Copper related toxic effects on cellular protein metabolism in human astrocytes

INTRODUCTION: Copper overload due to a defect in the ATPase 7B mediated copper excretion within hepatocytes produces the phenotype of Wilson disease. The overload of hepatocytes with copper results in necrotic liver cells and is accompanied by a high concentration of blood copper levels. That occurs to be the reason for increasing neurological copper concentration. Although copper is linked to oxidation, there are no data on the direct copper related effects in human brain cells. AIM: To test the copper induced changes in protein oxidation in human astrocyte like cells. METHODS: We used U87 cells as model for human astrocytes. Cells were treated with increasing concentrations of copper(II)-chloride in Dulbeccos minimal essential medium. Subsequently, at different time points we investigated: cellular growth, cellular survival under copper treatment, the concentration of oxidized tryptophane in GADPH in vitro as well as the carbonyl concentration and the concentration of oxidized proteins in vivo in U87 glial cells. RESULTS: The viability of cells decreased with both increasing copper concentration and duration of treatment. The concentration of oxidized proteins was directly correlated to the increase of copper concentration and duration of exposure. CONCLUSION: These observations demonstrate the similarities between copper treatment and treatment with other commonly used oxidants, including hydrogen peroxide. Furthermore, the vulnerability of astrocytes towards copper exposure could be demonstrated. Therefore, these data give further insights into understanding of copper metabolism, which in turn is important to reveal the exact pathological mechanism in copper related diseases such as Wilson disease.     

Anti-fibrosclerotic effects of shock wave therapy in lipedema and cellulite

In vivo measurements in 26 female patients with lipedema and cellulite parameters were carried out before and after therapy by means of complex physical decongestive therapy (CPDT) including manual lymph drainage and compression as main components and/or shock wave therapy (SWT). Oxidative stress parameters of blood serum and biomechanic skin properties/smoothening of dermis and hypodermis surface were evaluated. Oxidative stress in lipedema and cellulite was demonstrated by increased serum concentrations of malondialdehyde (MDA) and plasma protein carbonyls compared with healthy control persons. Both MDA and protein carbonyls in blood plasma decreased after serial shock wave application and CPDT. The SWT itself and CPDT itself lead to MDA release from edematous tissue into the plasma. Obviously both therapy types, SWT and CPDT, mitigate oxidative stress in lipedema and cellulite. In parallel SWT improved significantly the biomechanic skin properties leading to smoothening of dermis and hypodermis surface. Significant correlation between MDA depletion of edematous and lipid enriched dermis and improvement of mechanic skin properties was demonstrated. From these findings it is concluded, that a release of lipid peroxidation (LPO) products from edematous dermis is an important sclerosis-preventing effect of SWT and/or CPDT in lipedema and cellulite. Expression of factors stimulating angiogenesis and lymphangiogenesis such as VEGF was not induced by SWT and/or CPDT and, therefore, not involved in beneficial effects by SWT and/or CPDT.     

Role of 4-hydroxynonenal in the hemozoin-mediated inhibition of differentiation of human monocytes to dendritic cells induced by GM-CSF/IL-4

In falciparum malaria, rupture of parasitized RBC liberates hemozoin (HZ), polymerized heme that contains and generates lipoperoxidation products. In HZ and HZ-loaded monocytes 4-HNE attained approx. 50 and 15 microM, respectively. In malaria, HZ-loaded monocytes are precursors of dendritic cells (DC). Here, the role of 4-HNE as inhibitor of DC differentiation was examined. 4-HNE in HZ was quantified after derivatization by HPLC. DC were differentiated in vitro from human monocytes supplemented with GM-CSF/IL-4 and analyzed for surface antigens and 4-HNE-adducts by FACScan after labelling with specific antibodies. HZ-loading, or treatment with 4-HNE induced large numbers of 4-HNE-protein-adducts on the monocyte membrane. As low as 10 nM 4-HNE inhibited up-regulation of functionally important DC differentiation markers. 1 microM 4-HNE elicited inhibition of up-regulation of DC differentiation markers as follows: MHC-class I and II, -29% and -40%; CD1a, -16%; CD40, -25%; CD54, -27%; and CD83 (the most important DC differentiation marker), -45%, with no signs of apoptosis. The sequence of additions was important, as the inhibitory effect was reduced when 4-HNE was added after GM-CSF/IL-4, indicating that GM-CSF/IL-4 receptors could be modified by 4-HNE. In conclusion, inhibition of DC differentiation by 4-HNE may play a role in malaria immunodepression.     

Gradient of rigidity in the lamellipodia of migrating cells revealed by atomic force microscopy

Changes in mechanical properties of the cytoplasm have been implicated in cell motility, but there is little information about these properties in specific regions of the cell at specific stages of the cell migration process. Fish epidermal keratocytes with their stable shape and steady motion represent an ideal system to elucidate temporal and spatial dynamics of the mechanical state of the cytoplasm. As the shape of the cell does not change during motion and actin network in the lamellipodia is nearly stationary with respect to the substrate, the spatial changes in the direction from the front to the rear of the cell reflect temporal changes in the actin network after its assembly at the leading edge. We have utilized atomic force microscopy to determine the rigidity of fish keratocyte lamellipodia as a function of time/distance from the leading edge. Although vertical thickness remained nearly constant throughout the lamellipodia, the rigidity exhibited a gradual but significant decrease from the front to the rear of the lamellipodia. The rigidity profile resembled closely the actin density profile, suggesting that the dynamics of rigidity are due to actin depolymerization. The decrease of rigidity may play a role in facilitating the contraction of the actin-myosin network at the lamellipodium/cell body transition zone.     

Light-induced conformational changes of cyanobacterial phytochrome Cph1 probed by limited proteolysis and autophosphorylation

Photoreceptor chromoproteins undergo light-induced conformational changes that result in a modulation of protein interaction and enzymatic activity. Bacterial phytochromes such as Cph1 from the cyanobacterium Synechocystis PCC 6803 are light-regulated histidine kinases in which the light signal is transferred from the N-terminal chromophore module to the C-terminal kinase module. In this study, purified recombinant Cph1 was subjected to limited proteolysis using trypsin and endoproteinase Glu-C (V8). Cleavage sites of chromopeptide fragments were determined by MALDI-TOF and micro-HPLC on-line with tandem mass spectrometry in an ion trap mass spectrometer. Trypsin produced three major chromopeptides, termed F1 (S56 to R520), F2 (T64 to R472), and F3 (L81 to R472). F1 was produced only in the far-red absorbing form Pfr within 15 min and remained stable up to >1 h; F2 and F3 were obtained in the red-light absorbing form Pr within ca. 5-10 min. When F1 was photoconverted to Pr in the presence of trypsin, this fragment degraded to F2 and F3 within 1-2 min. On size exclusion chromatography, F1 eluted as a dimer in the Pfr and as a monomer in the Pr form, whereas F2 and F3 behaved always as monomers, irrespective of the light conditions. These and other results are discussed in the context of light-dependent subunit interactions, in which amino acids 473-520 within the PHY domain are required for chromophore-module subunit interaction within the homodimer. V8 proteolysis yielded five major chromopeptides, F4 (T17 to N449), F5 (T17 to E335), F6 (T17 to E323), F7 (unknown sequence), and F8 (tentatively L121 to E323). F6 and F8 were formed in the Pr form, whereas F4, F5, and F7 were preferentially formed in the Pfr form. Three amino acids next to specific cleavage sites, R520, R472, and E323, were altered by site-directed mutagenesis. The mutants were analyzed by UV-vis spectroscopy, size exclusion chromatography, and autophosphorylation. Histidine kinase activity was low in R472A, R520P, and R520A; in all mutants, the ratio of phosphorylation intensity between Pr and Pfr was reduced. Thus, light regulation of autophosphorylation is negatively affected in all mutants. In R472P, E323P, and E323D, the phosphorylation intensity of the Pfr form exceeded that of the wild-type control. This result shows that the histidine kinase activity of Cph1 is actively inhibited by photoconversion into Pfr.     

GTP analogue inhibits polymerization and GTPase activity of the bacterial protein FtsZ without affecting its eukaryotic homologue tubulin

The prokaryotic tubulin homologue FtsZ plays a key role in bacterial cell division. Selective inhibitors of the GTP-dependent polymerization of FtsZ are expected to result in a new class of antibacterial agents. One of the challenges is to identify compounds which do not affect the function of tubulin and various other GTPases in eukaryotic cells. We have designed a novel inhibitor of FtsZ polymerization based on the structure of the natural substrate GTP. The inhibitory activity of 8-bromoguanosine 5'-triphosphate (BrGTP) was characterized by a coupled assay, which allows simultaneous detection of the extent of polymerization (via light scattering) and GTPase activity (via release of inorganic phosphate). We found that BrGTP acts as a competitive inhibitor of both FtsZ polymerization and GTPase activity with a Ki for GTPase activity of 31.8 +/- 4.1 microM. The observation that BrGTP seems not to inhibit tubulin assembly suggests a structural difference of the GTP-binding pockets of FtsZ and tubulin.     

SORCS1 and SORCS3 control energy balance and orexigenic peptide production

SORCS1 and SORCS3 are two related sorting receptors expressed in neurons of the arcuate nucleus of the hypothalamus. Using mouse models with individual or dual receptor deficiencies, we document a previously unknown function of these receptors in central control of metabolism. Specifically, SORCS1 and SORCS3 act as intracellular trafficking receptors for tropomyosin‐related kinase B to attenuate signaling by brain‐derived neurotrophic factor, a potent regulator of energy homeostasis. Loss of the joint action of SORCS1 and SORCS3 in mutant mice results in excessive production of the orexigenic neuropeptide agouti‐related peptide and in a state of chronic energy excess characterized by enhanced food intake, decreased locomotor activity, diminished usage of lipids as metabolic fuel, and increased adiposity, albeit at overall reduced body weight. Our findings highlight a novel concept in regulation of the melanocortin system and the role played by trafficking receptors SORCS1 and SORCS3 in this process.