Heme dioxygenases catalyze the oxidation of l-tryptophan to N-formylkynurenine (NFK), the first and rate-limiting step in tryptophan catabolism. Although recent progress has been made on early stages in the mechanism, there is currently no experimental data on the mechanism of product (NFK) formation. In this work, we have used mass spectrometry to examine product formation in a number of dioxygenases. In addition to NFK formation (m/z = 237), the data identify a species (m/z = 221) that is consistent with insertion of a single atom of oxygen into the substrate during O2-driven turnover. The fragmentation pattern for this m/z = 221 species is consistent with a cyclic amino acetal structure; independent chemical synthesis of the 3a-hydroxypyrroloindole-2-carboxylic acid compound is in agreement with this assignment. Labeling experiments with 18O2 confirm the origin of the oxygen atom as arising from O2-dependent turnover. These data suggest that the dioxygenases use a ring-opening mechanism during NFK formation, rather than Criegee or dioxetane mechanisms as previously proposed.

(2015) Molecular sensing with hyperpolarized 129Xe using switchable chemical exchange relaxation transfer.

Due to low fluorine background signal in vivo, 19F is a good marker to study the fate of exogenous molecules by magnetic resonance imaging (MRI) using equilibrium nuclear spin polarization schemes. Since 19F MRI applications require high sensitivity, it can be important to assess experimental feasibility during the design stage already by estimating the minimum detectable fluorine concentration. Here we propose a simple method for the calibration of MRI hardware, providing sensitivity estimates for a given scanner and coil configuration. An experimental “calibration factor” to account for variations in coil configuration and hardware set-up is specified. Once it has been determined in a calibration experiment, the sensitivity of an experiment or, alternatively, the minimum number of required spins or the minimum marker concentration can be estimated without the need for a pilot experiment. The definition of this calibration factor is derived based on standard equations for the sensitivity in magnetic resonance, yet the method is not restricted by the limited validity of these equations, since additional instrument-dependent factors are implicitly included during calibration. The method is demonstrated using MR spectroscopy and imaging experiments with different 19F samples, both paramagnetically and susceptibility broadened, to approximate a range of realistic environments.

Summary. A variety of the indirect method of measuring the electric conductivity of the cytoplasm is described which is reduced to the double point measurement of the cell suspension conductivity at high and low frequency. An empirical dependence is shown on which this method has been based. This dependence has been verified for the suspension of erythrocytes in 0.9 % aqueous solution of NaC1 as well as for human blood. The measuring frequencies -~n and Fw for this type of suspension were defined. The agreement of the specific conductivity of cytoplasm isolated from erythrocytes (~1 = 5.36 c~) with the data achieved by the described method is good.Zusammen/assung. In der Arbeit wird eine Variation der indirekten YleBmethode der elektrischen Leitfahigkeit des Zytoplasma dargestellt, welche ausschlieBlich die Zweipunktmessung der Leitfahigkeit der Zellsnspension bei niedriger und hoher Frequenz umfaBt. Es wurde die empirische Abhangigkeit angegeben, die die Grundlage der Methode bildet.Diese Abhangigkeit wurde fiir die Erythrozytensuspension in 0,9 % waBriger NaC1-LSsung und fiir das menschliche Blut geprfift. Ffir diese Art der Suspension wurden die Me~-frequenzen, die Niederfrequenz und die Hochfrequenz, bestimmt. Es wurde eine gute ~ber-einstimmung der spezifiscben Leitfahigkeit des aus den Erythrozyten isolierten Zytoplasmas ~ul = 5,36m~] mit den mi~ Hilfe der vorgeschlagenen Methode erhaltenen Angaben erzielt. \ era/ EinfiihrungDie elektrischen Eigenschaften der Zellen und die mit ihnen verbundenen Erscheinungen spielen eine wiehtige Rolle in vielen biologisehen Prozessen, die sieh in lebenden Organismen abspielen. Es entsteht also die Notwendigkeit, die elektrisehen Gr6$en zu messen sowohl aus wissensehaftlichen Grfinden wie auch wegen der M6glichkeit ihrer praktisehen Ausnutzung, z. B. bei der medizinischen Diagnostik.Die elektrische Leitf~higkeit des Zytoplasma, mit der wit uns in dieser Arbeit befassen, kann grunds~tzlich nur auf zwei Arten unmittelbar bestimmt werden: mit Hilfe yon Mikroelektroden, die man in das Innere der Zelle einfiihren mu$, oder durch die Messung des pr~parierten Zytoplasmas. Die M/~ngel und EinsehrKnkungen der beiden Methoden sind offensichtlieh.

Paramagnetic gadolinium ions (GdIII), complexed within DOTA-based chelates, have become useful tools to increase the magnetic resonance imaging (MRI) contrast in tissues of interest. Recently, “on/off” probes serving as 19F·MRI biosensors for target enzymes have emerged that utilize the increase in transverse (T2∗ or T2) relaxation times upon cleavage of the paramagnetic GdIII centre. Molecular 19F·MRI has the advantage of high specificity due to the lack of background signal but suffers from low signal intensity that leads to low spatial resolution and long recording times. In this work, an “on/off” probe concept is introduced that utilizes responsive deactivation of paramagnetic relaxation enhancement (PRE) to generate 19F longitudinal (T1) relaxation contrast for accelerated molecular MRI. The probe concept is applied to matrix metalloproteinases (MMPs), a class of enzymes linked with many inflammatory diseases and cancer that modify bioactive extracellular substrates. The presence of these biomarkers in extracellular space makes MMPs an accessible target for responsive PRE deactivation probes. Responsive PRE deactivation in a 19F biosensor probe, selective for MMP-2 and MMP-9, is shown to enable molecular MRI contrast at significantly reduced experimental times compared to previous methods. PRE deactivation was caused by MMP through cleavage of a protease substrate that served as a linker between the fluorine-containing moiety and a paramagnetic GdIII-bound DOTA complex. Ultrashort echo time (UTE) MRI and, alternatively, short echo times in standard gradient echo (GE) MRI were employed to cope with the fast 19F transverse relaxation of the PRE active probe in its “on-state.” Upon responsive PRE deactivation, the 19F·MRI signal from the “off-state” probe diminished, thereby indicating the presence of the target enzyme through the associated negative MRI contrast. Null point 1H·MRI, obtainable within a short time course, was employed to identify false-positive 19F·MRI responses caused by dilution of the contrast agent.