Discrimination of Human Pathogen Clostridium Species Especially of the Heterogeneous C. sporogenes and C. botulinum by MALDI-TOF Mass Spectrometry.

Clostridium species cause several local and systemic diseases. Conventional identification of these microorganisms is in part laborious, not always reliable, time consuming or does not always distinguish different species, i.e.

Rapid identification of oral Actinomyces species cultivated from subgingival biofilm by MALDI-TOF-MS.

Background: Actinomyces are a common part of the residential flora of the human intestinal tract, genitourinary system and skin. Isolation and identification of Actinomyces by conventional methods is often difficult and time consuming. In recent years, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) has become a rapid and simple method to identify bacteria.

Proteomic and functional consequences of hexokinase deficiency in glucose-repressible Kluyveromyces lactis.

The analysis of glucose signaling in the Crabtree-positive eukaryotic model organism Saccharomyces cerevisiae has disclosed a dual role of its hexokinase ScHxk2, which acts as a glycolytic enzyme and key signal transducer adapting central metabolism to glucose availability. In order to identify evolutionarily conserved characteristics of hexokinase structure and function, the cellular response of the Crabtree-negative yeast Kluyveromyces lactis to rag5 null mutation and concomitant deficiency of its unique hexokinase KlHxk1 was analyzed by means of difference gel electrophoresis. In total, 2,851 fluorescent spots containing different protein species were detected in the master gel representing all of the K.

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.

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.

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

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.

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, Murray's lung injury severity score (Murray's LISS) and interleukins in BALF were gathered.

Oral brush biopsy analysis by matrix assisted laser desorption/ionisation-time of flight mass spectrometry profiling--a pilot study.

Oral squamous cell carcinomas (OSCCs) often present as advanced tumours requiring aggressive local and regional therapy and result in significant functional impairment. The objective is to develop pre-symptomatic screening detection of OSCC by a brush biopsy method which is less invasive than the conventional biopsy for histology. Given the molecular heterogeneity of oral cancer, it is unlikely that even a panel of tumour markers would provide accurate diagnosis.

Phenotypic heterogeneity of Streptococcus mutans in dentin.

Information concerning phenotypic heterogeneity of Streptococcus mutans in carious dentin is sparse. Matrix-assisted laser-desorption/ionization-time-of-flight mass-spectrometry (MALDI-TOF-MS) facilitates the phenotypic differentiation of bacteria to the subspecies level. To verify a supposed influence of restorative treatment on the phenotypic heterogeneity of S.

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 curcumin's potency as an Glo1 inhibitor.

Identification and characterization of CaApe2--a neutral arginine/alanine/leucine-specific metallo-aminopeptidase from Candida albicans.

The proteolytic potential of the pathogenic fungus Candida albicans was evaluated by the identification and functional characterization of a peptidolytic enzyme isolated from the cell wall of the microorganism. Determination of basic structural and kinetic data identified a neutral arginine/alanine/leucine-specific metallo-aminopeptidase of unknown function termed CaApe2, which is encoded by ORF CaO19.5197 (GenBank RefSeq XM_705313).

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.

Rapid identification of viridans streptococci by mass spectrometric discrimination.

Viridans streptococci (VS) are responsible for several systemic diseases, such as endocarditis, abscesses, and septicemia. Unfortunately, species identification by conventional methods seems to be more difficult than species identification of other groups of bacteria. The aim of the present study was to evaluate the use of cell matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF-MS) for the rapid identification of 10 different species of VS.

A novel form of 6-phosphofructokinase. Identification and functional relevance of a third type of subunit in Pichia pastoris.

Classically, 6-phosphofructokinases are homo- and hetero-oligomeric enzymes consisting of alpha subunits and alpha/beta subunits, respectively. Herein, we describe a new form of 6-phosphofructokinase (Pfk) present in several Pichia species, which is composed of three different types of subunit, alpha, beta, and gamma. The sequence of the gamma subunit shows no similarity to classic Pfk subunits or to other known protein sequences.

Differentiation of mutans streptococci by intact cell matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

It is difficult to distinguish mutans streptococci on the species level, and even more so on the subspecies level. Intact cell matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) (ICM) was applied to reference strains of five of the species of the mutans group (Streptococcus criceti, Streptococcus downei, Streptococcus mutans, Streptococcus ratti, Streptococcus sobrinus), nonmutans streptococci (Streptococcus oralis, Streptococcus mitis, Streptococcus salivarius, and Streptococcus sanguinis), and 177 mutans streptococci isolated from saliva of 10 children. From the analysis of the reference strains, readily distinguishable ICM mass spectra were obtained for the different species.

6-phosphofructokinase from Pichia pastoris: purification, kinetic and molecular characterization of the enzyme.

6-Phosphofructokinase from Pichia pastoris was purified for the first time to homogeneity applying seven steps, including pseudo-affinity dye-ligand chromatography on Procion Blue H-5R-Sepharose. The specific activity of the purified enzyme was about 80 U/mg. It behaves as a typically allosteric 6-phosphofructokinase exhibiting activation by AMP and fructose 2,6-bis(phosphate), inhibition by ATP and cooperativity to fructose 6-phosphate.

Human leptin forms complexes with alpha 2-macroglobulin which are recognized by the alpha 2-macroglobulin receptor/low density lipoprotein receptor-related protein.

Objective: To identify binding proteins of leptin in human plasma.

Methods: Binding was evaluated by electrophoresis, size exclusion chromatography (SEC), Western blotting, and radioisotope labeling. Quantification of leptin and the different forms of alpha2-macroglobulin (alpha2-M) was performed by ELISA.

Purification and characterization of phosphofructokinase from the yeast Kluyveromyces lactis.

Phosphofructokinase from Kluyveromyces lactis was purified by 180-fold enrichment, elaborating the following steps: cell disruption, polyethylene glycol precipitation, affinity chromatography, size exclusion chromatography on Sepharose 6B and on Bio-Sil SEC 400 and ion exchange chromatography. The homogeneous enzyme exhibits a molecular mass of 845 +/- 20 kDa as determined by sedimentation equilibrium measurements and a specific activity of 100 units/mg protein. The apparent sedimentation coefficient was found to be s20,c = 20.

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-bisphosphatase 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.

Bistability and damped oscillations in the fructose 6-phosphate/fructose 1,6-bisphosphate cycle in cell-free extracts from rat liver.

The dynamics of the fructose 6-phosphate/fructose 1,6-bisphosphate substrate cycle was investigated in cell-free extracts from rat liver. Under conditions of continuous substrate supply different types of stationary states and damped oscillations were observed experimentally and found to be in qualitative agreement with theoretical predictions. Changing the adenylate energy charge of the substrate supply, bistability was shown to be related to irreversible transitions between functionally different branches of stable stationary states.

Irreversible transitions in the 6-phosphofructokinase/fructose 1,6-bisphosphatase cycle.

The dynamics of the fructose 6-phosphate fructose-1,6-bisphosphate cycle operating in an open and homogeneous system reconstituted from purified enzymes was extensively studied. In addition to 6-phosphofructokinase and fructose-1,6-bisphosphatase, pyruvate kinase, adenylate kinae and glucose-6-phosphate isomerase were involved. In that multi-enzyme system, the main source of non-linearity is the reciprocal effect of AMP on the activities of 6-phosphofructokinase and fructose-1,6-bisphosphatase.

Yeast phosphofructokinase: kinetic characterization of a substrate-imprinted enzyme conformation.

Intramolecular cross-linking of octameric yeast phosphofructokinase was applied to study the effect of substrate-imprinted conformational changes on the regulatory properties of the enzyme:Cross-linking performed in the presence of fructose 6-phosphate yields a substrate-imprinted enzyme species the affinity of which towards this substrate is significantly higher than that of native phosphofructokinase and of the enzyme cross-linked in the absence of fructose 6-phosphate. The enzyme cross-linked in the presence of fructose 6-phosphate does not exhibit cooperativity with respect to this substrate but is still activated by AMP and by fructose 2,6-bisphosphate. This activation consists in an increase of substrate affinity with respect to fructose 6-phosphate.

Fructose-2,6-bisphosphate metabolism in permeabilized yeast cells.

Several methods for the permeabilization of Saccharomyces cerevisiae M1 were compared. Cells were permeabilized in the presence of 3% toluene/mercaptoethanol, and the activities of 6-phosphofructo-2-kinase, fructose-2,6-bisphosphatase and alkaline phosphatase were measured during growth of yeast on glucose. In the exponential phase of growth, the specific activities of 6-phosphofructo-2-kinase and fructose-2,6-bisphosphatase decrease significantly.

Regulation of the fructose 6-phosphate/fructose 2,6-bisphosphate cycle by enzyme phosphorylation and sn-glycerol 3-phosphate.

The regulation of the Fru-6-P/Fru-2,6-P2 cycle by the cooperation of allosteric and covalent mechanisms was investigated in a reconstituted enzyme system under in vitro conditions. Phosphorylation of the bifunctional enzyme exerts a much stronger effect than sn-glycerol 3-phosphate in lowering the quasi-stationary concentration of fructose 2,6-bisphosphate and in increasing the critical concentration of the fructose phosphates, respectively. However, sn-glycerol 3-phosphate is able to strongly amplify the decrease of the quasi-stationary concentration of fructose 2,6-bisphosphate due to phosphorylation.

A hysteretic cycle in glucose 6-phosphate metabolism observed in a cell-free yeast extract.

The dynamics of a partial glycolytic reaction sequence which converts glucose 6-phosphate to triose phosphates is described. The study was performed with cell-free extracts from baker's yeast harvested in the logarithmic and stationary growth phases. The experiments are based on a flow-through reactor supplied with the desalted cell-free extract as well as glucose 6-phosphate, ATP and phosphoenolpyruvate.