamin D₃.     

Binding characteristics of a membrane receptor that recognizes 1α25-dihydroxyvitamin D3 and its epimer, 1β,25-dihydroxyvitamin D3

The Steroid hormon 1α, @5-Dihydroxyvitamin D₃ has been shown to expert rapid effect (15 s to 5 min) in osteoblast. These occur in osteoblast-like cells lacking the nuclear vitamin D receptor, ROS 24/1, suggesting that a separate signalling system mediates the rapid action. These non-genomic action include rapid activation of phospholipase C and opening of calcium channels, pointing to a membrane localization of this signalling system. Previous studies have shown that the 1β epimer of 1α25-dihydroxyvitamina D₃ can block these rapid action, indicating that the 1β epimer may bind to the recptor responsible for the rapid action sin a competative manner. We have assessed the displacement of ³H-1α,25dihydroxyvitamin D₃ by vitamin D compounds, as well as the apparent dissociation constant of 1α25-dihydroxyvitamin D₃ and its 1β epimer for the memberane receptor in membrane prepration from ROS 24/1 cells. Increasing concentrations of 1α25-dihydroxyvitamin D₃, 7.25 nM to 725 nM, displaced ³H-1α25-dihydrxyvitamin D₃ from the membranes with 725 nM of the hormone displacing 40–49% of the radioactivity. Similarly, 1β,25-dihydroxyvitamin D₃, 7.25 nM and 72.5 nM, displaced 1α25-dihydroxyvitamin D₃ binding while 25-hydroxyvitamin D₃, 7.25 nM, did not. The apparent dissociation constant (K_D) for 1α25-dihydroxyvitamin D₃ was detrermined from displacement of ³H-1α25-dihydroxyvitamin D₃ yielding a value of 8.1 × 10^?7 M by Scatchard analysis. The K_D for the 1β epimer determine from displacement of ³H-1α25-dihydroxyvitamin D₃ was 4.8 × 10^?7 M. The data suggest the presence of a receptor on the membrane of ROS 24/1 cells that reconize 1α25-dihydroxyvitamin D₃ and its 1β epimer, but not 25-dihydroxyvitamin D₃. Its ability to reconize the 1β epimer which appears to be a specific anagonist of the rapid effect of the hormone suggests that these studies may be the initial steps in the isolation and characterization of the signalling system mediating the rapid action of vitamin D.     

Relationship between β4 hydrogen bond and β6 hydrophobic interactions during aggregate, fiber or crystal formation in oversaturated solutions of hemoglobin A and S

Oversaturated deoxy-α₂β₂^T4V aggregated instantly without a delay time, which is in contrast to the delay time before the generation of fibers of deoxy-HbS and deoxy-α₂β₂^E6V,D73H. Solubility of deoxy-α₂β₂^T4V was ∼10-fold lower than that of deoxy-HbS and was similar to oxy- and deoxy-α₂β₂^E6V,T4V. These results indicate that β4Val in HbA in the oxy and deoxy forms with or without β6Val facilitates hydrophobic interaction of the A-helix with the EF helix of adjacent molecules without forming a β4/β73 hydrogen bond. Deoxy-HbA generated crystals following aggregation as does HbC-Harlem(α₂β₂^E6V,D73N), while α₂β₂^T4V and α₂β₂^D73H as well as HbS, α₂β₂^E6V,D73H and α₂β₂^E6V,T4V in the oxy and deoxy forms did not form crystals, indicating in addition to the strength of β6 amino acid hydrophobicity that the synergism between the β4Thr hydrogen bond and β6 hydrophobic interaction free energies on the A-helix play a critical role in formation of fibers versus crystalline nuclei during phase transition.     

Studies Towards the Large Scale Chemical Synthesis of the Precursors of Ribonucleosides-3′,4′,5′,5″- 2H 4 and -2′,3′,4′,5′,5″- 2H 5

Abstract

A summary delineating the large scale synthetic studies to prepare labeled precursors of ribonucleosides-3′,4′,5′,5″- ²H ₄ and -2′,3′,4′,5′,5″- ²H ₅ from D-glucose is presented. The recycling of deuterium-labeled by-products has been devised to give a high overall yield of the intermediates and an expedient protocol has been elaborated for the conversion of 3-O-benzyl-α,β-D-allofuranose-3,4-d ₂ 6 to 1-O-methyl-3-O-benzyl-2-O-t-butyldimethylsilyl-α,β-D-ribofuranose-3,4,5,5′-d ₄ 16 (precursor of ribonucleosides-3′,4′,5′,5″- ²H ₄ ) or to 1-O-methyl-3,5-di-O-benzyl-α,β-D-ribofuranose-3,4,5,5′-d ₄ 18 (precursor of ribonucleosides-3′,4′,5′,5″- ²H ₄ ).     

Pharmacological Characterisation of [35S]-GTPγS Binding to Chinese Hamster Ovary Cell Membranes Stably Expressing Cloned Human 5-HT1D Receptor Subtypes

Abstract

[³⁵S]-GTPγS binding has been used to study the function of cloned human 5-HT_1D receptor subtypes stably expressed in chinese hamster ovary (CHO) cells. 5-HT stimulated [³⁵S]-GTPγS binding to membranes from cells expressing 5-HT_1Dα or 5-HT_1Dβ receptors. In membranes containing 5-HT_1Dβ receptors, 5-CT and sumatriptan stimulated binding to a similar extent as 5-HT while yohimbine, metergoline and 8-OHDPAT were partial agonists. The order of potency for agonists was 5-CT > 5-HT > metergoline > sumatriptan > yohimbine > 8-OHDPAT. The stimulation of binding by 5-HT in membranes containing 5-HT_1Dβ receptors was potently antagonised by methiothepin (pA₂ 8.9 ± 0.1). The overall pharmacological profile for the human 5-HT_1Dβ receptor, defined using [³⁵S]-GTPγS binding, agreed well with that reported for inhibition of forskolin-stimulated adenylyl cyclase. In addition, methiothepin and ketanserin inhibited basal [³⁵S]-GTPγS binding to membranes containing 5-HT_1Dα or 5-HT_1Dβ receptors, suggesting that these compounds show negative efficacy at 5-HT_1D receptor subtypes. The data show that [³⁵S]-GTPγS binding is a suitable method for studying the interaction between cloned human 5-HT_1D receptors and G-proteins.     

Polyhydroxylated steroid compounds from the Far Eastern starfish <Emphasis Type="Italic">Distolasterias nipon</Emphasis>

Two new steroid glycosides: distolasteroside D₆, (24S)-24-O-(β-D-xylopyranosyl)-5α-cholestane-3β,6α,8,15β,16β,24-hexaol, and distolasteroside D₇, (22E,24R)-24-O-(β-D-xylopyranosyl)-5α-cholest-22-ene-3β,6α,8,15β,24-pentaol were isolated along with the previously known distolasterosides D₁, D₂, and D₃, echinasteroside C, and (25S)-5α-cholestane-3β4β,6α,7α,8,15α,16β,26-octaol from the Far Eastern starfish Distolasterias nipon. The structures of new compounds were elucidated by NMR spectroscopy and MALDI TOF mass spectrometry. Like neurotrophins, distolasterosides D₁, D₂, and D₃ were shown to induce neuroblast differentiation in a mouse neuroblastoma C1300 cell culture.     

Mineralocorticoids modulate the expression of the β-3 subunit of the Na+, K+-ATPase in the renal collecting duct

Renal sodium reabsorption depends on the activity of the Na⁺,K⁺-ATPase α/β heterodimer. Four α (α_1–4) and 3 β (β_1–3) subunit isoforms have been described. It is accepted that renal tubule cells express α₁/β₁ dimers. Aldosterone stimulates Na⁺,K⁺-ATPase activity and may modulate α₁/β₁ expression. However, some studies suggest the presence of β₃ in the kidney. We hypothesized that the β₃ isoform of the Na⁺,K⁺-ATPase is expressed in tubular cells of the distal nephron, and modulated by mineralocorticoids. We found that β₃ is highly expressed in collecting duct of rodents, and that mineralocorticoids decreased the expression of β₃. Thus, we describe a novel molecular mechanism of sodium pump modulation that may contribute to the effects of mineralocorticoids on sodium reabsorption.     

Binding of 1α,25‐dihydroxyvitamin D3 to annexin II: Effect of vitamin D metabolites and calcium

We have recently reported that annexin II serves as a membrane receptor for 1α,25‐(OH)₂D₃ and mediates the rapid effect of the hormone on intracellular calcium. The purpose of these studies was to characterize the binding of the hormone to annexin II, determine the specificity of binding, and assess the effect of calcium on binding. The binding of [¹⁴C]‐1α,25‐(OH)₂D₃ bromoacetate to purified annexin II was inhibited by 1α,25‐(OH)₂D₃ in a concentration‐dependent manner. Binding of the radiolabeled ligand to annexin II was markedly diminished by 1α,25‐(OH)₂D₃ at 24 μM, 18 μM, and 12 μM and blunted by 6 μM and 3 μM. At a concentration of 12 μM, 1β,25‐(OH)₂D₃ also diminished the binding of [¹⁴C]‐1α,25‐(OH)₂D₃ bromoacetate to annexin II, but cholecalciferol, 25‐(OH)D₃, and 24,25‐(OH)₂D₃ did not. Saturation analyses of the binding of [³H]‐1α,25‐(OH)₂D₃ to purified annexin II showed a K_D of 5.5 × 10⁻⁹ M, whereas [³H]‐1β,25‐(OH)₂D₃ exhibited a K_D of 6.0 × 10⁻⁹ M. Calcium, which binds to the carboxy terminal domain of annexin II, had a concentration‐dependent effect on [¹⁴C]‐1α,25‐(OH)₂D₃ bromoacetate binding to annexin II, with 600 nM calcium being able to inhibit binding of the radiolabeled analog. The inhibitory effect of calcium was prevented by EDTA. Homocysteine, which binds to the amino terminal domain of annexin II, had no effect on the binding of the bromoacetate analog to the protein. The data indicate that 1α,25‐(OH)₂D₃ binding to annexin II is specific and suggest that the binding site may be located on the carboxy terminal domain of the protein. The ability of 1β,25‐(OH)₂D₃ to inhibit the binding of [¹⁴C]‐1α,25(OH)₂D₃ bromoacetate to annexin II provides a biochemical explanation for the ability of the 1β‐epimer to inhibit the rapid actions of the hormone in vitro. J. Cell. Biochem. 80:259–265, 2000. © 2000 Wiley‐Liss, Inc.     

Retrospective review of locally set tolerances for VMAT prostate patient specific QA using the COMPASS® system

PurposeThis study retrospectively reviewed locally set pass rates/tolerances for COMPASS^® pre-treatment quality assurance results for RapidArc prostate plans to determine if these are appropriate. This was performed via quantifying the agreement between treatment planning system calculations and measurements based on absolute dose comparisons (3% tolerance for all dose points) and global gamma index assessment (3%/3 mm criterion for 97% of points).MethodSeventy-three prostate one-arc RapidArc plans, delivered by four dosimetrically matched linacs, were measured using the MatriXX Evolution two-dimensional array and analysed using COMPASS^® (v.3, IBA Dosimetry). For the planning target volumes (PTV) considered, the D_99%, D_50%, D_1% and D_Mean differences were analysed. The percentage volume with gamma greater than 1, average gamma and D_Mean difference were investigated for all structures. Nine plans were also assessed across the linac fleet to investigate potential linac dependence of results.Results and ConclusionsRegarding PTV D_Mean differences, all plans fell within the 3% tolerance and mostly within 2%, although there was a relatively small systematic difference. The absolute percentage differences of average and median doses suggested a weak linac dependence of the results which was found to be clinically insignificant. New stricter tolerances were established both for dose comparisons and gamma evaluation. Correlation between the gamma pass rates and the differences in the D_99%, D_50% and D_1% was found to be moderate suggesting that gamma analysis in isolation has questionable clinical meaning and should only be used to indicate outliers for further analysis.     

Tracking,gating, free-breathing,which technique to use for lung stereotactic treatments? A dosimetric comparison

BackgroundThe management of breath-induced tumor motion is a major challenge for lung stereotactic body radiation therapy (SBRT). Three techniques are currently available for these treatments: tracking (T), gating (G) and free-breathing (FB).AimTo evaluate the dosimetric differences between these three treatment techniques for lung SBRT.Materials and methodsPretreatment 4DCT data were acquired for 10 patients and sorted into 10 phases of a breathing cycle, such as 0% and 50% phases defined respectively as the inhalation and exhalation maximum. GTV_ph, PTV_ph (=GTV_ph + 3 mm) and the ipsilateral lung were contoured on each phase.For the tracking technique, 9 fixed fields were adjusted to each PTV_ph for the 10 phases. The gating technique was studied with 3 exhalation phases (40%, 50% and 60%). For the free-breathing technique, ITV_FB was created from a sum of all GTV_ph and a 3 mm margin was added to define a PTV_FB. Fields were adjusted to PTV_FB and dose distributions were calculated on the average intensity projection (AIP) CT. Then, the beam arrangement with the same monitor units was planned on each CT phase.The 3 modalities were evaluated using DVHs of each GTV_ph, the homogeneity index and the volume of the ipsilateral lung receiving 20 Gy (V_20Gy).ResultsThe FB system improved the target coverage by increasing D_mean (75.87_(T)–76.08_(G)–77.49_(FB)Gy). Target coverage was slightly more homogeneous, too (HI: 0.17_{(T and G)}–0.15_(FB)). But the lung was better protected with the tracking system (V_20Gy: 3.82_(T)–4.96_(G)–6.34_(FB)%).ConclusionsEvery technique provides plans with a good target coverage and lung protection. While irradiation with free-breathing increases doses to GTV, irradiation with the tracking technique spares better the lung but can dramatically increase the treatment complexity.     

Synthesis and glycosidation of 1-deoxy-1-[(2,2-diacylvinyl)amino]-d-fructoses

The reactions of 1-amino-1-deoxy-d-fructose acetate (1) with methyl 3-methoxy-2-methoxycarbonylacrylate and 5-methoxymethylene-2,2-dimethyl-1,3-dioxane-4,6-dione in the presence of a base afforded 1-deoxy-1-[(2,2-dimethoxycarbonylvinyl)amino]- (2 and 1-deoxy-1-[(2,2-dimethyl-4,6-dioxo-1,3-dioxan-5-ylidenemethyl)amino]-d-fructose (3), respectively, in high yields. 1-Deoxy-1-[(4,4-dimethyl-2,6-dioxocyclohexylidenemethyl)amino]-d-fructose (4) was obtained (85%) by a transamination reaction between 1 and 5,5-dimethyl-2-phenylaminomethylene-1,3-cyclohexanedione in the presence of Et₃N. The isomeric composition of equilibrium solutions of 1–4 was established by ¹³C-n.m.r. spectroscopy. For all the compounds, the β-pyranose form was the main component in D₂O; the α-furanose, the β-furanose, and, for 1, the α-pyranose forms, were also present. The major constituents of 2 in (CD₃)₂SO solution were the β- and the α-furanose forms. Acetylation of 2 afforded the tetra-acetates of the α- and β-furanose forms, the 3,4,6-triacetates of the α- and β-furanose forms, the 3,4,5-triacetate of the β-pyranose form, and 2,3,4,5,6-penta-O-acetyl-1-deoxy-1-[(2,2-dimethoxycarbonylvinyl)amino]-d-arabino-hex-1-enitol. Glycosidation of 2 with MeOHHCl afforded a mixture of methyl 1-deoxy-1-[(2,2-dimethoxycarbonylvinyl)amino]-α- (11α) and -β-d-fructofuranoside (11β), and methyl 1-deoxy-1-[(2,2-dimethoxycarbonylvinyl)-amino]-β-d-fructopyranoside (13). Compounds 11α and 13 were isolated as their tri-acetates (12 and 14, respectively). Deacetylation and removal of the N-protecting group of 12 gave methyl 1-amino-1-deoxy-α-d-fructofuranoside (∼54% from 2).     

Metabolism of prostaglandin D2 in isolated rat lung: the stereospecific formation of 9α, 11β-prostaglandin F2 from prostaglandin D2

The metabolic transformation of exogenous prostaglandin D₂ was investigated in isolated perfused rat lung. Dose-dependent formation (2–150 ng) of 9α,11β-prostaglandin F₂, corresponding to about 0.1% of the perfused dose of prostaglandinD₂, was observed by specific radioimmunoassay both in the perfusate and in lung tissue after a 5-min perfusion. To investigate the reason for this low conversion ratio, we analyzed the metabolites of tritium-labeled 9α,11β-prostaglandin F₂ and prostaglandin D₂ by boric acid-impregnated TLC and HPLC. By 5 min after the start of perfusion, 9α,11β-prostaglandin F₂ disappeared completely from the perfusate and the major product formed remained unchanged during the remainder of the 30-min perfusion. The major product was separated by TLC and identified as 13,14-dihydro-15-keto-9α,11β-prostaglandin F₂ by GC/MS. In contrast, pulmonary breakdown of prostaglandin D₂ was slow and two major metabolites in the perfusate increased with time, each representing 56% and 11% of the total radioactivity at the end of the perfusion. The major product (56%) was identified as 13,14-dihydro-15-ketoprostaglandin D₂ and the minor one (11%) was tentatively identified as 13,14-dihydro-15-keto-9α,11β-prostaglandin F₂ based on the results from radioimmunoassays, TLC, HPLC, and the time course of pulmonary breakdown. These results demonstrate that the metabolism of prostaglandin D₂ in rat lung involves at least two pathways, one by 15-hydroxyprostaglandin dehydrogenase and the other by 11-ketoreductase, and that the 9α,11β-prostaglandin F₂ formed is rapidly metabolized to 13,14-dihydro-15-keto-9α,11β-prostaglandin F₂.     

Unique assignment of inter-subunit association in GABAA α1β3γ2 receptors determined by molecular modeling

Recent publications defined requirements for inter-subunit contacts in a benzodiazepine-sensitive GABA_A receptor (GABA_ARα1β3γ2). There is strong evidence that the heteropentameric receptor contains two α1, two β3, and one γ2 subunit. However, the available data do not distinguish two possibilities: When viewed clockwise from an extracellular viewpoint the subunits could be arranged in either γ2β3α1β3α1 or γ2α1β3α1β3 configurations. Here we use molecular modeling to thread the relevant GABA_AR subunit sequences onto a template of homopentameric subunits in the crystal structure of the acetylcholine binding protein (AChBP). The GABA_A sequences are known to have 15-18% identity with the acetylcholine binding protein and nearly all residues that are conserved within the nAChR family are present in AChBP. The correctly aligned GABA_A sequences were threaded onto the AChBP template in the γ2β3α1β3α1 or γ2α1β3α1β3  arrangements. Only the γ2α1β3α1β3 arrangement satisfied three known criteria: (1) α1 His¹⁰² binds at the γ2 subunit interface in proximity to γ2 residues Thr¹⁴², Phe⁷⁷, and Met¹³⁰; (2) α1 residues 80-100 bind near γ2 residues 91-104; and (3) α1 residues 58-67 bind near the β3 subunit interface. In addition to predicting the most likely inter-subunit arrangement, the model predicts which residues form the GABA and benzodiazepine binding sites.