Wolfgang Schellenberger

Curcumin inhibits glyoxalase 1: a possible link to its anti-inflammatory and anti-tumor activity.

Background Glyoxalases (Glo1 and Glo2) are involved in the glycolytic pathway by detoxifying the reactive methylglyoxal (MGO) into D-lactate in a two-step reaction using glutathione (GSH) as cofactor. Inhibitors of glyoxalases are considered as anti-inflammatory and anti-carcinogenic agents. The recent finding that various polyphenols modulate Glo1 activity has prompted us to assess curcumins potency as an Glo1 inhibitor. Methodology/Principal Findings Cultures of whole blood cells and tumor cell lines (PC-3, JIM-1, MDA-MD 231 and 1321N1) were set up to investigate the effect of selected polyphenols, including curcumin, on the LPS-induced cytokine production (cytometric bead-based array), cell proliferation (WST-1 assay), cytosolic Glo1 and Glo2 enzymatic activity, apoptosis/necrosis (annexin V-FITC/propidium iodide staining; flow cytometric analysis) as well as GSH and ATP content. Results of enzyme kinetics revealed that curcumin, compared to the polyphenols quercetin, myricetin, kaempferol, luteolin and rutin, elicited a stronger competitive inhibitory effect on Glo1 (Ki = 5.1±1.4 µM). Applying a whole blood assay, IC50 values of pro-inflammatory cytokine release (TNF-α, IL-6, IL-8, IL-1β) were found to be positively correlated with the Ki-values of the aforementioned polyphenols. Moreover, whereas curcumin was found to hamper the growth of breast cancer (JIMT-1, MDA-MB-231), prostate cancer PC-3 and brain astrocytoma 1321N1 cells, no effect on growth or vitality of human primary hepatocytes was elucidated. Curcumin decreased D-lactate release by tumor cells, another clue for inhibition of intracellular Glo1. Conclusions/Significance The results described herein provide new insights into curcumins biological activities as they indicate that inhibition of Glo1 by curcumin may result in non-tolerable levels of MGO and GSH, which, in turn, modulate various metabolic cellular pathways including depletion of cellular ATP and GSH content. This may account for curcumins potency as an anti-inflammatory and anti-tumor agent. The findings support the use of curcumin as a potential therapeutic agent.

Similarity of activation of yeast phosphofructokinase by AMP and fructose-2,6-bisphosphate

Phosphofructokinase from yeast is effectively activated by AMP and fructose-2,6-bisphosphate by increasing the affinity of the enzyme to fructose-6-phosphate and the maximum activity toward this substrate. The enzyme is activated by AMP and fructose-2, 6-bisphosphate both at high and at low concentrations of ATP. The half maximum stimulation concentrations of AMP and fructose-2, 6-bisphosphate are about 200 microM and 2 microM, respectively. At saturating concentrations of AMP and fructose-2, 6-bisphosphate similar maximum activities were observed in the dependence of enzyme activity on the concentrations of fructose-6-phosphate. The fructose-6-phosphate affinity is more enhanced by fructose-2, 6-bisphosphate than by AMP.

Ethyl pyruvate and ethyl lactate down-regulate the production of pro-inflammatory cytokines and modulate expression of immune receptors

Esters of alpha-oxo-carbonic acids such as ethyl pyruvate (EP) have been demonstrated to exert inhibitory effects on the production of anti-inflammatory cytokines. So far, there is no information about effects, if any, of ethyl lactate (EL), an obviously inactive analogue of EP, on inflammatory immune responses. In the present study, we provide evidence that the anti-inflammatory action of alpha-oxo-carbonic acid esters is mediated by inhibition of glyoxalases (Glo), cytosolic enzymes that catalyse the conversion of alpha-oxo-aldehydes such as methylglyoxal (MGO) into the corresponding alpha-hydroxy acids using glutathione as a cofactor. In vitro enzyme activity measurements revealed the inhibition of human Glo1 by alpha-oxo-carbonic acid esters, whilst alpha-hydroxy-carbonic acid esters such as EL were not inhibitory. In contrast, both EP and EL were shown to suppress the Lipopolysaccharide (LPS)-induced production of pro-inflammatory cytokines such as tumor necrosis factor-alpha, interleukin (IL)-1beta, IL-6 and IL-8 from human immunocompetent cells, and modulated the expression of the immune receptors HLA-DR, CD14 and CD91 on human monocytes. Here, we show a crossing link between glyoxalases and the immune system. The results described herein introduce glyoxalases as a possible target for therapeutic approaches of immune suppression.

A step towards the discrimination of beta-lactamase-producing clinical isolates of Enterobacteriaceae and Pseudomonas aeruginosa by MALDI-TOF mass spectrometry.

Summary Background Matrix-Assisted Laser-Desorption/Ionization Time of Flight Mass Spectrometry (MALDI-TOF MS) has already proven to be a powerful tool for species identification in microbiological laboratories. As adequate and rapid screening methods for antibiotic resistance are crucially needed, the present study investigated the discrimination potential of MALDI-TOF MS among extended-spectrum-beta-lactamase (ESBL) or metallo-beta-lactamases- (MBL) producing and the nonproducing strains of Escherichia coli (n=19), Klebsiella pneumoniae (n=19), and Pseudomonas aeruginosa (n=38), respectively. Material/Methods We used a MALDI-TOF MS protocol, usually applied for species identification, in order to integrate a screening method for beta-lactamases into the routine species identification workflow. The acquired spectra were analyzed by visual inspection, statistical similarity analysis and support vector machine (SVM) classification algorithms. Results Neither visual inspection nor mathematical similarity analysis allowed discrimination between spectra of beta-lactamase-producing and the nonproducing strains, but classification within a species by SVM-based algorithms could achieve a correct classification rate of up to 70%. Conclusions This shows that MALDI-TOF MS has definite potential to discriminate antibiotic-resistant strains due to ESBL and MBL production from nonproducing strains, but this performance is not yet sufficiently reliable for routine microbiological diagnostics.

In vitro demonstration of alternate stationary states in an open enzyme system containing phosphofructokinase.

Abstract The kinetic properties of an open reconstituted enzyme system are investigated with the aim of demonstrating experimentally hysteretic transitions between alternate stationary states. The approach is based on a stirred flow-through reactor containing phosphofructokinase and pyruvate kinase entrapped in polyacrylamide gel. Through the reactor is pumped a solution containing fructose 6-phosphate, ATP, and phosphoenol pyruvate as well as adenylate kinase and glucose-6-phosphate isomerase. The latter two enzymes are in excess in respect to phosphofructokinase and pyruvate kinase. According to theoretical predictions the existence of multiple stationary states could be shown experimentally within precisely definable parameters. Switches between alternate stationary states have been caused by perturbations of flow rates and of reactant concentrations.

Discrimination of Enterobacteriaceae and Non-fermenting Gram Negative Bacilli by MALDI-TOF Mass Spectrometry

Discrimination of Enterobacteriaceae and Non-fermenting Gram Negative Bacilli by MALDI-TOF Mass Spectrometry Matrix assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) has proven to be an effective identification tool in medical microbiology. Discrimination to subspecies or serovar level has been found to be challenging using commercially available identification software. By forming our own reference database and using alternative analysis methods, we could reliably identify all implemented Enterobacteriaceae and non-fermenting gram negative bacilli by MALDI-TOF MS and even succeeded to distinguish Shigella sonnei from Escherichia coli (E. coli) and Salmonella enterica spp. enterica serovar Enteritidis from Salmonella enterica spp. enterica serovar Typhimurium. Furthermore, the method showed the ability to separate Enterohemorrhagic E. coli (EHEC) and Enteropathogenic E. coli (EPEC) from non-enteropathogenic E. coli.

Sustained oscillations in a reconstituted enzyme system containing phosphofructokinase and fructose 1,6-bisphosphatase.

In a reconstituted open and homogeneous enzyme system containing phosphofructokinase, fructose 1,6-bisphosphatase, pyruvate kinase, adenylate kinase, and glucose-6-phosphate isomerase sustained oscillations could experimentally be generated. The approach is based on a stirred flow-through reaction chamber. The periodic motions of the reactants are mainly caused by the antagonistic allosteric effects of the adenine nucleotides on the activities of the phosphofructokinase and fructose 1,6-bisphosphatase.

Binding of fructose-6-phosphate to phosphofructokinase from yeast

Abstract Yeast phosphofructokinase binds one molecule of fructose-6-phosphate per subunit. The binding curve exhibits sigmoidality and yields a good fit to an equation derived from the kinetic model as developed previously for this enzyme. The results show that the allosteric kinetic response of the enzyme to fructose-6-phosphate is due to cooperativity of the binding process.

Oral brush biopsy analysis by MALDI-ToF Mass Spectrometry for early cancer diagnosis

OBJECTIVES Intact cell peptidome profiling (ICPP) with MALDI-ToF Mass Spectrometry holds promise as a non-invasive method to detect head and neck squamous cell carcinoma (HNSCC) objectively, which may significantly improve the early diagnosis of oral cancer. The present study was designed to discriminate between tumour samples and non-cancer controls (healthy mucosa and oral lesions) by analysing complete spectral patterns of intact cells using MALDI-ToF MS. MATERIALS AND METHODS In the first step, a database consisting of 26 patients suffering from HNSCC was established by taking brush biopsy samples of the diseased area and of the healthy buccal mucosa of the respective contralateral area. After performing MALDI-ToF MS on these samples, classification analysis was used as the basis for further classification of an additional 26 blinded samples including HNSCC, oral lesions and healthy mucosa. RESULTS By analysing spectral patterns of the blinded samples, all cancerous lesions were defined accurately. One incorrect evaluation (false positive) occurred in the lesion cohort, leading to a sensitivity of 100%, a specificity of 93% and an overall accuracy of 96.5%. CONCLUSION ICPP using MALDI-ToF MS is able to distinguish between healthy and cancerous mucosa and between oral lesions and oral cancer with excellent sensitivity and specificity, which may lead to more accurate early diagnosis of HNSCC.

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.