Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “PROTOPLASM”

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 865 records · Page 48Linked to original sources
PubMed1980

Method for preparing cultures of central neurons: cytochemical and immunochemical studies.

We report a simplified method for culturing fetal central nervous system cells predominantly inducing neurons that grow, differentiate, and live in vitro for as long as 10 weeks. These central nervous system cells form a confluent cell culture in which about 80% of the cells are fully differentiated neurons producing interconnecting axons and dendrite processes and live upon a sparse underlying population of fibrillary and protoplasmic astrocytes, oligodendrocytes, and fibroblasts. Morphological and cytochemical characteristics of these cell types, based on immunofluorescent cell specific markers and silver staining of neurons, are presented.

Animals↗
PubMed1981

Fracture-label:O cytochemistry of freeze-fracture faces in the erythrocyte membrane.

A method--"fracture label"--is described for the cytochemical labeling of the membrane faces produced by freeze-fracture. Human erythrocytes embedded in a crosslinked matrix are frozen, fractured in liquid nitrogen, thawed, labeled, and cut into thin sections. Electron microscope observation of the fracture faces shows preferential partition of concanavalin A binding sites with the inner half of the membrane. This signifies that, during freeze-fracture, binding sites are dragged from the outer surface across the outer ("exoplasmic") half of the membrane and retained on the protoplasmic fracture face (face P). The fracture process results in exposure of new anionic sites on face P. Fracture-label can be applied to the cytochemical characterization of the cellular components exposed by freeze-fracture of isolated cells and tissues.

Colloids↗
PubMed1982

Immunocytochemical demonstration of monoamine oxidase B in brain astrocytes and serotonergic neurons.

An antiserum to monoamine oxidase B (MAO-B) was used to define the distribution of this metabolic enzyme in the adult rat brain immunocytochemically. MAO-B is specifically located in two major central nervous system cell classes, astrocytes and serotonin-containing neurons. Double-immunofluorescence experiments using antisera to glial fibrillary acidic protein and MAO-B showed that both protoplasmic and fibrillary astrocytes throughout the brain contain MAO-B, whereas oligodendrocytes do not contain the enzyme. Areas lacking a blood-brain barrier, such as the specialized circumventricular organs, also contain MAO-B-positive cells. A double-immunofluorescence experiment using antisera to serotonin and MAO-B enabled the positive identification of neurons containing both molecules. The catecholamine-containing neurons of the brain did not contain detectable amounts of MAO-B. The specific distribution of MAO-B in the adult central nervous system indicates that the role of MAO-B in monoamine metabolism may be more specifically defined than previously believed.

Animals↗
PubMed1986

Loss of intercalated membrane particles by treatment with phorbols.

Because brief exposure to phorbol esters renders normal cells vulnerable to deformation and cytolysis by lymphocytes, it was postulated that these tumor promoters might cause a hitherto unrecognized physical alteration in membrane architecture. To investigate this possibility, four tissue culture cell lines (K-562 erythroleukemia cells, melanoma cells, N1121 adult fibroblasts, and normal fetal fibroblasts) and three blood cell types (lymphocytes, monocytes, and platelets) were subjected to freeze-fracture analysis before and after brief treatment with phorbol myristate acetate. Phorbol myristate acetate caused a 50% reduction of intramembranous particles associated with the external leaflet (E face) of the plasma membrane of every cell except platelets. In contrast, no change in size or number of intramembranous particles associated with the protoplasmic membrane leaflet (P face) was evident. Since the platelet membrane is known to be turned "inside out," as regards the partition coefficient of the intramembranous particles, the disparity between the results obtained with platelets and other cells may serve to determine the nature of intramembranous particles affected by phorbols. Also, since phorbols affect primarily glycolipids and/or glycoproteins anchored in the external membrane leaflet, these findings may provide a useful tool for future exploration of membrane structure.

Cell Membrane↗
PubMed1987

Localization of beta-adrenergic receptors on differentiated cells of the central nervous system in culture.

Hypothalamic neurons and cortical protoplasmic (type 1) astrocytes in dissociated cultures of rat brain express binding sites for antibodies specific for epitopes related to the beta-adrenergic receptor. Undifferentiated glial progenitor cells of the rat optic nerve do not detectably bind these antibodies, but both of the progeny [oligodendroglia and process-bearing (type 2) astrocytes] express immunologically identified beta-adrenergic receptors. Levels of receptor expression are variable: not all cells of each type express receptors nor is expression uniform on a given cell. The data suggest that cells of the central nervous system express beta-adrenergic receptors but that levels of expression may be determined by the differentiated state of the cells.

Animals↗
PubMed1987

Brief occurrence of a population of presynaptic intramembrane particles coincides with transmission of a nerve impulse.

Small pieces of Torpedo electric organ were cryofixed at 1-ms time intervals in a liquid medium at -190 degrees C before, during, and after the passage of a single nerve impulse. In contrast to studies using this or other preparations, these experiments were done without 4-aminopyridine or other drugs that potentiate transmitter release. Freeze-fracture replicas were made from the most superficial layers of the tissue, where the rate of cooling was rapid enough to retain ultrastructure in the absence of chemical fixation. We found that the transmission of an impulse was accompanied by the momentary appearance of a population of large intramembrane particles in both the protoplasmic (P) and the external (E) leaflets of the presynaptic plasma membrane. The change was very brief, appearing soon after the stimulus artifact. It lasted for 2-3 ms. Large pits denoting vesicle openings at the presynaptic membrane were found in a small proportion of nerve terminals; their number did not increase during transmission of the nerve impulse. Reducing the temperature from 16 to 5 degrees C slowed the time course of both the electrophysiological response and the change in intramembrane particles. The number of large particles did not increase when stimulation was applied in a low-Ca medium, a condition where the nerve terminals were still depolarized by the action potential but did not release the neurotransmitter. From these and other observations, we conclude that this transient change of intramembrane particles is closely linked to the mechanism of acetylcholine release at the nerve-electroplaque junction.

Animals↗
PubMed1995

D-serine, an endogenous synaptic modulator: localization to astrocytes and glutamate-stimulated release.

Using an antibody highly specific for D-serine conjugated to glutaraldehyde, we have localized endogenous D-serine in rat brain. Highest levels of D-serine immunoreactivity occur in the gray matter of the cerebral cortex, hippocampus, anterior olfactory nucleus, olfactory tubercle, and amygdala. Localizations of D-serine immunoreactivity correlate closely with those of D-serine binding to the glycine modulatory site of the N-methyl-D-aspartate (NMDA) receptor as visualized by autoradiography and are inversely correlated to the presence of D-amino acid oxidase. D-Serine is enriched in process-bearing glial cells in neuropil with the morphology of protoplasmic astrocytes. In glial cultures of rat cerebral cortex, D-serine is enriched in type 2 astrocytes. The release of D-serine from these cultures is stimulated by agonists of non-NMDA glutamate receptors, suggesting a mechanism by which astrocyte-derived D-serine could modulate neurotransmission. D-Serine appears to be the endogenous ligand for the glycine site of NMDA receptors.

Animals↗
PubMed1996

Muramic lactam in peptidoglycan of Bacillus subtilis spores is required for spore outgrowth but not for spore dehydration or heat resistance.

Bacterial endospores derive much of their longevity and resistance properties from the relative dehydration of their protoplasts. The spore cortex, a peptidoglycan structure surrounding the protoplasm, maintains, and is postulated to have a role in attaining, protoplast dehydration. A structural modification unique to the spore cortex is the removal of all or part of the peptide side chains from the majority of the muramic acid residues and the conversion of 50% of the muramic acid to muramic lactam. A mutation in the cwlD gene of Bacillus subtilis, predicted to encode a muramoyl-L-alanine amidase, results in the production of spores containing no muramic lactam. These spores have normally dehydrated protoplasts but are unable to complete the germination/ outgrowth process to produce viable cells. Addition of germinants resulted in the triggering of germination with loss of spore refractility and the release of dipicolinic acid but no degradation of cortex peptidoglycan. Germination in the presence of lysozyme allowed the cwlD spores to produce viable cells and showed that they have normal heat resistance properties. These results (i) suggest that a mechanical activity of the cortex peptidoglycan is not required for the generation of protoplast dehydration but rather that it simply serves as a static structure to maintain dehydration, (ii) demonstrate that degradation of cortex peptidoglycan is not required for spore solute release or partial spore core rehydration during germination, (iii) indicate that muramic lactam is a major specificity determinant of germination lytic enzymes, and (iv) suggest the mechanism by which the spore cortex is degraded during germination while the germ cell wall is left intact.

Bacillus subtilis↗
PubMed2000

Pentameric assembly of a neuronal glutamate transporter.

Freeze-fracture electron microscopy was used to study the structure of a human neuronal glutamate transporter (EAAT3). EAAT3 was expressed in Xenopus laevis oocytes, and its function was correlated with the total number of transporters in the plasma membrane of the same cells. Function was assayed as the maximum charge moved in response to a series of transmembrane voltage pulses. The number of transporters in the plasma membrane was determined from the density of a distinct 10-nm freeze-fracture particle, which appeared in the protoplasmic face only after EAAT3 expression. The linear correlation between EAAT3 maximum carrier-mediated charge and the total number of the 10-nm particles suggested that this particle represented functional EAAT3 in the plasma membrane. The cross-sectional area of EAAT3 in the plasma membrane (48 +/- 5 nm(2)) predicted 35 +/- 3 transmembrane alpha-helices in the transporter complex. This information along with secondary structure models (6-10 transmembrane alpha-helices) suggested an oligomeric state for EAAT3. EAAT3 particles were pentagonal in shape in which five domains could be identified. They exhibited fivefold symmetry because they appeared as equilateral pentagons and the angle at the vertices was 110 degrees. Each domain appeared to contribute to an extracellular mass that projects approximately 3 nm into the extracellular space. Projections from all five domains taper toward an axis passing through the center of the pentagon, giving the transporter complex the appearance of a penton-based pyramid. The pentameric structure of EAAT3 offers new insights into its function as both a glutamate transporter and a glutamate-gated chloride channel.

Amino Acid Transport System X-AG↗
PubMed1996

Kinetics and specificity of a H+/amino acid transporter from Arabidopsis thaliana.

The amino acid transporter AAP1/NAT2 recently cloned from Arabidopsis thaliana was expressed in Xenopus oocytes, and we used electrophysiological, radiotracer flux, and electron microscopic methods to characterize the biophysical properties, kinetics, and specificity of the transporter. Uptake of alanine was H(+)-dependent increasing from 14 pmol/oocyte/h at 0.032 microM H+ to 370 pmol/oocyte/h at 10 microM H+. AAP1 was electrogenic; there was an amino acid-induced depolarization of the oocyte plasma membrane and net inward currents through the transporter due to the transport of amino acids favoring neutral amino acids with shortside chains. The maximal current (imax) for alanine, proline, glutamine, histidine, and glutamate was voltage and [H+]o-dependent. Similarly, the imaxH was voltage and [amino acid]o-dependent. The imax for both H+ and amino acid were dependent on the concentrations of their respective cosubstrates, suggesting that both ligands bind randomly to the transporter. The K0.5 of the transporter for amino acids decreased as [H+]o increased and was lower at negative membrane potentials. The K0.5 for H+ was relatively voltage-independent and decreased as [amino acid]o increased. This positive cooperativity suggests that the transporter operates via a simultaneous mechanism. The Hill coefficients n for amino acids and H+ were > 1, suggesting that the transporter has more than one binding site for both H+ and amino acid. Freeze-fracture electron microscopy was used to estimate the number of transporters expressed in the plasma membrane of oocytes. The density of particles on the protoplasmic face of the plasma membrane of oocytes expressing AAP1 increased approximately 5-fold above water-injected controls and corresponded to a turnover number 350 to 800 s-1.

Amino Acid Transport Systems↗
PubMed1997

Five transmembrane helices form the sugar pathway through the Na+/glucose cotransporter.

To test the hypothesis that the C-terminal half of the Na+/glucose cotransporter (SGLT1) contains the sugar permeation pathway, a cDNA construct (C5) coding for rabbit SGLT1 amino acids 407-662, helices 10-14, was expressed in Xenopus oocytes. Expression and function of C5 was followed by Western blotting, electron microscopy, radioactive tracer, and electrophysiological methods. The C5 protein was synthesized in 20-fold higher levels than SGLT1. The particle density in the protoplasmic face of the oocyte plasma membrane increased 2-fold after C5-cRNA injection compared with noninjected oocytes. The diameters of the C5 particles were heterogeneous (4.8 +/- 0.3, 7.1 +/- 1.2, and 10.3 +/- 0.8 nm) in contrast to the endogenous particles (7.6 +/- 1.2 nm). C5 increased the alpha-methyl-D-glucopyranoside (alphaMDG) uptake up to 20-fold above that of noninjected oocytes and showed an apparent K0.5alphaMDG of 50 mM and a turnover of approximately 660 s-1. Influx was independent of Na+ with transport characteristics similar to those of SGLT1 in the absence of Na+: 1) selective (alphaMDG > D-glucose > D-galactose >> L-glucose approximately D-mannose), 2) inhibited by phloretin, KiPT = approximately 500 microM, and 3) insensitive to phlorizin. These results indicate that C5 behaves as a specific low affinity glucose uniporter. Preliminary studies with three additional constructs, hC5 (the human equivalent of C5), hC4 (human SGLT1 amino acids 407-648, helices 10-13), and hN13 (amino acids 1-648, helices 1-13), further suggest that helices 10-13 form the sugar permeation pathway for SGLT1.

Animals↗
PubMed1997

Thyroid Na+/I- symporter. Mechanism, stoichiometry, and specificity.

The rat thyroid Na+/I- symporter (NIS) was expressed in Xenopus laevis oocytes and characterized using electrophysiological, tracer uptake, and electron microscopic methods. NIS activity was found to be electrogenic and Na+-dependent (Na+ >> Li+ >> H+). The apparent affinity constants for Na+ and I- were 28 +/- 3 mM and 33 +/- 9 microM, respectively. Stoichiometry of Na+/anion cotransport was 2:1. NIS was capable of transporting a wide variety of anions (I-, ClO3-, SCN-, SeCN-, NO3-, Br-, BF4-, IO4-, BrO3-, but perchlorate (ClO4-) was not transported. In the absence of anion substrate, NIS exhibited a Na+-dependent leak current (approximately 35% of maximum substrate-induced current) with an apparent Na+ affinity of 74 +/- 14 mM and a Hill coefficient (n) of 1. In response to step voltage changes, NIS exhibited current transients that relaxed with a time constant of 8-14 ms. Presteady-state charge movements (integral of the current transients) versus voltage relations obey a Boltzmann relation. The voltage for half-maximal charge translocation (V0.5) was -15 +/- 3 mV, and the apparent valence of the movable charge was 1. Total charge was insensitive to [Na+]o, but V0.5 shifted to more negative potentials as [Na+]o was reduced. NIS charge movements are attributed to the conformational changes of the empty transporter within the membrane electric field. The turnover rate of NIS was >/=22 s-1 in the Na+ uniport mode and >/=36 s-1 in the Na+/I- cotransport mode. Transporter density in the plasma membrane was determined using freeze-fracture electron microscopy. Expression of NIS in oocytes led to a approximately 2. 5-fold increase in the density of plasma membrane protoplasmic face intramembrane particles. On the basis of the kinetic results, we propose an ordered simultaneous transport mechanism in which the binding of Na+ to NIS occurs first.

Animals↗
PubMed1999

Number of subunits comprising the epithelial sodium channel.

The human epithelial sodium channel (hENaC) is a hetero-oligomeric complex composed of three subunits, alpha, beta, and gamma. Understanding the structure and function of this channel and its abnormal behavior in disease requires knowledge of the number of subunits that comprise the channel complex. We used freeze-fracture electron microscopy and electrophysiological methods to evaluate the number of subunits in the ENaC complex expressed in Xenopus laevis oocytes. In oocytes expressing wild-type hENaC (alpha, beta, and gamma subunits), clusters of particles appeared in the protoplasmic face of the plasma membrane. The total number of particles in the clusters was consistent with the whole-cell amiloride-sensitive current measured in the same cells. The size frequency histogram for the particles in the clusters suggested the presence of an integral membrane protein complex composed of 17 +/- 2 transmembrane alpha-helices. Because each ENaC subunit has two putative transmembrane helices, these data suggest that in the oocyte plasma membrane, the ENaC complex is composed of eight or nine subunits. At high magnification, individual ENaC particles exhibited a near-square geometry. Functional studies using wild-type alphabeta-hENaC coexpressed with gamma-hENaC mutants, which rendered the functional channel differentially sensitive to methanethiosulfonate reagents and cadmium, suggested that the functional channel complex contains more than one gamma subunit. These data suggest that functional ENaC consists of eight or nine subunits of which a minimum of two are gamma subunits.

Animals↗
PubMed2000

Correlation of tight junction morphology with the expression of tight junction proteins in blood-brain barrier endothelial cells.

Endothelial cells of the blood-brain barrier form complex tight junctions, which are more frequently associated with the protoplasmic (P-face) than with the exocytoplasmic (E-face) membrane leaflet. The association of tight junctional particles with either membrane leaflet is a result of the expression of various claudins, which are transmembrane constituents of tight junction strands. Mammalian brain endothelial tight junctions exhibit an almost balanced distribution of particles and lose this morphology and barrier function in vitro. Since it was shown that the brain endothelial tight junctions of submammalian species form P-face-associated tight junctions of the epithelial type, the question of which molecular composition underlies the morphological differences and how do these brain endothelial cells behave in vitro arose. Therefore, rat and chicken brain endothelial cells were investigated for the expression of junctional proteins in vivo and in vitro and for the morphology of the tight junctions. In order to visualize morphological differences, the complexity and the P-face association of tight junctions were quantified. Rat and chicken brain endothelial cells form tight junctions which are positive for claudin-1, claudin-5, occludin and ZO-1. In agreement with the higher P-face association of tight junctions in vivo, chicken brain endothelia exhibited a slightly stronger labeling for claudin-1 at membrane contacts. Brain endothelial cells of both species showed a significant alteration of tight junctions in vitro, indicating a loss of barrier function. Rat endothelial cells showed a characteristic switch of tight junction particles from the P-face to the E-face, accompanied by the loss of claudin-1 in immunofluorescence labeling. In contrast, chicken brain endothelial cells did not show such a switch of particles, although they also lost claudin-1 in culture. These results demonstrate that the maintenance of rat and chicken endothelial barrier function depends on the brain microenvironment. Interestingly, the alteration of tight junctions is different in rat and chicken. This implies that the rat and chicken brain endothelial tight junctions are regulated differently.

Animals↗
PubMed2001

Muscle plasma membrane changes in dystrophin gene exon 52 knockout mouse.

Changes in muscle plasma membranes in mice lacking exon 52 of the dystrophin gene (mdx52 mouse) were studied using the freeze-fracture technique. The extensor digitorum longus (EDL) muscle plasma membrane of the mdx52 mouse at 8 weeks of age showed significantly increased caveola density (p < 0.05 by two-tailed t-test) and significantly decreased densities of intramembranous particles (IMPs), orthogonal arrays (OAs) and orthogonal array subunit particles (OASPs) (p < 0.05 by two-tailed t-test, p < 0.01 by Wilcoxon rank-sum test, p < 0.05 by two-tailed t-test, respectively) on the protoplasmic face when compared with those of control EDL muscles. These changes are more similar to those seen in DMD than those in the mdx mouse at the same age as reported previously. Thus, the gene abnormality in the different exon of the mouse dystrophin gene seems to induce somewhat different changes in the muscle plasma membrane.

Animals↗
PubMedDate not supplied

High resolution shadowing of Mycobacterium leprae.

Metal shadow casting techniques for transmission electron microscopic examination was used to determine the morphological characteristics of Mycobacterium leprae in untreated and treated patients. This technique is used to visualize bacterial surface structures by thermal evaporation of platinum alloys under moderate vacuum. This method gives a high contrast image at relatively low resolution and is useful for correlating micro-morphology quantitatively to early therapeutic effects of anti-leprosy drugs. Using these techniques in untreated cases, the surface structures of M. leprae were uniformly filled with relatively homogenous protoplasm surrounded by a cell wall. Most of the bacilli had thick cell walls with prominent banded and fibrous structures on the surface of the cell body. The cell wall was not detached in any of the solid bacilli in untreated cases. The bacilli varied in size and some of them were swollen in their mid-portion. Some bacilli were very short and completely filled with cytoplasm; therefore, these short bacilli were counted as solid bacilli in electron microscopic morphological index (EM-MI) determination. During treatment, mainly the cytoplasms of the bacilli were affected, and degeneration was observed. Ultrastructurally, the cytoplasm was shrunken and detached from the cell wall indicating mild degeneration. After moderate degeneration, the cytoplasm appeared fragmented. In advanced degeneration, all structures except the cell walls collapsed completely and no fibrous or band structures were visible on the surfaces of the cell walls. Therefore, these bacilli were counted as non-solid bacilli for EM-MI determination. This study shows that transmission electron shadowing gives more accurate counts than standard light microscopy of intact M. leprae bacilli in patient specimens.

Humans↗
PubMed1955

Studies on the fine structure of the mammalian testis. I. Differentiation of the spermatids in the cat (Felis domestica).

The differentiation of cat spermatids was studied in thin sections examined with the electron microscope. The Golgi complex of the spermatid consists of a central aggregation of minute vacuoles, partially surrounded by a lamellar arrangement of flattened vesicles. In the formation of the acrosome, one or more moderately dense homogeneous granules arise within vacuoles of the Golgi complex. The coalescence of these vacuoles and their contained granules gives rise to a single acrosomal granule within a sizable membrane-limited vacuole, termed the acrosomal vesicle. This adheres to the nuclear membrane and later becomes closely applied to the anterior two-thirds of the elongating nucleus to form a closed bilaminar head cap. The substance of the acrosomal granule occupies the narrow cleft between the membranous layers of the cap. The caudal sheath is comprised of many straight filaments extending backward from a ring which encircles the nucleus at the posterior margin of the head cap. Attention is directed to the frequent occurrence of pairs of spermatids joined by a protoplasmic bridge and the origin and possible significance of this relationship are discussed.

Acrosome↗
PubMed1955

On artifacts appearing in the histochemical fixation of glycogen.

1. Fixation artifacts associated with glycogen translocation are prevalent in tissues of parenchymatous type and scarce or non-existent in tissues of loose type. 2. Liver tissue treated with M/3 NaOH solution before fixation did not show an uneven distribution of glycogen. This was interpreted as indicating that the liver, a tissue of parenchymatous type, was changed, so to speak, into a loose type of tissue by alkali treatment. 3. The so called Alkohol-flucht of glycogen was produced in Yoshida's ascites tumor cells by a procedure which changed a loose type of tissue into a parenchymatous one, that is, by packing the tumor cells tightly. 4. The translocation of glycogen in cells appeared to occur when the fixatives penetrated the cells rapidly from a single direction, but failed to occur when the cells were attacked by the fixative from all directions. 5. In dried smears of Yoshida's ascites tumor cells and bone marrow cells, the glycogen particles are translocated to the peripheral regions of the cells, and coalesce there. The production of these artifacts is related in some way to the physicochemical properties of the protoplasm and plasma membrane of the cells.

Artifacts↗