bluuebunny/crossref_metadata_2025 huggingface dataset:
Characterization of nuclear membranes and endoplasmic reticulum isolated from plant tissue.
Authors: Philipp E I , Franke W W , Keenan T W , Stadler J , Jarasch E D
Published: May 2004

Nuclei, nuclear membranes and rough endoplasmic reticulum (rER) were isolated from onion root tips and stems. Structural preservation and purity of the fractions was determined by electron microscopic and biochemical methods. Gross compositional data (protein, phospholipid, nonpolar lipids, sterols, RNA, DNA), phospholipid and fatty acid patterns, enzyme activities (ATPases, ADPase, IDPase, glucose-6-phosphatase, 5'-nucleotidase, acid phosphatase, and NADH- and NADPH-cytochrome C reductases), and cytochrome contents were determined. A stable, high salt-resistant attachment of some DNA with the nuclear membrane was observed as well as the association of some RNA with high salt-treated nuclear and rER membranes. The phospholipid pattern was identical for both nuclear and rER membranes and showed a predominance of lecithin (about 60%) and phosphatidyl ethanolamine (20-24%). Special care was necessary to minimize lipid degradation by phospholipases during isolations. Nonpolar lipids, mostly sterols and triglycerides, accounted for 35-45% of the membrane lipids. Sterol contents were relatively high in both membrane fractions (molar ratios of sterols to phospholipids ranged from 0.12 to 0.43). Sitosterol accounted for about 80% of the total sterols. Palmitic, oleic, and linoleic acids were the most prevalent acids in membrane-bound lipids as well as in storage lipids and occurred in similar proportions in phospholipids, triglycerides and free fatty acids of the membrane. About 80% of the fatty acids in membrane phospholipids and triglycerides were unsaturated. A cytochrome of the b5 type was characterized in these membranes, but P-450-like cytochromes could not be detected. Both NADH and NADPH-cytochrome c reductases were found in nuclear and rER membranes and appeared to be enriched in rER membranes. Among the phosphatases, Mg2+-ATPase and, to lesser extents, ADPase, IDPase and acid phosphatase activities occurred in the fractions, but significant amounts of monovalent ion-stimulated ATPase, 5'-nucleotidase and glucose-6-phosphatase activities did not. The results obtained emphasize that the close biochemical similarities noted between rER and nuclear membranes of animal cells extend to these fractions from plant cells.
Spindle and kinetochore morphology of Dictyostelium discoideum.
Authors: Moens P B
Published: May 2004

The metaphase spindle of haploid Dictyostelium discoideum (n = 7) is 2 mum long. It consists of some 20 microtubules which seem continuous between the spindle pole bodies and there are about 20 chromosomal microtubules at each end of the spindle. During anaphase the central spindle elongates and the chromosomal microtubules shorten. The spindle length and structure at this stage suggests that lengthening is caused by elongation as well as parallel sliding of the nonchromosomal microtubules. The nuclear envelope remains mostly intact during mitosis, and nuclear separation through medial constriction takes place when the spindle is 6 mum long. Cytokinesis occurs when the spindle is 10 mum long. At that time the kinetochores double in size. During interphase, the spindle pole body separates from the nucleus to a distance of 0.7 mum, and it returns at the onset of the next prophase when it becomes functionally double, thereby starting the formation of a central spindle. When comparing mitosis in the cellular slime molds Polysphondylium violaceum and D. discoideum, several similarities and some differences are apparent.
Mitotic chromosome condensation in the sperm nucleus during post fertilization maturation division in urechis eggs
Authors: Das NK , Barker C
Published: May 2004

Changes in the morphology of the sperm nucleus in the egg cytoplasm are mong the immediate events in nucleocytoplasmic interactions during early embryogenesis. Soon after its entrance into the egg cytoplasm, the sperm nucleus of various organisms increases in size with the transformation of condensed chromatin to a diffuse state, resembling the chromatin of an interphase nucleus (2, 13, 15, 16). This is followed by a close association or fusion of male and female pronuclei (2, 13, 15, 16). Cytoplasmic influences on nuclear morphology have also been demonstrated clearly in nuclear transplantation and cell fusion studies (10, 11). Reactivation of the nucleus, such as the transplanted brain nucleus in Xenopus egg cytoplasm or the hen erythrocyte nucleus in interphase cytoplasm of HeLa cells, is accompanied by nuclear enlargement and chromatin dispersion (10, 11). However, premature mitotic-like chromosome condensation takes place in the nuclei of sperm or interphase cells fused with mitotic cells (9, 12). Thus, chromosome dispersion and condensation seem to depend on the state of the cytoplasm in which the nucleus is present. These observations imply that the initial morphological changes in the sperm nucleus after fertilization may very well be dependent on the state of maturation of eggs at the time of sperm entry. Unfertilized eggs of Urechis caupo, a marine echiuroid worm, are stored at the diakinesis stage. These eggs complete maturation division after insemination and this is followed by fusion of male and female pronuclei (5, 8). Therefore, Urechis caupo is a suitable organism in which to study the response of the sperm nucleus to the changing state of the egg cytoplasm during and after postfertilization maturation division.
Fine filaments in lymphatic endothelial cells
Authors: Lauweryns JM , Baert J , De-Loecker W
Published: May 2004

Several and various types of cells contain fine cytoplasmic filaments closely resembling the myofilaments of muscle cells (2, 18, 23, 24). In many of these cells and especially when cultured, it has been demonstrated that some of these filaments react with heavy meromyosin (HMM) in the same way as do the actin filaments of muscle cells (3, 6 7). This suggests that these filaments may be actinoid and form part of a contractile system. As fine intracytoplasmic filaments do occur in lymphatic endothelial cells (2, 14), we undertook an electron microscope investigation of their fine structure and their reaction on incubation with HMM and EDTA. We postulated that lymphatic endothelial cells possess a contractile filamentous system to which these filaments belong.
Analysis of the thylakoid outer surface. Coupling factor is limited to unstacked membrane regions.
Authors: Miller K R , Staehelin L A
Published: May 2004

The structure of the spinach thylakoid outer surface has been examined by deepetching, a technique which exposes the true surfaces of biological membranes by sublimination of frozen dilute buffer. The membrane surface is covered with large (150 A average diameter) and small (90 A average diameter) particles. Approximately 30% of the large particles can be removed under conditions reported to selectively remove carboxydismutase from the membrane surface. The remaining large particles can be removed only under conditions which cause a loss of coupling factor activity. When purified coupling factor is readded to membranes from which all coupling factor activity has been removed, large particles reappear, indicating that they represent coupling factor molecules. Since the number of particles and the amount of ATPase activity in the reconstituted and control membranes were the same, coupling factor molecules may be attached to specific binding sites. Analysis of antibody labeling experiments, enzyme assays, and experiments involving the unstacking and restacking of thylakoid membranes indicate that coupling factor is excluded from regions of membrane stacking (grana) and is present only in unstacked membrane regions. The exclusion of coupling factor from grana, which are known to be centers of intense photosynthetic activity, strongly suggests that the mechanism coupling electron transport to photophosphorylation is indirect. In addition to the large and small particles, in some cases regularly spaced ridges are visible on the outer surface after unstacking. Coupling factor binding sites seem to be excluded from regions where these structures occur.
Mating in Chlamydomonas: a system for the study of specific cell adhesion. I. Ultrastructural and electrophoretic analyses of flagellar surface components involved in adhesion.
Authors: Snell W J
Published: May 2004

To determine the ultrastructural and biochemical bases for flagellar adhesiveness in the mating reaction in Chlamydomonas, gametic and vegetative flagella and flagellar membranes were studied by use of electron microscope and electrophoretic procedures. Negative staining with uranyl acetate revealed no differences in gametic and vegetative flagellar surfaces; both had flagellar membranes, flagellar sheaths, and similar numbers and distributions of mastigonemes. Freezecleave procedures suggested that there may be a greater density of intramembranous particles on the B faces of gametic flagellar membranes than on the B faces of vegetative flagellar membranes. Gamone, the adhesive material that gametes release into their medium, was demonstrated, on the basis of ultrastructural and biochemical analyses, to be composed of flagellar surface components, i.e., membrane vesicles and mastigonemes. Comparison of vegetative (nonadhesive) and gametic (adhesive) "gamones" by use of SDS polyacrylamide gel electrophoresis showed both preparations to be composed of membrane, mastigoneme, and some microtubule proteins, as well as several unidentified protein and carbohydrate-staining components. However, there was an additional protein of approximately 70,000 mol wt in gametic gamone which was not present in vegetative gamone. When gametic gamone was separated into a membrane and a mastigoneme fraction on CSCl gradients, only the membrane fraction had isoagglutinating activity; the mastigoneme fraction was inactive, suggesting that mastigonemes are not involved in adhesion.
Intercellular adhesive selectivity. II. Properties of embryonic chick liver cell-cell adhesion.
Authors: McGuire E J
Published: May 2004

Studies directed at understanding the molecular basis of liver cell homotypic adhesion are presented. An assay which measures the rate of adhesion of isotopically labeled (32PO4) embryonic chick liver cells to liver cell aggregates, described in a companion paper, has been used to investigate the problem of intercellular adhesive selectivity. Cation requirements, the effects of various inhibitors of metabolism and protein synthesis, of chelators (EDTA and EGTA), and the effects of temperature on liver cell adhesion are reported. Two mechanisms of inhibition of liver intercellular adhesion are suggested. One involves destruction of cell-surface adhesion receptors (sensitivity to proteases); the other is an energy-dependent step which may involve alterations in plasma membrane conformation and/or membrane fluidity. Finally, a model is suggested for liver cell-cell adhesion that incorporates the early tissue selectivity of intercellular adhesion previously reported, followed by a multistep process which leads to histogenic aggregation.
Morphological changes in the neuritic growth cone and target neuron during synaptic junction development in culture.
Authors: Rees R P , Bunge M B , Bunge R P
Published: May 2004

Our object was to characterize the morphological changes occurring in pre- and postsynaptic elements during their initial contact and subsequent maturation into typical synaptic profiles. Neurons from superior cervical ganglia (SCG) of perinatal rats were freed of their supporting cells and established as isolated cells in culture. To these were added explants of embryonic rat thoracic spinal cord to allow interaction between outgrowing cord neurites and the isolated autonomic neurons. Time of initial contact was assessed by light microscopy; at timed intervals thereafter, cultures were fixed for electron microscopy. Upon contact, growth cone filopodia became extensively applied to the SCG neuronal plasmalemma and manifested numerous punctate regions in which the apposing plasma membranes were separated by only 7-10 nm. The Golgi apparatus of the target neuron hypertrophied, and its production of coated vesicles increased. Similar vesicles were seen in continuity with the SCG plasmalemma near the close contact site; their apparent contribution of a region of postsynaptic membrane with undercoating was considered to be the first definitive sign of synapse formation. Tracer work with peroxidase and ferritin confirmed that the traffic of coated vesicles within the neuronal soma is largely from Golgi region to somal surface. Subsequent to the appearance of postsynaptic density, the form and content of the growth cone was altered by the loss of filopodia and the appearance of synaptic vesicles which gradually became clustered opposite the postsynaptic density. As the synapse matured, synaptic vesicles increased in number, cleft width and content increased, presynaptic density appeared, branched membranous reticulum became greatly diminished, and most lysosomal structures disappeared. Coated vesicles continued to be associated with the postsynaptic membrane at all stages of maturation. The incorporation of Golgi-derived vesicles into discrete regions of the cell membrane could provide the mechanism for confining specific characteristics of the neuronal membrane to the synaptic region.
Immunoferritin determination of the distribution of (Na+ + K+) ATPase over the plasma membranes of renal convoluted tubules. II. Proximal segment.
Authors: Kyte J
Published: May 2004

The distribution of (Na+ + K+) ATPase over the plasma membranes of the proximal convoluted tubule from canine renal cortex has been determined. Ultrathin frozen sections of this tissue were stained with rabbit antibodies to this enzyme and ferritin-conjugated goat antirabbit gamma-globulin. It is demonstrated that high concentrations of this enzyme uniformly line the intercellular spaces of this epithelium. The consequences of this observation are discussed in terms of the low resistant tight junctions of these tubules and the isotonic fluid transport which they support. Furthermore, antibodies to (Na+ + K+) ATPase recognize an antigen on the luminal surfaces of the tubules within the brush border. It is proposed that the enzyme is present in this region of the plasma membrane as well, although at much lower concentration. To further substantiate this conclusion, a brush border fraction has been purified from rabbit kidney and been shown to contain significant (Na+ + K+) ATPase. These results contradict earlier conclusions about the location of (Na+ + K+) ATPase in this tissue.
Studies of muscle proteins in embryonic myocardial cells of cardiac lethal mutant mexican axolotls (Ambystoma mexicanum) by use of heavy meromyosin binding and sodium dodecyl sulfate polyacrylamide gel electrophoresis.
Authors: Lemanski L F , Mooseker M S , Peachey L D , Iyengar M R
Published: May 2004

In the Mexican axolotl Ambystoma mexicanum recessive mutant gene c, by way of abnormal inductive processes from surrounding tissues, results in an absence of embryonic heart function. The lack of contractions in mutant heart cells apparently results from their inability to form normally organized myofibrils, even though a few actin-like (60-A) and myosin-like (150-A) filaments are present. Amorphous "proteinaceous" collections are often visible. In the present study, heavy meromyosin (HMM) treatment of mutant heart tissue greatly increases the number of thin filaments and decorates them in the usual fashion, confirming that they are actin. The amorphous collections disappear with the addition of HMM. In addition, an analysis of the constituent proteins of normal and mutant embryonic hearts and other tissues is made by sodium dodecyl sulfate (SDS) gel electrophoresis. These experiments are in full agreement with the morphological and HMM binding studies. The gels show distinct 42,000-dalton bands for both normal and mutant hearts, supporting the presence of normal actin. During early developmental stages (Harrison's stage 34) the cardiac tissues in normal and mutant siblings have indistinguishable banding patterns, but with increasing development several differences appear. Myosin heavy chain (200,000 daltons) increases substantially in normal hearts during development but very little in mutants. Even so the quantity of 200,000-dalton protein in mutant hearts is significantly more than in any of the nonmuscle tissues studied (i.e. gut, liver, brain). Unlike normal hearts, the mutant hearts lack a prominent 34,000-dalton band, indicating that if mutants contain muscle tropomyosin at all, it is present in drastically reduced amounts. Also, mutant hearts retain large amounts of yolk proteins at stages when the platelets have virtually disappeared from normal hearts. The morphologies and electrophoresis patterns of skeletal muscle from normal and mutant siblings are identical, confirming that gene c affects only heart muscle differentiation and not skeletal muscle. The results of the study suggest that the precardiac mesoderm in cardiac lethal mutant axolotl embryos initiates but then fails to complete its differentiation into functional muscle tissue. It appears that this single gene mutation, by way of abnormal inductive processes, affects the accumulation and organization of several different muscle proteins, including actin, myosin, and tropomyosin.
Glycine-specific synapses in rat spinal cord. Identification by electron microscope autoradiography.
Authors: Price D L , Stocks A , Griffin J W , Young A , Peck K
Published: May 2004

Glycine, an inhibitory transmitter in spinal cord, is taken up into specific nerve terminals by means of a unique high-affinity uptake system. In this study, [3H]glycine was directly microinjected into rat ventral horn in vivo and electron microscope autoradiography used to localize the label in various anatomic compartments. Quantiative analysis showed that [3H]glycine labeled a high proportion of axosomatic and axodendritic synapses which presumably act to inhibit spinal motor neurons.
Membrane specializations of dentritic spines and glia in the weaver mouse cerebellum: a freeze-fracture study.
Authors: Hanna R B , Hirano A , Pappas G D
Published: May 2004

Electron microscopy of thin-sectioned and freeze-fractured preparations of the cerebellum of the weaver mouse indicates that the dendritic spines are morphologically identical to those of their normal littermates. The weaver dendritic spines have been characterized as "unattached" since the synaptic input from the parallel fibers is absent (8-10). The entire region around the dendritic spines is taken up by astrocytic processes in the weaver. The outer fracture face of a normal dendritic spine contains aggregations of 10-nm wide particles in the immediate postsynaptic region. Similar particle aggregations occur in the unattached spines of the weaver. Freeze-fracture preparations reveal rectilinear arrays of particles, having a 7-nm center-to-center distance in the glial membranes. Rectilinear arrays are apparently distributed throughout the astrocyte membrane.
Presence of histones in Aspergillus nidulans.
Authors: Felden R A , Sanders M M , Morris N R
Published: May 2004

Five major histone proteins have been extracted from chromatin isolated from purified nuclei of the fungus, Aspergillus nidulans. These proteins had chromatographic properties which were similar to reference calf thymus histones and were purified to electrophoretic homegeneity by gel chromatography of Bio-Gel P10, Bio-Gel P60, and Sephadex G-100. Electrophoresis of these proteins in three different systems (urea-starch, urea-acetic acid polyacrylamide, and discontinuous SDS polyacrylamide) showed that the A. nidulans histones H3 and H4 were nearly identical to calf thymus H3 and H4 with respect to net charge and molecular weight criteria, whereas the fungal histones H1, H2a and H2b were similar but not identical to the corresponding calf thymus histones. Amino acid analysis of A. nidulans histones H2a, H2b, and H4 showed them to be closely related to the homologous calf thymus histones. The mobility patterns of A. nidulans ribosomal basic proteins in three different electrophoretic systems were distinctly different from those of the fungal histones.
Effect of thiol-oxidation of glutathione with diamide on corneal endothelial function, junctional complexes, and microfilaments.
Authors: Edelhauser H F , Van Horn D L , Miller P , Pederson H J
Published: May 2004

Intracellular-reduced glutathione (GSH) was removed by thiol-oxidation with diamide during in vitro perfusion of the corneal endothelium. By 15 min the normal mosaic-like pattern of the endothelial cells was disrupted by serpentine-like lines of cell separation at the cell juntions. After 45 min of perfusion, infividual clusters of cells formed cup-shaped islands. The resultant exposure of Descemet's membrane to the perfusion solution resulted in corneal swelling. Transmission electron microscopy revealed that the endothelial cells separated at the apical junctions and that the microfilaments in the apical cytoplasm of cells formed dense bands, whereas the other subcellular organelles were normal in appearance. The change in cellular shape may be due to loss of cellular adhesion which results in the condensation of the microfilaments or contraction of the microfilaments. The addition of glucose to the perfusate prevented the diamide effect, and the diamide effect could be reversed upon removal and perfusion of a glutathione bicarbonate Ringer's solution. These results suggest that the ratio of reduced to oxidized glutathione in the endothelial cells plays a role in the maintenance of the endothelial cell barrier function.
The role of actin in the temperature-dependent gelation and contraction of extracts of Acanthamoeba.
Authors: Pollard T D
Published: May 2004

The temperature-dependent assembly and the interaction of Acanthamoeba contractile proteins have been studied in a crude extract. A cold extract of soluble proteins from Acanthamoeba castellanii is prepared by homogenizing the cells in a sucrose-ATP-ethyleneglycol-bis-(beta-aminoethyl ether) N,N'-tetraacetic acid buffer and centrifuging at 136,000 g for 1 h. When this supernate of soluble proteins is warmed to room temperature, it forms a solid gel. Upon standing at room temperature, the gel slowly contracts and squeezes out soluble components. The rates of gelation and contraction are both highly temperature dependent, with activation energies of about 20 kcal per mol. Gel formation is dependent upon the presence of ATP and Mg++. Low concentrations of Ca++ accelerate the contractile phase of this phenomenon. The major protein component of the gel is actin. It is associated with myosin, cofactor, a high molecular weight protein tentatively identfied as actin-binding protein, and several other unidentified proteins. Actin has been purified from these gels and was found to be capable of forming a solid gel when polymerized in the presence of ATP, MgCl3, and KCL. The rate of purified actin polymerication is very temperature dependent and is accelerated by the addition of fragments of muscle actin filaments. These data suggest that Acanthamoeba contractile proteins have a dual role in the cell; they may generate the forces for cellular movements and also act as cytoskeletal elements by controlling the consistency of the cytoplasm.
Scanning electron microscopy of in vitro chemically transformed mouse embryo cells.
Authors: Malick L E , Langenbach R
Published: May 2004

A cloned nontumorigenic control cell line of C3H mouse embryo cells (C3H/1OT1/2CL8) and two cell lines derived from it by treatment in vitro with 7,12-dimethylbenz(a)anthracene (DMBA) or 3-methylcholanthrene (MCA) were studied by scanning electron microscopy. Confluent control cells were polygonal in shape and extensively flattened with smooth surfaces. Both in vitro transformants were pleomorphic to fusiform in shape, thicker than the control cells, and lacked contact inhibition. Microvilli of variable length and small marginal ruffles were characteristic surface alterations of the MCA-transformed cells, while blebs and numerous cytoplasmic strands extending between cells were typical of the DMBA transformant. Inoculation of the DMBA-transformed cells into C3H mice and re-establishment of cells from one of the subsequent fibrosarcomas in culture revealed an increased number of microvilli on the surface of the cells and an alteration in growth pattern. Other surface characteristics remained the same. A possible relationship between surface topography and outer membrane glycolipids is discussed.
Membrane flow during pinocytosis. A stereologic analysis.
Authors: Steinman R M , Brodie S E , Cohn Z A
Published: May 2004

HRP has been used as a cytochemical marker for a sterelogic analysis of pinocytic vesicles and secondary lysosomes in cultivated macrophages and L cells. Evidence is presented that the diaminobenzidine technique (a) detects all vaculoes containing encyme and (b) distinguishes between incoming pinocytic vesicles and those which have fused with pre-existing lysosomes to form secondary lososomes. The HRP reactive pinocytic vesicle spaces fills completely within 5 min after exposure to enzyme, while the secondary lysosome compartment is saturated in 45--60 min. The size distribution of sectioned (profile) vaculoe diameters was measured at equilibrium and converted to actual (spherical) dimensions using a technique modified from Dr. S. D. Wicksell. The most important findings in this study have to do with the rate at which pinocytosed fluid and surface membrane move into the cell and on their subsequent fate. Each minute macrophages form at least 125 pinocytic vesicles having a fractional vol of 0.43% of the cell's volume and a fractional area of 3.1% of the cell's surface area. The fractional volume and surface area flux rates for L cells were 0.05% and 0.8% per minute respectively. Macrophages and L cells thus interiorize the equivalent of their cell surface area every 33 and 125 min. During a 3-period, the size of the secondary lysosome compartment remains constant and represents 2.5% of the cell volume and 18% of the surface area. Each hour, therefore, the volume and surface area of incoming vesicles is 10 times greater than the dimensions of the secondary lysosomes in both macrophages and L cells. This implies a rapid reduction in vesicle size during the formation of the secondary lysosome and the egress of pinocytosed fluid from the vacuole and the cell. In addition, we postulate that membrane components of the vacuole are subsequently recycled back to the cell surface.
Functional significance of the variations in the geometrical organization of tight junction networks.
Authors: Hull B E , Staehelin L A
Published: May 2004

Using freeze-fracture techniques, we have examined the morpholog of tight junction networks found along the length of the alimentary tract of Xenopus laevis before and after metamorphosis. We have developed the hypothesis, based on these observations, that the geometrical organization of the network determined by the stress-induced shape changes normally experienced by the cells linked by the network. Consistent with this theory, tight junctions can be classified into two distinct types of network organization which differ in their response normal and experimentally induced stress conditions: (a) loosely interconnected networks which can stretch or compress extensively under tension, thereby adapting to stress changes in the tissue; and (b) evenly cross-linked networks which retain their basic morphology under normal stress conditions. The absorptive cells of the large intestine as well as the mucous cells of the gastrointestine or stomach are sealed by the first, flexible type of tight junction. The second type of junctional organization, the evenly cross-connected network, is found between absorptive cells of the small intestine and ciliated cells of the esophagus, and reflects in its constant morphology the relative stability of the apical region of both of these cell types. Networks intermediate between these two types arise when a cell which would normally form a lossely interconnected network borders a cell which tends to form a more evenly cross-linked network, as is found in the esophagus where ciliated and goblet cells adjoin. Despite the change in the animal's diet during metamorphosis from herbivorous to carnivorous, the basic gemetrical organization of the networks associated with each tissue of the alimentary tract remains the same.
Isolation and electrophoretic analysis of nucleoli, phenol-soluble nuclear proteins, and outer cyst walls from Acanthamoeba castellanii during encystation initiation.
Authors: Rubin R W , Hill M C , Hepworth P , Boehmer J
Published: May 2004

A technique is described for isolating nuceoli from Acanthamoeba castellanii. Nuclei isolated by a modification of the technique of F. J. Chlapowski and R. N. Band (1971) are sonicated in a surcrose-Tris-MgSO4-KC1-Triton X-100 buffer and centrifuged on a linear sucrose gradient extending from 1.3 M to 1.5 M with a 2.6 M cushion, at 41000 rpm for 90 min. The only apparent contaminants in the nucleolar preparation are outer cyst walls. A procedure is described for the isolation of chemically pure outer cyst walls, and a comparison of the proteins with the nucleolar preparation reveals that outer cyst walls represent negligible contaminants. The ultrastructure of these isolated nucleoli examined with transmission electron microscopy is found to be identical with that of nucleoli from whole cells, fixed in an identical manner. The 50 nucleolar proteins separated by SDS gel electrophoresis have been examined throughout the growth cycle of Acanthamoeba and into the strat of induced encystment, at which time 10 protein bands disappear, 11 bands are observed to decrease, and 8 are seen to increase in concentration. Phenol-soluble proteins are extracted from the nucleolus which correspond to 29 of the 50 nucleolar proteins, with 17 of these proteins corresponding to nucleolar proteins that change at the onset of encystment. Thes nucleolar proteins are also compared with those of rat liver nucleoli by gel electrophoresis, resulting in the observation that extremely few protein homologies exist between the two. Numerous quantitative and qualitative changes in the gel pattern of phenol-soluble nuclear proteins during early and late log phase growth and the onset of stationary phase were also observed.
Concanavalin A induces microtubule assembly and specific granule discharge in human polymorphonuclear leukocytes.
Authors: Hoffstein S , Soberman R , Goldstein I , Weissmann G
Published: May 2004

Human neutrophils stimulated by concanavalin A (Con A, 100 microng/ml) contained markedly enhanced numbers of microtubules and discharged peroxidase-negative (specific) but not peroxidase-position (azurophile) granules. Release of lysozyme from specific granules was dose and time dependent, could be inhibitied by alpha-methyl-D-mannoside, and enhanced by cytochalasin B. Many microtubules were associated with internalized plasma membrane bearing Con A binding sites.
In vivo induction of tight junction proliferation in rat liver.
Authors: Montesano R , Gabbiani G , Perrelet A , Orci L
Published: May 2004

The chronic administration of phalloidin induces an extensive development of tight junctions between rat hepatocytes. The junctional strands lose their predominantly parallel orientation with respect to the canalicular lumen and extend abluminally in irregular patterns which cover large membrane areas at considerable distance from the bile canaliculi. These changes indicate both proliferation and provide further evidence that these junctions are not permanent differentiations of the cell membrane.
Involvement of vesicle coat material in casein secretion and surface regeneration.
Authors: Franke W W , Lüder M R , Kartenbeck J , Zerban H , Keenan T W
Published: May 2004

The ultrastructure of the apical zone of lactating rat mammary epithelial cells was studied with emphasis on vesicle coat structures. Typical 40-60 nm ID "coated vesicles" were abundant, frequently associated with the internal filamentous plasma membrane coat or in direct continuity with secretory vesicles (SV) or plasma membrane proper. Bristle coats partially or totally covered membranes of secretory vesicles identified by their casein micelle content. This coat survived SV isolation. Exocytotic fusion of SV membranes and release of the casein micelles was observed. Frequently, regularly arranged bristle coat structures were identified in those regions of the plasma membrane that were involved in exocytotic processes. Both coated and uncoated surfaces of the casein-containing vesicles, as well as typical "coated vesicles", were frequently associated with microtubules and/or microfilaments. We suggest that coat materials of vesicles are related or identical to components of the internal coat of the surface membrane and that new plasma membrane and associated internal coat is produced concomitantly by fusion and integration of bristle coat moieties. Postexocytotic association of secreted casein micelles with the cell surface, mediated by finely filamentous extensions, provided a marker for the integrated vesicle membrane. An arrangement of SV with the inner surface of the plasma membrane is described which is characterized by regularly spaced, heabily stained membrane to membrane cross-bridges (pre-exocytotic attachment plaques). Such membrane-interconnecting elements may represent a form of coat structure important to recognition and interaction of membrane surfaces.
Intranuclear complexes in a copper-tolerant green alga.
Authors: Silverberg B A , Stokes P M , Ferstenberg L B
Published: May 2004

The results of ultrastructural studies and transmission electron microscope microanalysis of two Scenedesmus strains experimentally exposed to copper sulfate are presented. A fine-structural examination of the cells revealed the presence of nuclear inclusions in the form of central dense-core complexes. Cytoplasmic structures resembling the intranuclear inclusions were occasionally found in the cells. TEM-X-ray microanalysis of these structures has provided evidence that the inclusions contain copper. It is concluded that their presence may be regarded as a detoxifying mechanism.
Cautionary note on the use of [methyl-3H]thymidine to measure rates of chloroplast DNA synthesis in Chlamydomonas reinhardtii.
Authors: Dashe S W , Howell S H
Published: May 2004

Commercial [methyl-3H]thymidine preparations tested here contain about a 0.2% contaminant which is rapidly incorporated into Chlamydomonas DNA. This contaminant obscures the measurement of the rate of chloroplast DNA synthesis when methyl-labeled preparations are used. Such contaminants are not present in ring-labeled (either 3H or 14C) thymidine preparations. In ring-labeled thymidine preparations, a slower incorporation rate commensurate with cell density is observed. These slower, long-term incorporation kinetics would be expected for the utilization of bona fide thymidine into chloroplast DNA.
Rhodopsin in the rod outer segment plasma membrane.
Authors: Basinger S , Bok D , Hall M
Published: May 2004

Isolated frog retinas were incubated in vitro with a 4-h pulse of [3H]leucine, then chased for 32 h with a nonradioactive amino acid mixture. At the end of the incubation, light and electron microscope autoradiograms were prepared from some of the retinas. The autoradiograms revealed: (a) intense radioactivity in the basal disks of the rod outer segments, (b) diffuse label evenly distributed throughout the rod outer segments, and (c) a high concentration of label in the entire rod outer segment plasma membrane. Incubation under identical conditions, but with puromycin added, significantly inhibited the labeling of all of these components. To identify the labeled proteins, purified outer segments from the remaining retinas were analyzed biochemically by SDS disc gel electrophoresis and gel filtration chromatography. SDS gel electrophoresis showed that about 90% of the total rod outer segment radioactivity chromatographed coincident with visual pigment, suggesting that the radiolabeled protein in the plasma membrane is visual pigment. Gel filtration chromatography demonstrated that the radiolabeled protein co-chromatographed with rhodopsin rather than opsin, and that the newly synthesized visual pigment is both the basal disks and the plasma membrane is present in the native configuration.
Subcellular localization of cellulases in auxin-treated pea.
Authors: Bal A K , Verma D P , Byrne H , Maclachlan G A
Published: May 2004

Two forms of cellulase, buffer soluble (BS) and buffer insoluble (BI), are induced as a result of auxin treatment of dark-grown pea epicotyls. These two cellulases have been purified to homogeneity. Antibodies raised against the purified cellulases were conjugated with ferritin and were used to localize the two cellulases. Tissue sections were fixed in cold paraformaldehyde-glutaraldehyde and incubated for 1 h in the ferritin conjugates. The sections were washed with continuous shaking for 18 h and subsequently postfixed in osmium tetroxide. Tissue incubated in unconjugated ferritin was used as a control. A major part of BI cellulase is localized at the inner surface of the cell wall in close association with microfibrils. BS cellulase is localized mainly within the distended endoplasmic reticulum. Gogli complex and plasma membrane appear to be completely devoid of any cellulase activity. These observations are consistent with cytochemical localization and biochemical data on the distribution of these two cellulases among various cell and membrane fractions.
Filopodia of spreading 3T3 cells. Do they have a substrate-exploring function?
Authors: Albrecht-Buehler G
Published: May 2004

Freshly plated 3T3 cells send out radial projections or filopodia. We observed cells which happended to settle on glass near the borderline of a gold-plated area. When some of the filopodia contacted the gold-plated area and others the glass substratum and remained attached for a few minutes, lamellipodia then extended preferentially toward the gold-plated area. 1-2 h later, most of the cells were found in the gold-plated area. When the filopodia of a spreading 3T3 cell contacted another already spread 3T3 cell and also the glass substratum, the first lamellipodia extended preferentially towards the glass. These observations suggest a directionally differentiated extension of lamellipodia after the filopodia of a spreading 3T3 cell have contacted different substrates in their environment. Before filopodia contact a substrate, they perform a rapid "scanning" motion. Therefore, we suggest that the filopodia of a spreading 3T3 cell serve as organs which explore the nonfluid environment and react to a certain quality of the substrate that is presently unknown. Subsequently, they mediate the extension of lamellipodia into the direction in which this quality is found. The described phenomena are reversibly inhibited by Cytochalasin B at concentrations above 5 mug/ml although filopodia are produced.
Characterization of cytoplasmic and nuclear genomes in the colorless alga Polytoma. II. General characterization of organelle nucleic acids.
Authors: Siu C , Swift H , Chiang K
Published: May 2004

Polytoma obtusum has a main band DNA (alpha) with a buoyant density in CsC1 of rho = 1.711 g/ml and a light DNA satellite (beta) with rho = 1.682 g/ml. beta-DNA was substantially enriched in a fraction containing small leucoplast fragments and some mitochondria, which was obtained in a pellet sedimenting between 3,000 g and 5,000 g. A crude mitochondrial pellet was also obtained by sedimenting at 12,000 g to recover particulates remaining in the supernate after 10 min at 5,000 g. This fraction contained a third DNA component (gamma) with rho = 1.714 g/ml. We have concluded that the leucoplasts of P. obtusum contain the beta-DNA (1.6882) and the mitochondria possess the gamma-component (1.714). Two distinct classess of ribosomes were isolated and separated by sucrose density gradients, a major 79S species and a minor species at 75S. The major species possessed the 25S and 18S ribosomal RNA (rRNA), characteristic of cytoplasmic ribosomes, and these particles co-sedimented in sucrose gradients with the 79S cytoplasmic ribosomes of Chlamydomonas reinhardtii. The minor species was present in about 2% of the total ribosomal population but showed an eight-to-ninefold enrichment in the leucoplast pellet, suggesting that it was of organelle origin. These 73S particles had RNA components migrating very closely with the 18S and 25S species of the 79S ribosomes, but the base composition of the rRNA from these two classes of ribosomes was significantly different; the rRNA from the 79S ribosomes had a G+C mole ratio of 50.0%, while the rRNA from the 73S class had a ratio of 47.5%. By comparison, chloroplast ribosomes of C. reinhardtii were found to sediment at 70S and contain rRNA molecules of 23S and 16S, with a G + C content of 51.0%. These findings support the concept that the Polytoma leucoplast possesses characteristic genetic and protein-forming systems.
Pressure-induced depolymerization of spindle microtubules. III. Differential stability in HeLa cells.
Authors: Salmon E D , Goode D , Maugel T K , Bonar D B
Published: May 2004

Evidence from light microscopy (principally polarization microscopy) has demonstrated that hydrostatic pressure can reversibly inhibit mitosis by rapidly depolymerizing the spindle fiber microtubules. We have confirmed this finding in ultrastructural studies of mitotic HeLa cells incubated at 37 degrees C and pressurized at 680 atm (10,000 psi). Althouth there are many spindle microtubules in the cells at atmospheric pressure, electron micographs of cells pressurized for 10 min (and fixed while under pressure in a Landau-Thibodeau chamber) show few microtubules. Pressure has a differential effect on the various types of spindle microtubules. Astral and interpolar MTs appear to be completely depolymerized in pressurized cells, but occasional groups of kinetochore fiber microtubules are seen. Surprisingly, the length and density of microtubules of the stem bodies and midbody of telophase cells appear unchanged by pressurization. In cells fixed 10 min after pressure was released, microtubules were again abundant, the density often appearing to be higher than in control cells. Reorganization seems incomplete, however, since many of the microtubules are randomly oriented. Unexpectedly, kinetochores appeared diffuse and were difficult to identify in sections of pressurized cells. Even after 10 min of recovery at atmospheric pressure, their structure was less distinct than in unpressurized cells.
Reflexive gap junctions. Gap junctions between processing arising from the same ovarian decidual cell.
Authors: Herr J C
Published: May 2004

Ajacent processes on ovarian decidual cells were shown by electron microscopy to form gap junctions with one another. Micrographs of tissues preserved with lanthanum included in the fixative confirm the hexagonal array and 2-4 nm gap which characterize gap junctions. It is suggested that these gap junctions may play a role in the process of merocrine secretion from the peduncular processes of ovarian decidual cells. The term reflexive gap junction is introduced to describe gap junctions between adjacent processes from the same cell.
Topographical differences in the distribution of surface coat components and intramembrane particles. A cytochemical and freeze-fracture study in culture forms of Trypanosoma cruzi.
Authors: Martínez-Palomo A , DeSouza W , Gonzalez-Robles A
Published: May 2004

A regional specialization of the cell surface of T. cruzi culture forms was found at the cytostome as a localized thick surface coat rich in carbohydrate-containing components. The prominent surface coat was located over a region of the plasma membrane where intramembranous particles were exceedingly low in number. In turn, the particle-poor region was related to specialized submembrane fibrils not present under other regions of the plasma membrane. The cystostome region provides a striking example of a stable regional differentiation of the plasma membrane, involving the outer surface, the membrane interior, and the underlying cytoplasm. In addition, independence of Con A receptors, colloidal iron binding sites, and ruthenium red-stainable surface components from membrane particles was demonstrated at the flagellar membrane.
Conservation of ribosomal RNA during compensatory renal hypertrophy. A major mechanism in RNA accretion.
Authors: Melvin W T , Kumar A , Malt R A
Published: May 2004

After removal of one mouse kidney, compensatory hypertrophy in the remaining kidney is marked in 2 days by a 20% average increase in ribosomal RNA (rRNA) per cell. Both 28S and 18S RNA are conserved during the initial stages of compensatory renal hypertrophy to an extent sufficient to account for the rest of the observed accumulation of rRNA. Like some cultured cells, the kidney conserves rRNA during physiological growth.
Hepatic organelle interaction. IV. Mechanism of succinate enhancement of formaldehyde accumulation from endoplasmic reticulum N-dealkylations.
Authors: Denk H , Moldeus P W , Schulz R A , Schenkman J B , Keyes S R , Cinti D L
Published: May 2004

Further evidence for organelle interaction during drug metabolism by the liver is presented. The apparent stimulation by succinate of formaldehyde accumulation in the medium, which was reported to occur with liver slices and homogenates as well as with mitochondria plus microsomes, has been shown to be the result of succinate inhibition of mitochondrial aldehyde dehydrogenase. The mechanism of succinate inhibition is shown to be by reverse electron transport, and an increase in the NADH to NAD+ ratio in the mitochondria; the aldehyde dehydrogenase requires the oxidized form of the pyridine nucleotide as its cofactor. Studies on in vitro N-demethylation by liver microsomes and endoplasmic reticulum segments which cosediment with the mitochondria indicate that formaldehyde produced by the mixed function oxidase is handled differently from formaldehyde added to the medium. The latter is mainly retained in the medium containing 5 mM semicarbazide, while the generated formaldehyde is more than 50% consumed by the mitochondria. Electron microscopy has indicated that the microsomes and the endoplasmic reticulum fragments have a tendency to align themselves close to the mitochondria when present in the same medium. Consequently, it is possible that formaldehyde released to the medium adjacent to the mitochondria, as by N-demethylation, would be exposed to semicarbazide for shorter periods than that added directly to the medium. In agreement with this suggestion, complexing of formaldehyde with semicarbazide was observed spectroscopically not to be an extremely rapid reaction even at 37 degrees C. This is believed to be the reason for the greater extent of consumption of formaldehyde generated by the endoplasmic reticulum.
Orderly arrangement of ribosomes in the embryogenic callus tissue of Corylus avellana L.
Authors: Vujicić R , Radojević L J , Nesković M
Published: May 2004

Cultured callus tissue of hazel (Corylus avellana L.), which has the potency of somatic embryogenesis, was used for the study of cell ultrastructure in the course of callus growth and embryoid formation. The meristematic cells of this tissue exhibit a specific organization of rough endoplasmic reticulum (RER), stacked into extensive parallel sheets. The membranes of the aggregated RER are associated with orderly arrays of bound ribosomes. The high regularity of the alignment of the attached ribosomes seems to be influenced by the distance between the two neighbouring membranes in the RER aggregate. The RER aggregates with orderly attached ribosomes are more frequently found in callus cells and in early embryogenesis than in the advanced stages of embryo development.