Search

Introduction: Integration of psychological services into pediatric primary care is increasingly common, but models of integration vary with regard to their level of coordination, colocation, and integration. High-integration models may provide some distinct advantages, such as preventative care and brief consultation for subclinical behavior concerns; however, psychologists face barriers to seeking reimbursement for these services. Alternatives to traditional psychotherapy and psychological testing codes, specifically Health & Behavior (H&B) codes, have been proposed as 1 method for supporting integrated care. The aim of this study was to investigate the relationships between psychologists' reported billing practices, reimbursement rates, and model of integration in pediatric primary care., Method: As part of a larger survey study, 55 psychologists working in pediatric primary care reported on characteristics of their practice's model of integration, billing practices, and frequency of reimbursement for consultative services., Results: Compared with those who categorized their integrated care model as colocated, psychologists who endorsed working in integrated models reported a significantly higher usage of H&B codes and more frequent reimbursement for consultations. Overall, use of H&B codes was associated with higher reported levels of coordination and integration., Discussion: Survey results showed a clear pattern of higher integration being associated with greater utilization of H&B codes and better reimbursement for consultation activities. These results underscore the importance of establishing and maintaining billing and reimbursement systems that adequately support integrated care. (PsycINFO Database Record, ((c) 2018 APA, all rights reserved).)

The Rohingya of Myanmar are a severely persecuted minority who form one of the largest groups of stateless people; thousands of them reside in refugee camps in southeastern Bangladesh. There has been little research into the mental health consequences of persecution, war, and other historical trauma endured by the Rohingya; nor has the role of daily environmental stressors associated with continued displacement, statelessness, and life in the refugee camps, been thoroughly researched. This cross-sectional study examined: trauma history, daily environmental stressors, and mental health outcomes for 148 Rohingya adults residing in Kutupalong and Nayapara refugee camps in Bangladesh. Results indicated high levels of mental health concerns: posttraumatic stress disorder (PTSD), depression, somatic complaints, and associated functional impairment. Participants also endorsed local idioms of distress, including somatic complaints and concerns associated with spirit possession. The study also found very high levels of daily environmental stressors associated with life in the camps, including problems with food, lack of freedom of movement, and concerns regarding safety. Regression and associated mediation analyses indicated that, while there was a direct effect of trauma exposure on mental health outcomes (PTSD symptoms), daily environmental stressors partially mediated this relationship. Depression symptoms were associated with daily stressors, but not prior trauma exposure. These findings indicate that daily stressors play a pivotal role in mental health outcomes of populations affected by collective violence and statelessness. It is, therefore, important to consider the role and effects of environmental stressors associated with life in refugee camps on the mental health and psychosocial well-being of stateless populations such as the Rohingya, living in protracted humanitarian environments.

Most small molecules are unable to rapidly traverse the outer membrane of Gram-negative bacteria and accumulate inside these cells, making the discovery of much-needed drugs against these pathogens challenging. Current understanding of the physicochemical properties that dictate small-molecule accumulation in Gram-negative bacteria is largely based on retrospective analyses of antibacterial agents, which suggest that polarity and molecular weight are key factors. Here we assess the ability of over 180 diverse compounds to accumulate in Escherichia coli. Computational analysis of the results reveals major differences from the retrospective studies, namely that the small molecules that are most likely to accumulate contain an amine, are amphiphilic and rigid, and have low globularity. These guidelines were then applied to convert deoxynybomycin, a natural product that is active only against Gram-positive organisms, into an antibiotic with activity against a diverse panel of multi-drug-resistant Gram-negative pathogens. We anticipate that these findings will aid in the discovery and development of antibiotics against Gram-negative bacteria.

Objective: To assess parents' perceptions and use of time-out (TO) in contrast to empirical indications and examine the relationship between reported implementation procedures and perceived effectiveness., Methods: We surveyed parents of preschool and school-age children (n = 401, aged 15 months to 10 years) at well-child visits with regard to their awareness, perception, and usage of TO. Parents were specifically surveyed regarding TO components that have been empirically evaluated or pertain directly to its underlying behavioral principles. Descriptive analyses, group comparisons, and correlational analyses were used to characterize responses and evaluate the relationship between TO administration variation and perceived effectiveness., Results: Most parents (76.8%) reported using TO in response to misbehavior, but a large majority of these parents (84.9%) reported implementing TO in a manner counter to empirical evidence. Parents who endorsed TO as effective varied significantly from those who did not on key implementation components (eg, use of a single warning). Further, several reported implementation practices were correlated with perceived effectiveness and challenging child behavior. For example, requiring a child to be calm before ending TO was positively correlated with perceived effectiveness., Conclusions: These results cement TO as a widely disseminated practice but cast doubt on the fidelity with which it is typically implemented. Better methods of educating parents on evidence-based discipline are needed., (Copyright © 2016 Academic Pediatric Association. Published by Elsevier Inc. All rights reserved.)

Opioids are widely used to treat millions suffering from pain, but their analgesic utility is limited due to associated side effects. Herein we report the development and evaluation of a chemical probe exhibiting analgesia and reduced opioid-induced side effects. This compound, kurkinorin (5), is a potent and selective μ-opioid receptor (MOR) agonist (EC 50 = 1.2 nM, >8000 μ/κ selectivity). 5 is a biased activator of MOR-induced G-protein signaling over β-arrestin-2 recruitment. Metadynamics simulations of 5's binding to a MOR crystal structure suggest energetically preferred binding modes that differ from crystallographic ligands. In vivo studies with 5 demonstrate centrally mediated antinociception, significantly reduced rewarding effects, tolerance, and sedation. We propose that this novel MOR agonist may represent a valuable tool in distinguishing the pathways involved in MOR-induced analgesia from its side effects.

Thiazolinyl imine reductases catalyze the NADPH-dependent reduction of a thiazoline to a thiazolidine, a required step in the formation of the siderophores yersiniabactin (Yersinia spp.) and pyochelin (Pseudomonas aeruginosa). These stand-alone nonribosomal peptide tailoring domains are structural homologues of sugar oxidoreductases. Two closed structures of the thiazolinyl imine reductase from Yersinia enterocolitica (Irp3) are presented here: an NADP(+)-bound structure to 1.45 Å resolution and a holo structure to 1.28 Å resolution with NADP(+) and a substrate analogue bound. Michaelis-Menten kinetics were measured using the same substrate analogue and the homologue from P. aeruginosa, PchG. The data presented here support the hypothesis that tyrosine 128 is the likely general acid residue for catalysis and also highlight the phosphopantetheine tunnel for tethering of the substrate to the nonribosomal peptide synthetase module during assembly line biosynthesis of the siderophore.

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.

Background: Research suggests that multimedia-based interventions possess advantages for disseminating safe and effective methods of behavior management to parents in pediatric primary care; however, little is known about their utilization in real-life settings. In order to maximize the impact of multimedia resources, more knowledge regarding dissemination and implementation is needed., Objective: To examine provider and parental perception and utilization of videos designed to communicate evidence-based parenting strategies for disruptive behavior. Videos were available in clinic and online., Results: Both provider and parent perceptions of the videos were largely positive. However, of 240 parents surveyed, only 33% were aware of the availability of videos subsequent to a well-child visit. Parents were unlikely to view the videos if they did not do so as part of their child's health care visit., Conclusion: Multimedia interventions for behavior management are likely to be well received, but systematic methods of implementation are needed. Further study of dissemination of multimedia interventions is merited., (© The Author(s) 2015.)

A total of 219 clinical isolates of Campylobacter spp. including 180 Campylobacter jejuni and 39 Campylobacter coli were assessed for in vitro antimicrobial susceptibility. Resistance among C. coli was higher for ciprofloxacin (41% versus 30.80%), erythromycin (12.80% versus 3.90%) and lower for tetracycline (53.80% versus 64.60%) compared to C. jejuni., (Crown Copyright © 2015. Published by Elsevier Inc. All rights reserved.)

To synthesise stabilised mimics of InsP8, the most phosphorylated inositol phosphate signalling molecule in Nature, we replaced its two diphosphate (PP) groups with either phosphonoacetate (PA) or methylenebisphosphonate (PCP) groups. Utility of the PA and PCP analogues was verified by structural and biochemical analyses of their interactions with enzymes of InsP8 metabolism.

Objective: The objective was to test whether Behavioral Family Systems Therapy for Diabetes (BFST-D), an evidence-based family therapy, produces individual changes in depressive symptoms for adolescents with type 1 diabetes in suboptimal glycemic control (HbA(1c) ≥9.0% [≥74.9 mmol/mol])., Research Design and Methods: Data were from a randomized controlled trial (RCT) comparing two modes of BFST-D delivery: in clinic versus Internet videoconferencing. There were no significant differences between groups in the RCT, so groups were collapsed into a within-group prepost design for secondary analyses. A multiple regression analysis was performed to test for mediation of treatment outcomes by changes in family processes., Results: Significant improvements in glycemic control, depressive symptoms, and family functioning were found from pre- to posttreatment. A multiple regression analysis for within-subject mediation indicated that improvements in depressive symptoms were partially mediated by improvements in parent-youth conflict; however, family process changes did not mediate diabetes health outcomes., Conclusions: In addition to improving treatment adherence and glycemic control, BFST-D has collateral benefits on depressive symptoms., (© 2015 by the American Diabetes Association. Readers may use this article as long as the work is properly cited, the use is educational and not for profit, and the work is not altered.)

Synthetic compounds open up new avenues to interrogate and manipulate intracellular Ca2+ signalling pathways. They may ultimately lead to drug-like analogues to intervene in disease. Recent advances in chemical biology tools available to probe Ca2+ signalling are described, with a particular focus on those synthetic analogues from our group that have enhanced biological understanding or represent a step towards more drug-like molecules. Adenophostin (AdA) is the most potent known agonist at the inositol 1,4,5-trisphosphate receptor (IP3R) and synthetic analogues provide a binding model for receptor activation and channel opening. 2-O-Modified inositol 1,4,5-trisphosphate (IP3) derivatives that are partial agonists at the IP3R reveal key conformational changes of the receptor upon ligand binding. Biphenyl polyphosphates illustrate that simple non-inositol surrogates can be engineered to give prototype IP3R agonists or antagonists and act as templates for protein co-crystallization. Cyclic adenosine 5'-diphosphoribose (cADPR) can be selectively modified using total synthesis, generating chemically and biologically stable tools to investigate Ca2+ release via the ryanodine receptor (RyR) and to interfere with cADPR synthesis and degradation. The first neutral analogues with a synthetic pyrophosphate bioisostere surprisingly retain the ability to release Ca2+, suggesting a new route to membrane-permeant tools. Adenosine 5'-diphosphoribose (ADPR) activates the Ca2+-, Na+- and K+-permeable transient receptor potential melastatin 2 (TRPM2) cation channel. Synthetic ADPR analogues provide the first structure-activity relationship (SAR) for this emerging messenger and the first functional antagonists. An analogue based on the nicotinic acid motif of nicotinic acid adenine dinucleotide phosphate (NAADP) antagonizes NAADP-mediated Ca2+ release in vitro and is effective in vivo against induced heart arrhythmia and autoimmune disease, illustrating the therapeutic potential of targeted small molecules.

The aggregation of the microtubule-associated protein tau is a seminal event in many neurodegenerative diseases, including Alzheimer's disease. The inhibition or reversal of tau aggregation is therefore a potential therapeutic strategy for these diseases. Fungal natural products have proven to be a rich source of useful compounds having wide varieties of biological activities. We have previously screened Aspergillus nidulans secondary metabolites for their ability to inhibit tau aggregation in vitro using an arachidonic acid polymerization protocol. One aggregation inhibitor identified was asperbenzaldehyde, an intermediate in azaphilone biosynthesis. We therefore tested 11 azaphilone derivatives to determine their tau assembly inhibition properties in vitro. All compounds tested inhibited tau filament assembly to some extent, and four of the 11 compounds had the advantageous property of disassembling preformed tau aggregates in a dose-dependent fashion. The addition of these compounds to the tau aggregates reduced both the total length and number of tau polymers. The most potent compounds were tested in in vitro reactions to determine whether they interfere with tau's normal function of stabilizing microtubules (MTs). We found that they did not completely inhibit MT assembly in the presence of tau. These derivatives are very promising lead compounds for tau aggregation inhibitors and, more excitingly, for compounds that can disassemble pre-existing tau filaments. They also represent a new class of anti-tau aggregation compounds with a novel structural scaffold.

Purpose: The aim of this study was to analyze microstructural corneal changes following phacoemulsification of cataract with intraocular lens insertion in diabetes mellitus (DM)., Methods: Prospective study of consecutive patients undergoing phacoemulsification with intraocular lens insertion, over a period of 6 months. Age, gender, serum glycosylated hemoglobin A1c levels, and effective phacoemulsification time were recorded. Anterior segment optical coherence tomography and corneal in vivo confocal microscopy were performed preoperatively and 1-month postoperatively. Subbasal nerve plexus (SBN) density, basal epithelial cell density, and endothelial cell density (ECD) were quantitatively analyzed., Results: Twenty-eight patients with type 2 DM (71.2 ± 7.6 years) and 23 healthy controls (mean age, 74.4 ± 7.4 years) were recruited. Mean hemoglobin A1c level was 7.5% in patients with DM and 5.7% in the control group (P < 0.0001). A significant reduction in SBN density was noted postoperatively in both DM (P < 0.0001) and non-DM groups (P < 0.0001). Patients with DM had a lower mean SBN density compared with non-DM group, before (P < 0.0001) and after surgery (P < 0.0001). Longer effective phacoemulsification time correlated with a greater decrease in ECD postoperatively in the whole cohort (P < 0.0001), and in the control (P = 0.002) and DM (P = 0.039) groups independently. There was no significant difference in preoperative ECD between the DM group (2254 +/- 426 cells/mm(2)) and non-DM group (2384 +/- 438 cells/mm(2); P = 0.2). [corrected]., Conclusions: Subbasal nerve density is reduced after cataract surgery in patients with and without DM. However, lower initial SBN density in patients with DM may predispose them to develop diabetic keratopathy. DM does not predispose to greater endothelial loss following phacoemulsification.

This study examined therapeutic mechanisms of action at the single-participant level in a behavior therapy (BT) for youth depression. By controlling for non-specific early responses, identifying potential mechanisms of action a priori, taking frequent measures of hypothesized mechanisms and dependent variables, rigorously evaluating internal validity, and using a variety of analytic methods, a unique model for analysis of potential mediators was created. Eleven children (M age = 9.84) meeting criteria on the Children's Depression Rating Scale-Revised (M = 55.36) and Children's Depression Inventory (M = 23.45) received non-directive therapy (NDT), followed by BT for those still displaying significant symptoms. Four participants (36%) had a clinically significant response to NDT. For the remaining seven, statistically significant changes in depressive symptoms and family interactions during the BT interval were found at the group level. At the single-participant level, evidence suggesting that outcome was at least partially mediated by changes in treatment targets was obtained for four of seven (57%). As the field further embraces efforts to learn not only whether treatments work but also how they work, the single-participant approach to evaluating mediators provides a useful framework for evaluating theories of therapeutic change., (© The Author(s) 2014.)

The pattern recognition receptor RIG-I is critical for Type-I interferon production. However, the global regulation of RIG-I signaling is only partially understood. Using a human genome-wide RNAi-screen, we identified 226 novel regulatory proteins of RIG-I mediated interferon-β production. Furthermore, the screen identified a metabolic pathway that synthesizes the inositol pyrophosphate 1-IP7 as a previously unrecognized positive regulator of interferon production. Detailed genetic and biochemical experiments demonstrated that the kinase activities of IPPK, PPIP5K1 and PPIP5K2 (which convert IP5 to1-IP7) were critical for both interferon induction, and the control of cellular infection by Sendai and influenza A viruses. Conversely, ectopically expressed inositol pyrophosphate-hydrolases DIPPs attenuated interferon transcription. Mechanistic experiments in intact cells revealed that the expression of IPPK, PPIP5K1 and PPIP5K2 was needed for the phosphorylation and activation of IRF3, a transcription factor for interferon. The addition of purified individual inositol pyrophosphates to a cell free reconstituted RIG-I signaling assay further identified 1-IP7 as an essential component required for IRF3 activation. The inositol pyrophosphate may act by β-phosphoryl transfer, since its action was not recapitulated by a synthetic phosphonoacetate analogue of 1-IP7. This study thus identified several novel regulators of RIG-I, and a new role for inositol pyrophosphates in augmenting innate immune responses to viral infection that may have therapeutic applications.

When applied extracellularly, myo-inositol hexakisphosphate (InsP6 ) and myo-inositol pentakisphosphate (InsP5 ) can inhibit the growth and proliferation of tumour cells. There is debate about whether these effects result from interactions of InsP6 and InsP5 with intracellular or extracellular targets. We synthesised FAM-InsP5 , a fluorescent conjugate of InsP5 that allows direct visualisation of its interaction with cells. FAM-InsP5 was internalised by H1229 tumour cells, a finding that supports earlier reports that externally applied inositol phosphates can-perhaps surprisingly-enter into cells. Close examination of the process of FAM-InsP5 uptake suggests a mechanism of non-receptor-mediated endocytosis, which is blocked at 4 °C and probably involves interaction of the ligand with the glycocalyx. However, our results are difficult to reconcile with antiproliferative mechanisms that require direct interactions of externally applied InsP5 or InsP6 with cytosolic proteins, because internalised FAM-InsP5 appears in lysosomes and apparently does not enter the cytoplasm. Studies using FAM-InsP5 are less difficult and time-consuming than experiments using InsP5 or InsP6 , a factor that allowed us to analyse cellular uptake across a range of human cell types, identifying strong cell-specific differences., (Copyright © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Transformations that selectively modify the furan ring present in a variety of naturals products would be useful in the synthesis of biological probes but remain largely underexplored. The neoclerodane diterpene salvinorin A, isolated from Salvia divinorum, is an example of a furan-containing natural product. Following selective bromination of salvinorin A, Suzuki-Miyaura and Sonogashira couplings were accomplished in moderate to good yields without hydrolyzing the labile C-2 acetate or altering the stereochemistry of the epimerizable centers.

The myo-inositol phosphates (InsPs) are specific signalling metabolites ubiquitous in eukaryotic cells. Although Ins(1,3,4,5,6)P(5) is the second most abundant member of the InsPs family, its certain biological roles are far from being elucidated, in part due to the large number of species formed by Ins(1,3,4,5,6)P(5) in the presence of metal ions. In light of this, we have strived in the past to make a complete and at the same time "biological-user-friendly" description of the Ins(1,3,4,5,6)P(5) chemistry with mono and multivalent cations. In this work we expand these studies focusing on the inframolecular aspects of its protonation equilibria and the microscopic details of its coordination behaviour towards biologically relevant metal ions. We present here a systematic study of the Ins(1,3,4,5,6)P(5) intrinsic acid-base processes, in a non-interacting medium, and over a wide pH range, analyzing the (31)P NMR curves by means of a model based on the Cluster Expansion Method. In addition, we have used a computational approach to analyse the energetic and structural features of the protonation and conformational changes of Ins(1,3,4,5,6)P(5), and how they are influenced by the presence of two physiologically relevant cations, Na(+) and Mg(2+).

IP(3)R (IP(3) [inositol 1,4,5-trisphosphate] receptors) and ryanodine receptors are the most widely expressed intracellular Ca(2+) channels and both are regulated by thiol reagents. In DT40 cells stably expressing single subtypes of mammalian IP(3)R, low concentrations of thimerosal (also known as thiomersal), which oxidizes thiols to form a thiomercurylethyl complex, increased the sensitivity of IP(3)-evoked Ca(2+) release via IP(3)R1 and IP(3)R2, but inhibited IP(3)R3. Activation of IP(3)R is initiated by IP(3) binding to the IBC (IP(3)-binding core; residues 224-604) and proceeds via re-arrangement of an interface between the IBC and SD (suppressor domain; residues 1-223). Thimerosal (100 μM) stimulated IP(3) binding to the isolated NT (N-terminal; residues 1-604) of IP(3)R1 and IP(3)R2, but not to that of IP(3)R3. Binding of a competitive antagonist (heparin) or partial agonist (dimeric-IP(3)) to NT1 was unaffected by thiomersal, suggesting that the effect of thimerosal is specifically related to IP(3)R activation. IP(3) binding to NT1 in which all cysteine residues were replaced by alanine was insensitive to thimerosal, so too were NT1 in which cysteine residues were replaced in either the SD or IBC. This demonstrates that thimerosal interacts directly with cysteine in both the SD and IBC. Chimaeric proteins in which the SD of the IP(3)R was replaced by the structurally related A domain of a ryanodine receptor were functional, but thimerosal inhibited both IP(3) binding to the chimaeric NT and IP(3)-evoked Ca(2+) release from the chimaeric IP(3)R. This is the first systematic analysis of the effects of a thiol reagent on each IP(3)R subtype. We conclude that thimerosal selectively sensitizes IP(3)R1 and IP(3)R2 to IP(3) by modifying cysteine residues within both the SD and IBC and thereby stabilizing an active conformation of the receptor.

Acid hydrolysis of myo-inositol 1,3,5-orthoesters, apart from orthoformates, exclusively affords the corresponding 2-O-acyl myo-inositol products via a 1,2-bridged five-membered ring dioxolanylium ion intermediate observed by NMR spectroscopy. These C-2-substituted inositol derivatives provide valuable precursors for rapid and highly efficient routes to 2-O-acyl inositol 1,3,4,5,6-pentakisphosphates and myo-inositol 1,3,4,5,6-pentakisphosphate with biologically interesting and anticancer properties. Deuterium incorporation into the α-methylene group of such alkyl ester products (2-O-C(O)CD2R), when the analogous alkyl orthoester is treated with deuterated acid, is established utilizing the novel orthoester myo-inositol 1,3,5-orthobutyrate as an example. Such deuterated ester products provide intermediates for deuterium-labeled synthetic analogues. Investigation into this selective formation of 2-O-ester products and the deuterium incorporation is presented with proposed mechanisms from NMR experiments.

Most animal cells express mixtures of the three subtypes of inositol 1,4,5-trisphosphate receptor (IP(3)R) encoded by vertebrate genomes. Activation of each subtype by different agonists has not hitherto been examined in cells expressing defined homogenous populations of IP(3)R. Here we measure Ca(2+) release evoked by synthetic analogues of IP(3) using a Ca(2+) indicator within the lumen of the endoplasmic reticulum of permeabilized DT40 cells stably expressing single subtypes of mammalian IP(3)R. Phosphorylation of (1,4,5)IP(3) to (1,3,4,5)IP(4) reduced potency by ~100-fold. Relative to (1,4,5)IP(3), the potencies of IP(3) analogues modified at the 1-position (malachite green (1,4,5)IP(3)), 2-position (2-deoxy(1,4,5)IP(3)) or 3-position (3-deoxy(1,4,5)IP(3), (1,3,4,5)IP(4)) were similar for each IP(3)R subtype. The potency of an analogue, (1,4,6)IP(3), in which the orientations of the 2- and 3-hydroxyl groups were inverted, was also reduced similarly for all three IP(3)R subtypes. Most analogues of IP(3) interact similarly with the three IP(3)R subtypes, but the decrease in potency accompanying removal of the 1-phosphate from (1,4,5)IP(3) was least for IP(3)R3. Addition of a large chromophore (malachite green) to the 1-phosphate of (1,4,5)IP(3) only modestly reduced potency suggesting that similar analogues could be used to measure (1,4,5)IP(3) binding optically. These data provide the first structure-activity analyses of key IP(3) analogues using homogenous populations of each mammalian IP(3)R subtype. They demonstrate broadly similar structure-activity relationships for all mammalian IP(3)R subtypes and establish the potential utility of (1,4,5)IP(3) analogues with chromophores attached to the 1-position.

Inositol 1,4,5-trisphosphate receptors (IP3R) are intracellular Ca(2+) channels. Most animal cells express mixtures of the three IP3R subtypes encoded by vertebrate genomes. Adenophostin A (AdA) is the most potent naturally occurring agonist of IP3R and it shares with IP3 the essential features of all IP3R agonists, namely structures equivalent to the 4,5-bisphosphate and 6-hydroxyl of IP3. The two essential phosphate groups contribute to closure of the clam-like IP3-binding core (IBC), and thereby IP3R activation, by binding to each of its sides (the α- and β-domains). Regulation of the three subtypes of IP3R by AdA and its analogues has not been examined in cells expressing defined homogenous populations of IP3R. We measured Ca(2+) release evoked by synthetic adenophostin A (AdA) and its analogues in permeabilized DT40 cells devoid of native IP3R and stably expressing single subtypes of mammalian IP3R. The determinants of high-affinity binding of AdA and its analogues were indistinguishable for each IP3R subtype. The results are consistent with a cation-π interaction between the adenine of AdA and a conserved arginine within the IBC α-domain contributing to closure of the IBC. The two complementary contacts between AdA and the α-domain (cation-π interaction and 3″-phosphate) allow activation of IP3R by an analogue of AdA (3″-dephospho-AdA) that lacks a phosphate group equivalent to the essential 5-phosphate of IP3. These data provide the first structure-activity analyses of key AdA analogues using homogenous populations of all mammalian IP3R subtypes. They demonstrate that differences in the Ca(2+) signals evoked by AdA analogues are unlikely to be due to selective regulation of IP3R subtypes.

A potential extracellular target for inositol phosphates and analogues with anticancer properties is identified. Proteins from detergent-solubilised HeLa cell lysates bound to a novel affinity column of myo -inositol 1,3,4,5,6-pentakisphosphate (Ins P 5 ) coupled to Affigel-10. One high-affinity ligand was fibrinogen Bβ. Inositol phosphates and analogues were able to elute purified fibrinogen from this matrix. Ins P 5 and the inositol phosphate mimic biphenyl 2,3',4,5',6-pentakisphosphate (BiPh P 5 ) bind fibrinogen in vitro , and block the effects of fibrinogen in A549 cell-based assays of proliferation and migration. They are also able to prevent the fibrinogen-mediated activation of phosphatidylinositol 3-kinase. These effects of fibrinogen appear to be mediated through the intercellular adhesion molecule-1 (ICAM-1), as cells not expressing ICAM-1 fail to respond. In contrast, myo -inositol hexakisphosphate and the epimeric scyllo -inositol 1,2,3,4,5-pentakisphosphate were without effect. These findings are consistent with earlier reports that higher inositol phosphates have anticancer properties. This new mechanism of action and target for these extracellular inositol phosphates to have their effects allows a re-evaluation of earlier data.

We synthesised analogues of diphosphoinositol polyphosphates (PP-InsPs) in which the diphosphate is replaced by an α-phosphonoacetic acid (PA) ester. Structural analysis revealed that 5-PA-InsP(5) mimics 5-PP-InsP(5) binding to the kinase domain of PPIP5K2; both molecules were phosphorylated by the enzyme. PA-InsPs are promising candidates for further studies into the biology of PP-InsPs.

Phosphoinositides regulate many cellular processes, and cellular levels are controlled by kinases and phosphatases. SHIP2 (SH2 (Src homology 2)-domain-containing inositol-phosphatase-2) plays a critical role in phosphoinositide signaling, cleaving the 5-phosphate from phosphatidylinositol 3,4,5-trisphosphate. SHIP2 is thought to be involved in type-2 diabetes and obesity, conditions that could therefore be open to pharmacological modulation of the enzyme. However, rational design of SHIP2 inhibitors has been limited by the absence of a high-resolution structure. Here, we present a 2.1 Å resolution crystal structure of the phosphatase domain of SHIP2 bound to the synthetic ligand biphenyl 2,3',4,5',6-pentakisphosphate (BiPh(2,3',4,5',6)P(5)). BiPh(2,3',4,5',6)P(5) is not a SHIP2 substrate but inhibits Ins(1,3,4,5)P(4) hydrolysis with an IC(50) of 24.8 ± 3.0 μM, (K(m) for Ins(1,3,4,5)P(4) is 215 ± 28 μM). Molecular dynamics simulations suggest that when BiPh(2,3',4,5',6)P(5) binds to SHIP2, a flexible loop folds over and encloses the ligand. Compounds targeting such a closed conformation might therefore deliver SHIP2-specific drugs.

The inositol phosphates are an abundant but poorly understood group of organic phosphorus compounds found widely in the environment. Four stereoisomers of inositol hexakisphosphate (IP(6)) occur, although for three of these (scyllo, neo, and D-chiro) the origins, dynamics, and biological function remain unknown, due in large part to analytical limitations in their measurement in environmental samples. We synthesized authentic neo- and D-chiro-IP(6) and used them to identify signals from these compounds in three soils from the Falkland Islands. Both compounds resisted hypobromite oxidation and gave quantifiable (31)P NMR signals at δ = 6.67 ppm (equatorial phosphate groups of the 4-equatorial/2-axial conformer of neo-IP(6)) and δ = 6.48 ppm (equatorial phosphate groups of the 2-equatorial/4-axial conformer of D-chiro-IP(6)) in soil extracts. Inositol hexakisphosphate accounted for 46-54% of the soil organic phosphorus, of which the four stereoisomers constituted, on average, 55.9% (myo), 32.8% (scyllo), 6.1% (neo), and 5.2% (D-chiro). Reappraisal of the literature based on the new signal assignments revealed that neo- and D-chiro-IP(6) occur widely in both terrestrial and aquatic ecosystems. These results confirm that the inositol phosphates can constitute a considerable fraction of the organic phosphorus in soils and reveal the prevalence of neo- and D-chiro-IP(6) in the environment. The hypobromite oxidation and solution (31)P NMR spectroscopy procedure allows the simultaneous quantification of all four IP(6) stereoisomers in environmental samples and provides a platform for research into the origins and ecological significance of these enigmatic compounds., (© 2012 American Chemical Society)

Simultaneous and molecularly selective parts-per-billion detection of benzene, toluene, and xylenes (BTX) using a thermal desorption (TD)-FTIR hollow waveguide (HWG) trace gas sensor is demonstrated here for the first time combining laboratory calibration with real-world sample analysis in field. A calibration range of 100-1000 ppb analyte/N(2) was developed and applied for predicting the concentration of blinded environmental air samples within the same concentration range, and demonstrate close agreement with the validation method used here, GC-FID. The analyte concentration prediction capability of the TD-FTIR-HWG trace gas sensor also compares well with the industrial standard and other experimental techniques including GC-PID, ultrafast GC-FID, and GC-DMS, which were simultaneously operated in the field. With the advent of a quantum cascade laser with emission frequencies specifically tailored to efficiently overlap benzene absorption as the most relevant analyte, the overall sensor footprint could be considerably reduced to ultimately yield hand-held trace gas sensors facilitating direct and real-time detection of BTX in air down to low ppb levels.

Background and Purpose: Adenophostin A (AdA) is a potent agonist of inositol 1,4,5-trisphosphate receptors (IP(3) R). AdA shares with IP(3) the essential features of all IP(3) R agonists, namely structures equivalent to the 4,5-bisphosphate and 6-hydroxyl of IP(3) , but the basis of its increased affinity is unclear. Hitherto, the 2'-phosphate of AdA has been thought to provide a supra-optimal mimic of the 1-phosphate of IP(3) ., Experimental Approach: We examined the structural determinants of AdA binding to type 1 IP(3) R (IP(3) R1). Chemical synthesis and mutational analysis of IP(3) R1 were combined with (3) H-IP(3) binding to full-length IP(3) R1 and its N-terminal fragments, and Ca(2+) release assays from recombinant IP(3) R1 expressed in DT40 cells., Key Results: Adenophostin A is at least 12-fold more potent than IP(3) in functional assays, and the IP(3) -binding core (IBC, residues 224-604 of IP(3) R1) is sufficient for this high-affinity binding of AdA. Removal of the 2'-phosphate from AdA (to give 2'-dephospho-AdA) had significantly lesser effects on its affinity for the IBC than did removal of the 1-phosphate from IP(3) (to give inositol 4,5-bisphosphate). Mutation of the only residue (R568) that interacts directly with the 1-phosphate of IP(3) decreased similarly (by ~30-fold) the affinity for IP(3) and AdA, but mutating R504, which has been proposed to form a cation-π interaction with the adenine of AdA, more profoundly reduced the affinity of IP(3) R for AdA (353-fold) than for IP(3) (13-fold)., Conclusions and Implications: The 2'-phosphate of AdA is not a major determinant of its high affinity. R504 in the receptor, most likely via a cation-π interaction, contributes specifically to AdA binding., (© 2010 The Authors. British Journal of Pharmacology © 2010 The British Pharmacological Society.)

Inositol 1,4,5-trisphosphate (IP(3)) receptors (IP(3)R) are intracellular Ca(2+) channels. Their opening is initiated by binding of IP(3) to the IP(3)-binding core (IBC; residues 224-604 of IP(3)R1) and transmitted to the pore via the suppressor domain (SD; residues 1-223). The major conformational changes leading to IP(3)R activation occur within the N terminus (NT; residues 1-604). We therefore developed a high-throughput fluorescence polarization (FP) assay using a newly synthesized analog of IP(3), fluorescein isothiocyanate (FITC)-IP(3), to examine the thermodynamics of IP(3) and adenophostin A binding to the NT and IBC. Using both single-channel recording and the FP assay, we demonstrate that FITC-IP(3) is a high-affinity partial agonist of the IP(3)R. Conventional [(3)H]IP(3) and FP assays provide similar estimates of the K(D) for both IP(3) and adenophostin A in cytosol-like medium at 4 degrees C. They further establish that the isolated IBC retains the ability of full-length IP(3)R to bind adenophostin A with approximately 10-fold greater affinity than IP(3). By examining the reversible effects of temperature on ligand binding, we established that favorable entropy changes (T Delta S) account for the greater affinities of both ligands for the IBC relative to the NT and for the greater affinity of adenophostin A relative to IP(3). The two agonists differ more substantially in the relative contribution of Delta H and T Delta S to binding to the IBC relative to the NT. This suggests that different initial binding events drive the IP(3)R on convergent pathways toward a similar open state.

Inositol 1,4,5-trisphosphate receptors (IP(3)Rs) are ubiquitous intracellular Ca2+ channels. IP(3) binding to the IP(3)-binding core (IBC) near the N terminus initiates conformational changes that lead to opening of a pore. The mechanisms underlying this process are unresolved. We synthesized 2-O-modified IP(3) analogs that are partial agonists of IP(3)R. These are similar to IP(3) in their interactions with the IBC, but they are less effective than IP(3) in rearranging the relationship between the IBC and the N-terminal suppressor domain (SD), and they open the channel at slower rates. IP(3)R with a mutation in the SD occupying a position similar to the 2-O substituent of the partial agonists has a reduced open probability that is similar for full and partial agonists. Bulky or charged substituents from either the ligand or the SD therefore block obligatory coupling of the IBC and the SD. Analysis of DeltaG for ligand binding shows that IP(3) is recognized by the IBC and conformational changes then propagate entirely via the SD to the pore.

Ca(2+) release by d-myo-inositol 1,4,5-trisphosphate receptors (IP(3)Rs) is widely considered to require the vicinal 4,5-bisphosphate motif of IP(3), with P-5 and P-4 engaging the alpha and beta domains of the binding site; using synthesis and mutagenesis we show that the adenine of synthetic glyconucleotides, through an interaction with Arg504, can replace the interaction of either P-1 or P-5 with the alpha-domain producing, respectively, the most potent bisphosphate agonist yet synthesised and the first agonist of IP(3)R without a vicinal bisphosphate motif; this will stimulate new approaches to IP(3)R ligand design.

Acid hydrolysis of myo-inositol 1,3,5-orthobenzoate leads regioselectively to 2-O-benzoyl-myo-inositol via a 1,2-bridged 2'-phenyl-1',3'-dioxolan-2'-ylium ion observed by 1H and 13C NMR spectroscopy, providing the precursor for a highly efficient route to the anticancer agent myo-inositol 1,3,4,5,6-pentakisphosphate.

myo-Inositol 1,3,4,5,6-pentakisphosphate (Ins(1,3,4,5,6)P(5)), an inositol polyphosphate of emerging significance in cellular signalling, and its C-2 epimer scyllo-inositol pentakisphosphate (scyllo-InsP(5)) were synthesised from the same myo-inositol-based precursor. Potentiometric and NMR titrations show that both pentakisphosphates undergo a conformational ring-flip at higher pH, beginning at pH 8 for scyllo-InsP(5) and pH 9 for Ins(1,3,4,5,6)P(5). Over the physiological pH range, however, the conformation of the inositol rings and the microprotonation patterns of the phosphate groups in Ins(1,3,4,5,6)P(5) and scyllo-InsP(5) are similar. Thus, scyllo-InsP(5) should be a useful tool for identifying biologically relevant actions of Ins(1,3,4,5,6)P(5), mediated by specific binding sites, and distinguishing them from nonspecific electrostatic effects. We also demonstrate that, although scyllo-InsP(5) and Ins(1,3,4,5,6)P(5) are both hydrolysed by multiple inositol polyphosphate phosphatase (MINPP), scyllo-InsP(5) is not dephosphorylated by PTEN or phosphorylated by Ins(1,3,4,5,6)P(5) 2-kinases. This finding both reinforces the value of scyllo-InsP(5) as a biological control and shows that the axial 2-OH group of Ins(1,3,4,5,6)P(5) plays a part in substrate recognition by PTEN and the Ins(1,3,4,5,6)P(5) 2-kinases.

Background: To assess the efficacy of Celecoxib, a cyclo-oxygenase 2 (COX-2) inhibitor, as prophylaxis for cystoid macular oedema after routine cataract surgery., Methods: A prospective, randomized, double-blind placebo-controlled trial of 69 hospital patients undergoing cataract surgery. Celecoxib 200 mg twice daily or placebo was given immediately after surgery for 14 days. Optical coherence tomography was used to quantify macular thickness before surgery and on day 1, week 2 and week 6 after surgery., Results: Sixty-nine patients were enrolled, of which 33 received placebo and 36 received active drug. Clinically apparent cystoid macular oedema occurred in four of the treatment group and two of the placebo group (P = 0.68). No difference in best-corrected visual acuity was seen at 6 weeks (P = 0.37). Covariate analysis of the results at 2 weeks and 6 weeks showed a macular thickness of 3% less in the treatment group compared with placebo (P = 0.050)., Conclusion: Celecoxib may decrease macular thickening following routine cataract surgery at 2 and 6 weeks after surgery as measured by Stratus OCT III. No difference in best-corrected visual acuity or clinically apparent cystoid macular oedema was seen. Further investigation of COX-2 inhibitors in a larger prospective randomized trial is required.

Purpose: To investigate correlations between optical coherence tomography macular thickness measurements and visual acuity after cataract surgery., Methods: Sixty-two patients underwent routine cataract surgery as part of a randomized clinical trial of oral Cox-2 inhibitor prophylaxis of cystoid macular oedema. Optical coherence tomography was used to quantify several parameters of macular thickness. Optical coherence tomography measurements were obtained before surgery, day one, week two and week six after surgery. These measurements were then correlated with logMAR best-corrected visual acuity., Results: Optical coherence tomography macular thickness parameters increased after surgery by up to 20%. A significant correlation was identified between foveal minimum macular thickness and best-corrected visual acuity at day one and week six after surgery. Other macular parameters failed to show any significant correlation. At day one and week six, the 10 patients with greatest macular thickness had significantly lower visual acuity than the other patients., Conclusion: In this study routine cataract surgery caused an increase in macular thickness. Some significant positive correlations between macular thickness and best-corrected visual acuity were found, although not for all parameters or time points. There may be a threshold relationship between degree of foveal anatomic change and significant loss of visual acuity.

Ins(1,4,5,6)P4, a biologically active cell constituent, was recently advocated as a substrate of human Ins(3,4,5,6)P4 1-kinase (hITPK1), because stereochemical factors were believed relatively unimportant to specificity [Miller, G.J., Wilson, M.P., Majerus, P.W. and Hurley, J.H. (2005) Specificity determinants in inositol polyphosphate synthesis: crystal structure of inositol 1,3,4-triphosphate 5/6-kinase. Mol. Cell. 18, 201-212]. Contrarily, we provide three examples of hITPK1 stereospecificity. hITPK1 phosphorylates only the 1-hydroxyl of both Ins(3,5,6)P3 and the meso-compound, Ins(4,5,6)P3. Moreover, hITPK1 has >13,000-fold preference for Ins(3,4,5,6)P4 over its enantiomer, Ins(1,4,5,6)P4. The biological significance of hITPK1 being stereospecific, and not physiologically phosphorylating Ins(1,4,5,6)P4, is reinforced by our demonstrating that Ins(1,4,5,6)P4 is phosphorylated (K(m) = 0.18 microM) by inositolphosphate-multikinase.

The purpose of this study was to investigate the antiangiogenic and in vivo properties of the recently identified phosphatidylinositol 3-kinase (PI3K)/Akt inhibitor Inositol(1,3,4,5,6) pentakisphosphate [Ins(1,3,4,5,6)P5]. Because activation of the PI3K/Akt pathway is a crucial step in some of the events leading to angiogenesis, the effect of Ins(1,3,4,5,6)P5 on basic fibroblast growth factor (FGF-2)-induced Akt phosphorylation, cell survival, motility, and tubulogenesis in vitro was tested in human umbilical vein endothelial cells (HUVEC). The effect of Ins(1,3,4,5,6)P5 on FGF-2-induced angiogenesis in vivo was evaluated using s.c. implanted Matrigel in mice. In addition, the effect of Ins(1,3,4,5,6)P5 on growth of ovarian carcinoma SKOV-3 xenograft was tested. Here, we show that FGF-2 induces Akt phosphorylation in HUVEC resulting in antiapoptotic effect in serum-deprived cells and increase in cellular motility. Ins(1,3,4,5,6)P5 blocks FGF-2-mediated Akt phosphorylation and inhibits both survival and migration in HUVEC. Moreover, Ins(1,3,4,5,6)P5 inhibits the FGF-2-mediated capillary tube formation of HUVEC plated on Matrigel and the FGF-2-induced angiogenic reaction in BALB/c mice. Finally, Ins(1,3,4,5,6)P5 blocks the s.c. growth of SKOV-3 xenografted in nude mice to the same extent than cisplatin and it completely inhibits Akt phosphorylation in vivo. These data definitively identify the Akt inhibitor Ins(1,3,4,5,6)P5 as a specific antiangiogenic and antitumor factor. Inappropriate activation of the PI3K/Akt pathway has been linked to the development of several diseases, including cancer, making this pathway an attractive target for therapeutic strategies. In this respect, Ins(1,3,4,5,6)P5, a water-soluble, natural compound with specific proapoptotic and antiangiogenic properties, might result in successful anticancer therapeutic strategies.

In this work, we report the synthesis and characterization of a series of hexagonal nanostructured platinum/tin/tellurium inorganic/surfactant composites. The composites are formed through solution-phase self-assembly of SnTe4(4-) Zintl clusters, which are cross-linked with platinum salts in the presence of a cetyltriethylammonium cationic structure directing agent. The cross-linking utilizes various combinations of Pt(II) and Pt(IV) salts. Low-angle X-ray diffraction indicates that all composites form hexagonal honeycomb (p6mm) structures. A combination of elemental analysis and XANES is used to describe the composition and oxidation states within the composites. We find that the extent of tin telluride self-oligomerization and the platinum:tin telluride ratio both vary, indicating that the composite compensates for different platinum oxidation states by tuning the inorganic composition. Near-IR/visible reflectance spectroscopy and UPS can be used to measure both band gaps and absolute band energies. The results show that while moving from all Pt(II) to all Pt(IV) increases the band gap from 0.6 to 0.8 eV, it increases the absolute valence and conduction band energies by almost a full electronvolt. AC impedance spectroscopy further reveals that the conductivities of the materials can be tuned from 0.009 to 0.003 Omega(-1).cm(-1). Additionally, a capacitance arising from the periodic nanoscale organic domains was observed. The conductivity and band gap were used to estimate carrier mobilities in these composites. Chemical tuning of the electronic properties within related nanostructured composites is a useful tool for designing applications that exploit the properties of nanostructured semiconductors.

The levels of inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] in the cytoplasm are tightly regulated by two enzymes, Ins(1,4,5)P3 3-kinase and type I Ins(1,4,5)P3 5-phosphatase. The catalytic domain of Ins(1,4,5)P3 3-kinase (isoenzymes A, B and C) is restricted to approximately 275 amino acids at the C-terminal end. We were interested in understanding the catalytic mechanism of this key family of enzymes in order to exploit this in inhibitor design. We expressed the catalytic domain of rat Ins(1,4,5)P3 3-kinase A in Escherichia coli as a His- and S-tagged fusion protein. The purified enzyme was used in an Ins(1,4,5)P3 kinase assay to phosphorylate a series of inositol phosphate analogues with three or four phosphate groups. A synthetic route to D-2-deoxy-Ins(1,4,5)P3 was devised. D-2-Deoxy-Ins(1,4,5)P3 and D-3-deoxy-Ins(1,4,6)P3 were potent inhibitors of the enzyme, with IC50 values in the micromolar range. Amongst all analogues tested, only D-2-deoxy-Ins(1,4,5)P3 appears to be a good substrate of the Ins(1,4,5)P3 3-kinase. Therefore, the axial 2-hydroxy group of Ins(1,4,5)P3 is not involved in recognition of the substrate nor does it participate in the phosphorylation mechanism of Ins(1,4,5)P3. In contrast, the equatorial 3-hydroxy function must be present in that configuration for phosphorylation to occur. Our data indicate the importance of the 3-hydroxy function in the mechanism of inositol trisphosphate phosphorylation rather than in substrate binding.

In this work, we examine the phase stability of both uncoated and alumina-coated zirconia nanoparticles using in-situ X-ray diffraction. By tracking structural changes in these particles, we seek to understand how changing interfacial bonding affects the kinetics of amorphous zirconia crystallization and the kinetics of grain growth in both initially amorphous and initially crystalline zirconia nanocrystals. Activation energies associated with crystallization are calculated using nonisothermal kinetic methods. The crystallization of the uncoated amorphous zirconia colloids has an activation energy of 117 +/- 13 kJ/mol, while that for the alumina-coated amorphous colloids is 185 +/- 28 kJ/mol. This increase in activation energy is attributed to inhibition of atomic rearrangement imparted by the alumina coating. The kinetics of grain growth are also studied with nonisothermal kinetic methods. The alumina coating again dramatically affects the activation energies. For colloids that were coated with alumina when they were in an amorphous structure, the coating imparts a 5x increase in the activation energy for grain growth (33 +/- 8 versus 150 +/- 30 kJ/mol). This increase shows that the alumina coating inhibits zirconia cores from coarsening. When the colloids are synthesized in the tetragonal phase and then coated with alumina, the effect of surface coating on coarsening kinetics is even more dramatic. In this case, a 10x increase in activation energies, from 28 +/- 3 kJ/mol for the uncoated particles to 300 +/- 25 kJ/mol for the alumina-coated crystallites, is found. The results show that one can alter phase stability in colloidal systems by using surface coatings and interfacial energy to dramatically change the kinetic barriers to structural rearrangement.

Previous studies have shown that adenophostin A is a potent initiator of the activation of SOCs (store-operated Ca2+ channels) in rat hepatocytes, and have suggested that, of the two subtypes of Ins(1,4,5)P3 receptor predominantly present in rat hepatocytes [Ins(1,4,5)P3R1 (type I receptor) and Ins(1,4,5)P3R2 (type II receptor)], Ins(1,4,5)P3R1s are required for SOC activation. We compared the abilities of Ins(1,4,6)P3 [with higher apparent affinity for Ins(1,4,5)P3R1] and Ins(1,3,6)P3 and Ins(1,2,4,5)P4 [with higher apparent affinities for Ins(1,4,5)P3R2] to activate SOCs. The Ins(1,4,5)P3 analogues were microinjected into single cells together with fura 2, and dose-response curves for the activation of Ca2+ inflow and Ca2+ release from intracellular stores obtained for each analogue. The concentration of Ins(1,4,6)P3 which gave half-maximal stimulation of Ca2+ inflow was substantially lower than that which gave half-maximal stimulation of Ca2+ release. By contrast, for Ins(1,3,6)P3 and Ins(1,2,4,5)P3, the concentration which gave half-maximal stimulation of Ca2+ inflow was substantially higher than that which gave half-maximal stimulation of Ca2+ release. The distribution of Ins(1,4,5)P3R1 and Ins(1,4,5)P3R2 in rat hepatocytes cultured under the same conditions as those employed for the measurement of Ca2+ inflow and release was determined by immunofluorescence. Ins(1,4,5)-P3R1s were found predominantly at the cell periphery, whereas Ins(1,4,5)P3R2s were found at the cell periphery, the cell interior and nucleus. It is concluded that the idea that a small region of the endoplasmic reticulum enriched in Ins(1,4,5)P3R1 is required for the activation of SOCs is consistent with the present results for hepatocytes.

2-O-(2-Aminoethyl)-Ins(1,4,5)P(3), (5), a novel derivative of the Ca(2+)-mobilising second messenger d-myo-inositol 1,4,5-trisphosphate [Ins(1,4,5)P(3)], was synthesised from myo-inositol. 5 was found to be a potent mobiliser of intracellular Ca(2+), and an Ins(1,4,5)P(3) affinity matrix synthesised from 5 was effective at selectively binding N-terminal fragments of the Ins(1,4,5)P(3) receptor containing the intact Ins(1,4,5)P(3) binding site. The microprotonation scheme for 5 was resolved and the related constants were determined in comparison with Ins(1,4,5)P(3) and another reactive Ins(1,4,5)P(3) analogue 1-O-(2-aminoethyl-1-phospho)-Ins(4,5)P(2), (2a), by potentiometric and NMR titration methods. The (31)P and (1)H NMR titration curves for compound 5 and Ins(1,4,5)P(3) are remarkably close, indicating analogous acid-base properties and intramolecular interactions for the two compounds. The 1-phosphate-modified Ins(1,4,5)P(3) derivative 2a, on the contrary, behaves as a bisphosphorylated rather than a trisphosphorylated inositol. Thus, 5 is a new reactive Ins(1,4,5)P(3) analogue of considerable potential for investigation of the chemical biology of Ins(1,4,5)P(3)-mediated cellular signalling.

The design and synthesis of dimeric versions of the intracellular signaling molecule d-myo-inositol 1,4,5-trisphosphate [Ins(1,4,5)P(3)] are reported. Ins(1,4,5)P(3) dimers in a range of sizes were constructed by conjugation of a partially protected 2-O-(2-aminoethyl)-Ins(1,4,5)P(3) intermediate with activated oligo- and poly(ethylene glycol) (PEG) tethers, to give benzyl-protected dimers with amide or carbamate linkages. After deprotection, the resulting water-soluble Ins(1,4,5)P(3) dimers were purified by ion-exchange chromatography. The interaction of the Ins(1,4,5)P(3) dimers with tetrameric Ins(1,4,5)P(3) receptors was explored, using equilibrium [(3)H]Ins(1,4,5)P(3)-binding to membranes from cerebellum, and (45)Ca(2+)-release from permeabilized hepatocytes. The results showed that dimers, even when they incorporate large PEG tethers, interact potently with Ins(1,4,5)P(3) receptors, and that the shorter dimers are more potent than Ins(1,4,5)P(3) itself. A very small dimer, consisting of two Ins(1,4,5)P(3) motifs joined by a short N,N'-diethylurea spacer, was synthesized. Preliminary studies of (45)Ca(2+) release from the intracellular stores of permeabilized hepatocytes showed this shortest dimer to be almost as potent as adenophostin A, the most potent Ins(1,4,5)P(3) receptor ligand known. Possible interpretations of this result are considered in relation to the recently disclosed X-ray crystal structure of the type 1 Ins(1,4,5)P(3) receptor core binding domain.

New and rapid syntheses of the enantiomeric intracellular signalling molecules d-myo-inositol 1,4,5,6-tetrakisphosphate (1 a) and D-myo-inositol 3,4,5,6-tetrakisphosphate (1 b) are described. The synthetic strategy employs the novel butane-2,3-diacetal-protected (BDA-protected) myo-inositol (+/-)-3 ab, directly accessible from myo-inositol on a large scale, and an optical resolution with diastereoisomeric (R)-(-)-acetylmandelate esters. The X-ray crystal structure of (+/-)-4, an unusual side product of acid-catalysed reaction of myo-inositol with butanedione is also presented, and the absolute configurations of 1 a and 1 b are definitively assigned by conversion of key precursors into (+)-bornesitol and L-iditol hexaacetate, respectively. Biological activity of synthetic 1 b was confirmed in comparison with the natural polyphosphate.

The synthesis of a series of adenophostin A analogues modified at C-6 and C-2 of adenine is described. The target compounds were synthesized by a convergent route involving a modified Vorbrüggen condensation of either 6-chloropurine or 2,6-dichloropurine with a protected disaccharide, yielding two versatile intermediates capable of undergoing substitution with a range of nucleophiles. The new analogues showed a range of abilities to mobilize Ca(2+) from the intracellular stores of permeabilized hepatocytes and are among the first totally synthetic compounds to approach the activity of adenophostin A. In agreement with the biological results, docking studies of adenophostin A using the recently reported X-ray crystal structure of the type 1 Ins(1,4,5)P(3) receptor binding core suggested that, in likely binding modes of adenophostin A, the area around N(6) may be relatively open, identifying this region of the adenophostin A molecule as a promising target for further elaboration. The docking results also point to specific interactions involving residues within the binding domain of the Ins(1,4,5)P(3) receptor that may be involved in the molecular recognition of the adenophostins.