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Selected AbstractsHeat Shock Protein-27 Is Upregulated in the Temporal Cortex of Patients with EpilepsyEPILEPSIA, Issue 12 2004Hans-J Bidmon Summary:,Purpose: Heat shock protein-27 (HSP-27) belongs to the group of small heat shock proteins that become induced in response to various pathologic conditions. HSP-27 has been shown to protect cells and subcellular structures, particularly mitochondria, and serves as a carrier for estradiol. It is a reliable marker for tissues affected by oxidative stress. Oxidative stress and related cellular defence mechanisms are currently thought to play a major role during experimentally induced epileptic neuropathology. We addressed the question whether HSP-27 becomes induced in the neocortex resected from patients with pharmacoresistant epilepsy. Methods: Human epileptic temporal neocortex was obtained during neurosurgery, and control tissue was obtained at autopsy from subjects without known neurologic diseases. The tissues were either frozen for Western blot analysis or fixed in Zamboni's fixative for the topographic detection of HSP-27 at the cellular level by means of immunohistochemistry. Results: HSP-27 was highly expressed in all epilepsy specimens and in the cortex of a patient who died in the final stage of multiple sclerosis (positive control), whereas only low amounts of HSP-27 were detectable in control brains. In epilepsy patients, HSP-27 was present in astrocytes and in the walls of blood vessels. The intracortical distribution patterns varied strongly among the epilepsy specimens. Conclusions: These results demonstrate that HSP-27 becomes induced in response to epileptic pathology. Although the functional aspects of HSP-27 induction during human epilepsy have yet to be elucidated, it can be concluded that HSP-27 is a marker for cortical regions in which a stress response has been caused by seizures. [source] Construction of Recombinant Escherichia coli Catalysts which Simultaneously Express an (S)-Oxynitrilase and Different Nitrilase Variants for the Synthesis of (S)-Mandelic Acid and (S)-Mandelic Amide from Benzaldehyde and CyanideADVANCED SYNTHESIS & CATALYSIS (PREVIOUSLY: JOURNAL FUER PRAKTISCHE CHEMIE), Issue 10 2009Olga Sosedov Abstract Recombinant Escherichia coli strains were constructed which simultaneously expressed the genes encoding the (S)-oxynitrilase from cassava (Manihot esculenta) together with the wild-type or a mutant variant of the arylacetonitrilase from Pseudomonas fluorescens EBC191 in a single organism under the control of a rhamnose-inducible promoter. The whole cell catalysts obtained converted benzaldehyde and potassium cyanide in aqueous media at pH,5.2 mainly to (S)-mandelic acid and/or (S)-mandelic amide and synthesized only low amounts of the corresponding (R)-enantiomers. The conversion of benzaldehyde and potassium cyanide (KCN) by a whole-cell catalyst simultaneously expressing the (S)-oxynitrilase and the wild-type nitrilase resulted in a ratio of (S)-mandelic acid to (S)-mandelic amide of about 4:3. This could be explained by the strong nitrile hydratase activity of the wild-type nitrilase with (S)-mandelonitrile as substrate. The relative proportion of (S)-mandelic amide formed in this system was significantly increased by coexpressing the (S)-oxynitrilase with a carboxy-terminally truncated variant of the nitrilase. This whole-cell catalyst converted benzaldehyde and KCN to mandelic amide and mandelic acid in a ratio of about 9:1. The ee of the (S)-mandelic amide formed was calculated to be >95%. [source] Shotgun proteomic analysis of the microsomal fraction of eukaryotic cells using a two-dimensional reversed-phase×ion-pair reversed-phase HPLC setupJOURNAL OF SEPARATION SCIENCE, JSS, Issue 8 2009Martin Wörner Abstract A RP×IP-RP HPLC separation scheme was combined with on-line ESI-IT tandem MS or off-line MALDI tandem TOF MS and applied to the analysis of eukaryotic subcellular proteomes. Previous proteomic studies [1] were complemented by the approval of the approach to eukaryotic proteomes using the fission yeast Schizosaccharomyces pombe. The major focus was set to the analysis of primary human hepatocyte microsomes, representing a compartment of high interest due to its involvement in xenobiotic detoxification and cholesterol homeostasis. Of the 588 proteins identified from two donors, 24% are involved in cholesterol homeostasis or xenobiotic/lipid metabolism. Up to 50% of the identified proteins belong to the group of membrane proteins, difficult to investigate using gel-based proteomic approaches. We further demonstrated the reproducibility and comparability of the approach and reduced the amount of sample load by almost 70% with only minor loss of information about the proteins identified in the samples. The presented study clearly demonstrates the good applicability of the experimental setup to the analysis of subcellular proteomes including large membrane fractions, where only low amounts of sample material are available. [source] Coronin is involved in uptake of Mycobacterium bovis BCG in human macrophages but not in phagosome maintenanceCELLULAR MICROBIOLOGY, Issue 12 2001Stephanie Schüller By applying density gradient electrophoresis (DGE) to human macrophages infected with Mycobacterium bovis BCG, we were able to separate three different bacterial fractions representing arrested phagosomes, phagolysosomes and mycobacterial clumps. After further purification of the phagosomal population, we found that isolated phagosomes containing live BCG were arrested in maturation as they exhibited only low amounts of the lysosomal glycoprotein LAMP-1 and processing of the lysosomal hydrolase cathepsin D was blocked. In addition, low amounts of MHC class I and class II molecules and the absence of HLA-DM suggest sequestration of mycobacterial phagosomes from antigen-processing pathways. We further investigated the involvement of the actin-binding protein coronin in intracellular survival of mycobacteria and showed that human coronin, as well as F-actin, were associated with early stages of mycobacterial phagocytosis but not with phagosome maintenance. Therefore, we conclude that the unique DGE migration pattern of arrested phagosomes is not as a result of retention of coronin, but that there are other proteins or lipids responsible for the block in maturation in human macrophages. [source] | ||||||||||