Doxorubicin increases intracellular hydrogen peroxide in PC3 prostate cancer cells

We studied the effect of doxorubicin on the production of hydrogen peroxide by PC3 human prostate cancer cells, using a sensitive assay based on aminotriazole-mediated inhibition of catalase. PC3 cells exposed to increasing concentrations of doxorubicin had an increase in intracellular hydrogen peroxide that was concentration-dependent up to 1 microM doxorubicin. The apparent hydrogen peroxide concentration in the PC3 cells was 13 +/- 4 pM under basal steady-state conditions and increased to 51 +/- 13 pM after exposure to 1 microM doxorubicin for 30 min. The level of hydrogen peroxide in the medium as measured by Amplex Red did not increase as a result of doxorubicin treatment. PC3 cells overexpressing catalase were no more resistant to doxorubicin cytotoxicity as compared to non-transduced wild-type cells; therefore, the exact role of hydrogen peroxide in anthracycline cytotoxicity remains unproven. This study demonstrates that a specific oxidative event associated with the exposure of PC3 human prostate cancer cells to anthracyclines results in an increase in intracellular hydrogen peroxide.     

Studies of the retrogradation process for various starch gels using Raman spectroscopy

The retrogradation of untreated wild-type starches (potato, maize, and wheat), waxy maize starches, and one pregelatinized, modified amylose-rich starch was investigated continuously using Raman spectroscopy. The method detects conformational changes due to the multi-stage retrogradation, the rate of which differs between the starches. The pregelatinized, modified amylose-rich starch shows all stages of retrogradation in the course of its Raman spectra. In comparison to amylose, the retrogradation of amylopectin is faster at the beginning of the measurements and slower in the later stages. The untreated starches can be ranked in the order of their rate of retrogradation as follows: potato>maize>wheat.     

Mass spectrometric analysis of single identified neurons of an insect

The combination of retrograde labelling with dextran-tetramethylrhodamine and MALDI-TOF mass spectrometry was used to analyse for the first time the peptidome of a series of morphologically identified single neurosecretory cells of an insect. Eight postero-lateral cells of the metathoracic ganglion of the American cockroach, Periplaneta americana, were used to demonstrate that: (1) the complete dissection procedure can be documented and (2) the mass spectrometric analysis of the dissected somata results in highly reproducible mass spectra. In total, 21 FMRFamide-related peptides were detected in each of the postero-lateral cells which release their neurosecretions via thoracic perisympathetic organs. Direct analysis of these neurohemal organs confirmed the co-storage of FMRFamide-related peptides. Two additional abundant peptides from thoracic perisympathetic organs which were not detectable in the postero-lateral cells were characterized using ESI-Q-TOF MS/MS. De novo sequencing yielded two related peptides (FERL/IEamides) without any similarity with known peptide families of insects.     

Lateral spike conduction velocity in the visual cortex affects spatial range of synchronization and receptive field size without visual experience: a learning model with spiking neurons

Classical receptive fields (cRF) increase in size from the retina to higher visual centers. The present work shows how temporal properties, in particular lateral spike velocity and spike input correlation, can affect cRF size and position without visual experience. We demonstrate how these properties are related to the spatial range of cortical synchronization if Hebbian learning dominates early development. For this, a largely reduced model of two successive levels of the visual cortex is developed (e.g., areas V1 and V2). It consists of retinotopic networks of spiking neurons with constant spike velocity in lateral connections. Feedforward connections between level 1 and 2 are additive and determine cRF size and shape, while lateral connections within level 1 are modulatory and affect the cortical range of synchronization. Input during development is mimicked by spike trains with spatially homogeneous properties and a confined temporal correlation width. During learning, the homogeneous lateral coupling shrinks to limited coupling structures defining synchronization and related association fields (AF). The size of level-1 synchronization fields determines the lateral coupling range of developing level-1-to-2 connections and, thus, the size of level-2 cRFs, even if the feedforward connections have distance-independent delays. AFs and cRFs increase with spike velocity in the lateral network and temporal correlation width of the input. Our results suggest that AF size of V1 and cRF size of V2 neurons are confined during learning by the temporal width of input correlations and the spike velocity in lateral connections without the need of visual experience. During learning from visual experience, a similar influence of AF size on the cRF size may be operative at successive levels of processing, including other parts of the visual system.     

Myeloperoxidase is involved in H2O2-induced apoptosis of HL-60 human leukemia cells

We examined the mechanism of H(2)O(2)-induced cytotoxicity and its relationship to oxidation in human leukemia cells. The HL-60 promyelocytic leukemia cell line was sensitive to H(2)O(2), and at concentrations up to about 20-25 micrometer, the killing was mediated by apoptosis. There was limited evidence of lipid peroxidation, suggesting that the effects of H(2)O(2) do not involve hydroxyl radical. When HL-60 cells were exposed to H(2)O(2) in the presence of the spin trap alpha-(4-pyridyl-1-oxide)-N-tert-butylnitrone (POBN), we detected a 12-line electron paramagnetic resonance spectrum assigned to the POBN/POBN(.) N-centered spin adduct previously described in peroxidase-containing cell-free systems. Generation of this radical by HL-60 cells had the same H(2)O(2) concentration dependence as initiation of apoptosis. In contrast, studies with the K562 human erythroleukemia cell line, which is often used for comparison with the HL-60, and with high passaged HL-60 cells (spent HL-60) studied under the same conditions failed to generate POBN(.). Cellular levels of antioxidant enzymes superoxide dismutase, glutathione peroxidase, and catalase did not explain the differences between these cell lines. Interestingly, the K562 and spent HL-60 cells, which did not generate the radical, also failed to undergo H(2)O(2)-induced apoptosis. Based on this we reasoned that the difference in H(2)O(2)-induced apoptosis might be due to the enzyme myeloperoxidase. Only the apoptosis-manifesting HL-60 cells contained appreciable immunoreactive protein or enzymatic activity of this cellular enzyme. When HL-60 cells were incubated with methimazole or 4-aminobenzoic acid hydrazide, which are inhibitors of myeloperoxidase, they no longer underwent H(2)O(2)-induced apoptosis. Hypochlorous acid stimulated apoptosis in both HL-60 and spent HL-60 cells, indicating that another oxidant generated by myeloperoxidase induces apoptosis and that it may be the direct mediator of H(2)O(2)-induced apoptosis. Taken together these observations indicate that H(2)O(2)-induced apoptosis in the HL-60 human leukemia cell is mediated by myeloperoxidase and is linked to a non-Fenton oxidative event marked by POBN(.).     

Evidence that proximal multiple mutations in Big Blue transgenic mice are dependent events

To characterize the nature of multiple mutations in the tissues of an intact animal, the Big Blue transgenic mouse mutation detection system was used to examine 1459 mutants from eight normal tissues and 507 mutants from 11 tumors. Multiple mutations occurred and predominantly doublet mutants were identified (i.e. two mutations within one mutant lacI gene), but multiplets of up to five mutations were observed. The frequency of doublets in normal tissues and spontaneous tumors from p53-deficient mice was enhanced to the same degree (660 and 667 fold, respectively) over that expected for two independent mutational events. Doublets, multiplets and singlets have similar patterns of mutation. The distance between mutations in doublets fits an exponential distribution, not that expected for randomly spaced events, suggesting that many doublets occur in rapid succession within the same cell cycle.     

Specificity of the binding site of the sialic acid-binding lectin from ovine placenta, deduced from interactions with synthetic analogues

The specificity of the sialic acid-binding lectin from ovine placenta was examined in detail by haemagglutination inhibition assays applying a panel of 32 synthetic sialic acid analogues. The carboxylic acid group is a prerequisite for the interaction with the lectin, the -anomer of the methyl glycoside is only a little more effective as an inhibitor than the -anomer and the most potent inhibitor was 9-deoxy-10-carboxylic acid Neu5Ac, followed by 4-oxo-Neu5Ac. In contrast to the majority of known sialic acid-binding lectins, the N-acetyl group of Neu5Ac is not indispensable for binding, neither is the hydroxyl group at C-9 since substitutions at this carbon atom are well tolerated. Furthermore, all sulfur-containing substituents at C-9 enhanced the affinity of the lectin. This is the first sialic acid-binding lectin found to strongly bind thio derivatives.