Search

Atetrahydroisoquinoline (THIQ) core is able to mimic the A and B rings of 2-methoxyestradiol (2ME2), an endogenous estrogen metabolite that demonstrates promising anticancer properties primarily by disrupting microtubule dynamic instability parameters, but has very poor pharmaceutical properties that can be improved by sulfamoylation. The nonsteroidal THIQ-based microtubule disruptor 2-(3-bromo-4,5-dimethoxybenzyl)-7-methoxy-6-sulfamoyloxy-1,2,3,4-tetrahydro isoquinoline (STX3451), with enhanced pharmacokinetic and pharmacodynamic profiles, was explored for the first time in radition biology. We investigated whether 24 h pre-treatment with STX3451 could pre-sensitize MCF-7 and MDA-MB-231 breast cancer cells to radiation. This regimen showed a clear increase in cytotoxicity compared to the individual modalities, results that were contiguous in spectrophotometric analysis, flow cytometric quantification of apoptosis induction, clonogenic studies and microscopy techniques. Drug pre-treatment increased radiation-induced DNA damage, with statistically more double-strand (ds) DNA breaks demonstrated. The latter could be due to the induction of a radiation-sensitive metaphase block or the increased levels of reactive oxygen species, both evident after compound exposure. STX3451 pre -exposure may also delay DNA repair mechanisms, as the DNA damage response element ataxia telangiectasia mutated (ATM) was depressed. These in vitro finding s may translate into in vivo models, with the ultimate aim of reducing both radiation and drug doses for maximal clinical effect with minimal adverse effects.

Sodium/potassium-transporting adenosine triphosphatase (Na+/K+-ATPase) is one of the most abundant cell membrane proteins and is essential for eukaryotes. Endogenous negative regulators have long been postulated to play an important role in regulating the activity and stability of Na+/K+-ATPase, but characterization of these regulators has been elusive. Mechanisms of regulating Na+/K+-ATPase homeostatic turnover are unknown. Here, we report that 5-diphosphoinositol 1,2,3,4,6-pentakisphosphate (5-InsP7), generated by inositol hexakisphosphate kinase 1 (IP6K1), promotes physiological endocytosis and downstream degradation of Na+/K+-ATPase-α1. Deletion of IP6K1 elicits a twofold enrichment of Na+/K+-ATPase-α1 in plasma membranes of multiple tissues and cell types. Using a suite of synthetic chemical biology tools, we found that 5-InsP7 binds the RhoGAP domain of phosphatidylinositol 3-kinase (PI3K) p85α to disinhibit its interaction with Na+/K+-ATPase-α1. This recruits adaptor protein 2 (AP2) and triggers the clathrin-mediated endocytosis of Na+/K+-ATPase-α1. Our study identifies 5-InsP7 as an endogenous negative regulator of Na+/K+-ATPase-α1.

Diphosphoinositol phosphates (PP-InsPs) are an evolutionarily ancient group of signalling molecules that are essential to cellular and organismal homeostasis. As the detailed mechanisms of PP-InsP signalling begin to emerge, synthetic analogues of PP-InsPs containing stabilised mimics of the labile diphosphate group can provide valuable investigational tools. We synthesised 5-PCF2Am-InsP5 (1), a novel fluorinated phosphonate analogue of 5-PP-InsP5, and obtained an X-ray crystal structure of 1 in complex with diphosphoinositol pentakisphosphate kinase 2 (PPIP5K2). 5-PCF2Am-InsP5 binds to the kinase domain of PPIP5K2 in a similar orientation to that of the natural substrate 5-PP-InsP5 and the PCF2Am structure can mimic many aspects of the diphosphate group in 5-PP-InsP5. We propose that 1, the structural and electronic properties of which are in some ways complementary to those of existing phosphonoacetate and methylenebisphosphonate analogues of 5-PP-InsP5, may be a useful addition to the expanding array of chemical tools for the investigation of signalling by PP-InsPs. The PCF2Am group may also deserve attention for wider application as a diphosphate mimic.

Graphical abstract, The inositol phosphates, InsP5 and InsP6, have recently been identified as binding partners of fibrinogen, which is critically involved in hemostasis by crosslinking activated platelets at sites of vascular injury. Here, we investigated the putative physiological role of this interaction and found that platelets increase their InsP6 concentration upon stimulation with the PLC-activating agonists thrombin, collagen I and ADP and present a fraction of it at the outer plasma membrane. Cone and plate analysis in whole blood revealed that InsP6 specifically increases platelet aggregate size. This effect is fibrinogen-dependent, since it is inhibited by an antibody that blocks fibrinogen binding to platelets. Furthermore, InsP6 has only an effect on aggregate size of washed platelets when fibrinogen is present, while it has no influence in presence of von Willebrand factor or collagen. By employing blind docking studies we predicted the binding site for InsP6 at the bundle between the γ and β helical subunit of fibrinogen. Since InsP6 is unable to directly activate platelets and it did not exhibit an effect on thrombin formation or fibrin structure, our data indicate that InsP6 might be a hemostatic agent that is produced by platelets upon stimulation with PLC-activating agonists to promote platelet aggregation by supporting crosslinking of fibrinogen and activated platelets.

Aims and objectives Methods and materials Results Conclusion Personal information References, Aims and objectives: A cavity is defined by the Fleischner Society Glossary of Terms for Thoracic Imaging as "a gas-filled space, seen as a lucency or low-attenuation area, within pulmonary consolidation, a mass or a nodule" [1]. It is well to remember that cavities are...

Aims and objectives Methods and materials Results Conclusion Personal information References, Aims and objectives: Radiologists arefrequently confronted with pulmonary nodules and masses in everyday practice. However, cavitation of such a nodule or mass, and especially solitary cavitation, is not seen that often. A cavity is defined by the Fleischner Society...

No description provided Copyright 2008 JM Godfrey, KT Trotman, KG Chalmers, R Moroney, B Potter, F Farrelly, M Beverland. No part of this article may be reproduced by any means without the written consent of the publisher.

The synthesis of several fluorinated analogues of thiamine and precursors has been undertaken, all of which exhibit weak activity against E.Coli. A route to 3-fluoro-(R)-alanine has been explored. The first synthesis of the enantiomeric fluorosuccinic acids has been accomplished, and a stereochemical analysis has shown that synthesis via esterified malate precursors using diethylamino- sulphurtrifluoride gives inversion of configuration, whereas prep- aration via aspartic acid and polyhydrogen fluoride in pyridine gives retention of configuration. The circular dichroism spectra of the fluorosuccinic acids are anomalous, being the first example of such behaviour in a-substituted carboxylic acids and derivatives. A previously assigned configuration of a Pseudomonal metabolite ( + )- fluorosuccinic acid has been corrected. The fluorosuccinic acids are not substrates for malic enzyme, but are potent inhibitors of fumarase. Using (2S)-fluorosuccinic acid as a mechanistic probe the fumarase reaction is identified as a bimolecular E2 elimination. A stereochemical analysis of enzymic phosphoryl transfer reactions using CD spectroscopy has been investigated, and a new synthesis of inorganic pyrophosphate used to prepare P1=[(S)- 160, 170, 180]-pyrophosphate whose 31P n.m.r. spectrum is discussed. The first example of 31 p- 17 o coupling as observed by 31 P n.m.r. is demonstrated.- The magnitude of the 31P- 180 isotope shift is dependent on the nature of the phosphorus to oxygen bond. An elegant method for establishing the isotopic configuration of oxygen chiral phosphates by n.m.r. has been developed, requiring the synthesis of D-glucose-6[(S) - 160, 170, 180]-phosphate, adenosine-5'- [(S)- 160, 170, 180]-phosphate, and their six membered cyclic phosphate triesters. Chemical cyclisation of these molecules occurs both with racemisation and inversion of configuration at phosphorus. The chemical synthesis of adenosine-5'[ (S) - 160, 170, 180]- triphosphate has been achieved and exploited to demonstrate that hexokinase phosphoryl transfer proceeds with inversion of config- uration at phosphorus. Pyruvate kinase phosphoryl transfer from 2 [(S)- 160, 170, 180]-phospho- (R)-glycerate also proceeds with inversion of configuration, as does phosphofructokinase phosphoryl transfer from sn-glycerol-3[ (5)- 160, 170, 180]-phosphate.

• the interplay between values and evidence in screening policy • methods of an ongoing systematic review • examples of disinvestment decisions

Histone deacetylases (HDACs) 1, 2 and 3 form the catalytic subunit of several large transcriptional repression complexes. Unexpectedly, the enzymatic activity of HDACs in these complexes has been shown to be regulated by inositol phosphates, which bind in a pocket sandwiched between the HDAC and co-repressor proteins. However, the actual mechanism of activation remains poorly understood. Here we have elucidated the stereochemical requirements for binding and activation by inositol phosphates, demonstrating that activation requires three adjacent phosphate groups and that other positions on the inositol ring can tolerate bulky substituents. We also demonstrate that there is allosteric communication between the inositol-binding site and the active site. The crystal structure of the HDAC1:MTA1 complex bound to a novel peptide-based inhibitor and to inositol hexaphosphate suggests a molecular basis of substrate recognition, and an entropically driven allosteric mechanism of activation.

Tumor neo-angiogenesis is regulated, in part, by the hypoxia-inducible gene HIF1.. Evidence suggests HIF1 associates with polymerized microtubules and traffics to the nucleus. This study investigated the role of HIF1 in mediating the antitumor activity of two steroid-based sulfamate ester microtubule disruptors, STX140 and STX243, in vitro and in vivo. The effects of STX140, STX243 and the parental compound 2-methoxyestradiol (STX66) on HIF1α and HIF2α protein expression were assessed in vitro in MCF-7 and MDA-MB-231 cells cultured under hypoxia. More pertinently, their effects were examined on HIF1-regulated genes in vivo in mice bearing MCF-7 or MDA-MB-231 tumors. The level of mRNA expression of Vascular Endothelial Growth Factor (VEGF), Glucose Transporter 1 (GLUTI), Phosphoglycerate Kinase (PGK), ATP-binding cassette sub-family B member 1 (ABCB1) and Carbonic Anhydrase IX (CAIX) was quantified by Real-time Polymerase Chain Reaction (RT-PCR). Despite inhibiting nuclear HIF1 protein accumulation under hypoxia in vitro, STX140 and STX243 did not significantly regulate the expression of four out of five HIF1α-regulated genes in vitro and in vivo. Only CAIX mRNA expression was down-regulated both in vitro and in vivo. Immunoblot analysis showed that STX140 and STX243 reduced CAIX protein expression in vitro. These compounds had no effect on HIF2α translocation. The potential for inhibition of CAIX by STX140 and STX243 was examined by docking the ligands to the active site in comparison with a known sulfamate-based inhibitor. Microtubule disruption and antitumor activity of STX140 and STX243 is most likely HIF1-independent and may, at least in part, be mediated by inhibition of CAIX expression and activity.

Adenophostin A, the most potent known agonist of inositol 1,4, 5-trisphosphate (InsP(3)) receptors, stimulated (45)Ca(2+) release from the intracellular stores of permeabilized hepatocytes. The concentration of adenophostin A causing the half-maximal effect (EC(50)) was 7.1+/-0.5 nM, whereas the EC(50) for InsP(3) was 177+/-26 nM; both responses were positively co-operative. In rapid superfusion analyses of (45)Ca(2+) release from the intracellular stores of immobilized hepatocytes, maximal concentrations of adenophostin A or InsP(3) evoked indistinguishable patterns of Ca(2+) release. The Ca(2+) release evoked by both agonists peaked at the same maximal rate after about 375 ms and the activity of the receptors then decayed to a stable, partially (60%) inactivated state with a half-time (t(1/2)) of 318+/-29 ms for adenophostin A and 321+/-22 ms for InsP(3). Dissociation rates were measured by recording rates of InsP(3)-receptor channel closure after rapid removal of agonist. The rate of adenophostin A dissociation (t(1/2), 840+/-195 ms) was only 2-fold slower than that of InsP(3) (t(1/2), 436+/-48 ms). We conclude that slow dissociation of adenophostin A from InsP(3) receptors does not underlie either its high-affinity binding or the reported differences in the Ca(2+) signals evoked by InsP(3) and adenophostin A in intact cells.

The binding of von Willebrand factor (vWF) to subendothelium constitutes an important initial step in the process of platelet adhesion to exposed subendothelium following blood vessel injury. We previously demonstrated that vWF is present in human vascular subendothelium and recently found that a 150 kd vWF-binding protein, which we extracted from subendothelium, is type VI collagen. Although we have established that vWF and type VI collagen bind in vitro, it is not known whether these two proteins are associated in the vascular subendothelium in situ. We, therefore, 1) investigated the morphological effects of our biochemical extraction procedure on human umbilical veins by scanning and transmission electron microscopy, 2) analyzed the subendothelial extract by immunofluorescence for the presence of vWF and collagens and by electron microscopy for morphological characteristics, and 3) localized vWF and type VI collagen in subendothelium by immunofluorescence and by single- and double-label immunoelectron microscopic studies with protein A-conjugated gold particles. We found that the surface exposed following de-endothelialization is composed of microfibrils and contains very little fibrillar collagen. The subendothelium is stripped after sodium dodecyl sulfate-urea extraction, and the extract itself contains immunoreactive vWF and type VI collagen but no immunoreactive type I or III fibrillar collagens. Immunofluorescence and immunoelectron microscopic studies showed that vWF and type VI collagen are both present in subendothelium, where both co-localized to microfibrils. In conclusion, vWF that binds to type VI collagen in vitro, also co-localizes with type VI collagen in subendothelium, where both are associated with microfibrils. Type VI collagen, therefore appears to serve as a biologically significant binding site for vWF in vivo and may thereby play a role in mediating platelet adhesion to exposed subendothelium following vascular injury.

Document uit de collectie Chemische Procestechnologie

The oligonucleotide d(GGsAATTCC) containing the recognition sequence of the Eco R1 restriction endonuclease with a phosphorothioate group at the site of cleavage was synthesized by two approaches both based on the polymer support phosphoroamidite method. Only the Rp-diastereomer was cleaved by Eco R1, at a rate approximately 20 times slower than the all-phosphate-containing octamer. To study the interaction of restriction endonucleases with phosphorothioate internucleotidic linkages double stranded Mi3mp2 DNA was prepared in which the (+)strand contained only phosphate groups but the (-)strand phosphate groups as well as base specifically introduced phosphorothioate groups of the Rp-configuration. This hybrid DNA was cleaved by several restriction enzymes. The results obtained allow the classification of these enzymes into three groups: those which produce nicked DNA as an isolatable intermediate, those where the nicked DNA is the final product and a third where only linearised DNA can be detected. Particularly the second class of enzymes should be of interest for the manipulation of DNA.

The effect of endotoxin and immune complexes on monocyte lysosomal enzyme activity has been studied in patients with Crohn's disease and ulcerative colitis. Immune complexes made at equivalence or in antigen excess caused a rapid fall in intracellular activity of the enzyme N'acetyl-beta-D-glucosaminidase which was significantly greater than that seen when cells were incubated with medium alone. Endotoxin had no effect on intracellular enzyme activity but there was a significant elevation of activity in the supernatants. Cells from patients with inflammatory bowel disease were no different to cells from healthy control subjects in their response to either endotoxin or immune complexes.

The metabolism of the third component of complement (C3) has been investigated in four patients with ulcerative colitis, three patients with Crohn's disease and seven control subjects, using radioiodinated C3 prepared from fresh human plasma. Both the fractional catabolic rate and synthesis rate of C3 were increased in the patients with inflammatory bowel disease, although the serum-C3 levels were normal or raised. The results suggest that complement activation may play a role in the pathogenesis of mucosal inflammation in these diseases.

In single mouse pancreatic acinar cells the effects of intracellular infusion of inositol 1,4,5-trisphosphate (InsP3) or the non-metabolizable InsP3 analogue inositol 1,4,5-triphosphorothioate (InsPS3) have been investigated using a wide range of concentrations. Different types of cytosolic Ca2+ fluctuation patterns (monitored as Ca(2+)-dependent Cl- current in patch-clamp whole-cell recording experiments) could be generated by InsP3 or InsPS3, dependent on concentration, resembling those previously shown to be evoked by varying degrees of receptor activation in these cells. Low InsPS3 concentrations evoked repetitive local Ca2+ spikes whereas at relatively high concentrations repetitive Ca2+ waves were produced. In the presence of intracellular citrate a much lower messenger level was sufficient to generate waves. The InsP3 concentration determines whether the cytosolic Ca2+ signals are local or global.

Blank

In this work, myo-inositol phosphates and pyrophosphates were investigated to facilitate further understanding of their chemical biology and biological functions. The synthesis of novel carbohydrate-based polyphosphate analogues of lower inositol phosphates, including the second messenger ᴅ-myo-inositol 1,4,5-trisphosphate [Ins(1,4,5)P₃], was undertaken, and novel routes for some naturally-occurring inositol pyrophosphates were designed. Synthetic routes for the meso myo-inositol pyrophosphates 5-PP-InsP₅ (5-IP₇) and 5- PP-InsP₄, as well as both the racemic and chiral versions of D-1,5-(PP)₂-InsP₄ (D-1,5-IP₈), were successfully executed. These approaches incorporated the hitherto little-used methylsulfonylethyl (MSE)-protecting group, allowing its versatility to be explored. Final products were then used with collaborators to investigate: substrate binding to the yeast diphosphoinositol polyphosphate phosphohydrolase through a structural biology approach; the potential scope of a novel pyrophosphate-monitoring, europium-based luminescence assay; and structure-activity (SAR) aspects of the inositol 1,4,5,6-tetrakisphosphate binding site in the histone deacetylase 3 - SMRT corepressor (HDAC3-SMRT) complex. The SAR investigation for HDAC3-SMRT featured seven glucose-based polyphosphate compounds (two known, five novel) that were designed, synthesised and biologically evaluated. The ʟ-glucose-based ligands were found to be the first carbohydrate-based ligands able to activate the complex, while their ᴅ-glucose-based enantiomers were inactive. A binding site region suspected to be able to accommodate steric bulk was explored, and it also was concluded that inositol pyrophosphates are unlikely to be physiologically relevant endogenous activators for the HDAC3-SMRT complex. A further six novel glucose-based polyphosphates were synthesised to investigate calcium release via the Ins(1,4,5)P₃ receptor (InsP₃R). Two ligands, α-ᴅ-glucopyranosyl 1,3,4-trisphosphate and β-ᴅ-glucopyranosyl 1,3,4-trisphosphate, were full agonists, the latter equipotent to Ins(1,4,5)P₃ itself. These two ligands were also substrates for Arabidopsis inositol tetrakisphosphate kinase-1 (ITPK1). Although unconfirmed, it is proposed that ITPK1 phosphorylates the 6-position primary hydroxyl. Both ligands were uniquely co-crystallised with Ins(1,4,5)P₃ 3-kinase. The crystallographicallysolved ternary complexes also revealed the primary hydroxyl group to be well-situated for potential phosphorylation. Two of the InsP₃R-study ligands, methyl α-ʟ-glucopyranoside 2,3,6-trisphosphate and methyl α-ʟ-glucopyranoside 2,4,6-trisphosphate, were low-affinity, low-efficacy partial agonists. From the presumed binding mode of the latter, structurally-inspired myo-inositol pyrophosphate-containing compounds were designed, targeting a potential higher-affinity partial agonist or antagonist. Using techniques earlier developed, three novel inositol pyrophosphates were synthesized, 4-PP-Ins(5)P, 5-PPIns(4)P and 4-PP-Ins(1,5)₂, which are the first examples of an Ins(1,4,5)P₃ pyrophosphate analogue modified at the 4,5-vicinal bisphosphate pharmacophore. Biological evaluation of these compounds is currently underway.

Purpose: To retrospectively evaluate the outcome and toxicity in patients receiving high-dose (>69 Gy) adjuvant radiotherapy (HD-ART) and the impact of androgen deprivation therapy (ADT). Methods and Materials: Between 1999 and 2008, 225 node-negative patients were referred for HD-ART with or without ADT to two large academic institutions. Indications for HD-ART were extracapsular extension, seminal vesicle invasion (SVI), and/or positive surgical margins at radical prostatectomy (RP). A dose of at least 69.1 Gy was prescribed to the prostate bed and seminal vesicle bed. The ADT consisted of a luteinizing hormone-releasing hormone analog. The duration and indication of ADT was left at the discretion of the treating physician. The effect of HD-ART and ADT on biochemical (bRFS) and clinical (cRFS) relapse-free survival was examined through univariate and multivariate analysis, with correction for known patient-and treatment-related variables. Interaction terms were introduced to evaluate effect modification. Results: After a median follow-up time of 5 years, the 7-year bRFS and cRFS were 84% and 88%, respectively. On multivariate analysis, the addition of ADT was independently associated with an improved bRFS (hazard ratio [HR] 0.4, p = 0.02) and cRFS (HR 0.2, p = 0.008). Higher Gleason scores and SVI were associated with decreased bRFS and cRFS. Alymphadenectomy at the time of RP independently improved cRFS (HR 0.09, p = 0.009). The 7-year probability of late Grade 2-3 toxicity was 29% and 5% for genitourinary (GU) and gastrointestinal (GI) symptoms, respectively. The absolute incidence of Grade 3 toxicity was