Diazepam inhibits cell respiration and induces fragmentation of mitochondrial reticulum

AbstractDiazepam (70–150 μg/ml) significantly inhibits oxygen consumption by pig kidney embryo cells and causes the cellular ATP level to fall. The maximum inhibitory effect develops after 1.5–2.5 h of diazepam treatment. In isolated mitochondria diazepam inhibits respiration in state 2 and 3u with glutamate and in state 3u with succinate. Ethylrhodamine staining and electron microscopic study reveal fragmentation of mitochondria in living cells

Automated image segmentation for detecting cell spreading for metastasizing assessments of cancer development

The automated segmentation of cells in microscopic images is an open research problem that has important implications for studies of the developmental and cancer processes based on in vitro models. In this paper, we present the approach for segmentation of the DIC images of cultured cells using G-neighbor smoothing followed by Kauwahara filtering and local standard deviation approach for boundary detection. NIH FIJI/ImageJ tools are used to create the ground truth dataset. The results of this work indicate that detection of cell boundaries using segmentation approach even in the case of realistic measurement conditions is a challenging problem

Microvesicles and intercellular communication in the context of parasitism

There is a rapidly growing body of evidence that production of microvesicles (MVs) is a universal feature of cellular life. MVs can incorporate microRNA (miRNA), mRNA, mtDNA, DNA and retrotransposons, camouflage viruses/viral components from immune surveillance, and transfer cargo between cells. These properties make MVs an essential player in intercellular communication. Increasing evidence supports the notion that MVs can also act as long-distance vehicles for RNA molecules and participate in metabolic synchronization and reprogramming eukaryotic cells including stem and germinal cells. MV ability to carry on DNA and their general distribution makes them attractive candidates for horizontal gene transfer, particularly between multi-cellular organisms and their parasites; this suggests important implications for the co-evolution of parasites and their hosts. In this review, we provide current understanding of the roles played by MVs in intracellular pathogens and parasitic infections. We also discuss the possible role of MVs in co-infection and host shifting

Non-linear Dose Response of Lymphocyte Cell Lines to Microtubule Inhibitors

Microtubule (MT) inhibitors show anti-cancer activity in a wide range of tumors in vitro and demonstrate high clinical efficacy. To date they are routinely included into many chemotherapeutic regimens. While the mechanisms of MT inhibitors’ interactions with tubulin have been well-established, the relationship between their concentration and effect on neoplastic cells is not completely understood. The common notion is that tumor cells are most vulnerable during division and all MT inhibitors block them in mitosis and induce mitotic checkpoint-associated cell death. At the same time multiple evidence of more subtle effects of lower doses of MT inhibitors on cell physiology exist. The extent of efficacy of the low-dose MT inhibitor treatment and the mechanisms of resulting cell death currently present a critical issue in oncology. The prospect of MT inhibitor dose reduction is promising as protocols at higher concentration have multiple side effects. We assessed cell cycle changes and cell death induced by MT inhibitors (paclitaxel, nocodazole, and vinorelbine) on human lymphoid B-cell lines in a broad concentration range. All inhibitors had similar accumulation effects and demonstrated “trigger” concentrations that induce cell accumulation in G2/M phase. Concentrations slightly below the “trigger” promoted cell accumulation in sub-G1 phase. Multi-label analysis of live cells showed that the sub-G1 population is heterogeneous and may include cells that are still viable after 24 h of treatment. Effects observed were similar for cells expressing Tat-protein. Thus cell cycle progression and cell death are differentially affected by high and low MT inhibitor concentrations

Non-centrosomal MTs play a crucial role in organization of MT array in interphase fibroblasts

Microtubules in interphase fibroblast-like cells are thought to be organized in a radial array growing from a centrosome-based microtubule-organizing center (MTOC) to the cell edges. However, many morphogenetic processes require the asymmetry of the microtubules (MT) array. One of the possible mechanisms of this asymmetry could be the presence of non-centrosomal microtubules in different intracellular areas. To evaluate the role of centrosome-born and non-centrosomal microtubules in the organization of microtubule array in motile 3T3 fibroblasts, we have performed the high-throughput analysis of microtubule growth in different functional zones of the cell and distinguished three subpopulations of growing microtubules (centrosome-born, marginal and inner cytoplasmic). Centrosome as an active microtubule-organizing center was absent in half of the cell population. However, these cells do not show any difference in microtubule growth pattern. In cells with active centrosome, it was constantly forming short (ephemeral) MTs, and ~15–20 MT per minute grow outwards for a distance >1 µm. Almost no persistent growth of microtubules was observed in these cells with the average growth length of 5–6 µm and duration of growth periods within 30 s. However, the number of growing ends increased towards cell margin, especially towards the active edges. We found the peripheral cytoplasmic foci of microtubule growth there. During recovery from nocodazole treatment microtubules started to grow around the centrosome in a normal way and independently in all the cell areas. Within 5 minutes microtubules continued to grow mainly near the cell edge. Thus, our data confirm the negligible role of centrosome as MTOC in 3T3 fibroblasts and propose a model of non-centrosomal microtubules as major players that create the cell asymmetry in the cells with a mesenchymal type of motility. We suggest that increased density of dynamic microtubules near the active lamellum could be supported by microtubule-based microtubule nucleation

Deterministic mechanical model of T-killer cell polarization reproduces the wandering of aim between simultaneously engaged targets

T-killer cells of the immune system eliminate virus-infected and tumorous cells through direct cell-cell interactions. Reorientation of the killing apparatus inside the T cell to the T-cell interface with the target cell ensures specificity of the immune response. The killing apparatus can also oscillate next to the cell-cell interface. When two target cells are engaged by the T cell simultaneously, the killing apparatus can oscillate between the two interface areas. This oscillation is one of the most striking examples of cell movements that give the microscopist an unmechanistic impression of the cell's fidgety indecision. We have constructed a three-dimensional, numerical biomechanical model of the molecular-motor-driven microtubule cytoskeleton that positions the killing apparatus. The model demonstrates that the cortical pulling mechanism is indeed capable of orienting the killing apparatus into the functional position under a range of conditions. The model also predicts experimentally testable limitations of this commonly hypothesized mechanism of T-cell polarization. After the reorientation, the numerical solution exhibits complex, multidirectional, multiperiodic, and sustained oscillations in the absence of any external guidance or stochasticity. These computational results demonstrate that the strikingly animate wandering of aim in T-killer cells has a purely mechanical and deterministic explanation. © 2009 Kim, Maly

TO DIE OR NOT TO DIE—REGULATED CELL DEATH AND SURVIVAL IN CYANOBACTERIA

Regulated cell death (RCD) is central to the development, integrity, and functionality of multicellular organisms. In the last decade, evidence has accumulated that RCD is a universal phenomenon in all life domains. Cyanobacteria are of specific interest due to their importance in aquatic and terrestrial habitats and their role as primary producers in global nutrient cycling. Current knowledge on cyanobacterial RCD is based mainly on biochemical and morphological observations, often by methods directly transferred from vertebrate research and with limited understanding of the molecular genetic basis. However, the metabolism of different cyanobacteria groups relies on photosynthesis and nitrogen fixation, whereas mitochondria are the central executioner of cell death in vertebrates. Moreover, cyanobacteria chosen as biological models in RCD studies are mainly colonial or filamentous multicellular organisms. On the other hand, unicellular cyanobacteria have regulated programs of cellular survival (RCS) such as chlorosis and post-chlorosis resuscitation. The co-existence of different genetically regulated programs in cyanobacterial populations may have been a top engine in life diversification. Development of cyanobacteria-specific methods for identification and characterization of RCD and wider use of single-cell analysis combined with intelligent image-based cell sorting and metagenomics would shed more light on the underlying molecular mechanisms and help us to address the complex colonial interactions during these events. In this review, we focus on the functional implications of RCD in cyanobacterial communities

Non-linear Dose Response of Lymphocyte Cell Lines to Microtubule Inhibitors

Microtubule (MT) inhibitors show anti-cancer activity in a wide range of tumors in vitro and demonstrate high clinical efficacy. To date they are routinely included into many chemotherapeutic regimens. While the mechanisms of MT inhibitors' interactions with tubulin have been well-established, the relationship between their concentration and effect on neoplastic cells is not completely understood. The common notion is that tumor cells are most vulnerable during division and all MT inhibitors block them in mitosis and induce mitotic checkpoint-associated cell death. At the same time multiple evidence of more subtle effects of lower doses of MT inhibitors on cell physiology exist. The extent of efficacy of the low-dose MT inhibitor treatment and the mechanisms of resulting cell death currently present a critical issue in oncology. The prospect of MT inhibitor dose reduction is promising as protocols at higher concentration have multiple side effects. We assessed cell cycle changes and cell death induced by MT inhibitors (paclitaxel, nocodazole, and vinorelbine) on human lymphoid B-cell lines in a broad concentration range. All inhibitors had similar accumulation effects and demonstrated "trigger" concentrations that induce cell accumulation in G2/M phase. Concentrations slightly below the "trigger" promoted cell accumulation in sub-G1 phase. Multi-label analysis of live cells showed that the sub-G1 population is heterogeneous and may include cells that are still viable after 24 h of treatment. Effects observed were similar for cells expressing Tat-protein. Thus cell cycle progression and cell death are differentially affected by high and low MT inhibitor concentrations

Optimization of flow cytometric detection and cell sorting of transgenic Plasmodium parasites using interchangeable optical filters

Background: Malaria remains a major cause of morbidity and mortality worldwide. Flow cytometry-based assays that take advantage of fluorescent protein (FP)-expressing malaria parasites have proven to be valuable tools for quantification and sorting of specific subpopulations of parasite-infected red blood cells. However, identification of rare subpopulations of parasites using green fluorescent protein (GFP) labelling is complicated by autofluorescence (AF) of red blood cells and low signal from transgenic parasites. It has been suggested that cell sorting yield could be improved by using filters that precisely match the emission spectrum of GFP. Methods: Detection of transgenic Plasmodium falciparum parasites expressing either tdTomato or GFP was performed using a flow cytometer with interchangeable optical filters. Parasitaemia was evaluated using different optical filters and, after optimization of optics, the GFP-expressing parasites were sorted and analysed by microscopy after cytospin preparation and by imaging cytometry. Results: A new approach to evaluate filter performance in flow cytometry using two-dimensional dot blot was developed. By selecting optical filters with narrow bandpass (BP) and maximum position of filter emission close to GFP maximum emission in the FL1 channel (510/20, 512/20 and 517/20; dichroics 502LP and 466LP), AF was markedly decreased and signal-background improve dramatically. Sorting of GFP-expressing parasite populations in infected red blood cells at 90 or 95% purity with these filters resulted in 50-150% increased yield when compared to the standard filter set-up. The purity of the sorted population was confirmed using imaging cytometry and microscopy of cytospin preparations of sorted red blood cells infected with transgenic malaria parasites. Discussion Filter optimization is particularly important for applications where the FP signal and percentage of positive events are relatively low, such as analysis of parasite-infected samples with in the intention of gene-expression profiling and analysis. The approach outlined here results in substantially improved yield of GFP-expressing parasites, and requires decreased sorting time in comparison to standard methods. It is anticipated that this protocol will be useful for a wide range of applications involving rare events

Specific and reliable detection of Myosin 1C isoform A by RTqPCR in prostate cancer cells

Background Prostate cancer (PC) diagnostics and treatment often present a challenging task due to cancer subtype heterogeneity and differential disease progression in patient subgroups. Hence, the critical issue is finding a reliable and sensitive diagnostic and prognostic PC marker, especially for cases of biopsies with low percentages of cancer cells. Isoform A of myosin 1C was shown to be expressed in PC cells and responsible for their invasive properties, however, its feasibility for diagnostic purposes remains to be elucidated. Methods To verify the role of myosin 1C isoform A mRNA expression as a putative prostate cancer marker we performed RT qPCR normalized by three reference genes (GAPDH, YWHAZ, HPRT1) on PC3, RWPE-1, LNCaP and 22Rv1 cell lines. Myosin 1C isoform A detection specificity was confirmed by immunofluorescence staining, cancer and non-cancer prostate cell lines were immunophenotyped by flow cytometry. Results Median normalized mRNA expression level of myosin 1C isoform A in PC cells (PC3 and 22Rv1) is two orders of magnitude higher compared to RWPE-1 cells, which functionally correspond to benign prostate cells. Myosin 1C isoform A expression allows PC cell detection even at a dilution ratio of 1:1000 cancer to non-cancer cells. At the protein level, the mean fluorescence intensity of myosin 1C isoform A staining in PC3 nuclei was only twice as high as in RWPE-1, while the immunophenotypes of both cell lines were similar (CD44+/CD90-/CD133-/CD57-/CD24+-). Conclusions We report a distinct difference in myosin 1C isoform A mRNA levels in malignant (PC3) and benign (RWPE-1) prostate cell lines and suggest a combination of three reference genes for accurate data normalization. For the first time we provide an immunophenotype comparison of RWPE-1 and PC3 cells and demonstrate that RT qPCR analysis of MYO 1C A using appropriate reference genes is sufficient for PC detection even in low-abundance cancer specimens