Jochen Frenzel

Calorimetric, 13C NMR, and 31P NMR studies on the interaction of some phenothiazine derivatives with dipalmitoyl phosphatidylcholine model membranes.

1. The dipalmitoyl phosphatidylcholine/water system was employed to study the interaction of phenothiazines with model membranes. In particular the effects of the drugs upon the lipid phase transition were examined using differential scanning calorimetry and NMR spectroscopy. The studied phenothiazines have peripheral (diethazine) or central (chlorpromazine) properties. 2. Both drugs were observed to lower the phase transition temperature of dipalmitoyl phosphatidylcholine. The molar activity of chlorpromazine is somewhat higher than of diethazine. At low concentrations the drugs affect the dipalmitoyl phosphatidylcholine pretransition endotherm. 3. In the 13C NMR spectra of the drug-containing samples the signal of the trimethylammonium group of dipalmitoyl phosphatidylcholine is broadened, whereas a narrowing of the signal of the fatty-acid chain methylene groups is observed. Further, addition of the phenothiazines causes higher values of the effective chemical shift anisotropy of the 31P in the phosphate group, in comparison to the pure dipalmitoyl phosphatidylcholine sample. 4. The results obtained by three different techniques indicate a higher fluidity in the fatty-acid chain region and a mobility reduction of the polar headgroup of the dipalmitoyl phosphatidylcholine molecules in the presence of the phenothiazines. These phenomena can be well accounted for by a model for the incorporation of the phenothiazines in the dipalmitoyl phosphatidyl-choline bilayer, in which the dialkylaminoalkyl chains are located near the polar headgroups and the ring system does not penetrate far beyond the glycerol backbone into the hydrocarbon phase.

Fructose inhibits apoptosis induced by reoxygenation in rat hepatocytes by decreasing reactive oxygen species via stabilization of the glutathione pool

Oxidative stress induces apoptosis in liver parenchymal cells. The present study demonstrates that the substitution of fructose for glucose as sole carbon source in the incubation medium reduced apoptosis due to reoxygenation up to 50% in cultured rat hepatocytes. This anti-apoptotic action of fructose cannot be explained by the effects of this sugar on the intracellular ATP concentration and the ATP/ADP ratio. Rather, the suppression of apoptosis by fructose seems to be a consequence of remarkably higher intracellular levels of glutathione observed during reoxygenation in fructose-fed hepatocytes in contrast to glucose-fed ones. With fructose as substrate, the generation of excess reactive oxygen species (ROS) during the initial phase of reoxygenation was strongly reduced. With respect to ROS reduction and stabilization of the cellular glutathione pool fructose was found as efficient as a pretreatment of glucose fed cells with N-acetyl-L-cysteine. The enhanced metabolization of ROS by the glutathione/glutathione peroxidase system in fructose-cultured hepatocytes under reoxygenation was expected to improve their mitochondrial status so that late events in the apoptotic pathway are suppressed. This could be confirmed by the reduced release of cytochrome c from mitochondria into the cytosol as well as by the observed decrease of caspase-3 activity during reoxygenation.

Pyruvate protects glucose-deprived Müller cells from nitric oxide-induced oxidative stress by radical scavenging.

The cellular defense of Müller cells against oxidative and nitrosative stress was examined after the addition of the nitric oxide donor papanonoate. Glucose concentrations of ≥550 μM efficiently protected the Müller cells from cell death by maintaining high ATP and glutathione and allowing only a moderate increase of free radicals. Fluorescence‐activated cell sorting (FACS) analysis showed that 22% of the cells underwent apoptosis whereas necrosis was strongly suppressed. Under glucose deprivation, the intracellular concentration of ATP declined to 15% after 1 h; glutathione dropped to 50% within 2 h after papanonoate addition. Both the number of cells containing excess free radicals and the mean concentration of free radicals increased twofold at 0.5–2 h of incubation with papanonoate. Cell death switched from prevailing apoptosis to massive necrosis and cell viability decreased drastically. Several metabolites of glycolysis, gluconeogenesis, and the pentose phosphate pathway were tested with respect to their capability to protect the stressed Müller cells. 2 mM pyruvate was found to enhance cell viability 1.6‐fold predominantly by reducing the necrotic cell demise. It could be shown that pyruvate did not act by improving the energy status of Müller cells but by scavenging excess free radicals. Inhibition of the monocarboxylate transporters in Müller cells by α‐cyano‐4‐hydroxycinnamate abolished this effect. Other 2‐ketoacids, like oxalacetate, 2‐ketoglutarate and 2‐ketobutyrate had a similar protecting effect as pyruvate. Lactate, glutamate, 2‐deoxyglucose, and ribose 5‐phosphate did not protect Müller cells against oxidative and nitrosative stress.

Outcome Prediction in Pneumonia Induced ALI/ARDS by Clinical Features and Peptide Patterns of BALF Determined by Mass Spectrometry

Background Peptide patterns of bronchoalveolar lavage fluid (BALF) were assumed to reflect the complex pathology of acute lung injury (ALI)/acute respiratory distress syndrome (ARDS) better than clinical and inflammatory parameters and may be superior for outcome prediction. Methodology/Principal Findings A training group of patients suffering from ALI/ARDS was compiled from equal numbers of survivors and nonsurvivors. Clinical history, ventilation parameters, Murrays lung injury severity score (Murrays LISS) and interleukins in BALF were gathered. In addition, samples of bronchoalveolar lavage fluid were analyzed by means of hydrophobic chromatography and MALDI-ToF mass spectrometry (MALDI-ToF MS). Receiver operating characteristic (ROC) analysis for each clinical and cytokine parameter revealed interleukin-6>interleukin-8>diabetes mellitus>Murrays LISS as the best outcome predictors. Outcome predicted on the basis of BALF levels of interleukin-6 resulted in 79.4% accuracy, 82.7% sensitivity and 76.1% specificity (area under the ROC curve, AUC, 0.853). Both clinical parameters and cytokines as well as peptide patterns determined by MALDI-ToF MS were analyzed by classification and regression tree (CART) analysis and support vector machine (SVM) algorithms. CART analysis including Murrays LISS, interleukin-6 and interleukin-8 in combination was correct in 78.0%. MALDI-ToF MS of BALF peptides did not reveal a single identifiable biomarker for ARDS. However, classification of patients was successfully achieved based on the entire peptide pattern analyzed using SVM. This method resulted in 90% accuracy, 93.3% sensitivity and 86.7% specificity following a 10-fold cross validation (AUC = 0.953). Subsequent validation of the optimized SVM algorithm with a test group of patients with unknown prognosis yielded 87.5% accuracy, 83.3% sensitivity and 90.0% specificity. Conclusions/Significance MALDI-ToF MS peptide patterns of BALF, evaluated by appropriate mathematical methods can be of value in predicting outcome in pneumonia induced ALI/ARDS.

Fructose 2,6‐bisphosphate induces irreversible transitions in cell‐free extracts of rat liver

The effect of fructose 2,6‐bisphosphate on the dynamics of the 6‐phosphofructo‐1‐kinase/fructose‐1,6‐bisphos‐phatase cycle is investigated in a cell‐free extract of rat liver under steady‐state conditions. Bistability emerges on the basis of the reciprocal allosteric modulation of 6‐phosphofructo‐1‐kinase and fructose‐1,6‐bisphosphatase. Under conditions of bistability fructose 2,6‐bisphosphate may cause transitions between alternative steady states. However, in contrast to what is frequently observed in bistable systems, within a broad range of experimental conditions these transitions proceed irreversibly from states with high ATP to states characterized by low ATP.