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Effects of acetone, methanol, or paraformaldehyde on cellular structure, visualized by reflection contrast microscopy and transmission and scanning electron microscopy.

The authors recently showed variable subcellular immunoreactivity of the Bcl-2 and Bax proteins after fixation of cell monolayers with acetone, methanol, or paraformaldehyde (PF) followed by methanol (PF/methanol). Here, the authors demonstrate by reflection contrast microscopy and transmission electron microscopy that acetone or methanol fixation result in complete loss of integrity of intracellular structures in contrast with PF or glutaraldehyde fixation. Scanning electron microscopy revealed poor preservation of plasma membrane integrity after fixation in acetone or methanol. Fixation with PF before methanol reduced damage to intracellular and plasma membranes. In addition, Western blot analysis demonstrated loss of Bcl-2 and Bax protein during acetone or methanol fixation, whereas PF fixation before methanol permeabilization markedly reduced this loss. For studies on the intracellular localization of soluble or unknown types of antigen, the authors discourage the use of acetone and methanol as single fixatives.

Acetone↗

Effects of ionizing radiation on cellular structures, induced instability and carcinogenesis.

Ionizing radiation is perhaps the most extensively studied human carcinogen. There have been a number of epidemiological studies on human populations exposed to radiation for medical or occupational reasons, as a result of protracted environmental exposures due to radiation accidents, or after atomic bombings. As a result of these studies exposure to ionizing radiation has been unambiguously linked to cancer causation. While cancer induction is the primary concern and the most important somatic effect of exposure to ionizing radiation, potential health risks do not only involve neoplastic diseases but also somatic mutations that might contribute to birth defects and ocular maladies, and heritable mutations that might impact on disease risks in future generations. Consequantly it is important we understand the long-term health risks associated with exposure to ionizing radiation.

Cell Transformation, Neoplastic↗

Universality in two-dimensional cellular structures evolving by cell division and disappearance.

The dynamics of two-dimensional cellular networks is written in terms of coupled population equations, which describe how the population of s-sided cells is affected by cell division and disappearance. In these equations the effect of the rest of the foam on the disappearing or dividing cell is treated as a local mean field. Under not too restrictive conditions, the equilibrium distribution P(s) of cells satisfies a linear difference equation of order two or higher. The population equations are asymptotically integrable. The asymptotic integrability implies a "universal" distribution P(s) approximately Cs-kZs for large values of s, which is also the Boltzmann distribution associated with the maximum entropy inference. Asymptotic integrability of the population equations is absent in a global mean-field approximation. The importance of short-range topological information to control the evolution of foams is thus confirmed.

Apoptosis↗

Steric interactions in cellular structures formed in a water/oil/surfactant/cosurfactant mixture.

Dynamics of dense microemulsion droplets forming ordered cellular phases as temperature increases are investigated by neutron spin echo spectroscopy (NSE). The shape fluctuations of droplets are shown using a specific contrast. Their relaxation time tau(f) is obtained by analysis of the NSE curves, which also reveals the short-range Brownian motion (diffusion constant D0) of the "caged" droplets. The thermal dependence of D0 and tau(f) supports the notion of entropically driven interdroplet steric repulsion stabilizing the cellular phases.

Journal Article↗

[Estimation of the randomness of the overlapping of cellular structures].

Methods for estimation of randomness of mutual overlaps of cells, their parts, and intracellular structures are developed. Two methods are proposed: a "method of areas" based on the estimation of the area of overlaps of randomly located figures, and a "method of contours" based on the evaluation of the number of intersections of linear structures located randomly. Examples are given as applied to cells and cellular nuclei, in particular during contact inhibition of cellular movement.

Animals↗

Community structure, cellular rRNA content, and activity of sulfate-reducing bacteria in marine arctic sediments.

The community structure of sulfate-reducing bacteria (SRB) of a marine Arctic sediment (Smeerenburgfjorden, Svalbard) was characterized by both fluorescence in situ hybridization (FISH) and rRNA slot blot hybridization by using group- and genus-specific 16S rRNA-targeted oligonucleotide probes. The SRB community was dominated by members of the Desulfosarcina-Desulfococcus group. This group accounted for up to 73% of the SRB detected and up to 70% of the SRB rRNA detected. The predominance was shown to be a common feature for different stations along the coast of Svalbard. In a top-to-bottom approach we aimed to further resolve the composition of this large group of SRB by using probes for cultivated genera. While this approach failed, directed cloning of probe-targeted genes encoding 16S rRNA was successful and resulted in sequences which were all affiliated with the Desulfosarcina-Desulfococcus group. A group of clone sequences (group SVAL1) most closely related to Desulfosarcina variabilis (91.2% sequence similarity) was dominant and was shown to be most abundant in situ, accounting for up to 54. 8% of the total SRB detected. A comparison of the two methods used for quantification showed that FISH and rRNA slot blot hybridization gave comparable results. Furthermore, a combination of the two methods allowed us to calculate specific cellular rRNA contents with respect to localization in the sediment profile. The rRNA contents of Desulfosarcina-Desulfococcus cells were highest in the first 5 mm of the sediment (0.9 and 1.4 fg, respectively) and decreased steeply with depth, indicating that maximal metabolic activity occurred close to the surface. Based on SRB cell numbers, cellular sulfate reduction rates were calculated. The rates were highest in the surface layer (0.14 fmol cell(-1) day(-1)), decreased by a factor of 3 within the first 2 cm, and were relatively constant in deeper layers.

Arctic Regions↗

On the ontogeny of aldolase isozymes and their interactions with cellular structure.

In an endeavour to extend the available information on the biological significance of the interactions between aldolase and cellular ultrastructure, the extent of association has been studied in the tissues of the mouse during the major stages of development from embryo to adult. Analysis of the isozyme status in these compartments and the latency of the enzyme during tissue differentiation was also effected. In all tissues investigated, a considerable variation in the degree of association of aldolase with structure was evident during development. Binding was particularly extensive in the early embryonic stages, but regardless of the tissue or the stage of differentiation, binding preference was directed towards A-type activity over the B- and C-type of enzyme. Substantial latent activity of aldolase was evident only in brain in the postnatal stages of development, and not in the other tissues or early stages of ontogeny. The significance of these ontogenic phenomena have been discussed, along with the physiological variations in individual tissues during maturation.

Aging↗

Alphaherpesvirus proteins related to herpes simplex virus type 1 ICP0 affect cellular structures and proteins.

The herpes simplex virus type 1 (HSV-1) immediate-early protein ICP0 interacts with several cellular proteins and induces the proteasome-dependent degradation of others during infection. In this study we show that ICP0 is required for the proteasome-dependent degradation of the ND10 protein Sp100 and, as with the other target proteins, the ICP0 RING finger domain is essential. Further, comparison of the kinetics and ICP0 domain requirements for the degradation of PMI and Sp100 suggests that a common mechanism is involved. Homologues of ICP0 are encoded by other members of the alphaherpesvirus family. These proteins show strong sequence homology to ICP0 within the RING finger domain but limited similarity elsewhere. Using transfection assays, we have shown that all the ICP0 homologues that we tested have significant effects on the immunofluorescence staining character of at least one of the proteins destabilized by ICP0, and by using a recombinant virus, we found that the equine herpesvirus ICP0 homologue induced the proteasome-dependent degradation of endogenous CENP-C and modified forms of PML and Sp100. However, in contrast to ICP0, the homologue proteins had no effect on the distribution of the ubiquitin-specific protease USP7 within the cell, consistent with their lack of a USP7 binding domain. We also found that ICP0 by itself could induce the abrogation of SUMO-1 conjugation and then the proteasome-dependent degradation of unmodified exogenous PML in transfected cells, thus demonstrating that other HSV-1 proteins are not required. Surprisingly, the ICP0 homologues were unable to cause these effects. Overall, these data suggest that the members of the ICP0 family of proteins may act via a similar mechanism or pathway involving their RING finger domain but that their intrinsic activities and effects on endogenous and exogenous proteins differ in detail.

Alphaherpesvirinae↗

Comparison of in vivo and ex vivo cellular structure in rabbit eyes detected by tandem scanning microscopy.

Using the tandem scanning microscope, in vivo confocal microscopic images of living eyes were compared to images obtained from ex vivo, freshly enucleated or fixed tissue in the rabbit. In the normal cornea, microscopic details of the superficial epithelium, basal lamina, stromal fibrocyte nuclei, nerves and endothelial cell borders were easily discernible. Removal of the eye from the intact animal resulted in loss of detail with distortion of the normal structural interrelationships within the corneal stroma whilst enhancing details of the corneal epithelium. Formalin fixation further enhanced details of the basal and suprabasal corneal epithelial cell nuclei and the stromal fibrocyte cell borders whilst inducing prominent brightly reflecting folds in the thickened stroma with concomitant enhancement of the edge contrast of the collagen lamellae. These changes appeared to be related, in part, to hydration of the cornea and artefactual pooling of water between structures that may enhance reflectivity by increasing the difference between the refractive index of the cellular and extracellular elements. We conclude that microscopic examination of ex vivo preparations of corneal tissue, although providing increased resolution similar to conventional light microscopic techniques, significantly altered the normal structural relationships and could lead to erroneous measurements of the physiological properties of the tissue as compared to in vivo microscopy of undisturbed, intact tissue.

Animals↗

Selective induction of cytokines in mouse brain infected with canine distemper virus: structural, cellular and temporal expression.

We have previously shown that, in experimentally inoculated mice, canine distemper virus (CDV), a neurotropic virus, selectively infects certain brain structures (hypothalamus, hippocampus, monoaminergic nuclei, etc). Here we demonstrate that tumor necrosis factor (TNF)-alpha, interleukin (IL)-1 beta and IL-6 transcripts are selectively expressed in these CDV-targeted structures, except in the dentate gyrus, where cytokines are induced without prior CDV replication. The time-course of TNF-alpha expression vs. viral replication in the hypothalamus was different from that in hippocampus. In addition, we show that a substantial number of neurons express TNF-alpha and IL-6. These findings provide new insights into the possible participation of cytokines in the neurological disorders triggered by CDV infection.

Animals↗

Diarthroses in the mouse: from different cellular structure to similar joint appearance.

The structure and function of two major joints, the humeroscapular and the squamoso-mandibular joints in mice, are compared. The specific roles that these two joints fulfill during early postnatal period are reflected in a different cellular organization observed in them. Following maturation, when similar functional needs are stressed upon these two joints, similar homologous structures are detected in both of them. A possible conceptual explanation for these observations and their relatedness to the human equivalent joints are discussed.

Animals↗

Cellular structure and biology of Dupuytren's disease.

Numerous studies support the idea that the myofibroblast is a key cell responsible for the tissue contraction in Dupuytren's disease. In vitro models have been developed to study the underlying cellular basis of myofibroblast differentiation and contraction. Studies suggest that the growth factor TGF-beta 1 combined with mechanical stress can promote the differentiation of fibroblasts into myofibroblasts. Agonists, such as LPA and thrombin, can promote the contraction of myofibroblasts through specific intracellular signaling pathways that regulate levels of phosphorylated myosin light chain. Agents that can affect these intracellular signaling pathways hold promise as a means to decrease contraction of the myofibroblast and of the palmar fascia in Dupuytren's disease. Finally, the recent finding that IFN-gamma can suppress both the differentiation of the myofibroblast and the generation of contractile force, together with preliminary clinical results using IFN-gamma, suggest the potential use of IFN-gamma for nonsurgical therapy of Dupuytren's disease. Future studies into the cellular basis of tissue contraction should provide alternative methods to improve management of Dupuytren's contracture.

Cell Differentiation↗

Actions of ultraviolet light on cellular structures.

Solar radiation is the primary source of human exposure to ultraviolet (UV) radiation. Overexposure without suitable protection (i.e., sunscreen and clothing) has been implicated in mutagenesis and the onset of skin cancer. These effects are believed to be initiated by UV-mediated cellular damage, with proteins and DNA as primary targets due to a combination of their UV absorption characteristics and their abundance in cells. UV radiation can mediate damage via two different mechanisms: (a) direct absorption of the incident light by the cellular components, resulting in excited state formation and subsequent chemical reaction, and (b) photosensitization mechanisms, where the light is absorbed by endogenous (or exogenous) sensitizers that are excited to their triplet states. The excited photosensitizers can induce cellular damage by two mechanisms: (a) electron transfer and hydrogen abstraction processes to yield free radicals (Type I); or (b) energy transfer with O2 to yield the reactive excited state, singlet oxygen (Type II). Direct UV absorption by DNA leads to dimers of nucleic acid bases including cyclobutane pyrimidine species and pyrimidine (6-4) pyrimidone compounds, together with their Dewar isomers. These three classes of dimers are implicated in the mutagenicity of UV radiation, which is typified by a high level of CC-->TT and C-->T transversions. Single base modifications can also occur via sensitized reactions including Type 1 and Type II processes. The main DNA product generated by (1)O2 is 8-oxo-Gua; this is a common lesion in DNA and is formed by a range of other oxidants in addition to UV. The majority of UV-induced protein damage appears to be mediated by (1)O2, which reacts preferentially with Trp, His, Tyr, Met, Cys and cystine side chains. Direct photo-oxidation reactions (particularly with short-wavelength UV) and radicals can also be formed via triplet excited states of some of these side chains. The initial products of (1)O2-mediated reactions are endoperoxides with the aromatic residues, and zwitterions with the sulfur-containing residues. These intermediates undergo a variety of further reactions, which can result in radical formation and ring-opening reactions; these result in significant yields of protein cross-links and aggregates, but little protein fragmentation. This review discusses the formation of these UV-induced modifications and their downstream consequences with particular reference to mutagenesis and alterations in protein structure and function.

Cell Transformation, Neoplastic↗

Characteristics of cellular structures derived by tissue culture of pre-implantation blastocysts of the pig.

Tissue mechanically dissociated from blastocysts of the pig around the time of implantation were found to produce, in culture, free-floating multicellular spheroids (trophospheres) and adherent monolayer cells. Ultrastructurally the two cellular layers of the trophospheres were very similar to those of the blastocyst but the trophosphere outer layer characteristically contained very large mitochondria with a vastly expanded matrix and few cristae. Similar mitochondria were also found in the monolayer cells. Using a monoclonal antibody specific for pig trophectoderm, it was found that about 20% of the monolayer cells, and some of the spheroids expressed this trophectodermal antigen. In the latter case the antigen was present only on the surface facing the medium. The spheroids were fluid-filled and occasionally grew inside each other. The monolayer cells were predominantly uninuclear but did form a number of binucleate cells and in older cultures the occasional cell with many nuclei could be seen. The spheroids and the monolayer cells had similar glycoprotein profiles indicating that they were composed of similar cell populations. A glycoprotein of apparent molecular weight 68,000 observed in both spheres and monolayers may represent pig placental alkaline phosphatase. Both trophospheres and monolayer cells were observed to interconvert steroid precursors. It is apparent that the trophospheres share many features of the blastocyst and may thus represent a valuable model system similar to those described in other species for the investigation of their biochemical physiological and immunological properties.

Animals↗

Structure, cellular distribution, antigenicity, and biological functions of Fonsecaea pedrosoi ceramide monohexosides.

Monohexosylceramides (CMHs, or cerebrosides) have been reported as membrane and cell wall constituents of both pathogenic and nonpathogenic fungi, presenting remarkable differences in their ceramide moiety compared to mammalian CMHs. Current evidence suggests that CMHs are involved in fungal differentiation and growth and contribute to host immune response. Here we describe a structural diversity between cerebrosides obtained from different forms of the human pathogen Fonsecaea pedrosoi. The major CMH species produced by conidial forms displayed the same structure previously demonstrated by our group for mycelia, an N-2'-hydroxyhexadecanoyl-1-beta-d-glucopyranosyl-9-methyl-4,8-sphingadienine. However, the major cerebroside species purified from sclerotic cells carries an additional hydroxyl group, bound to its long-chain base. The structural difference between cerebrosides from mycelial and sclerotic cells was apparently not relevant for their antigenicity, since they were both recognized at similar levels by sera from individuals with chromoblastomycosis and a monoclonal antibody to a conserved cerebroside structure. Preincubation of fungal cells with anti-CMH monoclonal antibodies had no effect on the interaction of F. pedrosoi sclerotic cells with murine macrophages. In contrast to what has been described for other fungal species, sclerotic bodies are resistant to the antifungal action of anti-CMH antibodies. Immunofluorescence analysis showed that recognition of sclerotic cells by these antibodies only occurs at cell wall regions in which melanization is not evident. Accordingly, melanin removal with alkali results in an increased reaction of fungal cells with anti-CMH antibodies. Our results indicate that cerebroside expression in F. pedrosoi cells is associated with dimorphism and melanin assembly on the fungal cell wall.

Animals↗

Cloning, structure, cellular localization, and possible function of the tumor suppressor gene lethal(3)malignant blood neoplasm-1 of Drosophila melanogaster.

The tumor suppressor gene, lethal(3)malignant blood neoplasm-1+, of Drosophila melanogaster is required for the differentiation of the phagocytic blood-cell type, the plasmatocyte. In the homozygously mutated state it causes the malignant transformation of these blood cells. We present here the cloning, sequencing, structure, and expression of the l(3)mbn-1+ gene during development. The cloned gene was identified by germ-line transformation, generation of revertants, and the detection of the corresponding mRNA in blood cells and other tissues. Homologies of the G-S-rich C-terminus of the putative MBN83 protein to human cytokeratins K1, K10, and mouse loricrin were found. The structure and possible function of the wild-type l(3)mbn-1+ gene are discussed.

Alleles↗

Linker region of nebulin family members plays an important role in targeting these molecules to cellular structures.

The nebulin family of actin-binding proteins plays an essential role in cytoskeletal dynamics and actin filament stability. All of the family members are modular proteins with their key defining structural feature being the presence of the 35-residue nebulin modules. The family members now include nebulin, nebulette, N-RAP, LASP-1, and LIM-nebulette. Nebulin and nebulette are associated with the thin filament/Z-line junction of striated muscle. LASP-1 and LIM-nebulette are found within focal adhesions, and N-RAP is associated with muscle cellular junctions. Although much investigation has focused on the role of the interactions between nebulin modules and actin, each of these proteins contains other domains that are essential for their cellular targeting and functions. The serine-rich linker region of nebulette has previously been shown to serve just such a purpose by targeting the association of the nebulin modules to the cardiac Z-line in cultured cardiomyocytes. In this report, we analyze the targeting functions of the homologous regions of LASP-1 and LIM-nebulette in their incorporation into focal adhesions. We have found that the linker region of LASP-1 is indeed important for its cellular localization and that the shortened linker region of LIM-nebulette drives the association of nebulin modules to focal adhesions.

Actinin↗

Assessment of fluorochromes for cellular structure and function studies by flow cytometry.

Because flow cytometry permits the analysis of individual whole cells, one of the key requirements in selecting a probe is its ability to target the site of interest into cells. In addition, dyes must possess ideal properties (ie extinction coefficient, Stoke's shift) rendering them appropriate for this methodology. Other characteristics, such as fluorescence quenching and energy transfer, inherent to the staining, provide numerous applications in flow cytometry. The available fluorophores used in flow cytometry are classified according to their cellular incorporation and binding. Thus, probes are presented and discussed as follows: 1) dyes of cellular components (DNA, RNA, proteins, lipids); 2) probes of membrane potential; 3) fluorophores that are sensitive to their microenvironment (pH, calcium, etc); and 4) those used for measurement of enzymatic activities into cells.

Animals↗