Dichloroacetate induces autophagy in colorectal cancer cells and tumours.

BACKGROUND: Dichloroacetate (DCA) has been found to have antitumour properties. METHODS: We investigated the cellular and metabolic responses to DCA treatment and recovery in human colorectal (HT29, HCT116 WT and HCT116 Bax-ko), prostate carcinoma cells (PC3) and HT29 xenografts by flow cytometry, western blotting, electron microscopy, (1)H and hyperpolarised (13)C-magnetic resonance spectroscopy. RESULTS: Increased expression of the autophagy markers LC3B II was observed following DCA treatment both in vitro and in vivo. We observed increased production of reactive oxygen species (ROS) and mTOR inhibition (decreased pS6 ribosomal protein and p4E-BP1 expression) as well as increased expression of MCT1 following DCA treatment. Steady-state lactate excretion and the apparent hyperpolarised [1-(13)C] pyruvate-to-lactate exchange rate (k(PL)) were decreased in DCA-treated cells, along with increased NAD(+)/NADH ratios and NAD(+). Steady-state lactate excretion and k(PL) returned to, or exceeded, control levels in cells recovered from DCA treatment, accompanied by increased NAD(+) and NADH. Reduced k(PL) with DCA treatment was found in HT29 tumour xenografts in vivo. CONCLUSIONS: DCA induces autophagy in cancer cells accompanied by ROS production and mTOR inhibition, reduced lactate excretion, reduced k(PL) and increased NAD(+)/NADH ratio. The observed cellular and metabolic changes recover on cessation of treatment.

Conceptual design of Thomson scattering diagnostics for the COMPASS-U tokamak.

The Thomson scattering (TS) diagnostic, one of the key diagnostics used on the tokamaks around the world, is planned for the COMPASS-U tokamak, which is recently under design and construction in the Institute of Plasma Physics in Prague, Czech Republic. This tokamak is supposed to be a world-unique, high magnetic field device with hot walls, allowing for the study of the plasma exhaust in advanced operational scenarios and testing cutting-edge technologies relevant to future fusion reactors, e.g., use of liquid metals. The core and edge TS systems are planned to be designed and operational, with a limited performance, already in the early stage of the tokamak operation. In this contribution, requirements and the most important constraints defining the TS system design are presented. The impact of both the possible collection lens location and spatial resolution on the plasma pedestal observation is simulated. Design considerations also take into account the high-resolution TS core and edge systems available from the COMPASS tokamak, which will be reused. The collection lenses will be newly built. Extension of the detection system will complete the plasma radius coverage in the future. The divertor TS is considered for later periods.

Thomson scattering synthetic diagnostic module for the Cherab framework.

This contribution presents a Thomson scattering module developed for the Raysect and Cherab framework. Detailed models of spectroscopic diagnostic systems can be created in the framework, which deliver synthetic data with high precision due to accurate physical treatment of ray propagation and radiation phenomena. The addition of the presented module will allow us to model Thomson scattering systems that can aid both data validation and design. Two examples of such application are given. The first example shows the application of the module on the COMPASS tokamak edge Thomson scattering diagnostic and experimental data. The second example shows the possibility to use the framework and the Thomson scattering module as a design support tool.

Microwave reflectometer for density profile and turbulence measurements on the COMPASS tokamak.

The fast microwave reflectometer system on the COMPASS tokamak consists of an O-mode polarized K-band (18 GHz-26 GHz), Ka-band (26 GHz-40 GHz), and a part of U-band (40 GHz-54 GHz). The plasma density profile from the edge plasma area is measured using a fast sweeping rate up of to 6 µs of the probing wave. The processing of the reflected signal is realized by the heterodyne detection configuration based on the I/Q modulator. Two different methods of dynamic calibration of the required linear sweep frequency, together with static frequency and dispersion calibration, were used. The electron density profile was reconstructed by a spectrogram-based method with four sweeps on average. The system has the capability to measure the mid-plane low-field side electron density profile in the density range from 4 × 1018 m-3 to 3.6 × 1019 m-3. Experimental results obtained on COMPASS discharges are presented to demonstrate the performance of the diagnostics.

Screening to prevent polyoma virus nephropathy in kidney transplantation: a cost analysis.

Polyoma virus nephropathy is an important cause of graft dysfunction in kidney transplant recipients and screening to prevent disease has been advocated. Although screening incurs new costs, our hypothesis is that savings from less immunosuppression in those with positive screening tests could pay for overall costs of screening. In 134 consecutive recipients, polyoma virus (positive decoy cells) was detected in the urine of 34 (25.4%) individuals over a 2-year follow-up. Of these 34, 11 had a plasma BK PCR of >7700 copies/mL. Immunosuppression was reduced stepwise in these patients until viral loads fell <1000/mL. Overall screening costs (including extra plasma PCR testing) were estimated at $33,450. Those with positive PCR had greater reductions in annual immunosuppression costs by year 2 ($6452 vs. $2799, p = 0.0015) compared to those with negative screens. At the end of the 2-year period, 61% of the screening costs were covered by less immunosuppressant costs. At the end of 30 months there were net savings. In summary, reductions in immunosuppression cover the cost of screening for polyoma viral infection. Longer-term follow-up is needed to ensure patient outcomes remain acceptable.

Fast density reconstruction of Li-BES signal on the COMPASS tokamak.

This article describes a fast and automatic reconstruction of the edge plasma electron density from the radiation of energetic Li atoms of the diagnostic beam on the COMPASS tokamak. Radiation is detected by using a CCD camera and by using an avalanche photo-diode system with a temporal resolution of 20 ms and 2 µs, respectively. Both systems are equipped with a 670.8 nm optical filter which corresponds to the lithium 1s22s1-1s22p1 transition. A theoretical model and a data processing procedure of a raw signal to obtain the density profile are described. The reconstruction algorithm provides the absolutely calibrated electron density profiles together with the measurement error estimated from relatively calibrated light profiles; the implementation is performed in Python. Time demanding operations of the code were optimized to provide reconstruction of a single profile within less than 10 ms which makes the code applicable for processing of a large amount of data. Thanks to this calculation speed, it is possible to reconstruct electron density profiles between two consecutive shots on the COMPASS tokamak with 2 µs time resolution.

Observation and evaluation of the alignment of Thomson scattering systems.

Concerning plasma diagnostics based on Thomson scattering (TS), precise adjustment and proper alignment is of great importance in order to provide reliable and accurate measurements. Any misalignment could result in an incorrectly determined plasma density or prevent the measurement with this type of diagnostic altogether. Suitable means of alignment monitoring should be integrated into each TS diagnostic system. Variations of commonly used methods are discussed in this article. Correlation of results from alignment control with performed measurements of vibrations on the COMPASS tokamak is presented. Various techniques of optimization of alignment monitoring are shown. The optimal technique, which could be accommodated during the construction of TS diagnostic systems in future fusion devices, is proposed.

Development of an ion beam detector for the atomic beam probe diagnostic.

The atomic beam probe diagnostic concept aims at measuring the edge magnetic field and through that edge current distribution in fusion plasmas by observing trajectories of an ion beam stemming from a diagnostic neutral beam. The diagnostic potentially has microsecond scale time resolution and can thus prove to be a powerful option to study fast changes in the edge plasma. A test detector has been installed on the COMPASS tokamak as an extension of the existing lithium beam diagnostic system. It employs a relatively simple concept of an array of conductive detection plates measuring the incident ion current, which is then amplified and converted to a voltage signal. The aim of the test detector is to experimentally examine the idea of the diagnostic and provide background data for design and installation of a final detector. Also, a numerical code based on the CUDA parallel computing platform has been developed for modeling lithium ion trajectories in the given COMPASS plasma discharges. We present the developments of the detector design and test measurements of the diagnostic performed both in a laboratory beam system and on the COMPASS tokamak.

Filamentary probe on the COMPASS tokamak.

This paper describes a new filamentary probe recently introduced on the COMPASS tokamak. It allows the measurement of electrostatic and magnetic properties of the filaments and their changes in dependence on distance from the separatrix in the region between a divertor and midplane. The probe head is mounted on a manipulator moving the probe radially on a shot-to-shot basis. This configuration is suitable for the long term statistical measurement of the plasma filaments and the measurement of their evolution during their propagation from the separatrix to the wall. The basics of the filamentary probe construction, the evolution of the plasma parameters, and first conditional averages of the plasma filaments in the scrape-off layer of the COMPASS tokamak during the L-mode regime are presented.

Fitting of the Thomson scattering density and temperature profiles on the COMPASS tokamak.

A new technique for fitting the full radial profiles of electron density and temperature obtained by the Thomson scattering diagnostic in H-mode discharges on the COMPASS tokamak is described. The technique combines the conventionally used modified hyperbolic tangent function for the edge transport barrier (pedestal) fitting and a modification of a Gaussian function for fitting the core plasma. Low number of parameters of this combined function and their straightforward interpretability and controllability provide a robust method for obtaining physically reasonable profile fits. Deconvolution with the diagnostic instrument function is applied on the profile fit, taking into account the dependence on the actual magnetic configuration.

Profile measurements of the electron temperature on the ASDEX Upgrade, COMPASS, and ISTTOK tokamak using Thomson scattering, triple, and ball-pen probes.

The ball-pen probe (BPP) technique is used successfully to make profile measurements of the electron temperature on the ASDEX Upgrade (Axially Symmetric Divertor Experiment), COMPASS (COMPact ASSembly), and ISTTOK (Instituto Superior Tecnico TOKamak) tokamak. The electron temperature is provided by a combination of the BPP potential (ΦBPP) and the floating potential (Vfl) of the Langmuir probe (LP), which is compared with the Thomson scattering diagnostic on ASDEX Upgrade and COMPASS. Excellent agreement between the two diagnostics is obtained for circular and diverted plasmas and different heating mechanisms (Ohmic, NBI, ECRH) in deuterium discharges with the same formula Te = (ΦBPP - Vfl)/2.2. The comparative measurements of the electron temperature using BPP/LP and triple probe (TP) techniques on the ISTTOK tokamak show good agreement of averaged values only inside the separatrix. It was also found that the TP provides the electron temperature with significantly higher standard deviation than BPP/LP. However, the resulting values of both techniques are well in the phase with the maximum of cross-correlation function being 0.8.

Subclasses of cyclic GMP-specific phosphodiesterase and their role in regulating the effects of atrial natriuretic factor.

Two subclasses of cyclic guanosine monophosphate (GMP)-specific phosphodiesterases were identified in vascular tissue from several beds. The activity of one subclass (phosphodiesterase IB) was stimulated severalfold by calmodulin and selectively inhibited by the phosphodiesterase inhibitor TCV-3B. The activity of the other subclass (phosphodiesterase IC) was not stimulated by calmodulin and was selectively inhibited by the phosphodiesterase inhibitor M&B 22,948. To assess the involvement of both subclasses in regulating cyclic GMP-dependent responses, the ability of TCV-3B and M&B 22,948 to potentiate the in vitro and in vivo responses to the endogenous guanylate cyclase stimulator atrial natriuretic factor (ANF) was evaluated. Both TCV-3B and M&B 22,948 relaxed isolated rabbit aortic and pulmonary artery rings and also potentiated the relaxant effect of ANF. In addition, both inhibitors produced small increases in urine flow and sodium excretion in anesthetized rats and potentiated the diuretic and natriuretic responses to exogenous ANF. M&B 22,948 (30 micrograms/kg/min) produced a threefold increase in the natriuretic response to simultaneously administered ANF, and TCV-3B (10 micrograms/kg/min) produced a twofold increase in the response to ANF. The results of the present experiments suggest that both the calmodulin-sensitive and calmodulin-insensitive subclasses of cyclic GMP-specific phosphodiesterase play a role in regulating the in vitro and in vivo response to ANF.

Progress in the development of inhibitors of SH2 domains.

SH2 domains are discrete structural motifs common to a variety of critical intracellular signaling proteins. Inhibitors of specific SH2 domains have become important therapeutic targets in the treatment and/or prevention of restenosis, cancers (including small cell lung), cardiovascular disease, osteoporosis, apoptosis among others. Considering the social and economic impact of these diseases significant attention has been focused on the development of potent and selective inhibitors of specific SH2 domains. In particular, considerable research has been performed on Src, PI 3-kinase, Grb2 and more recently, Lck. In this review, we will focus on progress in the development of inhibitors for these specific SH2 domains and evaluate potential future targets.

Nonpeptide angiotensin II receptor antagonists. 1. Synthesis and in vitro structure-activity relationships of 4-[[[(1H-pyrrol-1-ylacetyl)amino]phenyl]methyl]imidazole derivatives as angiotensin II receptor antagonists.

A novel series of non-biphenylyltetrazole angiotensin II receptor antagonists which contain a 1H-pyrrol-1-ylacetyl residue in place of the benzoyl residue in EXP 6803 have been developed. The receptor binding activity of several members of this new series was in the 10(-8) M range, which was better than that of EXP 6803. Introduction of a carboxylic acid moiety at the 2-position of the pyrrole ring enhanced the in vitro binding affinity at the receptor by 10-fold. Compounds containing an acetic acid (18) or a propionic acid residue (20) at the 5-position of the imidazole were more potent than the carboxylic acid analogue (24). The binding IC50 of the most potent compound 20 was 22 nM. Compounds 18, 20, and 24 in their best fit conformations were manually overlayed on that of the template conformation of EXP 6803 and EXP 8623, respectively. The synthesis and structure-activity relationship data are described.

Multiple binding modes for the receptor-bound conformations of cyclic AII agonists.

Angiotensin II, Asp-Arg-Val-Tyr-His-Pro-Phe, binds its receptor with a postulated turn centered at residue four. Analogs of angiotensin II which contain a disulfide bridge between the side chains of residues 3 and 5 retain significant activity consistent with this hypothesis. Incorporation of 4-mercaptoproline residues, a hybrid, or chimeric amino acid which combines the properties of proline and homocysteine, into either of these positions with analogous disulfide bridges allows retention of high affinity for the receptor. These more highly constrained bicyclic systems give new insight into the details of molecular recognition of residues 3-5 of angiotensin by the receptor. Retention of activity by the antiparallel dimer of [Sar1,Cys3,5]-AII in which the peptide backbone is held in an extended conformation was unexpected. Analysis of the conformational constraints imposed in these active analogs suggests that AII agonists bind to their receptor with different backbone conformations in the region of the central tyrosine residue.

Design of peptidomimetics that inhibit the association of phosphatidylinositol 3-kinase with platelet-derived growth factor-beta receptor and possess cellular activity.

Phosphorylated tyrosine residues of growth factor receptors that associate with intracellular proteins containing src-homology 2 (SH2) domains are integral components in several signal transduction pathways related to proliferative diseases such as cancer, atherosclerosis, and restenosis. In particular, a phosphorylated pentapeptide [pTyr751-Val-Pro-Met754-Leu (pTyr = phosphotyrosine)] derived from the primary sequence of platelet-derived growth factor-beta (PDGF-beta) receptor blocks the association of the C-terminal SH2 domain of the p85 subunit of phosphatidylinositol 3-kinase (PI 3-kinase) to PDGF-beta receptor with an IC50 of 0.445 +/- 0.047 microM. Further evaluation of the structure-activity relationships for pTyr751-Val-Pro-Met-Leu resulted in the design of smaller peptidomimetics with enhanced affinity including Ac-pTyr-Val-Ala-N(C6H13)2 (IC50 = 0.076 +/- 0.010 microM). In addition, the phosphotyrosine residue was replaced with a difluorophosphonate derivative [4-phosphono(difluoromethyl)phenylalanine (CF2Pmp)] which has been shown to be stable to cellular phosphatases. The extracellular administration of either CF2Pmp-Val-Pro-Met-Leu or Ac-CF2Pmp-Val-Pro-Met-NH2 in a whole cell assay resulted in specific inhibition of the PDGF-stimulated association from the C-terminal SH2 domain of the p85 subunit of PI 3-kinase to the PDGF-beta receptor in a dose-dependent manner. These compounds were also effective in inhibiting GLUT4 translocation, c-fos expression, and cell membrane ruffling in single-cell microinjection assay.

Synthesis and structure-activity relationships of 7-substituted 3-(2, 6-dichlorophenyl)-1,6-naphthyridin-2(1H)-ones as selective inhibitors of pp60(c-src).

7-substituted 3-(2,6-dichlorophenyl)-1,6-naphthyridin-2(1H)-ones are potent inhibitors of protein tyrosine kinases, with some selectivity for c-Src. The compounds were prepared by condensing 4, 6-diaminonicotinaldehyde with 2,6-dichlorophenylacetonitrile and selectively converting the 2- and 7-amino groups of the product to hydroxy and fluoro groups, respectively, by prolonged diazotization in 50% aqueous fluoboric acid. N-Methylation, followed by treatment with aliphatic diamines, aromatic amines, or their derived lithium anions, gave the desired compounds. Selected isomeric 1, 8-naphthyridin-2(1H)-ones were also prepared in order to evaluate the relative contributions of both ring A aza atoms of the related pyrido[2,3-d]pyrimidin-7(8H)-ones to the inhibitory activity. The compounds were evaluated for their ability to prevent phosphorylation of a model substrate by c-Src, FGF-1 receptor, and PDGF-beta receptor enzymes. Overall, there was a high degree of correlation of the activities against the different kinases, with c-Src being generally the most sensitive to structural changes. 1, 6-Naphthyridin-2(1H)-one analogues bearing basic aliphatic side chains [7-NH(CH(2))(n)()NRR, 7-NHPhO(CH(2))(n)()NRR, or 7-NHPhN(CH(2))(4)NMe] were the most potent against c-Src (IC(50)s of 10-80 nM), showing good selectivity with respect to PDGFR (10-300-fold) but less with respect to FGFR. The 1, 6-naphthyridin-2(1H)-ones showed broadly similar activity to the analogous pyrido[2,3-d]pyrimidin-7(8H)-ones, whereas the 1, 8-naphthyridin-2(1H)-ones were at least 10(3)-fold less potent. These results, indicating that the 3-aza atom in the pyrido[2, 3-d]pyrimidin-7(8H)-ones is mandatory, whereas the 1-aza atom is not, support the published binding model for these compounds to c-Src (J. Med. Chem. 1998, 41, 1752), where the 3-aza and 2-NH atoms form a bidentate H-bond donor-acceptor motif that interacts with Met341 and the 1-aza atom is not involved in specific binding interactions.

Derivatives of 5-[[1-(4'-carboxybenzyl)imidazolyl]methylidene]hydantoins as orally active angiotensin II receptor antagonists.

A series of 5-[[1-(4'-carboxybenzyl)imidazolyl]methylidene]hydantoins have been prepared and evaluated as in vitro and in vivo angiotensin II (Ang II) antagonists. Variation of substituents on the hydantoin ring leads to potent and selective Ang II antagonists with nanomolar IC50 values at the AT1 receptor and negligible affinity for the AT2 receptor. Preferred substituents include an n-butyl at R1 and an alkyl or heteroarylmethyl substituent at R2. The selection of the R2 substituent was guided in part by the calculation of its log P since a significant correlation was observed between CLOGP and AT1 binding affinity. The biphenyl tetrazole pharmacophore, common to a number of AT1 antagonists, could be replaced by, for example, a 4-carbomethoxyphenyl substituent resulting in potent Ang II antagonists both in vitro and in vivo. A representative compound of this series is 57, which reduced the mean arterial blood pressure of renal hypertensive rats by 40% at 30 mg/kg po and by 25% at 10 mg/kg po. In addition this compound was efficacious in the salt-deplete normotensive monkey model maximally decreasing blood pressure 27% at 10 mg/kg po. In summary, these compounds belong to a novel class of Ang II antagonists that lack the biphenyl tetrazole moiety yet display appreciable and long lasting oral activity.

Structure-activity relationships for 5-substituted 1-phenylbenzimidazoles as selective inhibitors of the platelet-derived growth factor receptor.

Following an earlier discovery of 1-phenylbenzimidazoles as ATP-site inhibitors of the platelet-derived growth factor receptor (PDGFR), further structure-activity relationships for analogues (particularly 5-substituted derivatives) are reported. The data are consistent with a binding model (constructed from the homology-modeled structure of the catalytic subunit of the PDGFR using protein kinase A as the template) in which the ligand binds in the relatively narrow ATP site, with the phenyl ring pointing toward the interior of the pocket and the 5-position of the benzimidazole ring toward the mouth of the pocket. The narrow binding pocket allows a maximum torsion angle between the phenyl and benzimidazole rings of about 40 degrees, consistent with that calculated (43.6 degrees) for the minimum-energy conformation of the unsubstituted free ligand. The inactivity of 7- or 2'-substituted analogues is consistent with the greater torsion angle (and thus larger ligand cross-section) of such substituted analogues. There is substantial bulk tolerance for 5-substituents, which protrude out of the mouth of the hydrophobic pocket, with the most effective analogues being those bearing weak bases. On the basis of this model, 5-OR derivatives bearing cationic side chains were prepared as soluble analogues, and these showed sub-micromolar potencies against the isolated PDGFR enzyme. They were also moderately effective inhibitors of autophosphorylation of PDGFR in rat aortic vascular smooth muscle cells, with IC50s in the range 0.1-1 microM.

Structure-activity relationships for 1-phenylbenzimidazoles as selective ATP site inhibitors of the platelet-derived growth factor receptor.

1-Phenylbenzimidazoles are shown to be a new class of ATP-site inhibitors of the platelet-derived growth factor receptor (PDGFR). Structure-activity relationships (SARs) are narrow, with closely related heterocycles being inactive. A systematic study of substituted 1-phenylbenzimidazoles showed clear SARs. Substituents at the 4'- and 3'-positions of the phenyl ring are tolerated but do not significantly improve activity, while substituents at the 2'-position abolish it. Substituents in the 2-, 4-, and 7-positions of the benzimidazole ring (with the exception of 4-OH) also abolish activity. Most substituents at the 5- and 6-positions maintain or increase activity, with the 5-OH, 5-OMe, 5-COMe, and 5-CO2Me analogues being >10-fold more potent than the parent 1-phenylbenzimidazole. The 5-OMe analogue was both the most potent inhibitor, and showed the highest selectivity (50-fold) between PDGFR and FGFR isolated enzymes, and also a moderately effective inhibitor (IC50 = 1.9 microM) of PDGF-stimulated PDGFR autophosphorylation in rat aorta smooth muscle cells.