Reduction of a tetrazolium salt, CTC, by intact HepG2 human hepatoma cells: subcellular localisation of reducing systems

AbstractCell-mediated reduction of tetrazolium salts, including MTT, XTT, MTS, NBT, NTV, INT, in the presence or absence of intermediate electron carriers is used as a convenient test for animal or bacterial cell viability. Bioreduction of tetrazolium is considered an alternative to a clonogenic assay and a thymidine incorporation assay. However, correlation between clonogenic potential and capacity to reduce tetrazolium has not been demonstrated convincingly. Moreover, despite a wide use of tetrazolium viability assays, the mechanism and subcellular localisation of reducing systems or species in viable intact cells have not been fully elucidated. We report evidence indicating that a tetrazolium salt CTC can be reduced in the presence as well as in the absence of an electron carrier by viable HepG2 human hepatoma cells. CTC-formazan is formed within or at the outer surface of plasma membranes. We hypothesise that in the presence of an electron carrier the electron donors active in the reduction of CTC are located in the intracellular compartment, as well as in plasma membranes. However, in the absence of an electron carrier, the reduction occurs primarily via a plasma membrane-associated enzymatic system or species

Various Aspects of Auditory Fatigue Caused by Listening to Loud Music

This chapter presents results of research on influence of auditory fatigue on some aspects of listening condition measured among various groups of listeners. Three experiments have been carried out. The aim of the first one was to find the influence of the kind of headphones used by young people on their hearing loss. The second experiment was concerning the temporary threshold shift (TTS) caused by the listening of loud musical signals after several time of sound exposure. The main interest of the third experiment was the detection ability of changes in spectrum of musical samples obtained after several time of listening to the loud music. It turned out that except for frequency of 4 kHz there is no relation between the types of preferred headphones and the shift of hearing threshold while for the frequency of 4 kHz, a statistically important influence of the headphone types on the threshold values was observed. The second and third experiments were carried out under conditions which normally exist in a studio or on the stage when the sound material is recorded and/or mixed. It turned out that after several loud music listening sessions the average value of temporary threshold shift reached more than 3 dB for 1 kHz and increased up to 6–7 dB with an exposure time of 120 min. On the basis of results obtained from the third experiment, it was found that the decrease in ability to detect the spectrum changes for longer noise exposure exists particularly for lower changes (of ±1.5 dB) and at all frequency regions under investigation. It may suggest that the hearing system gets tired for the region of higher frequencies faster than for other bands after listening to loud music. The results may also be influenced by the mental fatigue which occurred after several time duration of permanently played loud sounds, together with demanding tasks. Such conditions involving the mental engagement in a noisy environment, which is referred to the natural scenery of the studio work can significantly reduce the time of exhaustion which causes the decrease of accuracy in solving several tasks. It should be also noted that the tendencies observed within young people culture in listening loud music in order to be isolated from the environment is actually causing not the TTS phenomenon but permanent threshold shift (PTS)

UV-induced spectral shift and protonation of DNA fluorescent dye Hoechst 33258

DNA-bound Hoechst 33258 is readily excited with UV light and emits blue fluorescence, however, upon exposure to UV, the dye undergoes photobleaching as well as photoconversion to a blue-excited green-emitting form. We demonstrate that the UV-generated green-emitting form of Hoechst 33258 exhibits spectral properties very similar to the form of the dye that can be obtained by subjecting it to an acidic environment (pH 0.5–3.0). We also demonstrate that exposure of Hoechst 33258 to UV light (or hydrogen peroxide) leads to generation of the protonated (1+, 2+, 3+ and possibly the 4+) forms of the dye. Photoconversion of Hoechst 33258 has recently been exploited in single molecule localisation microscopy, thus understanding photophysics of this process can facilitate further development of high resolution optical imaging

Elucidating structure-function relationships from molecule-to-cell-to-tissue: from research modalities to clinical realities

The National Academy of Engineering selected ‘Imaging’ as one of the greatest engineering achievements of the 20th century (Greatest Engineering Achievements of the 20th Century. 2009 (cited 2008, November 10); available from: http://www.greatachievements.org/). The combination of different imaging modalities and technologies for mapping bimolecular and/or biological processes within single cells or even whole organs has extraordinary potential for revolutionizing the diagnosis and treatment of pathophysiological disorders, and thus for mitigating the significant social and economic costs associated with the clinical management of disease. Such integrated imaging approaches will eventually lead to individualized programs for disease prevention through advanced diagnosis, risk stratification and targeted cell therapies resulting in more successful and efficient health care. The goal of this article is to provide readers with a current update of selected of state-of-the-art imaging modalities which would likely to lead to improved clinical outcomes if employed in an integrated approach, including use of ultramicrosensors to detect reactive oxygen/nitrogen species in a single cell, use of electron tomography to visualize and characterize cellular organization in three dimensions (3D), and molecular imaging strategies to assess naturally occurring and therapeutic peripheral and myocardial angiogenesis using targeted radiolabeled tracers

Spatial heterogeneity of dynamics of H1 linker histone

Linker histone H1 participates in maintaining higher order chromatin structures. It is a dynamic protein that binds to DNA and exchanges rapidly with a mobile pool. Therefore, the dynamics of H1 were probed in the nuclei of intact, live cells, using an array of microscopy techniques: fluorescence recovery after photobleaching (FRAP), raster image correlation spectroscopy (RICS), fluorescence correlation spectroscopy (FCS), pair correlation functions (pCF) and fluorescence anisotropy. Combination of these techniques yielded information on H1 dynamics at small (1–100 μs: FCS, RICS, anisotropy), moderate (1–100 ms: FCS, RICS, pCF) and large (1–100 s: pCF and FRAP) time scales. These results indicate that the global movement of H1 in nuclei (at distances >1 µm) occurs at the time scale of seconds and is determined by processes other than diffusion. Moreover, a fraction of H1, which remains immobile at the time scale of tenths of seconds, is detectable. However, local (at distances <0.7 µm) H1 dynamics comprises a process occurring at a short (~3 ms) time scale and multiple processes occurring at longer (10–2,500 ms) scales. The former (fast) process (corresponding probably to H1 diffusion) is more pronounced in the nuclear regions characterized by low H1 concentration, but the latter (slow, attributable to H1 binding) in the regions of high H1 concentration. Furthermore, some regions in nuclei (possibly containing dense chromatin) may constitute barriers that impair or block movement of H1 histones within short (<1 µm) distances

Low Level Phosphorylation of Histone H2AX on Serine 139 (gammaH2AX) Is Not Associated with DNA Double-Strand Breaks

Phosphorylation of histone H2AX on serine 139 (γH2AX) is an early step in cellular response to a DNA double-strand break (DSB). γH2AX foci are generally regarded as markers of DSBs. A growing body of evidence demonstrates, however, that while induction of DSBs always brings about phosphorylation of histone H2AX, the reverse is not true - the presence of γH2AX foci should not be considered an unequivocal marker of DNA double-strand breaks. We studied DNA damage induced in A549 human lung adenocarcinoma cells by topoisomerase type I and II inhibitors (0.2 μM camptothecin, 10 μM etoposide or 0.2 μM mitoxantrone for 1 h), and using 3D high resolution quantitative confocal microscopy, assessed the number, size and the integrated intensity of immunofluorescence signals of individual γH2AX foci induced by these drugs. Also, investigated was spatial association between γH2AX foci and foci of 53BP1, the protein involved in DSB repair, both in relation to DNA replication sites (factories) as revealed by labeling nascent DNA with EdU. Extensive 3D and correlation data analysis demonstrated that γH2AX foci exhibit a wide range of sizes and levels of H2AX phosphorylation, and correlate differently with 53BP1 and DNA replication. This is the first report showing lack of a link between low level phosphorylation γH2AX sites and double-strand DNA breaks in cells exposed to topoisomerase I or II inhibitors. The data are discussed in terms of mechanisms that may be involved in formation of γH2AX sites of different sizes and intensities