Fine Mapping the Spatial Distribution and Concentration of Unlabeled Drugs within Tissue Micro-Compartments Using Imaging Mass Spectrometry

Readouts that define the physiological distributions of drugs in tissues are an unmet challenge and at best imprecise, but are needed in order to understand both the pharmacokinetic and pharmacodynamic properties associated with efficacy. Here we demonstrate that it is feasible to follow the in vivo transport of unlabeled drugs within specific organ and tissue compartments on a platform that applies MALDI imaging mass spectrometry to tissue sections characterized with high definition histology. We have tracked and quantified the distribution of an inhaled reference compound, tiotropium, within the lungs of dosed rats, using systematic point by point MS and MS/MS sampling at 200 µm intervals. By comparing drug ion distribution patterns in adjacent tissue sections, we observed that within 15 min following exposure, tiotropium parent MS ions (mass-to-charge; m/z 392.1) and fragmented daughter MS/MS ions (m/z 170.1 and 152.1) were dispersed in a concentration gradient (80 fmol-5 pmol) away from the central airways into the lung parenchyma and pleura. These drug levels agreed well with amounts detected in lung compartments by chemical extraction. Moreover, the simultaneous global definition of molecular ion signatures localized within 2-D tissue space provides accurate assignment of ion identities within histological landmarks, providing context to dynamic biological processes occurring at sites of drug presence. Our results highlight an important emerging technology allowing specific high resolution identification of unlabeled drugs at sites of in vivo uptake and retention.     

Endorphin mediated increase in pain threshold induced by long-lasting exercise in rats

Rats were trained to run spontaneously, without stress, in running wheels. The running activity increased gradually and could reach a plateau of 7 km/night after 3–4 weeks. During the first hour of running in the dark phase the squeak threshold increased significantly and remained high in the morning. The degree of increased threshold was correlated to the amount of running activity. The squeak threshold declined during the following 6 hours of inactivity. A rapid decrease in threshold occurred after naloxone (1–2 mg/kg i.p.). It is suggested that long-lasting muscle exercise (e.g. jogging), acupuncture, and low frequency electrical stimulation of afferent nerve fibres produce discharges in muscle afferents which influence central endorphin mechanics giving analgetic effects.     

EPR Monitored Redox Titration of the Cofactors of Saccharomyces cerevisiae Nar1

Electron Paramagnetic Resonance (EPR) monitored redox titrations are a powerful method to determine the midpoint potential of cofactors in proteins and to identify and quantify the cofactors in their detectable redox state.The technique is complementary to direct electrochemistry (voltammetry) approaches, as it does not offer information on electron transfer rates, but does establish the identity and redox state of the cofactors in the protein under study. The technique is widely applicable to any protein containing an electron paramagnetic resonance (EPR) detectable cofactor.A typical titration requires 2 ml protein with a cofactor concentration in the range of 1-100 µM. The protein is titrated with a chemical reductant (sodium dithionite) or oxidant (potassium ferricyanide) in order to poise the sample at a certain potential. A platinum wire and a Ag/AgCl reference electrode are connected to a voltmeter to measure the potential of the protein solution. A set of 13 different redox mediators is used to equilibrate between the redox cofactors of the protein and the electrodes. Samples are drawn at different potentials and the Electron Paramagnetic Resonance spectra, characteristic for the different redox cofactors in the protein, are measured. The plot of the signal intensity versus the sample potential is analyzed using the Nernst equation in order to determine the midpoint potential of the cofactor.     

The generation and subsequent fate of glutathionyl radicals in biological systems

Horseradish peroxidase-catalyzed oxidation of p-phenetidine in the presence of either glutathione (GSH), cysteine, or N-acetylcysteine led to the production of the appropriate thioyl radical which could be observed using EPR spectroscopy in conjunction with the spin trap 5,5-dimethyl-1-pyrroline-N-oxide. This confirms earlier work using acetaminophen (Ross, D., Albano, E., Nilsson, U., and Moldéus, P. (1984) Biochem. Biophys. Res. Commun. 125, 109-115). The further reactions of glutathionyl radicals (GS.), generated during horseradish peroxidase-catalyzed oxidation of p-phenetidine and acetaminophen in the presence of GSH, were investigated by following kinetics of oxygen uptake and oxidized glutathione (GSSG) formation. Oxygen uptake and GSSG generation were dependent on the concentration of GSH but above that which was required for maximal interaction with the primary amine or phenoxy radical generated during peroxidatic oxidation of p-phenetidine or acetaminophen, suggesting that a secondary GSH-dependent process was responsible for oxygen uptake and GSSG production. GSSG was the only product of thiol oxidation detected during peroxidatic oxidation of p-phenetidine or acetaminophen in the presence of GSH, but under nitrogen saturation conditions its production was reduced to 8 and 33% of the corresponding amounts obtained under aerobic conditions in the cases of p-phenetidine and acetaminophen, respectively. Nitrogen saturation conditions did not affect horseradish peroxidase-catalyzed metabolism. This shows that the main route of GSSG generation in such reactions is not by dimerization of GS. but via mechanism(s) involving oxygen consumption such as via GSSG-. or via GSOOH.     

Molecular mechanisms of ceramide-mediated CD95 clustering.

Receptor clustering has been suggested as a crucial mechanism to initiate receptor signaling. Here we show that ceramide in sphingolipid-rich membrane rafts mediates clustering of CD95. Neutralization of surface ceramide or inhibition of its endogenous generation prevented CD95 clustering. Furthermore, application of ceramide at the cell surface triggered clustering of active but not inactive CD95. Apoptosis was inhibited by neutralization of surface ceramide or inhibition of ceramide release in vitro and in vivo. Thus, we conclude that surface ceramide mediates CD95 clustering, which is required for initiation of apoptosis, at least in some cell types.     

A [2Fe-2S] protein from the hyperthermophilic bacterium Aquifex aeolicus.

Overexpression in Escherichia coli of the fdx4 gene from Aquifex aeolicus has allowed isolation and characterization of the first hyperthermophilic [2Fe-2S](Scys)(4) protein, a homodimer of M = 2 x 12.4 kDa with one [2Fe-2S] cluster per subunit. This protein is undamaged by heating to 100 degrees C for at least three hours. The primary structure, in particular the characteristic distribution of the four cysteine ligands of the metal site, and the spectroscopic properties of the A. aeolicus protein relate it to well characterized [2Fe-2S] proteins from Clostridium pasteurianum and Azotobacter vinelandii. These proteins are also homologous to subunits or domains of hydrogenases and NADH-ubiquinone oxidoreductase (Complex I) of respiratory chains. The A. aeolicus [2Fe-2S] protein is thus representative of a presumably novel protein fold involved in a variety of functions in very diverse cellular backgrounds.