A system for accurate and automated injection of hyperpolarized substrate with minimal dead time and scalable volumes over a large range

Over recent years hyperpolarization by dissolution dynamic nuclear polarization has become an established technique for studying metabolism in vivo in animal models. Temporal signal plots obtained from the injected metabolite and daughter products, e.g. pyruvate and lactate, can be fitted to compartmental models to estimate kinetic rate constants. Modeling and physiological parameter estimation can be made more robust by consistent and reproducible injections through automation. An injection system previously developed by us was limited in the injectable volume to between 0.6 and 2.4 ml and injection was delayed due to a required syringe filling step. An improved MR-compatible injector system has been developed that measures the pH of injected substrate, uses flow control to reduce dead volume within the injection cannula and can be operated over a larger volume range. The delay time to injection has been minimized by removing the syringe filling step by use of a peristaltic pump. For 100 ll to 10.000 ml, the volume range typically used for mice to rabbits, the average delivered volume was 97.8% of the demand volume. The standard deviation of delivered volumes was 7 ll for 100 ll and 20 ll for 10.000 ml demand volumes (mean S.D. was 9 ul in this range). In three repeat injections through a fixed 0.96 mm O.D. tube the coefficient of variation for the area under the curve was 2%. For in vivo injections of hyperpolarized pyruvate in tumor-bearing rats, signal was first detected in the input femoral vein cannula at 3–4 s post-injection trigger signal and at 9–12 s in tumor tissue. The pH of the injected pyruvate was 7.1 ± 0.3 (mean ± S.D., n = 10). For small injection volumes, e.g. less than 100 ll, the internal diameter of the tubing contained within the peristaltic pump could be reduced to improve accuracy. Larger injection volumes are limited only by the size of the receiving vessel connected to the pump

Insights into the folding and activity of phosphoryl transfer enzymes

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Atomic details of near-transition state conformers for enzyme phosphoryl transfer revealed by MgF\u3csub\u3e3\u3c/sub\u3e− rather than by phosphoranes

Prior evidence supporting the direct observation of phosphorane intermediates in enzymatic phosphoryl transfer reactions was based on the interpretation of electron density corresponding to trigonal species bridging the donor and acceptor atoms. Close examination of the crystalline state of β-phosphoglucomutase, the archetypal phosphorane intermediate-containing enzyme, reveals that the trigonal species is not PO−3 , but is MgF−3 (trifluoromagnesate). Although MgF−3 complexes are transition state analogues rather than phosphoryl group transfer reaction intermediates, the presence of fluorine nuclei in near-transition state conformations offers new opportunities to explore the nature of the interactions, in particular the independent measures of local electrostatic and hydrogen-bonding distributions using 19F NMR. Measurements on three β-PGM-MgF−3 -sugar phosphate complexes show a remarkable relationship between NMR chemical shifts, primary isotope shifts, NOEs, cross hydrogen bond F⋯H-N scalar couplings, and the atomic positions determined from the highresolution crystal structure of the β-PGM-MgF3- -G6P complex. The measurements provide independent validation of the structural and isoelectronic MgF3− model of near-transition state conformations

Carbon monoxide releasing molecule A1 reduces myocardial damage after acute myocardial infarction in a porcine model

International audienceInfarct size is a major determinant of outcomes after acute myocardial infarction (AMI). Carbon monoxide releasing molecules (CORMs), which deliver nano-molar concentrations of carbon monoxide to tissues, have been shown to reduce infarct size in rodents. We evaluated efficacy and safety of CORM-A1 to reduce infarct size in a clinically relevant porcine model of AMI. We induced AMI in Yorkshire White pigs by inflating a coronary angioplasty balloon to completely occlude the left anterior descending artery for 60 minutes, followed by deflation of the balloon to mimic reperfusion. Fifteen minutes after balloon occlusion, animals were given an infusion of 4.27mM CORM-A1 (n=7) or sodium borate control (n=6) over 60 minutes. Infarct size, cardiac biomarkers, ejection fraction and hepatic and renal function were compared amongst the groups. Immunohistochemical analyses were performed to compare inflammation, cell proliferation and apoptosis between the groups. CORM-A1 treated animals had significant reduction in absolute infarct area (158+/-16 vs. 510+/-91 mm2, p<0.001) and infarct area corrected for area at risk (24.8+/-2.6% vs. 45.2+/-4.0%, p<0.0001). Biochemical markers of myocardial injury also tended to be lower and LV function tended to recover better in CORM-A1 treated group. There was no evidence of hepatic or renal toxicity with the doses used. The cardio-protective effects of CORM-A1 were associated with a significant reduction in cell proliferation and inflammation. CORM-A1 reduces infarct size and improves LV remodelling and function in a porcine model of reperfused MI via a reduction in inflammation. These potential cardio-protective effects of CORMs warrant further translational investigations

Two domains of a dual-function NarK protein are required for nitrate uptake, the first step of denitrification in Paracoccus pantotrophus

Uptake of nitrate into the cytoplasm is the first but least well understood step of denitrification; no gene has previously been identified to be necessary for this process. Upstream from the structural genes of the membrane-bound nitrate reductase (narGHJI) in Paracoccus pantotrophus there is a fusion of two genes, each homologous to members of the narK family. The single open reading frame is predicted to encode 24 transmembrane helices, comprising two domains, NarK1 and NarK2. Analysis of both the accumulation of intracellular nitrite and electron transport through the nitrate reductase enzyme in narK mutants reveals that NarK1 and NarK2 are both involved in nitrate uptake. Maximal rate of nitrate transport via NarK2 was dependent upon nitrite, indicating that NarK2 encodes a nitrate/nitrite antiporter. The uncouplers S13 and dinitrophenol showed that NarK2 was not dependent on the proton motive force for maximal nitrate transport activity. Nitrate transport via NarK1 was dependent on proton motive force, indicating that it is likely to be a nitrate/proton symporter. Low expression of membrane-bound nitrate reductase in narK mutants was counteracted by azide, which induced nitrate reductase expression only if the transcriptional activator NarR was present