Expression and characterization of endochitinase C from Serratia marcescens BJL200 and its purification by a one-step general chitinase purification method

In this study we cloned, expressed, purified, and charaterized chitinase C1 from Serratia marcescens strain BJL200. As expected, the BJL200-ChiC1 amino acid sequence of this strain was highly similar to sequences of ChiC1 identified in two other strains of S. marcescens. BJL200-ChiC1 was overproduced in E. coli by the T7 expression system, and purified by a one-step hydrophobic interaction chromatography (HIC) with phenyl-sepharose. BJL200-ChiA and BJL200-ChiB had an approximately 30-fold higher k(cat) and 15 fold-lower K(m) than BJL200-ChiC1 for the oligomeric substrate 4-methylumbelliferyl-beta-D-N-N'-N'-triacetylchitotrioside, while BJL200-ChiC1 was 10-15 times faster than BJL200-ChiB and BJL200-ChiA in degrading the polymeric substrate CM-chitin-RBV. BJL200-ChiC1 degradation of beta-chitin resulted in a range of different chito-oligosaccharides (GlcNAc)(2) (main product), GlcNAc, (GlcNAc)(3), (GlcNAc)(4), and (GlcNAc)(5), indicating endo activity. The purification method used for BJL200-ChiC1 in this study is generally applicable to family 18 chitinases and their mutants, including inactive mutants, some of which tend to bind almost irreversibly to chitin columns. The high specificity of the interaction with the (non-chitinous) column material is mediated by aromatic residues that occur in the substrate-binding clefts and surfaces of the enzymes.     

Analysis of mitochondrial generation and release of reactive oxygen species.

BACKGROUND: Reactive oxygen species (ROS) are mainly produced in mitochondria and are important contributors to many forms of cell death. ROS also function as second messengers within the cell and may constitute a signaling pathway from mitochondria to the cytoplasm and nucleus. The aim of the present study was to develop a protocol to detect changes in intra- and extramitochondrial releases of ROS, which could be used to analyze the role of mitochondria in cell signaling and cell death. METHODS: Fluorescence-based assays were used to measure (a) total production of ROS, (b) intramitochondrial ROS, (c) extramitochondrial hydrogen peroxide, and (d) superoxide outside inverted (inside-out) submitochondrial particles. ROS generation in the samples was increased or decreased by the addition of different substrates, enzymes, and inhibitors of the electron transport chain. RESULTS: The individual assays used were sensitive to increased (e.g., after addition of antimycin A; increased signal) and decreased (ROS scavenging; decreased signal) levels of ROS. In combination, the assays provided information about mitochondrial ROS generation and release dynamics from small samples of isolated mitochondria. CONCLUSIONS: The combination of fluorescent techniques described is a useful tool to study the role of ROS in cell death and in cellular redox signaling.     

A TGFβRII frameshift-mutation-derived CTL epitope recognised by HLA-A2-restricted CD8+ T cells

Microsatellite instability (MSI) is recognised as genome-wide alterations in repetitive DNA sequences caused by defects in the DNA mismatch repair machinery. Such mutation patterns have been found in almost all analysed malignancies from patients with hereditary non-polyposis colorectal cancer, and in approximately 15% of sporadic colorectal cancers. In cancers with the MSI phenotype, microsatellite-like sequences in coding regions of various cancer-related genes, including transforming growth factor beta receptor type II (TGF betaRII), are targets for mutations. The TGF betaRII gene harbours a 10-bp polyadenine tract, and mutations within this region are found in 90% of colorectal cancers with MSI. The frameshift mutations result in new amino acid sequences in the C-terminal part of the proteins, prematurely terminating where a novel stop codon appears. In this study we have defined new cytotoxic T lymphocyte (CTL) epitope (RLSSCVPVA), carrying a good HLA-A*0201 binding motif, and resulting from the most common frameshift mutation in TGF betaRII. A CTL line and several CTL clones were generated from an HLA-A2+ normal donor by repeated stimulation of T cells with dendritic cells pulsed with the peptide. One of the CTL clones was able to kill an HLA-A2+ colon cancer cell line harbouring mutant TGF betaRII. This epitope may be a valuable component in cancer vaccines directed at MSI-positive carcinomas.     

Beobachtungen und Fragen zur Biologie des Kolkraben (Corvus c. corax L.)

Ohne Zusammenfassung     

Die Hieracien der Umgebung von Seckau in Ober-Steiermark

Ohne Zusammenfassung     

Preparation and characterization of total,rough and smooth microsomes from the lung of control and methylcholanthrene-treated rats

Optimal conditions for the preparation of relatively pure microsomes and microsomal subfractions from rat lung have been determined. The most important of these conditions is homogenization of a 20% (w/v) suspension of lung tissue in 0.44 M sucrose/1% (w/v) bovine serum albumin with four up-and-down strokes at 440 rev./min in a Potter-Elvehjem homogenizer. The 10 000 × g supernatant prepared from this homogenate can be centrifuged at 105 000 × g to obtain total microsomes or subfractionated into rough and smooth microsomes on a Cs⁺-containing discontinuous sucrose gradient. The total, rough and smooth microsomes have been characterized in terms of their chemical composition, enzymatic activity, and morphology. These preparations should prove useful in studies of various enzymes in lung (e.g. benzpyrene monooxygenase, epoxide hydrase, enzymes of phospholipid and ascorbic acid synthesis) and in subfractionations designed to reveal heterogeneites in the lateral plane of the lung endoplasmic reticulum.     

Die Salzpflanzengesellschaften des Neusiedler Sees

Ohne Zusammenfassung     

Der mikrochemische Nachweis der Alkaloide in der Pflanze

Zusammenfassung Es wurde eine eindeutige sichere Methode ausgearbeitet, um das Kokain in der Pflanze mikrochemisch zu fassen und zu verfolgen. Der sicherste Nachweis läßt sich erbringen durch Mikrosublimation des Kokains und Nachweis im Sublimate mit Au Cl₃ + K Br oder Extraktion mit CHCl₃ + NH₃ und Reaktion mit Au Cl₃ + K Br. Daneben lassen sich noch verwenden die Benzoesäuresublimation und die direkte Reaktion im Schnitt, die jedoch nur bei Anwesenheit von größeren Mengen Kokain gute Resultate geben.     

Quantum-chemical and empirical calculations on phospholipids: I. The charge distribution of model headgroups of phospholipids obtained by a CNDO-APSG procedure

A modified CNDO-APSG procedure to obtain the charge distributions of the headgroups of phosphatidylserine, -ethanolamine and -choline as monomers and as constituents of hexagonal lattice is presented. The calculated charge distributions are typically zwitter-ionic. There is only a small difference between the charges for the monomer and as constituent of the lattice, from which it may be assumed that the main characteristics of the headgroups are the same in both cases.