The role of charged residues in the transmembrane helices of monocarboxylate transporter 1 and its ancillary protein basigin in determining plasma membrane expression and catalytic activity

Monocarboxylate transporters MCT1-MCT4 require basigin (CD147) or embigin (gp70), ancillary proteins with a glutamate residue in their single transmembrane (TM) domain, for plasma membrane (PM) expression and activity. Here we use site-directed mutagenesis and expression in COS cells or Xenopus oocytes to investigate whether this glutamate (Glu218 in basigin) may charge-pair with a positively charged TM-residue of MCT1. Such residues were predicted using a new molecular model of MCT1 based upon the published structure of the E. coli glycerol-3-phosphate transporter. No evidence was obtained for Arg306 (TM 8) of MCT1 and Glu218 of basigin forming a charge-pair; indeed E218Q-basigin could replace WT-basigin, although E218R-basigin was inactive. No PM expression of R306E-MCT1 or D302R-MCT1 was observed but D302R/R306D-MCT1 reached the PM, as did R306K-MCT1. However, both were catalytically inactive suggesting that Arg306 and Asp302 form a charge-pair in either orientation, but their precise geometry is essential for catalytic activity. Mutation of Arg86 to Glu or Gln within TM3 of MCT1 had no effect on plasma membrane expression or activity of MCT1. However, unlike WT-MCT1, these mutants enabled expression of E218R-basigin at the plasma membrane of COS cells. We propose that TM3 of MCT1 lies alongside the TM of basigin with Arg86 adjacent to Glu218 of basigin. Only when both these residues are positively charged (E218R-basigin with WT-MCT1) is this interaction prevented; all other residue pairings at these positions may be accommodated by charge-pairing or stabilization of unionized residues through hydrogen bonding or local distortion of the helical structure

Modeling payback from research into the efficacy of left-ventricular assist devices as destination therapy

Objectives: Ongoing developments in design have improved the outlook for left-ventricular assist device (LVAD) implantation as a therapy in end-stage heart failure. Nevertheless, early cost-effectiveness assessments, based on first-generation devices, have not been encouraging. Against this background, we set out (i) to examine the survival benefit that LVADs would need to generate before they could be deemed cost-effective; (ii) to provide insight into the likelihood that this benefit will be achieved; and (iii) from the perspective of a healthcare provider, to assess the value of discovering the actual size of this benefit by means of a Bayesian value of information analysis. Methods: Cost-effectiveness assessments are made from the perspective of the healthcare provider, using current UK norms for the value of a quality-adjusted life-year (QALY). The treatment model is grounded in published analyses of the Randomized Evaluation of Mechanical Assistance for the Treatment of Congestive Heart Failure (REMATCH) trial of first-generation LVADs, translated into a UK cost setting. The prospects for patient survival with second-generation devices is assessed using Bayesian prior distributions, elicited from a group of leading clinicians in the field. Results: Using established thresholds, cost-effectiveness probabilities under these priors are found to be low (.2 percent) for devices costing as much as £60,000. Sensitivity of the conclusions to both device cost and QALY valuation is examined. Conclusions: In the event that the price of the device in use would reduce to £40,000, the value of the survival information can readily justify investment in further trials

AR-C155858 is a potent inhibitor of monocarboxylate transporters MCT1 and MCT2 that binds to an intracellular site involving transmembrane helices 7–10

In the present study we characterize the properties of the potent MCT1 (monocarboxylate transporter 1) inhibitor AR-C155858. Inhibitor titrations of L-lactate transport by MCT1 in rat erythrocytes were used to determine the Ki value and number of AR-C155858-binding sites (Et) on MCT1 and the turnover number of the transporter (kcat). Derived values were 2.3±1.4 nM, 1.29±0.09 nmol per ml of packed cells and 12.2±1.1 s−1 respectively. When expressed in Xenopus laevis oocytes, MCT1 and MCT2 were potently inhibited by AR-C155858, whereas MCT4 was not. Inhibition of MCT1 was shown to be time-dependent, and the compound was also active when microinjected, suggesting that AR-C155858 probably enters the cell before binding to an intracellular site on MCT1. Measurement of the inhibitor sensitivity of several chimaeric transporters combining different domains of MCT1 and MCT4 revealed that the binding site for AR-C155858 is contained within the C-terminal half of MCT1, and involves TM (transmembrane) domains 7–10. This is consistent with previous data identifying Phe360 (in TM10) and Asp302 plus Arg306 (TM8) as key residues in substrate binding and translocation by MCT1. Measurement of the Km values of the chimaeras for L-lactate and pyruvate demonstrate that both the C- and N-terminal halves of the molecule influence transport kinetics consistent with our proposed molecular model of MCT1 and its translocation mechanism that requires Lys38 in TM1 in addition to Asp302 and Arg306 in TM8 [Wilson, Meredith, Bunnun, Sessions and Halestrap (2009) J. Biol. Chem. 284, 20011–20021]

The inhibition of monocarboxylate transporter 2 (MCT2) by AR-C155858 is modulated by the associated ancillary protein

In mammalian cells, MCTs (monocarboxylate transporters) require association with an ancillary protein to enable plasma membrane expression of the active transporter. Basigin is the preferred binding partner for MCT1, MCT3 and MCT4, and embigin for MCT2. In rat and rabbit erythrocytes, MCT1 is associated with embigin and basigin respectively, but its sensitivity to inhibition by AR-C155858 was found to be identical. Using RT (reverse transcription)–PCR, we have shown that Xenopus laevis oocytes contain endogenous basigin, but not embigin. Co-expression of exogenous embigin was without effect on either the expression of MCT1 or its inhibition by AR-C155858. In contrast, expression of active MCT2 at the plasma membrane of oocytes was significantly enhanced by co-expression of exogenous embigin. This additional transport activity was insensitive to inhibition by AR-C155858 unlike that by MCT2 expressed with endogenous basigin that was potently inhibited by AR-C155858. Chimaeras and C-terminal truncations of MCT1 and MCT2 were also expressed in oocytes in the presence and absence of exogenous embigin. L-Lactate Km values for these constructs were determined and revealed that the TM (transmembrane) domains of an MCT, most probably TM7–TM12, but not the C-terminus, are the major determinants of L-lactate affinity, whereas the associated ancillary protein has little or no effect. Inhibitor titrations of lactate transport by these constructs indicated that embigin modulates MCT2 sensitivity to AR-C155858 through interactions with both the intracellular C-terminus and TMs 3 and 6 of MCT2. The C-terminus of MCT2 was found to be essential for its expression with endogenous basigin

Safety of nifedipine GITS in stable angina: The ACTION trial

Aim: We describe the safety profile of nifedipine GITS as assessed from adverse events reported in the ACTION trial in which 7,665 patients with stable, symptomatic coronary artery disease were randomly assigned nifedipine GITS or placebo and followed for a mean of 4.9 years. Methods: All adverse events were coded using the COSTART coding dictionary. The incidence rate for each event was calculated as the number of patients with the event concerned divided by the total time 'at risk'. Hazard ratios comparing nifedipine with placebo and their 95% confidence intervals were obtained by Cox proportional-hazards analysis. Results: As reported previously, nifedipine significantly reduced the incidence of cardiovascular events and procedures [hazard ratio (HR) 0.89, 95% confidence interval (CI) 0.83-0.95]. Apart from the known side effects of nifedipine, which include peripheral oedema, vasodilatation, hypotension, asthenia, constipation, leg cramps, non-specific respiratory complaints, impotence and polyuria, and which were reported more frequently in patients assigned nifedipine, the incidence rates of most other adverse events were similar. There were no differences in the occurrence of gastrointestinal haemorrhage, myocardial infarction and suicide. The rate of occurrence of death or new cancer excluding non-melanoma skin cancer for patients with no history of cancer at baseline was 2.53/100 patient years for patients assigned nifedipine and 2.37/100 patient years for patients assigned placebo (HR 1.06, 95% CI 0.93-1.22). Conclusion: Overall nifedipine GITS was well tolerated by patients with stable symptomatic angina

Should beta-blocker therapy be reduced or withdrawn after an episode of decompensated heart failure? Results from COMET.

BACKGROUND: It is unclear whether beta-blocker therapy should be reduced or withdrawn in patients who develop acute decompensated heart failure (HF). We studied the relationship between changes in beta-blocker dose and outcome in patients surviving a HF hospitalisation in COMET. METHODS: Patients hospitalised for HF were subdivided on the basis of the beta-blocker dose administered at the visit following hospitalisation, compared to that administered before. RESULTS: In COMET, 752/3029 patients (25%, 361 carvedilol and 391 metoprolol) had a non-fatal HF hospitalisation while on study treatment. Of these, 61 patients (8%) had beta-blocker treatment withdrawn, 162 (22%) had a dose reduction and 529 (70%) were maintained on the same dose. One-and two-year cumulative mortality rates were 28.7% and 44.6% for patients withdrawn from study medication, 37.4% and 51.4% for those with a reduced dosage (n.s.) and 19.1% and 32.5% for those maintained on the same dose (HR,1.59; 95%CI, 1.28-1.98; p<0.001, compared to the others). The result remained significant in a multivariable model: (HR, 1.30; 95%CI, 1.02-1.66; p=0.0318). No interaction with the beneficial effects of carvedilol, compared to metoprolol, on outcome was observed (p=0.8436). CONCLUSIONS: HF hospitalisations are associated with a high subsequent mortality. The risk of death is higher in patients who discontinue beta-blocker therapy or have their dose reduced. The increase in mortality is only partially explained by the worse prognostic profile of these patients

Black hole candidate XTE J1752-223: Swift observations of canonical states during outburst

We present Swift broadband observations of the recently discovered black hole candidate, X-ray transient, XTE J1752-223, obtained over the period of outburst from October 2009 to June 2010. From Swift-UVOT data we confirm the presence of an optical counterpart which displays variability correlated, in the soft state, to the X-ray emission observed by Swift-XRT. The optical counterpart also displays hysteretical behaviour between the states not normally observed in the optical bands, suggesting a possible contribution from a synchrotron emitting jet to the optical emission in the rising hard state. We offer a purely phenomenological treatment of the spectra as an indication of the canonical spectral state of the source during different periods of the outburst. We find that the high energy hardness-intensity diagrams over two separate bands follows the canonical behavior, confirming the spectral states. Our XRT timing analysis shows that in the hard state there is significant variability below 10Hz which is more pronounced at low energies, while during the soft state the level of variability is consistent with being minimal. These properties of XTE J1752-223 support its candidacy as a black hole in the Galactic centre region.Comment: 8 pages, 8 figures; MNRAS in pres

The evolution of the terrestrial-terminating Irish Sea glacier during the last glaciation

Here we reconstruct the last advance to maximum limits and retreat of the Irish Sea Glacier (ISG), the only land‐terminating ice lobe of the western British Irish Ice Sheet. A series of reverse bedrock slopes rendered proglacial lakes endemic, forming time‐transgressive moraine‐ and bedrock‐dammed basins that evolved with ice marginal retreat. Combining, for the first time on glacial sediments, optically stimulated luminescence (OSL) bleaching profiles for cobbles with single grain and small aliquot OSL measurements on sands, has produced a coherent chronology from these heterogeneously bleached samples. This chronology constrains what is globally an early build‐up of ice during late Marine Isotope Stage 3 and Greenland Stadial (GS) 5, with ice margins reaching south Lancashire by 30 ± 1.2 ka, followed by a 120‐km advance at 28.3 ± 1.4 ka reaching its 26.5 ± 1.1 ka maximum extent during GS‐3. Early retreat during GS‐3 reflects piracy of ice sources shared with the Irish‐Sea Ice Stream (ISIS), starving the ISG. With ISG retreat, an opportunistic readvance of Welsh ice during GS‐2 rode over the ISG moraines occupying the space vacated, with ice margins oscillating within a substantial glacial over‐deepening. Our geomorphological chronosequence shows a glacial system forced by climate but mediated by piracy of ice sources shared with the ISIS, changing flow regimes and fronting environments

Composition of the pericellular matrix modulates the deformation behaviour of chondrocytes in articular cartilage under static loading

The aim was to assess the role of the composition changes in the pericellular matrix (PCM) for the chondrocyte deformation. For that, a three-dimensional finite element model with depth-dependent collagen density, fluid fraction, fixed charge density and collagen architecture, including parallel planes representing the split-lines, was created to model the extracellular matrix (ECM). The PCM was constructed similarly as the ECM, but the collagen fibrils were oriented parallel to the chondrocyte surfaces. The chondrocytes were modelled as poroelastic with swelling properties. Deformation behaviour of the cells was studied under 15% static compression. Due to the depth-dependent structure and composition of cartilage, axial cell strains were highly depth-dependent. An increase in the collagen content and fluid fraction in the PCMs increased the lateral cell strains, while an increase in the fixed charge density induced an inverse behaviour. Axial cell strains were only slightly affected by the changes in PCM composition. We conclude that the PCM composition plays a significant role in the deformation behaviour of chondrocytes, possibly modulating cartilage development, adaptation and degeneration. The development of cartilage repair materials could benefit from this information