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Intracellular Structures (intracellular + structure)
Selected AbstractsEvidence for a widespread involvement of NO in control of photogenesis in bioluminescent fishACTA ZOOLOGICA, Issue 4 2010Jenny Krönström Abstract Krönström, J. and Mallefet, J. 2009. Evidence for a widespread involvement of NO in control of photogenesis in bioluminescent fish. ,Acta Zoologica (Stockholm) 91: 474,483. The presence of nitric oxide synthase (NOS) and nerve fibres in the photophores of seven bioluminescent fish species (Hygophum benoiti, Myctophum punctatum, Electrona risso, Cyclothone braueri, Vinciguerria attenuata, Maurolicus muelleri and Porichthys notatus) with endogenous photocytes, were investigated. Antibodies directed against neuronal and inducible NOS (n and iNOS respectively) and NADPH-diaphorase activity were used to reveal the locations of NOS, while antibodies directed against acetylated tubulin were used to visualize nerve fibres. The nNOS antibody labelled structures in all investigated photophores except in the organs from P. notatus. The photocytes of P. notatus showed NADPH-diaphorase activity. In the myctophid species, NOS-like immunoreactivity was found in small intracellular structures of the photocytes and in nerve fibres reaching the photocytes. nNOS-positive fibres were also found among lens/filter cells in V. attenuata, and in M. muelleri the cytoplasm of lens/filter cells contained NOS-like material. In C. braueri, a cell type located at a collecting chamber for luminous products in the photophore contained NOS-like material. All photophores received an innervation reaching the photocytes, as well as other components including lens/filter areas. The results of this study comply with an involvement of nitric oxide in the control of bioluminescence in several fish species. [source] Large-scale screening of intracellular protein localization in living fission yeast cells by the use of a GFP-fusion genomic DNA libraryGENES TO CELLS, Issue 3 2000Da-Qiao Ding Background Intracellular localization is an important part of the characterization of a gene product. In an attempt to search for genes based on the intracellular localization of their products, we constructed a green fluorescent protein (GFP)-fusion genomic DNA library of S. pombe. Results We constructed the S. pombe GFP-fusion genomic DNA library by fusing, in all three reading frames, random fragments of genomic DNA to the 5, end of the GFP gene in such a way that expression of potential GFP-fusion proteins would be under the control of the own promoters contained in the genomic DNA fragments. Fission yeast cells were transformed with this plasmid library, and microscopic screening of 49 845 transformants yielded 6954 transformants which exhibited GFP fluorescence, of which 728 transformants showed fluorescence localized to distinct intracellular structures such as the nucleus, the nuclear membrane, and cytoskeletal structures. Plasmids were isolated from 516 of these transformants, and a determination of their DNA sequences identified 250 independent genes. The intracellular localizations of the 250 GFP-fusion constructs was categorized as an image database; using this database, DNA sequences can be searched for based on the localizations of their products. Conclusions A number of new intracellular structural components were found in this library. The library of GFP-fusion constructs also provides useful fluorescent markers for various intracellular structures and cellular activities, which can be readily used for microscopic observation in living cells. [source] Properties of cell penetrating peptides (CPPs)IUBMB LIFE, Issue 1 2006Alexandre Kerkis Abstract Different approaches have been developed for the introduction of macromolecules, proteins and DNA into target cells. Viral (retroviruses, lentiviruses, etc.) and nonviral (liposomes, bioballistics etc.) vectors as well as lipid particles have been tested as DNA delivery systems. However, all of them share several undesirable effects that are difficult to overcome, such as unwanted immunoresponse and limited cell targeting. The discovery of the cell penetrating peptides (CPPs) showing properties of macromolecules carriers and enhancers of viral vectors, opened new opportunities for the delivery of biologically active cargos, including therapeutically relevant genes into various cells and tissues. This review summarizes recent data about the best characterized CPPs as well as those sharing cell-penetrating and cargo delivery properties despite differing in the primary sequence. The putative mechanisms of CPPs penetration into cells and interaction with intracellular structures such as chromosomes, cytoskeleton and centrioles are addressed. We further discuss recent developments in overcoming the lack of cells specificity, one of the main obstacles for CPPs application in gene therapy. In particular, we review a newly discovered affinity of CPPs to actively proliferating cells. IUBMB Life, 58: 7 - 13, 2006 [source] Complex patterns of mitochondrial dynamics in human pancreatic cells revealed by fluorescent confocal imagingJOURNAL OF CELLULAR AND MOLECULAR MEDICINE, Issue 1-2 2010Andrey V. Kuznetsov Abstract Mitochondrial morphology and intracellular organization are tightly controlled by the processes of mitochondrial fission,fusion. Moreover, mitochondrial movement and redistribution provide a local ATP supply at cellular sites of particular demands. Here we analysed mitochondrial dynamics in isolated primary human pancreatic cells. Using real time confocal microscopy and mitochondria-specific fluorescent probes tetramethylrhodamine methyl ester and MitoTracker Green we documented complex and novel patterns of spatial and temporal organization of mitochondria, mitochondrial morphology and motility. The most commonly observed types of mitochondrial dynamics were (i) fast fission and fusion; (ii) small oscillating movements of the mitochondrial network; (iii) larger movements, including filament extension, retraction, fast (0.1,0.3 ,m/sec.) and frequent oscillating (back and forth) branching in the mitochondrial network; (iv) as well as combinations of these actions and (v) long-distance intracellular translocation of single spherical mitochondria or separated mitochondrial filaments with velocity up to 0.5 ,m/sec. Moreover, we show here for the first time, a formation of unusual mitochondrial shapes like rings, loops, and astonishingly even knots created from one or more mitochondrial filaments. These data demonstrate the presence of extensive heterogeneity in mitochondrial morphology and dynamics in living cells under primary culture conditions. In summary, this study reports new patterns of morphological changes and dynamic motion of mitochondria in human pancreatic cells, suggesting an important role of integrations of mitochondria with other intracellular structures and systems. [source] Changes in immune and enzyme histochemical phenotypes of cells in the intestinal mucosa of Atlantic salmon, Salmo salar L., with soybean meal-induced enteritisJOURNAL OF FISH DISEASES, Issue 2 2000A M Bakke-McKellep Extracted soybean meal (SBM) in the diet for Atlantic salmon, Salmo salar L., causes an inflammatory response in the distal intestine. The morphological changes of the epithelial cells and a characterization of the inflammatory cell infiltrate of the distal intestinal mucosa were studied using a panel of enzyme and immunohistochemical markers. The salmon (average body weight 927 g) used in the study were fed either a fishmeal-based diet (control diet) or a diet in which 30% of the fishmeal protein was replaced with SBM protein (SBM diet). In salmon fed SBM, there were markedly reduced enzyme reactivities in the distal intestinal epithelial cells, both in the brush border [5,-nucleotidase (5,N), Mg2+-ATPase, alkaline phosphatase (ALP) and leucine aminopeptidase (LAP)] and in the intracellular structures [alkaline and acid phosphatase, non-specific esterase (NSE) and alanine aminopeptidase (AAP)]. There appeared to be an increased presence of cells of monocytic lineage, including macrophages, as well as neutrophilic granulocytes and immunoglobulin (Ig) M in the lamina propria of the SBM-fed fish. The mid intestine showed little response to the diet. The results suggest that toxic/antigenic component(s) of SBM affect the differentiation of the distal intestinal epithelial cells and may help explain the reduced nutrient digestibilities previously reported in salmonids fed extracted SBM. [source] A guided tour into subcellular colocalization analysis in light microscopyJOURNAL OF MICROSCOPY, Issue 3 2006S. BOLTE Summary It is generally accepted that the functional compartmentalization of eukaryotic cells is reflected by the differential occurrence of proteins in their compartments. The location and physiological function of a protein are closely related; local information of a protein is thus crucial to understanding its role in biological processes. The visualization of proteins residing on intracellular structures by fluorescence microscopy has become a routine approach in cell biology and is increasingly used to assess their colocalization with well-characterized markers. However, image-analysis methods for colocalization studies are a field of contention and enigma. We have therefore undertaken to review the most currently used colocalization analysis methods, introducing the basic optical concepts important for image acquisition and subsequent analysis. We provide a summary of practical tips for image acquisition and treatment that should precede proper colocalization analysis. Furthermore, we discuss the application and feasibility of colocalization tools for various biological colocalization situations and discuss their respective strengths and weaknesses. We have created a novel toolbox for subcellular colocalization analysis under ImageJ, named JACoP, that integrates current global statistic methods and a novel object-based approach. [source] Mitochondrial displacements in response to nanomechanical forcesJOURNAL OF MOLECULAR RECOGNITION, Issue 1 2008Yaron R. Silberberg Abstract Mechanical stress affects and regulates many aspects of the cell, including morphology, growth, differentiation, gene expression and apoptosis. In this study we show how mechanical stress perturbs the intracellular structures of the cell and induces mechanical responses. In order to correlate mechanical perturbations to cellular responses, we used a combined fluorescence-atomic force microscope (AFM) to produce well defined nanomechanical perturbations of 10,nN while simultaneously tracking the real-time motion of fluorescently labelled mitochondria in live cells. The spatial displacement of the organelles in response to applied loads demonstrates the highly dynamic mechanical response of mitochondria in fibroblast cells. The average displacement of all mitochondrial structures analysed showed an increase of ,40%, post-perturbation (,160,nm in comparison to basal displacements of ,110,nm). These results show that local forces can produce organelle displacements at locations far from the initial point of contact (up to ,40,µm). In order to examine the role of the cytoskeleton in force transmission and its effect on mitochondrial displacements, both the actin and microtubule cytoskeleton were disrupted using Cytochalasin D and Nocodazole, respectively. Our results show that there is no significant change in mitochondrial displacement following indentation after such treatments. These results demonstrate the role of the cytoskeleton in force transmission through the cell and on mitochondrial displacements. In addition, it is suggested that care must be taken when performing mechanical experiments on living cells with the AFM, as these local mechanical perturbations may have significant structural and even biochemical effects on the cell. Copyright © 2008 John Wiley & Sons, Ltd. [source] X-ray imaging of various biological samples using a phase-contrast hard X-ray microscopeMICROSCOPY RESEARCH AND TECHNIQUE, Issue 9 2008Guk Bae Kim Abstract In this study, we visualized the internal structures of various bio-samples and found the optimum conditions of test samples for the 7 keV hard X-ray microscope of the Pohang light source. From the captured X-ray images, we could observe the intercellular and intracellular structures of dehydrated human cells and mouse tumor tissues without using any staining materials in a spatial resolution better than 100 nm. The metastasized lung tissue, which was several tens of micrometers in thickness, was found to be very well suited to this hard X-ray microscope system, because it is nearly impossible to observe such a nontransparent and thick sample with a high spatial resolution better than 100 nm using any microscopes such as a soft X-ray microscope, an optical microscope, or an electron microscope. Microsc. Res. Tech., 2008. © 2008 Wiley-Liss, Inc. [source] Characterization of tissue structure at varying length scales using temporal diffusion spectroscopy,NMR IN BIOMEDICINE, Issue 7 2010John C. Gore Abstract The concepts, theoretical behavior and experimental applications of temporal diffusion spectroscopy are reviewed and illustrated. Temporal diffusion spectra are obtained using oscillating-gradient waveforms in diffusion-weighted measurements, and represent the manner in which various spectral components of molecular velocity correlations vary in different geometrical structures that restrict or hinder free movements. Measurements made at different gradient frequencies reveal information on the scale of restrictions or hindrances to free diffusion, and the shape of a spectrum reveals the relative contributions of spatial restrictions at different distance scales. Such spectra differ from other so-called diffusion spectra which depict spatial frequencies and are defined at a fixed diffusion time. Experimentally, oscillating gradients at moderate frequency are more feasible for exploring restrictions at very short distances which, in tissues, correspond to structures smaller than cells. We describe the underlying concepts of temporal diffusion spectra and provide analytical expressions for the behavior of the diffusion coefficient as a function of gradient frequency in simple geometries with different dimensions. Diffusion in more complex model media that mimic tissues has been simulated using numerical methods. Experimental measurements of diffusion spectra have been obtained in suspensions of particles and cells, as well as in vivo in intact animals. An observation of particular interest is the increased contrast and heterogeneity observed in tumors using oscillating gradients at moderate frequency compared with conventional pulse gradient methods, and the potential for detecting changes in tumors early in their response to treatment. Computer simulations suggest that diffusion spectral measurements may be sensitive to intracellular structures, such as nuclear size, and that changes in tissue diffusion properties may be measured before there are changes in cell density. Copyright © 2010 John Wiley & Sons, Ltd. [source] | |||||||||||||