Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “Cellular Structures”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 973 records · Page 54Linked to original sources
PubMed2003

Cell-cycle-regulatory elements and the control of cell differentiation in the budding yeast.

The stable differentiation of cells into other cell types typically involves dramatic reorganization of cellular structures and functions. This often includes remodeling of the cell cycle and the apparatus that controls it. Here we review our understanding of the role and regulation of cell cycle control elements during cell differentiation in the yeast, Saccharomyces cerevisiae. Although the process of differentiation may be more overtly obvious in metazoan organisms, those systems are by nature more difficult to study at a mechanistic level. We consider the relatively well-understood mechanisms by which mating-type switching and the pheromone-induced differentiation of gametes are coupled to the cell cycle as well as the more obscure mechanisms that govern the remodeling of the cell cycle during meiosis and filamentous growth. In some cases, the cell cycle is a primary stimulus for differentiation whereas, in other cases, the signals that promote differentiation alter the cell cycle. Thus, despite relative simplicity of these processes in yeast, the nature of the interplay between the cell cycle and differentiation is diverse.

Cell Cycle↗
PubMed2024

Cytoskeletal mechanisms regulating attaching/effacing bacteria interactions with host cells: It takes a village to build the pedestal.

The actin cytoskeleton is a key cellular structure subverted by pathogens to infect and survive in or on host cells. Several pathogenic strains of Escherichia coli, such as enteropathogenic E. coli (EPEC) and enterohemorrhagic E. coli (EHEC), developed a unique mechanism to remodel the actin cytoskeleton that involves the assembly of actin filament-rich pedestals beneath the bacterial attachment sites. Actin pedestal assembly is driven by bacterial effectors injected into the host cells, and this structure is important for EPEC and EHEC colonization. While the interplay between bacterial effectors and the actin polymerization machinery of host cells is well-understood, how other mechanisms of actin filament remodelling regulate pedestal assembly and bacterial attachment are poorly investigated. This review discusses the gaps in our understanding of the complexity of the actin cytoskeletal remodelling during EPEC and EHEC infection. We describe possible roles of actin depolymerizing, crosslinking and motor proteins in pedestal dynamics, and bacterial interactions with the host cells. We also discuss the biological significance of pedestal assembly for bacterial infection.

Humans↗
PubMed1997

Evolution of the multi-tubulin hypothesis.

Microtubules are organized into diverse cellular structures in multicellular organisms. How is such diversity generated? Although highly conserved overall, variable regions within alpha- and beta-tubulins show divergence from other alpha- and beta-tubulins in the same species, but show conservation among different species. Such conservation raises the question of whether diversity in tubulin structure mediates diversity in microtubule organization. Recent studies probing the function of beta-tubulin isotypes in axonemes of insects suggest that tubulin structure, through interactions with extrinsic proteins, can direct the architecture and supramolecular organization of microtubules.

Animals↗
PubMed2004

Dynamic change in the distribution of alpha5beta1 integrin on isolated ventral membrane: effect of divalent cation species.

We investigated the spatial distribution of alpha5beta1 integrin in isolated ventral plasma membranes (VPMs) of human foreskin fibroblasts in order to elucidate how the interaction of integrin with cytoskeletal and extracellular matrix proteins might affect the integrin distribution. Cells were exposed to the jet of buffer to remove the apical surface and most of cellular organelles. After this treatment VPMs, which adhered to the glass surface, possessed the cellular structures such as fibronectin (FN) fibrils and actin stress fibers. The isolated VPMs thus prepared were employed without fixation to investigate the change in the integrin distribution. In isolated VPMs, alpha5beta1 integrin, labeled with Cy3-tagged anti-integrin antibody, was found to accumulate not only at the tips of stress fibers but also along FN fibrils extending from there. When divalent cations were removed with EDTA, the accumulated integrin was dispersed, and the original pattern of distribution was recovered upon restoration of divalent cations. Talin, an integrin-actin cytoskeleton linker protein, was found to accumulate only at the tips of stress fibers in isolated VPMs, but alpha5beta1 integrin did not exhibit strong accumulation there, indicating that talin played little role in integrin distribution in isolated VPMs. The amount of alpha-actinin associated with stress fibers was found to drastically decrease in isolated VPMs, which was presumably related to the failure of localization of integrin at the tips of stress fibers. It was also shown that the association of stress fibers to isolated VPMs seemed to be independent of accumulation of integrin.

Actinin↗
PubMed2005

Superficial and deep changes of cellular mechanical properties following cytoskeleton disassembly.

The cytoskeleton, composed of actin filaments, intermediate filaments, and microtubules, is a highly dynamic supramolecular network actively involved in many essential biological mechanisms such as cellular structure, transport, movements, differentiation, and signaling. As a first step to characterize the biophysical changes associated with cytoskeleton functions, we have developed finite elements models of the organization of the cell that has allowed us to interpret atomic force microscopy (AFM) data at a higher resolution than that in previous work. Thus, by assuming that living cells behave mechanically as multilayered structures, we have been able to identify superficial and deep effects that could be related to actin and microtubule disassembly, respectively. In Cos-7 cells, actin destabilization with Cytochalasin D induced a decrease of the visco-elasticity close to the membrane surface, while destabilizing microtubules with Nocodazole produced a stiffness decrease only in deeper parts of the cell. In both cases, these effects were reversible. Cell softening was measurable with AFM at concentrations of the destabilizing agents that did not induce detectable effects on the cytoskeleton network when viewing the cells with fluorescent confocal microscopy. All experimental results could be simulated by our models. This technology opens the door to the study of the biophysical properties of signaling domains extending from the cell surface to deeper parts of the cell.

Actins↗
PubMed1990

Immunocytochemical identification of cytoskeletal linkages to smooth muscle cell nuclei and mitochondria.

In avian smooth muscle cells, desmin-containing intermediate filaments (IFs) are a prominent component of the cytoskeleton and are readily seen in several domains, including the axial intermediate filament bundle (IFB). Both the nucleus and some of the mitochondria are partly surrounded by elements of the IFB. By using anti-desmin and protein-A-colloidal gold labeling, we have identified intermediate filaments that form linkages with the nuclear envelope and with mitochondria. These linkage regions seem to occupy a proportionately greater part of the mitochondrial surface than of the nuclear envelope. The existence of these linkages in smooth muscle cells is consistent with results that support similar linkages to mitochondria and other cellular structures in various cells that contain either vimentin or keratin IFs. These linkages could functionally restrain or assist in homeostatically restoring organelles to their normal position after the rearrangement that accompanies the substantial shortening of smooth muscle cells.

Animals↗
PubMed1992

Fertilization alters the orientation of pigment granule saltations in Arbacia eggs.

Unfertilized eggs of the sea urchin Arbacia punctulata contain pigment granules distributed throughout their cytoplasm. During the first 15 minutes after fertilization, these vesicles move out to the cortex where they become firmly anchored. We have used time-lapse video differential interference microscopy to analyze the motility of these organelles in unfertilized and fertilized Arbacia eggs. Pigment granules exhibit saltatory movement in both unfertilized and fertilized eggs. Quantitation of vesicle saltations before and after fertilization demonstrates that while there is no significant difference in the speed or path-length of vesicle movement, there is a dramatic change in the orientation of these saltations. Saltations in the unfertilized egg are very non-radial and are as likely to be directed toward the cortex as away. In contrast, saltations in the fertilized egg are more radially oriented and more likely to be cortically directed. This transition must reflect underlying changes in the cellular structures necessary for pigment granule saltations. The change in the orientation of pigment granule saltations following fertilization requires both a transient increase in the cytoplasmic concentration of Ca2+ and an elevation of cytoplasmic pH. Similarly, the ability of pigment granules to adhere to the cortex requires both the transient elevation of cytoplasmic Ca2+ and the alkalinization of the cytoplasm. As the reorganization of cortical actin at fertilization is regulated by these ionic fluxes, and both movement and adhesion are sensitive to cytochalasins, we hypothesize that the alterations in directed motility and adhesion reflect underlying changes in the actin cytoskeleton.

Animals↗
PubMed1992

Microtubule assembly protects the region 28-38 of the beta-tubulin subunit.

Polyclonal antibodies have been raised against the peptide 28-38 of the beta-subunit of the tubulin heterodimer in order to study the accessibility of this region in the tubulin heterodimer and in various tubulin assemblies. These antibodies were specific for all beta-tubulin subunits, except for beta'-tubulin isotypes, and did not recognize the alpha-tubulin subunit. The 28-38 region does not play a role in the interaction between the alpha- and beta-subunits since it was accessible to the antibodies on the native heterodimer. The accessibility of the antibodies was not modified by several microtubular poisons. In contrast, in all tubulin assemblies obtained in the presence of microtubular associated proteins, the region 28-38 was not available to the antibodies. These antibodies did not react with microtubules or tubulin spirals assembled either from microtubule proteins or from pure tubulin when these tubulin assemblies were probed in the absence of free tubulin after centrifugation on glass coverslips. In addition, antibodies failed to interact with the microtubule cytoskeleton in cultured Ptk2 cells indicating that the 28-38 region of beta-tubulin is also protected in cellular structures. These observations suggest that the 28-38 region of the beta-tubulin subunit is either located in a zone of interaction between two successive tubulin dimers within a protofilament or hidden by an allosteric conformational change which occurs during tubulin assembly.

Amino Acid Sequence↗
PubMed1976

Topological analysis of the brain stem of the axolotl Ambystoma mexicanum.

The ventricular sulcal pattern and the cellular structure of the brain stem of the axolotl Ambystoma mexicanum have been studied in transversely cut Nissl and Bodian stained serial sections. Six longitudinal sulci, the sulcus medianus inferior, the sulcus intermedius ventralis, the sulcus limitans, the sulcus intermedius dorsalis, the sulcus medianus superior and the sulcus lateralis mesencephali could be distinguished. A seventh groove, the sulcus isthmi, clearly deviates from the overall longitudinal pattern of the other sulci. Although most neuronal perikarya are contained within a diffuse periventricular gray, 19 cell masses could be delineated; seven of these are primary efferent or motor nuclei, four are primary afferent or sensory centers, four nuclei are considered as components of the reticular formation, and the remaining four cell masses can be interpreted as "relay" nuclei. In order to study the zonal pattern of the brain stem, this structure was subjected to a topological analysis (cf, Nieuwenhuys, '74 and fig. 13). This analysis yielded the following results. In the rhombencephalon the grisea are arranged in four longitudinal zones which, following Kuhlenbeck, have been termed area ventralis, area intermedioventralis, area intermediodorsalis and area dorsalis. Where present the sulcus intermedius ventralis, the sulcus limitans and the sulcus intermedius dorsalis mark the boundaries between these four morphological entities. The zonal areas in question coincide largely, but not entirely, with the so-called functional columns of Herrick and Johnston. The most obvious incongruity is that the area intermediodorsalis contains, in addition to the nucleus fasciculi solitarii and the nucleus visceralis secundarius, two non-visceral sensory cell masses, namely the nucleus vestibularis magnocellularis and the nucleus cerebelli. The four morphological zones delineated in the rhombencephalon cannot be distinguished in the mesencephalon and it is of particular importance that the sulcus limitans does not extend into this part of the brain. Functionally, however, the medial part of the tegmentum mesencephali may be considered the rostral extreme of the somatic motor column, whereas the tectum primarily represents a somatic sensory correlation area.

Ambystoma↗
PubMed1979

Topological analysis of the brain stem of the lungfish Lepidosiren paradoxa.

The ventricular sulcal pattern and the cellular structure of the brain stem of the lungfish Lepidosiren paradoxa have been studied in transversely cut Nissl and Bodian stained sections. Five longitudinal sulci, the sulcus medianus inferior, the sulcus intermedius ventralis, the sulcus limitans, the sulcus intermedius dorsalis and the sulcus medianus superior could be distinguished. In the isthmus region a number of obliquely oriented sulci is present. One of these, designated here as sulcus d, continues as a longitudinal groove into the mesencephalon. In Lepidosiren most neuronal perikarya are contained within a diffuse periventricular gray. However, 24 separate cell masses could be delineated. Six of these are primary efferent nuclei, six are primary afferent center, six nuclei are considered to be components of the reticular formation and the remaining six may be interpreted as "relay" nuclei. The distribution of the cell masses and their relations to the ventricular sulci were studied with the aid of the graphical reconstruction procedure termed tolopogical analysis (cf. Nieuwenhuys, '74, and fig. 11). This analysis yielded the following results. In the rhombencephalon the gray matter is arranged in four longitudinal columns or areas which have been termed the area ventralis, area intermedioventralis, area intermediodorsalis and area dorsalis. In many places the sulcus intermedius ventralis, the sulcus limitans and the sulcus intermedius dorsalis mark the boundaries between these morphological entities. These longitudinal areas coincide largely, but not entirely, with so-called functional columns of Herrick and Johnston. The most obvious incongruity is that the area intermediodorsalis contains, in addition to the nucleus of the solitary tract, two non-visceral sensory cell masses, namely the magnocellular and parvocellular vestibular nuclei. The four longitudinal zones cannot be distinguished in the mesencephalon nor can the sulcus limitans be recognized here. Functionally, however, the medial part of the tegmentum mesencephali may be considered the rostral extreme of the somatic motor column, whereas the remainder of the midbrain contains a number of somatic sensory centers.

Animals↗
PubMed1979

Topological analysis of the brain stem of the crossopterygian fish Latimeria chalumnae.

The ventricular sulcal pattern and the cellular structure of the brain stem of the single surviving crossopterygian species Latimeria chalumnae have been studied in transversely cut Nissl, Klüver-Barrera and Bodian stained serial sections. Five longitudinal sulci, the sulcus medianus inferior, the sulcus medianus superior, the sulcus intermedius ventralis, the sulcus limitans and the sulcus intermedius dorsalis could be delimited. The latter three of these sulci are confined to the rhombencephalon. The walls of the mesencephalon also display some longitudinal grooves, but none of these could be traced into continuity with any of the rhombencephalic sulci. Although the neuronal perikarya in many places show a diffuse arrangement, 27 cell masses could be delineated; eight of these are primary efferent nuclei, seven are primary afferent centers, seven nuclei are considered as components of the reticular formation, and the remaining five cell masses may be interpreted as "relay" nuclei. In order to study the zonal pattern of the brain stem, this structure was subjected to a topological analysis (cf., Nieuwenhuys, '74, and fig. 16). This analysis yielded the following results. The sulcus limitans divides the greater part of the rhombencephalon into a basal plate and an alar plate. In the basal plate the sulcus intermedius ventralis marks the boundary between an area ventralis and an area intermedioventralis. The area ventralis contains two somatic motor centers (i.e., the rostral end of the spinal motor column and the nucleus of IV) and most the entire rhombencephalic medial reticular formation. The latter may be primarily considered as a somatic motor coordinating center. The area intermedioventralis contains the visceral motor nuclei of V, VII, IX and X. However, the basal plate also harbours a number of non-motor centers, for example the sensory princeps nucleus of V and the inferior olive. The alar plate is subdivided by the sulcus intermedius dorsalis into an area intermediodorsalis and an area dorsalis. The area intermediolateralis is largely occupied by the common visceral sensory center of VII, IX and X; however, this area also contains a number of somatic sensory cell masses, as e.g. the nucleus descendens of V and the magnocellular vestibular nucleus. The area dorsalis is entirely occupied by two large lateral line centers. The cell masses in the isthmus region do not exhibit a clear-cut morphological pattern. As regards the mensencephalon, the medial part of the tegmentum, which contains a primary somatic motor center (the nucleus of III) and a somatic motor coordination center (the nucleus of the f.l.m.) may be considered a direct rostral continuation of the area ventralis. The remainder of the midbrain contains a number of somatic sensory centers of primary and higher order.

Animals↗
PubMed1980

Glial fibrillary acidic protein in the retina of the developing albino rat: an immunoperoxidase study of paraffin-embedded tissue.

The peroxidase-anti-peroxidase method was used on paraffin-embedded material to demonstrate the distribution of glial fibrillary acidic (GFA) protein, an astrocyte-specific protein, in the developing retina of the albino rat. At birth activity was scant and was confined to scattered, poorly differentiated cells in the inner retinal layers near the optic disc. At 3 days primitive astrocytes which displayed GFA protein activity were confined to the stratum opticum near the optic disc. With increasing age these cells were found at greater distances from the optic disc and began to assume the appearance of typical fibrous astrocytes. By 30 days the perikarya of these cells were confined almost exclusively to the region between the nerve fiber layer and the inner limiting membrane. The processes of these cells terminated either in suckerlike end-feet upon blood vessels or, to a lesser extent, ended in relation to axon fascicles of the nerve fiber layer. A second population of GFA protein-active cells existed as perivascular glia which were found upon vessels in the inner portion of the stratum opticum in young animals. In the mature retina perivascular glia were found on vessels throughout the stratum opticum and in the inner portion of the inner plexiform layer. Unequivocal staining of Müller cells or their processes was not obtained. The best staining was obtained with fixatives containing minimal concentrations of aldehydes, especially in tissue from younger animals. The fixative which gave the best preservation of cellular structure along with preservation of GFA protein antigenicity was Perfix (Fischer Scientific Company).

Animals↗
PubMed1986

GABA-immunoreactive neurons in the rat cerebellum: a light and electron microscope study.

An antibody raised against gamma - amino-butyric acid (GABA) coupled to bovine serum albumin with glutaraldehyde (Hodgson et al: J. Histochem. Cytochem. 33:229-239, '85) was used to localise immunocytochemically the presumptive GABAergic neuronal elements in the cerebellar cortex of the adult rat. employing the unlabelled antibody enzyme method with pre- and post-embedding immunocytochemical procedures, the following cellular structures were observed to be GABA-immunopositive in both the light and electron microscopes: the somata, dendrites, and axonal processes (including axon terminals) of stellate, basket, and Golgi neurons. In immunopositive neuronal somata and dendrites, the reaction product was found to be associated with all intracellular organelles and with the postsynaptic densities of synaptic junctions. Specific GABA-like immunoreactivity was also seen around outer mitochondrial membranes, microtubules, and neurofilaments, and coating synaptic vesicles in presynaptic axon terminals. In the pre-embedding procedure with dilutions of the antiserum between 1:1,000 and 1:2,000, the perikarya and dendrites of Purkinje cells were GABA-immunonegative, whereas at an antiserum dilution of 1:500 the somata of Purkinje cells were mildly GABA-immunoreactive. Purkinje cell axon terminals in the infra- and supraganglionic plexuses and in the deep cerebellar nuclei were always strongly immunopositive. Neuroglia were invariably GABA-immunonegative, as were the dendrites, axons (parallel fibres), and somata of granule cells. Mossy fibre and climbing fibre afferents were also immunonegative. The pattern of immunoreactivity obtained with this antiserum directed against the inhibitory neurotransmitter GABA was found to resemble closely the immunocytochemical distribution of GABA and of the GABA-synthesizing enzyme glutamic acid decarboxylase (GAD) as reported previously in other immunocytochemical investigations (Oertel et al. and Wu et al: Cytochemical Methods in Neuroanatomy. New York: A. R. Liss, '82; Seguela et al: Neuroscience 16:865-874, '85; Mugnaini and Oertel: GABA and Neuropeptides in the CNS. Handbook of Chemical Neuroanatomy, Vol. 4, Part I. Amsterdam: Elsevier, '85.

4-Aminobutyrate Transaminase↗
PubMed1990

Immunocytological localization of the highly polysialylated form of the neural cell adhesion molecule during development of the murine cerebellar cortex.

The expression of the highly polysialylated form of the neural cell adhesion molecule (N-CAM)--the so-called embryonic N-CAM (E-N-CAM)--was investigated in the developing and adult mouse cerebellar cortex by immunohistology and immunocytology at the light and electron microscopic levels. E-N-CAM was never (from embryonic day 14 to postnatal day 15) detectable in the germinal zone of neuroblasts destined to form or forming the external granular layer and was only observed once small cerebellar interneurons had become postmitotic before the beginning of granule cell migration. Granule cells expressed E-N-CAM on cell bodies, axons, and leading and trailing processes also during migration but ceased to reveal detectable levels of E-N-CAM at the end of migration after having reached their final position in the internal granular layer. Other cerebellar cell types, such as Purkinje cells, Bergmann glia, astrocytes, oligodendrocytes, and most prominently, stellate and basket cells, also expressed E-N-CAM, but became E-N-CAM-negative during the third and fourth postnatal weeks, coinciding with overt cessation of cerebellar histogenesis. Thus, except for neuroblasts, E-N-CAM appeared characteristic of growing and moving cellular structures, in agreement with the notion that the highly polysialylated form of N-CAM is less adhesive than the adult form.

Animals↗
PubMed1994

Noradrenergic innervation of vasopressin- and oxytocin-containing neurons in the hypothalamic paraventricular nucleus of the macaque monkey: quantitative analysis using double-label immunohistochemistry and confocal laser microscopy.

Previous reports on the rat and monkey hypothalamus have revealed a dense noradrenergic innervation within the hypothalamic paraventricular nucleus as assessed by dopamine-beta-hydroxylase immunohistochemistry. These single-label analyses were unable to delineate the cellular structures which receive this catecholaminergic innervation. Double-label preparations in the rat hypothalamic paraventricular nucleus have demonstrated synaptic interactions between noradrenergic varicosities and magnocellular neurons. However, the density and distribution of varicosities contacting chemically identified magnocellular neurons have not been assessed at the light or electron microscopic level. In this report, single-label immunohistochemistry was used to assess the morphology and distribution of vasopressin- and oxytocin-immunoreactive neurons within the macaque hypothalamic paraventricular nucleus. In addition, double-label immunohistochemistry was combined with confocal laser scanning microscopy to quantify the number of dopamine-beta-hydroxylase-immunoreactive varicosities in apposition to magnocellular neurons expressing vasopressin or oxytocin immunoreactivity. The morphology of chemically identified neurons was also compared to magnocellular neurons in the monkey hypothalamic paraventricular nucleus which were filled with Lucifer Yellow in order to assess the somatodendritic labeling of the immunohistochemical preparation. Qualitative assessment of immunohistochemically identified magnocellular cells indicated that vasopressin- and oxytocin-containing neurons are observed throughout the rostrocaudal extent of the monkey hypothalamic paraventricular nucleus, demarcating this structure from the surrounding anterior hypothalamus. The distribution of the two nonapeptides is complementary, with vasopressin-immunoreactive neurons having a greater somal volume and located in a more medial aspect of the mid and caudal hypothalamic paraventricular nucleus relative to oxytocin-immunoreactive perikarya. For the double-label preparations, a series of confocal optical sections was assessed through the total somal volume of vasopressin- and oxytocin-immunoreactive neurons along with the corresponding dopamine-beta-hydroxylase-immunoreactive varicosities in the same volume of tissue, generating a varicosity-to-neuron ratio which was further characterized morphologically to assess afferent input to the soma and proximal dendrites. Quantitative analysis revealed that vasopressin-immunoreactive neurons received approximately two thirds of their dopamine-beta-hydroxylase-immunoreactive varicosities in apposition to the proximal dendrites and one third in apposition to the somata. Furthermore, vasopressin-immunoreactive neurons received a greater innervation density than oxytocin-immunoreactive neurons, which did not have a differential distribution of varicosities on the proximal dendrites and somata. The distribution of dopamine-beta-hydroxylase-immunoreactive afferents on magnocellular neurons in the hypothalamic paraventricular nucleus may reflect a physiological role of this circuit in terms of preferential release of vasopressin from magnocellular neurons upon noradrenergic stimulation.

Animals↗
PubMed2005

Kinetics of the initial steps of G protein-coupled receptor-mediated cellular signaling revealed by single-molecule imaging.

We report on an in vivo single-molecule study of the signaling kinetics of G protein-coupled receptors (GPCR) performed using the neurokinin 1 receptor (NK1R) as a representative member. The NK1R signaling cascade is triggered by the specific binding of a fluorescently labeled agonist, substance P (SP). The diffusion of single receptor-ligand complexes in plasma membrane of living HEK 293 cells is imaged using fast single-molecule wide-field fluorescence microscopy at 100 ms time resolution. Diffusion trajectories are obtained which show intra- and intertrace heterogeneity in the diffusion mode. To investigate universal patterns in the diffusion trajectories we take the ligand-binding event as the common starting point. This synchronization allows us to observe changes in the character of the ligand-receptor-complex diffusion. Specifically, we find that the diffusion of ligand-receptor complexes is slowed down significantly and becomes more constrained as a function of time during the first 1000 ms. The decelerated and more constrained diffusion is attributed to an increasing interaction of the GPCR with cellular structures after the ligand-receptor complex is formed.

Cell Line↗
PubMed1988

A rapid flow cytometric method for bivariate bromodeoxyuridine/DNA analysis using simultaneous proteolytic enzyme digestion and acid denaturation.

This report describes an immunocytochemical procedure for the simultaneous quantification of bromodeoxyuridine (BrdUrd) incorporated into cellular DNA and total DNA content in individual cells in suspension. Improvement of existing methods was achieved by combining acid denaturation and proteolytic enzyme digestion (0.2 mg/ml pepsin in 2N HCl for 30 min at room temperature). Acid denaturation preceded by enzyme digestion resulted in a large amount of debris and the occurrence of naked nuclei. In contrast, the simultaneous denaturation/protein digestion procedure did not damage the cellular structure, is rapid and reproducible, and has cell recoveries of more than 85%. Although experimental conditions were tested on human cultured keratinocytes, this method also appeared applicable to bone marrow cells and cells obtained from solid tissues.

Bone Marrow↗
PubMed2006

Static magnetic fields affect cell size, shape, orientation, and membrane surface of human glioblastoma cells, as demonstrated by electron, optic, and atomic force microscopy.

BACKGROUND: It is common knowledge that static magnetic fields (SMF) do not interact with living cells; thus, fewer studies of SMF compared with variable magnetic fields are carried out. However, evidence demonstrated that SMF affect cellular structures. To investigate the effect of exposure to increasing doses of SMF on cell morphology, human glioblastoma cells were exposed to SMF ranging between 80 and 3,000 G (8 and 300 mT). METHODS: Cell morphology of human glioblastoma cells, derived from a primary culture, was studied by electron and optic microscopy. FITC-phalloidin staining of actin filaments was also investigated. Finally, cell surface structure changes were detected by atomic force microscopy. RESULTS: Scanning electron microscopy demonstrated a dose-dependent cell shape modification, progressive cell detachment, loss of the long villi, and appearance of membrane roughness and blebs. FITC-phalloidin staining confirmed the villi retention and cell dimension decrease. At 3,000 G, the appearance of apoptotic morphology was also observed by transmission electron microscopy. Cell exposed to SMF showed different orientation and alignment when compared with nonexposed cells. The atomic force microscopy of the exposed cells' membrane surfaces demonstrated the disappearance of the ordered surface ripples and furrows typical of the unexposed cells, and the occurrence of surface membrane corrugation at increasing dose exposure CONCLUSIONS: Our experimental procedures demonstrated that exposure to SMF affects not only cell size, shape, and orientation but also human glioblastoma cells' membrane surfaces.

Apoptosis↗