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PubMed2004

Calcium-dependent open/closed conformations and interfacial energy maps of reconstituted hemichannels.

Using an atomic force microscope, we have studied three-dimensional molecular topography and calcium-sensitive conformational changes of individual hemichannels. Full-length (non-truncated) Cx43 hemichannels (connexons), when reconstituted in lipid bilayer, appear as randomly distributed individual particles and clusters. They show a lack of preferential orientation of insertion into lipid membrane; in a single bilayer, connexons with protrusion of either the extracellular face or the large non-truncated cytoplasmic face are observed. Extracellular domains of these undocked hemichannels are structurally different from hemichannels in the docked gap junctional plaques examined after their exposure by force dissection or chemical dissection. Calcium induced a reversible change in the extracellular pore diameter. Hemichannels imaged in a physiological buffer with 1.8 mm Ca(+2) had the pore diameter of approximately 1.8 nm, consistent with the closed channel conformation. Reducing Ca(+2) concentration to approximately 1.4, 1, and 0 mm, which changes hemichannels from the closed to open conformation, increased the pore diameter to approximately 2.5 nm for approximately 27, 74, and 100% of hemichannels, respectively. Thus, open/close probability of the hemichannel appears to be [Ca(2+)]-dependent. Computational analysis of the atomic force microscopy phase mode imaging reveals a significantly higher interfacial energy for open hemichannels that results from the interactions between the atomic force microscope probe and the hydrophobic domains. Thus, hydrophobic extracellular domains of connexins regulate calcium-dependent conformational changes.

Animals↗
PubMed2004

Actin filament organization in aligned prefusion myoblasts.

The organization of the actin cytoskeleton in prefusion aligning myoblasts is likely to be important for their shape and interaction. We investigated actin filament organization and polarity by transmission electron microscopy (TEM) in these cells. About 84% of the filaments counted were either found in a subplasmalemma sheet up to 0.5 microm thick that was aligned with the long axis of the cell, or in protrusions. The remaining filaments were found in the cytoplasm, where they were randomly orientated and not organized into bundles. The polarity of the subplasmalemma filaments changed progressively from one end of the cell to the other. At the ends of the cells and in protrusions, the majority of filaments were organized such that their barbed ends faced the tip of the protrusion. We did not find any actin filament bundles or stress fibres in these cells. Time-lapse phase microscopy demonstrated that aligned cells were still actively migrating at the time of our TEM observations, and their direction of movement was restricted to the long axis of the cell group. The ability of these cells to locomote actively in the absence of actin filament bundles suggests that in these cells the subplasmalemma actin sheet contributes not only to cell shape but also to cell locomotion.

Actin Cytoskeleton↗
PubMed1997

Circular ruffle formation in rat basophilic leukemia cells in response to antigen stimulation.

Rat basophilic leukemia cells have previously been described to undergo striking cell surface changes after IgE-mediated stimulation of histamine secretion, whereby the dorsal surface loses its microvilli and acquires characteristic wavy ruffles. We have found using scanning electron microscopy, phase contrast and immunofluorescence, that a proportion of these cells also exhibit the formation of circular membrane ruffles on their dorsal surface after exposure to an IgE-directed secretagogue; some cells also develop circular membrane ruffles following stimulation by phorbol myristate acetate or by calcium ionophore A23187. A flattened morphology appears to be linked to circular membrane ruffle formation in that these ruffles were found in areas of presumed cell spreading which are largely devoid of intermediate filaments and displaced to one side of the cell's nucleus, and they were not observed on rounded cells. This is in contrast to the wavy ruffles which are found on the entire cell surface including the region overlying the nucleus, and which are observed in rounded cells as well as spread cells. Circular ruffle formation and secretion are triggered by similar concentrations of antigen, but the circular ruffles are formed more slowly and only become abundant at times after most of the histamine has been released. The circular membrane ruffles showed no obvious association with endocytosis, as detected using fluorescein isothiocyanate-dextran as a fluid phase marker. The position of accumulation of endocytotic vesicles occurring subsequent to secretion was not found to be related to the circular membrane ruffles, but was observed around the nucleus. Circular membrane ruffles contain F-actin, and their formation is prevented by cytochalasin D. At least three types of myosin, types I, II and V are present and presumably play a role in circular ruffle formation.

Actin Cytoskeleton↗
PubMed1991

Oxidative injury induces influx-dependent changes in intracellular calcium homeostasis.

Understanding of the mechanisms of cell injury and cell death is fundamental to the understanding of both protection against and initiation of cell injury and cell death. We subjected primary cultures of proximal tubular epithelium (PTE) from adult rats to an exogenous oxidative stress, generated by xanthine/xanthine oxidase (X/XOD), and studied its effect on the concentration of cytosolic ionized calcium ([Ca2+]i) by means of digital imaging fluorescence microscopy (DIFM) using a cytosolic calcium probe, fura-2. Exposure to 25 mU/ml X/XOD caused notable increases in [Ca2+]i detectable within 15 sec and increasing to micromolar levels with time. Experiments with Ca(2+)-free medium containing ethylene glycol-bis(beta-aminoethyl ether)N,N,N',N'-tetraacetic acid (EGTA) showed that the increase of [Ca2+]i was due to influx from the extracellular space. Smaller and slower increases in [Ca2+]i were seen after exposure to lower concentrations of X/XOD (5 and 10 mU/ml). PTE injury and killing were assessed by measuring the release of cytosolic lactate dehydrogenase (LDH), exclusion of trypan blue, and observation of morphologic changes. Exposures to the 25 mU/ml concentration of X/XOD caused significant LDH release after 2 hr and correlated with trypan blue staining of exposed cells. Again, lesser concentrations of X/XOD resulted in a slower release of smaller amounts of LDH, and thus delayed trypan blue staining. Cytoplasmic bleb formation was seen by phase microscopy within minutes of exposure to 25 mU/ml, followed by cell rounding, retraction, and disintegration. Transmission electron microscopy revealed a progression of changes characteristic of lethal cell injury, beginning with dilatation of the endoplasmic reticulum, detachment of ribosomes, condensation of mitochondria, and chromatin clumping and terminating with mitochondrial swelling and formation of intramitochondrial flocculent densities. These studies clearly show that notable increases of [Ca2+]i precede both sub-lethal and lethal changes in rat PTE. These results indicate that interventions designed to minimize or to accelerate calcium entry could be of importance in cell preservation or cell killing, respectively, and therefore to therapeutic strategies for myocardial infarction, stroke, or shock in the former instance and for cancers in the latter.

Animals↗
PubMed1999

On the phase problem in electron microscopy: the relationship between structure factors, exit waves, and HREM images.

In electron microscopy, the word phase is used for different physical phenomena, including crystallographic structure-factor phases and the electron wave phases. This has resulted in great confusion, as to whether the phase information is present or lost when an image is recorded. The aim of this paper is to solve this phase confusion problem by studying the relationships between structure factors, exit waves, and high-resolution electron microscopy (HREM) images. Three approaches are taken. First phases at different stages of the imaging processes are compared analytically for a crystal that can be considered a weak phase object (WPO). Then these different phases are calculated by the multi-slice method based on dynamical diffraction theory, and their numerical values are compared. Finally, the validity of the theoretical description is checked by comparison with experimental data on a real crystal, Ti(2)S. It is demonstrated that it is possible to obtain accurate structure factor-phases directly from HREM images by crystallographic image processing. The two major methods for structure determination from HREM images-exit wave reconstruction and crystallographic image processing-are compared. It is shown that the information utilised by the two methods as well as the results are essentially the same.

Crystallography↗
PubMed2006

Effect of follicular size on in vitro developmental competence of oocytes and viability of embryos after transfer in the dromedary (Camelus dromedarius).

The aim of this work was to determine the effect of follicle size on camel oocyte quality as measured by developmental competence in vitro and in vivo. Ovaries from a local slaughterhouse were dissected to obtain two classes of follicle size: small (3-6 mm) and large (>6 mm) follicles. Quality of the oocytes was assessed after in vitro maturation (IVM), in vitro fertilization (IVF) and in vitro culture (IVC) of cumulus oocyte complexes (COCs). All cultures were done in four replicates at 38.5 degrees C, under 5% CO(2) and high humidity (>95%). Only COCs with cumulus and homogenous (dark) cytoplasm were used. The COCs were matured for 28 h in TCM-199 medium supplemented with 10% heat-treated fetal calf serum (FCS), 10 ng/mL EGF, and 250 microM cysteamine. Nuclear maturation rate for each class of follicle size was determined by contrast phase microscopy in a sample of COCs (n=30) denuded, fixed and stained with aceto-orcein. In vitro fertilization was performed using fresh semen (0.5 x 10(6)spermatozoa/mL in modified TALP-solution). Fertilized oocytes were cultured in mKSOMaa, under 5% O(2) and 90% N(2). The percentage of COCs reaching metaphase II (MII) after 28 h of maturation was 87% (26/30) and 73% (22/30) for oocytes originating from large and small follicles, respectively (P>0.1). The rate of total cleavage (two cells to blastocyst stage) was greater (P<0.05) for oocytes originating from large follicles (72%; 116/162) than for those derived from small follicles (59%; 140/237). The percentage of fertilized oocytes reaching the blastocyst stage was 35% (57/162) and 20% (48/237) for oocytes collected from large and small follicles, respectively (P<0.05). The viability of in vitro-produced hatched blastocyst from the two groups (15 from 3 to 6mm follicle size and 22 from follicles >6 mm) was assessed by transfer to synchronized recipients. None of the hatched blastocysts from small follicles resulted in a pregnancy whereas 68% (15/22) of the transferred hatched embryos from large follicles developed into a 25-day pregnancy. Of the resulting 15 pregnancies, 53% (n=8) aborted (five between 2 and 4 months and three between 5 and 7 months of pregnancy). The remaining seven pregnant females gave birth to normal healthy offsprings (four females and three males). The present study shows that dromedary oocytes developmental competence is acquired late during the final phase of follicular development and this developmental ability translates into greater pregnancy rates after transfer of in vitro produced hatched blastocysts.

Animals↗
PubMed1965

Structure and composition of the Bacillus anthracis capsule.

Avakyan, A. A. (Academy of Medical Sciences, Moscow, USSR), L. N. Katz, K. N. Levina, and I. B. Pavlova. Structure and composition of the Bacillus anthracis capsule. J. Bacteriol. 90:1082-1095. 1965.-Observations by various methods of light microscopy (phase contrast, dark-field, and fluorescence) revealed the complex structure of the Bacillus anthracis capsule, which changes regularly during the growth cycle of the culture. Special cytological methods of staining the capsule made it possible to study its fine structure, which is not revealed by negative staining with India ink. For example, the capsule shows a membranelike outline, fine transverse lines, and interruptions and transverse septa traversing the entire capsule. By using cytochemical methods, it was found that the capsule has a stratified structure and that the various layers of the capsule differ as to the value of the isoelectric point, metachromatic ability, sensitivity to various enzymes, and, consequently, chemical composition. It was thus shown that the membranelike outline of the capsule consists of peptides and neutral mucopolysaccharides. The middle part of the capsule consists of a complex of substances of both polysaccharide and protein nature, and the inner part consists of acid mucopolysaccharides. Observation of the capsular forms of B. anthracis by means of an electron microscope revealed differences in the osmiophilia and submicroscopic structure of the membranelike outline and the middle and inner parts of the capsule. Immunochemical studies conducted by the fluorescent-antibody method revealed localization of antigens in different parts of the capsule, and made it possible to differentiate the capsular antigens according to their serum-staining ability and according of their relations to enzymes, i.e., their chemical composition. This paper concerns the possibility of studying the fine structure of bacterial capsules in fixed preparations, and the differences and similarities of the antigens of the capsule and cell wall of B. anthracis and of the related species, B. megaterium.

Bacillus anthracis↗
PubMedDate not supplied

Rat erythrocyte morphological changes after gavage dosing with 2-butoxyethanol: a comparison with the in vitro effects of butoxyacetic acid on rat and human erythrocytes.

Rats exposed to 2-butoxyethanol (2-BE) develop hemolysis preceded by red blood cell swelling and shape changes. In this study effects on red blood cell morphology of dosing rats with 2-BE by gavage were compared with the effects of incubation of rat erythrocytes in vitro with the principal metabolite of 2-BE, butoxyacetic acid (BAA). Morphology was assessed by bright-field and phase microscopy of Wright's stained blood smears and glutaraldehyde-fixed cells suspended in plasma or buffer. In vivo exposure to 2-BE resulted in stomatocytosis and spherocytosis in blood smears and cup-shaped cells and spherocytosis in the fixed samples. In vitro incubation with BAA produced erythrocytes with cup shapes, spherocytosis and red blood cell ghosts in fixed samples. The stomatocytes observed in the blood smears appear to be the morphological equivalents of the cup-shaped cells observed in fixed samples. A variable degree of echinocytosis was observed in blood smears from animals exposed to 2-BE and in the in vitro experiments with BAA. Stomatocytes, cup-shaped cells, and spherocytes are the principal morphological features of erythrocytes from rats exposed to 2-BE or in vitro exposure to BAA. In comparison, human red blood cells incubated with up to 2.0 mM BAA exhibited none of the morphological changes observed in rat erythrocytes. 2-Butoxyethanol in vivo and BAA in vitro cause similar changes in rat red blood cell morphology, adding further evidence to support the primary role of BAA in the hemolytic effect of 2-BE exposure in the rat.

Animals↗
PubMed2006

Parasites of free-ranging black howler monkeys (Alouatta pigra) from Belize and Mexico.

Parasites are important members of the ecological web within which an animal lives, and can be used as indicators of ecosystem health. However, few baseline parasitological data are available for free-ranging animals, particularly for the black howler monkey (Alouatta pigra). In this study a total of 283 fecal samples were collected from 50 individually identified A. pigra during 2003 and 2004 and examined for parasites. The samples were processed using standard quantitative centrifugation concentration techniques, with sugar and zinc sulfate used as flotation media. The slides were examined using bright-field and phase microscopy. Antigen capture enzyme-linked immunosorbent assays (ELISAs) were used to detect protozoa. Four parasites were detected: 1) Controrchis biliophilus (Dicrocoeliidae), 2) Trypanoxyuris minutus (Oxyuridae), 3) Giardia sp. (Hexamitidae), and 4) Entamoeba sp. (Endamoebidae). Controrchis biliophilus was detected in 80% (wet season) and 81% (dry season) of the A. pigra samples; Trypanoxyuris minutus was detected in 8% (wet season) and 27% (dry season) of samples; and Giardia sp. was detected in 40% (wet season) and 27% (dry season) of samples. For the first time, DNA from Giardia sp.-positive fecal samples was extracted from A. pigra. Alouatta pigra individuals that lived near human settlements in Belize were infected with Giardia duodenalis (syn. G. lamblia, G. intestinalis) Assemblages A and B. These results suggest that G. duodenalis is transmitted from people and/or domestic animals to A. pigra.

Alouatta↗
PubMed1983

Toxic effect of phenytoin on developing cortical neurons in culture.

Studies were undertaken to determine the effect of chronic phenytoin exposure on developing neurons. Cerebral cortex from 16-day fetal mice was utilized to prepare primary dissociated cell cultures. Phenytoin was added to the cultures 10 days after plating and the cultures were harvested on day 17. Cortical cultures were assayed for neuronal cell number by phase microscopy and for high-affinity uptake of 3H-labeled gamma-aminobutyric acid (GABA) by both radioautography and scintillation spectrometry. Neuronal cell counts demonstrated a highly significant decrement in the number of neurons in cultures exposed to phenytoin at 15, 25, and 50 micrograms/ml. 3H-GABA-labeled neurons constituted 13% of the neurons present in both control and phenytoin-exposed cultures. These data indicate that phenytoin is toxic to cortical neurons in culture and that GABAergic neurons are affected to the same extent as the total neuronal population.

Animals↗
PubMed1980

Connective tissue activation. XV. Stimulation of glycosaminoglycan and DNA synthesis by a polymorphonuclear leukocyte factor.

Human synovial fibroblasts in culture have been stimulated to augment hyaluronate synthesis and glucose utilization by connective tissue activating peptides (CTAP) extracted from human spleen, lymphocytes, platelets, granulocytes, and tumour cells. The platelet-derived mediator CTAP-III also stimulated DNA synthesis in synovial fibroblasts, but CTAP-I from lymphocytes and spleen did not. The present study demonstrates the mitogenic potential of a granulocyte mediator (CTAP-PMN). Normal granulocytes were prepared with Ficoll-diatrizoate gradients, platelet contamination being estimated by phase microscopy and by radioimmunoassay for the platelet-specific protein, beta-thromboglobulin. CTAP-PMN preparations derived from 4 x 10(7) cells/ml stimulated culture 3H-thymidine incorporation to 3.56 +/- 1.32 (SD) times control levels. Although exposure of preparations to thiols reduced their mitogenicity, CTAP-PMN was relatively heat-stable. SDS gel electrophoresis of active fractions suggested a molecular weight between 12,700 and 15,700 daltons. In double immunodiffusion, antisera to CTAP-III showed no reactivity with CTAP-PMN. CTAP-PMN or other granulocyte factors capable of stimulating fibroblast DNA synthesis may play a role in chronic proliferative synovitis or in other settings where exudative inflammation is accompanied by connective tissue growth.

Connective Tissue↗
PubMed1993

Tipping of flagellar agglutinins by gametes of Chlamydomonas reinhardtii.

The migration of cross-linked agglutinins to the gametic flagellar tips (tipping) is a hallmark of the Chlamydomonas mating reaction. In this study, an assay was developed to analyze the kinetics and biological requirements for the tipping response: isolated flagella from mt- gametes of C. reinhardtii were allowed to agglutinate to the immotile flagella of pf-18 mt+ gametes, and their migration to the tips was monitored by phase microscopy. The tipping process is shown to require both adhesion and elevated levels of cAMP. The cAMP may activate tipping motors directly. In addition, cAMP stimulates the recruitment of agglutinins to the flagellar surface to replace those inactivated by adhesion. These results are compared with previous studies on the tipping of flagellar surface proteins cross-linked by soluble ligands, and an integrated model is presented.

1-Methyl-3-isobutylxanthine↗
PubMed1987

Characterization of a slowly proliferative cell along the oligodendrocyte differentiation pathway.

A single bipotential glial progenitor cell of newborn rat optic nerve (the O-2A progenitor) characterized by its reactivity with antibodies to surface gangliosides (A2B5) and the presence of vimentin, can grow in microcultures in conditions which favor this progenitor's differentiation into oligodendrocytes. We selected at 8 days larger clones derived from such bipolar progenitors which had steadily proliferated on a layer of Type-1 astrocytes during the first week. Clonal growth and ratio of progenitor cells to oligodendrocytes was measured over the next two weeks by phase microscopy and double immunofluorescence labeling for the specific markers A2B5, GC (galactocerebroside, a surface marker for differentiated oligodendrocytes), O4 (a glycolipid marker of oligodendrocytes and some progenitors), GFAP (an astrocyte specific intermediate filament protein) and vimentin. Two types of clones were identified: type A clones (the majority), which were still slowly expanding at three weeks, and type B clones (the minority), which had stopped proliferating during the second week. In type A clones, some cells became GC positive multipolar oligodendrocytes, while other multipolar cells remained GC negative for days and expressed A2B5 and O4, but not GFAP or vimentin. Type B clones contained only GC positive, vimentin negative oligodendrocytes, which were generated during the second week and then decreased in number because of their restricted lifespan. Type B clones only developed in the presence of insulin or neurons. The number of GC negative cells in type A clones increased when insulin or neurons were deleted.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1975

Centrifugal elutriation: separation of spermatogenic cells on the basis of sedimentation velocity.

Various types of cells from the testes of mice and hamsters were separated according to differences in sedimentation velocity by centrifugal elutriation, a counterflow centrifugation technique. Approximately 3 times 10(8) cells, prepared from six mouse testes or from one hanster testis, were separated into 11 fractions in less than two hours as compared to the 4--5 hours required for sedimentation at unit gravity ("Staput"). Fractions enriched in elongated spermatids and spermatozoa (100%), stages 1--8 spermatids (69%) and pachytene spermatocytes (58%) were obtained from mouse testis dispersions. Similarly enriched fractions were obtained from hamster cells. A single fraction enriched in stages 1--8 spermatids (mouse) was prepared in less than 30 minutes. As many as 2 times 10(9) cells were separated in a single procedure. Spermatogenic cells exhibited no evidence of structural damage with trypan blud and phase microscopy, and recovery was essentially 100%. Centrifugal elutriation had no effect on sperm motility or on the plating efficiency of CHO cells.

Animals↗
PubMed2006

Analysis of changes in optical fibers during arc-fusion splicing by use of quantitative phase imaging.

A non-interferometric imaging technique in conjunction with Abel inversion is used to directly and quantitatively examine the changes in optical fibers due to the heating produced during arc-fusion splicing as a function of fusion arc parameters. Phase images in the vicinity of a fusion splice are obtained using Quantitative Phase Microscopy, allowing the refractive-index change to be reconstructed with high spatial resolution. This simple, nondestructive method confirms that, for a fixed arc current, while the fusion time increases, the refractive-index of both fiber cores within the fusion region decreases in magnitude, the core region broadens, and the axial gradient decreases.

Journal Article↗
PubMed1983

Cell surface and cytoskeletal antigens in cerebral cell cultures after chloroform-methanol delipidation.

Fixation and in situ total delipidation of nerve cells in monolayer cultures have been used for immunofluorescence visualization of cellular antigens. Cells grown on poly-L-lysine-coated coverslips are exposed to a chloroform/methanol (2/1 by vol) solvent mixture for 15 min at -65 degrees C. This step results in the fixation of cells and is accompanied by the complete extraction of cellular lipids. About 3.2% of the total 35S-methionine-labeled trichloroacetic acid-insoluble cellular material is extracted into the organic solvent mixture, demonstrating that aqueous soluble proteins are not significantly extracted under these conditions. A total loss of tetanus toxin binding sites, presumably reflecting the removal of polysialogangliosides from the cell surface, is observed. The accessibility of antibodies against tubulin and against intermediate filaments after this treatment has been investigated. Visualization of these cytoskeletal elements by indirect immunofluorescence and by phase microscopy suggests that the removal of cellular lipids does not affect the apparent cytoarchitecture and that extraction with these organic solvents does not interfere with the staining of tubulin and intermediate filaments, the latter being exclusively present in nonneuronal cells. The advantage of this method of cell fixation and lipid extraction for immunofluorescence of cells in monolayer culture is discussed.

Animals↗