bluuebunny/crossref_metadata_2025 huggingface dataset:
THE STRUCTURAL BASIS OF CILIARY BEND FORMATION
Authors: Warner Fred D. , Satir Peter
Published: May 2004

The sliding microtubule model of ciliary motility predicts that cumulative local displacement (Δl) of doublet microtubules relative to one another occurs only in bent regions of the axoneme. We have now tested this prediction by using the radial spokes which join the A subfiber of each doublet to the central sheath as markers of microtubule alignment to measure sliding displacements directly. Gill cilia from the mussel Elliptio complanatus have radial spokes lying in groups of three which repeat at 860 Å along the A subfiber. The spokes are aligned with the two rows of projections along each of the central microtubules that form the central sheath. The projections repeat at 143 Å and form a vernier with the radial spokes in the precise ratio of 6 projection repeats to 1 spoke group repeat. In straight regions of the axoneme, either proximal or distal to a bend, the relative position of spoke groups between any two doublets remains constant for the length of that region. However, in bent regions, the position of spoke groups changes systematically so that Δl (doublet 1 vs. 5) can be seen to accumulate at a maximum of 122 Å per successive 860-Å spoke repeat. Local contraction of microtubules is absent. In straight regions of the axoneme, the radial spokes lie in either of two basic configurations: (a) the parallel configuration where spokes 1–3 of each group are normal (90°) to subfiber A, and (b) the tilted spoke 3 configuration where spoke 3 forms an angle (θ) of 9–20°. Since considerable sliding of doublets relative to the central sheath (∼650 Å) has usually occurred in these regions, the spokes must be considered, functionally, as detached from the sheath projections. In bent regions of the axoneme, two additional spoke configurations occur where all three spokes of each group are tilted to a maximum of ± 33° from normal. Since the spoke angles do not lie on radii through the center of bend curvature, and Δl accumulates in the bend, the spokes must be considered as attached to the sheath when bending occurs. The observed radial spoke configurations strongly imply that there is a precise cycle of spoke detachment-reattachment to the central sheath which we conclude forms the main part of the mechanism converting active interdoublet sliding into local bending.
SUBSTRATE-ATTACHED GLYCOPROTEINS MEDIATING ADHESION OF NORMAL AND VIRUS-TRANSFORMED MOUSE FIBROBLASTS
Authors: Culp Lloyd A.
Published: May 2004

When BALB/c 3T3, simian virus 40 (SV40)-transformed 3T3 (SVT2), and revertant variants of the transformed cells are removed by EGTA treatment from the substrate on which they were grown, they leave behind a layer of glycoprotein which has been characterized biochemically (Terry, A. H. and L. A. Culp. 1974. Biochemistry. 13:414.)—substrate-attached material (SAM). The influence of SAM from normal and from transformed cells on cellular attachment to the substrate, morphology, movement, and growth has been examined. All three cell types displayed a 30% higher plating efficiency when grown on 3T3 SAM. The morphology of SVT2 colonies and of individual SVT2 cells was dramatically affected by growth on 3T3 SAM—the cells (a) were more highly spread on the substrate, (b) resisted crawling over neighboring cells, and (c) resisted movement away from the edge of colonies; SVT2 SAM was not effective in causing these changes. A cell-to-substrate attachment assay using thymidine-radiolabeled cells and untreated or SAM-coated cover slips was developed. SVT2 cells attached to 3T3 SAM- or SVT2 SAM-coated cover slips with a faster initial rate and to a higher saturation level than to untreated substrate, whereas 3T3 and revertant cells exhibited no preference; there was no species specificity in these cell-substrate attachment phenomena. Trypsin-released cells attached much more slowly than EGTA-released cells. 3T3 SAM, however, was not effective in lowering the saturation density of mass cultures of virus-transformed cells. These experiments suggest that the substrate-attached glycoproteins of normal cells affect the cellular adhesivity, morphology, movement, and perhaps growth patterns of virus-transformed cells—i.e., causing partial reversion of these properties of transformed cells to those found in contact-inhibited fibroblasts. A model for the involvement of substrate-attached glycoproteins in cell-to-substrate adhesion, and possibly cell-to-cell adhesion, has been proposed.
STUDIES ON THE DEPLETION AND ACCUMULATION OF MICROVILLI AND CHANGES IN THE TUBULOVESICULAR COMPARTMENT OF MOUSE PARIETAL CELLS IN RELATION TO GASTRIC ACID SECRETION
Authors: Ito S. , Schofield G. C.
Published: May 2004

Gastric parietal cells in mice present a spectrum of microscopic appearances due mainly to variations in the abundance of the tubular and vesicular component of the cytoplasm and in the size and number of microvilli lining the intracellular canaliculi. Differences in the range of forms among parietal cells of fasting versus fed mice were not especially striking, but cells with very numerous tubules and vesicles were more common after fasting. However, in mice treated with drugs or hormones that induce acid secretion, parietal cells were more uniform in appearance. There was a marked reduction of these cytoplasmic membranes and a concomitant increase in both the number and size of microvilli. Measurements of acid secretion in control animals and in animals treated with acid secretagogues indicated hydrogen ion secretion contemporaneous with depletion of the cytoplasmic tubulovesicular membranes and with increase of the microvilli. In mice with inhibited acid secretion, parietal cells showed an accumulation of cytoplasmic tubules and vesicles and reduction in the numbers of microvilli. Stereological methods were used to quantitate 10 different parietal cell compartments. Tracer studies with lanthanum did not reveal continuity between the tubules and the plasma membrane. However, there were regions of close apposition between the tubulovesicular membranes and the cell membrane of the canaliculus, and instances where cytoplasmic tubules extended from the cell into the core of enlarged microvilli.
THE CHEMICAL COMPOSITION OF THE CELL WALL OF CHLAMYDOMONAS GYMNOGAMA AND THE CONCEPT OF A PLANT CELL WALL PROTEIN
Authors: Miller David H. , Mellman Ira Seth , Lamport Derek T. A. , Miller Maureen
Published: May 2004

Cell walls of Chlamydomonas gymnogama, shed during sexual mating, were collected and analyzed. Ultrastructural examination indicates that the walls are free of cytoplasmic contamination and that they exhibit a regular lamellate structure. The walls are composed of glycoprotein rich in hydroxyproline. The hydroxyproline is linked glycosidically to a mixture of heterooligosaccharides composed of arabinose and galactose. Altogether, the glycoprotein complex accounts for at least 32% of the wall. The amino acid composition of the walls is extraordinarily similar in widely different plant species. The implications of these similarities as well as the widespread occurrence of these glycoproteins are discussed.
ISOLATION OF POSTSYNAPTIC DENSITIES FROM RAT BRAIN
Authors: Cotman C. W. , Banker G. , Churchill L. , Taylor D.
Published: May 2004

Most synapses in the central nervous system exhibit a prominent electron-opaque specialization of the postsynaptic plasma membrane called the postsynaptic density (PSD). We have developed a procedure for the isolation of PSDs which is based on their buoyant density and their insolubility in N-lauroyl sarcosinate. Treatment of synaptic membranes with this detergent solubilizes most plasma membranes and detaches PSDs from the plasma membrane so that they can be purified on a density gradient. Isolated PSDs appear structurally intact and exhibit those properties which characterize them in tissue. The isolated PSDs are of the size, shape, and electron opacity of those seen in tissue; they stain with both ethanolic phosphotungstic acid and bismuth iodide-uranyl lead and the fraction contains cyclic 3',5'-phosphodiesterase activity. Quantitative electron microscope analysis of the PSD fraction gives an estimated purity of better than 85%. Inasmuch as the PSD is associated primarily with dendritic excitatory synapses, our PSD fraction represents the distinctive plasma membrane specialization of this specific synaptic type in isolation.
PROTEINS OF THE POSTSYNAPTIC DENSITY
Authors: Banker G. , Churchill L. , Cotman C. W.
Published: May 2004

An analysis was made of the protein composition of a fraction of postsynaptic densities (PSDs) prepared from rat brain. Protein makes up 90% of the material in the PSD fraction. Two major polypeptide fractions are present, based on sodium dodecyl sulfate polyacrylamide gel electrophoresis. The major polypeptide fraction has a molecular weight of 53,000, makes up about 45% of the PSD protein, and comigrates on gels with a major polypeptide of the synaptic plasma membrane. The other polypeptide band has a molecular weight of 97,000, accounts for 17% of the PSD protein, and is not a prominent constituent of other fractions. Six other polypeptides of higher molecular weight (100,000–180,000) are consistently present in small amounts (3–9% each). The PSD fraction contains slightly greater amounts of polar amino acids and proline than the synaptic plasma membrane fraction, but no amino acid is usually prominent. The PSD apparently consists of a structural matrix formed primarily by a single polypeptide or class of polypeptides of 53,000 molecular weight. Small amounts of other specialized proteins are contained within this matrix.
THE ACCESSIBILITY OF BOVINE RHODOPSIN IN PHOTORECEPTOR MEMBRANES
Authors: Saari John C.
Published: May 2004

Bovine photoreceptor membranes have been treated with proteases to determine the accessibility of rhodopsin to these large, water soluble molecules. The polypeptides that remain associated with the membranous structure after proteolysis were detected by sodium dodecyl sulfate gel electrophoresis. Thermolysin and chymotrypsin degraded rhodopsin (apparent mol wt 35,000–36,000) to fragments of 29,000 and 23,000 apparent mol wt, respectively, without affecting the chromophoric absorption of the molecule or removing the region of the polypeptide carrying carbohydrate. The two fragments were isolated and their amino acid compositions were determined. They do not appear to be more hydrophobic than rhodopsin. Subtilisin, at low concentration and temperature, produced a fragment with the same molecular weight as that produced by thermolysin. At higher concentrations, subtilisin yields major fragments of mol wt 23,000 and 20,000 without affecting the chromophoric absorption. Two intermediate fragments of apparent mol wt 29,000 and 26,000 were detected during the course of this degradation. Carbohydrate is retained by all but the smallest fragment. Bleaching of the photoreceptor pigment did not appreciably alter any of the fragmentation patterns. Trypsin did not alter the molecular weight of rhodopsin under the conditions of this study. Approximately 35–45% of rhodopsin appears to be accessible to the aqueous environment and can be removed without affecting the chromophoric properties of the retinaldehyde-carrying region which remains bound to the membrane.
THE INTERACTION OF CATIONIC LIPOSOMES CONTAINING ENTRAPPED HORSERADISH PEROXIDASE WITH CELLS IN CULTURE
Authors: Magee Wayne E. , Goff Charles W. , Schoknecht Jean , Smith M. Diane , Cherian K.
Published: May 2004

Cationic liposomes composed of sphingomyelin, cholesterol, and stearylamine were prepared with horseradish peroxidase trapped inside. Stable particles were formed in which 10–12% of the enzymic activity appeared to be located at, or near, the outer surface of the liposome. Adsorption and uptake of liposomes by HeLa cells were followed cytochemically by electron microscopy and quantitated by enzyme assay and by the distribution and fate of particles labeled with [14C]cholesterol and [125I]horseradish peroxidase. The particles were adsorbed by HeLa cells at least 300 times as efficiently as was free horseradish peroxidase. Many of the particles remained at the cell surface, but numerous membrane-bound cytoplasmic inclusions were observed to contain peroxidase-staining material. In addition, many areas of the cell membrane gave a positive staining reaction. It was concluded that many particles (presumably the larger ones) did not gain access to the interior of the cells, many were phagocytized, and some enzyme was transferred to the cell membrane, perhaps as a result of fusion of the liposomal membrane with the cell membrane.
ISOLATION OF EPIDERMAL DESMOSOMES
Authors: Skerrow Christine J. , Matoltsy A. Gedeon
Published: May 2004

A method is reported for the isolation of desmosomes in a high yield and of a purity suitable for biochemical analysis. The procedure utilizes the selective solubilizing action of citric acid-sodium citrate (CASC) buffer, pH 2.6, on the non-cornified layers of cow nose epidermis, followed by discontinuous sucrose density gradient centrifugation. Electron microscopy with both thin sections of pellets and unfixed spread preparations reveals that after centrifugation, desmosomes are located mainly at the 55–60% sucrose interface. In the desmosome preparation thus obtained, the characteristic desmosome structure is well preserved, showing the midline, unit membranes, and dense plaques. Furthermore, removal of the epidermal filament bundles by the solubilizing action of CASC buffer has revealed a finely filamentous layer on the cytoplasmic surface of the plaques. The dimensions, location, and appearance of this layer correspond with those of the "connecting component" which has been previously suggested as being responsible for the attachment of epidermal filament bundles to the desmosome.
CHEMICAL CHARACTERIZATION OF ISOLATED EPIDERMAL DESMOSOMES
Authors: Skerrow Christine J. , Matoltsy A. Gedeon
Published: May 2004

Desmosomes, isolated from cow nose epidermis by a method utilizing citrate buffer pH 2.6 and density gradient centrifugation, have been analyzed and found to contain approximately 76% protein, 17% carbohydrate, and 10% lipid. Nonpolar amino acids predominate in desmosomal protein, representing 456 residues per 1,000. The sialic acid content is 5 nM/mg of protein. The lipid fraction is composed of approximately 40% cholesterol and 60% phospholipids.

Desmosomes are completely solubilized by incubation with 2% sodium dodecyl sulphate and 1% ß-mercaptoethanol. Gel electrophoresis of the denatured desmosomal proteins reveals 24 bands, with mobilities corresponding to a molecular weight range of 15,000–230,000 daltons. Seven of these are considered to be major bands, together constituting 81% of the desmosomal protein. Bands 1 and 2, of molecular weights 230,000 and 210,000 daltons, together comprise 28% by weight of the desmosome. It is suggested that these protein chains are located in the desmosomal plaque. Bands 3 and 4 are PAS-positive, constitute 23% of the desmosomal protein, and have apparent molecular weights of 140,000 and 120,000 daltons, respectively. At least part of this material must originate from the carbohydrate-containing layer which is demonstrated, by histochemistry, to be present in the desmosomal interspace. The possible nature and origin of the remaining major bands, of molecular weights 90,000, 75,000, and 60,000 daltons, are discussed.
THE EFFECTS OF HALOTHANE ON CULTURED MOUSE NEUROBLASTOMA CELLS
Authors: Hinkley Robert E. , Telser Alvin G.
Published: May 2004

Mouse neuroblastoma cells (clone NB2a) were cultured in the presence of 0.3–2.1% halothane in the gas phase for up to 72 h. Halothane inhibited neurite extension dose dependently and virtually abolished microspike formation even at the lowest concentration tested. These effects were completely reversible. Electron microscopy demonstrated that microfilaments measuring 40–80 Å in diameter are the only fibrous organelles visible within microspikes. When the cells were exposed to halothane, no microfilamentous complexes could be identified in any cells and the subcortical regions of neurites often appeared devoid of individual microfilaments. Microtubules were still present in neurites after exposure to halothane concentrations at which microfilaments disappeared. However, at concentrations above 1.0%, microtubules gradually appeared to decrease in number. Short-term experiments showed that existing neurites and microspikes rapidly retracted when suddenly exposed to culture medium equilibrated with 1.0% halothane and quickly reformed when the halothane was removed. The inhibition of neuroblastoma cell differentiation by halothane appears to be mediated by disruption of 40–80 Å diameter microfilaments.
THE VISUALIZATION OF CONCANAVALIN A BINDING SITES IN PURKINJE CELL SOMATA AND DENDRITES OF RAT CEREBELLUM
Authors: Wood John G. , McLaughlin Barbara J. , Barber Robert P.
Published: May 2004

The localization of concanavalin A (Con A) binding sites in Purkinje cell somata and dendrites has been studied using a peroxidase labeling technique. In the somata, the nuclear, Golgi, and endoplasmic reticulum (ER) membranes are rich in Con A binding sites. The hypolemmal cisternae, which are continuous with the ER from the soma and throughout the dendritic tree of Purkinje cells, are also rich in Con A binding sites. Other cisternae seen in these dendrites do not bind detectable amounts of Con A. The results suggest that a cisternal system, rich in carbohydrate, may be continuous from the nuclear envelope to distal dendritic segments of Purkinje cells. Such a system could play a role in the movement of materials from Purkinje somata to dendrites.
STUDIES OF EXCITABLE MEMBRANES
Authors: Rash John E. , Ellisman Mark H.
Published: May 2004

The neuromuscular junctions and nonjunctional sarcolemmas of mammalian skeletal muscle fibers were studied by conventional thin-section electron microscopy and freeze-fracture techniques. A modified acetylcholinesterase staining procedure that is compatible with light microscopy, conventional thin-section electron microscopy, and freeze-fracture techniques is described. Freeze-fracture replicas were utilized to visualize the internal macromolecular architecture of the nerve terminal membrane, the chemically excitable neuromuscular junction postsynaptic folds, and the electrically excitable nonjunctional sarcolemma. The nerve terminal membrane is characterized by two parallel rows of 100–110-Å particles which may be associated with synpatic vesicle fusion and release. On the postsynpatic folds, irregular rows of densely packed 110–140-Å particles were observed and evidence is assembled which indicates that these large transmembrane macromolecules may represent the morphological correlate for functional acetylcholine receptor activity in mammalian motor endplates. Differences in the size and distribution of particles in mammalian as compared with amphibian and fish postsynaptic junctional membranes are correlated with current biochemical and electron micrograph autoradiographic data. Orthogonal arrays of 60-Å particles were observed in the split postsynaptic sarcolemmas of many diaphragm myofibers. On the basis of differences in the number and distribution of these "square" arrays within the sarcolemmas, two classes of fibers were identified in the diaphragm. Subsequent confirmation of the fiber types as fast- and slow-twitch fibers (Ellisman et al. 1974. J. Cell Biol. 63[2, Pt. 2]:93 a. [Abstr.]) may indicate a possible role for the square arrays in the electrogenic mechanism. Experiments in progress involving specific labeling techniques are expected to permit positive identification of many of these intriguing transmembrane macromolecules.
INTRAMEMBRANE PARTICLE AGGREGATION IN ERYTHROCYTE GHOSTS
Authors: Elgsaeter Arnljot , Branton Daniel
Published: May 2004

We have used freeze-etching and SDS-polyacrylamide gel electrophoresis to study the conditions under which the intramembrane particles of the human erythrocyte ghost may be aggregated. The fibrous membrane protein, spectrin, can be almost entirely removed from erythrocyte ghosts with little or no change in the distribution of the particles. However, after spectrin depletion, particle aggregation in the plane of the membrane may be induced by conditions which cause little aggregation in freshly prepared ghosts. This suggests that the spectrin molecules form a molecular meshwork which limits the translational mobility of the erythrocyte membrane particles.
EFFECTS OF CALCIUM-CONTAINING FIXATION SOLUTIONS ON CHOLINERGIC SYNAPTIC VESICLES
Authors: Boyne Alan F. , Bohan Timothy P. , Williams Terence H.
Published: May 2004

Calcium (Ca)-containing fixation solutions applied to slices of electric organ of the electric ray, Narcine brasiliensis, have been shown to have three distinct ultrastructural effects on cholinergic synaptic vesicles of the nerve terminals.

(a) An electron-dense particle (EDS) is observed within the vesicle; the particle is seen in unosmicated, unstained tissues and can be removed from thin sections by Ca-chelating agents. It is concluded that the EDS represents Ca bound by the vesicle. It is suggested that the bound ATP of the vesicle provides anionic Ca binding sites.

(b) The vesicle membrane tends to ‘crinkle’ or collapse depending on the concentration of the other components of the fixative solution. The ‘crinkling’ or collapse are largely reversed by a wash step in the absence of Ca.

(c) The presence of Ca results in the appearance of a population of vesicles which form characteristic fusions or ‘tight’ junctions with the terminal membrane. This appears to be morphological evidence for the proposal, which has been frequently put forward, that Ca facilitates such a fusion before discharge of vesicle-bound transmitter.

With the discovery that the use of Ca-containing fixatives leads to the demonstration of a subpopulation of synaptic vesicles fused to the terminal membrane, we are led to propose that this is the ultrastructural location of the newly synthesized acetylcholine which has been shown by others to be preferentially released by stimulation.
CELL CONTACT DEPENDENCE OF SURFACE GALACTOSYLTRANSFERASE ACTIVITY AS A FUNCTION OF THE CELL CYCLE
Authors: Webb Glenda C. , Roth Stephen
Published: May 2004

Mitotic, nonmalignant Balb/c 3T3 cells exhibit endogenous, surface galactosyltransferase activity that does not require intercellular contact throughout the assay period. In this respect, mitotic 3T3 cells resemble malignant Balb/c 3T12 cells which similarly show no contact requirement for optimum transferase activity in any phase of their cell cycle.

Previously, it was shown that randomly growing populations of 3T3 cells have lower galactosyltransferase activity when assayed under conditions which decreased cell contact. This led to the conclusion that these normal (3T3) and malignant (3T12) cells differed in that intercellular contact is required for optimum activity of surface galactosyltransferases on the normal cell type.

The present data indicate that mitotic 3T3 cells may be capable of expressing enzyme activities exhibited at all times by malignant cells. That is, mitotic 3T3 cells and randomly growing 3T12 cells may readily catalyze galactosyltransferase reactions between enzymes and acceptors on the same cell. Interphase 3T3 cells, on the other hand, might require that enzymes glycosylate acceptors on adjacent cells. A model is proposed that suggests that changes in the spatial arrangement of surface enzymes and acceptors or variations in the fluidity of the cell membrane can account for this contact-related glycosylation.
GENETIC CONTROL OF CHLOROPHYLL BIOSYNTHESIS IN CHLAMYDOMONAS
Authors: Wang Wei-Yeh , Wang Wenan Lee , Boynton John E. , Gillham Nicholas W.
Published: May 2004

In this report we describe two nonallelic Mendelian protoporphyrin accumulating mutants brs-1 and brc-1. Results of experiments with these mutants lead us to postulate that porphyrin biosynthesis branches into light and dark steps between protoporphyrin-IX and magnesium protoporphyrin. We hypothesize that the brc locus controls a dark step while the brs locus either controls a step in the main pathway before the branch or mediates the preparation of the magnesium ion for its insertion into protoporphyrin-IX. The brs-1 mutant is thought to be light sensitive because a block prior to the branch point in the porphyrin pathway prevents chlorophyll formation in either the light or the dark. The brc-1 mutant, which also accumulates protoporphyrin in the dark, forms chlorophyll and chloroplast lamellae when transferred to the light, showing that function of the porphyrin pathway is normal in the light.
INFLUENCE OF EXTERNAL POTASSIUM ON THE SYNTHESIS AND DEPOSITION OF MATRIX COMPONENTS BY CHONDROCYTES IN VITRO
Authors: Daniel Jon C. , Kosher Robert A. , Hamos James E. , Lash James W.
Published: May 2004

The effect of a high external potassium concentration on the synthesis and deposition of matrix components by chondrocytes in cell culture was determined. There is a twofold increase in the amount of chondroitin 4- and 6-sulfate accumulated by chondrocytes grown in medium containing a high potassium concentration. There is also a comparable increase in the production of other sulfated glycosaminoglycans (GAG) including heparan sulfate and uncharacterized glycoprotein components. The twofold greater accumulation of GAG in the high potassium medium is primarily the result of a decrease in their rate of degradation. In spite of this increased accumulation of GAG, the cells in high potassium fail to elaborate appreciable quantities of visible matrix, although they do retain the typical chondrocytic polygonal morphology. Although most of the products are secreted into the culture medium in the high potassium environment, the cell layer retains the same amount of glycosaminoglycan as the control cultures. The inability of chondrocytes grown in high potassium to elaborate the typical hyaline cartilage matrix is not a consequence of an impairment in collagen synthesis, since there is no difference in the total amount of collagen synthesized by high potassium or control cultures. There is, however, a slight increase in the proportion of collagen that is secreted into the medium by chondrocytes in high potassium. Synthesis of the predominant cartilage matrix molecules is not sufficient in itself to ensure that these molecules will be assembled into a hyaline matrix.
Damage and repair of the peripheral myelin sheath and node of Ranvier after treatment with trypsin.
Authors: Yu R C , Bunge R P
Published: May 2004

Cultures of whole fetal rat sensory ganglia which had matured and myelinated in culture were treated for 1-3 h with a pulse of 0.2% trypsin. The tissue was observed during the period of treatment and during subsequent weeks using both light and electron microscopy. Within minutes after trypsin addition the matrix of the culture was altered and the nerve fascicles loosened. Progressive changes included the retraction of Schwann cell processes from the nodal region the detachment of the myelin-related paranodal Schwann cell loops from the axon, and lengthening of the nodal region as the axon was bared. The retraction of myelin from nodal stabilized several hours after trypsin withdrawal. Breakdown of the altered myelin segments was rare. There were no discernable changes in neurons or their processes after this exposure to trypsin. The partial repair which occured over a period of several weeks included the reattachment of paranodal Schwann cell loops to the axolemma and the insertion of new myelin segments where a substantial length of axolemma had been bared. The significance of these observations to the characterization of the Schwann cell-axolemmal junctions on myelinated nerve fibers is discussed. The dramatic degree of myelin change that can occur without concomitant myelin breakdown is particularly noted, as is the observation that these altered myelin segments are, in part, repaired.
An ultrastructural examination of the role of cell membrane surface coat material during neurulation.
Authors: Moran D , Rice R W
Published: May 2004

Data from neural crest cultures indicate that cell surface coat material (CSM) is directly involved in cellular migration and events surrounding differentiation. To investigate whether the CSM also has a morphogenetic role, embryos of the amphibian Ambystoma maculatum were examined ultrastructurally throughout the stages of neurulation. Segments of the neural axis were fixed in glutaraldehyde-containing Alcian blue 8GX, which reportedly enhances preservation of CSM, and were postfixed in OsO4 containing 1 percent lanthanum nitrate, which stains the CSM. The medial groove formed by the appearance of the neural ridges contains a large amount of CSM and numerous vesicles coated with lanthanum-positive material. In contrast, the lateral ridge surfaces are covered by a small amount of uniformly distributed CSM and a paucity of vesicles. As the ridges begin to fold there is a progressive increase in the amount of CSM within the presumptive neural tube region. Further convergence of the neural folds is accompanied by an increase of CSM at their leading edges. As the folds approximate each other, lanthanum-positive material physically bridges the gap. However, as the apposing tissue actually abuts to form the neural tube, no CSM is observed in the remaining interspace. The specific distribution and sequential accumulation of cell CSM during the events of neurulation strongly suggest its direct participation in the morphogenetic process.
Microtubules with 15 subunits in cockroach epidermal cells
Authors: Nagano T , Suzuki F
Published: May 2004

Since Ledbetter and Porter (1964) described the 13 subunits which are visible in cross sections of negatively stained plant microtubules, subsequent observations have generally confirmed this number. By using Mizuhira's fixative composed of tannic acid and glutaraldehyde, it is easyto demonstrate the subunits of microtublules without optical reinforcement Cytoplasmic microtubules and sperm axonemes, fixed with Mizuhira's fixtive, similarly show 13 subunits (Mizuhira's and Futaesaku, 1971, 1972; Futaesaky et al., 1972; Tilney et al., 1973). This paper will describe a particular type of microtubule in insect epidermal cells fixed with the above fixative. The number of the subunits is found to be 15 in tranverse sections.
Cytochemical demonstration of hydrogen peroxide in polymorphonuclear leukocyte phagosomes
Authors: Briggs RT , Karnovsky ML , Karnovsky MJ
Published: May 2004

Phagocytosis by polymorphonuclear leukocytes (PMN) is accompanied by specific morphological and metabolic events which may result in the killing of internalized micro-organism. Hydrogen peroxide is produced in increased amounts during phagocytosis (17) and in combination with myeloperoxidase and halide ions constitute a potent, microbicidal mechanism (8,9,11). There can be direct iodination of micro-organisms (10), or alternatively, other intermediate reaction products, i.e. chloramines and aldehydes (21), can exert a microbicidal effect. The H2O2-peroxidase-halide system is presumed to operate within the phagocytic vacuole (12,18). Myeloperoxidase, present in the primary granules of PMN, enters the phagocytic vacuole during degranulation (1,4,7), and halide ions are probably derived from the extracellular medium or are present in the PMN (see 11, 18). For the operation of this system in intact cells, the presence of H2O2 in the phagocytic vacuole is necessary, and indeed this has been suggested by the work of several investigators (12, 18, 21). In the present investigation, the diaminobenzidine reaction of Graham and Karnovsky (5), modified to utilize endogenous myeloperoxidase and hydrogen peroxide, has been applied to actively phagocytizing PMN to demonstrate cytochemically the presence of H2O2 in the phagocytic vacuole.
Regulation of DNA replication on subchromosomal units of mammalian cells.
Authors: Hand R
Published: May 2004

The regulation of DNA replication at a subchromosomal level in mammalian cells has been investigated. DNA fiber autoradiographs were prepared from mouse L-929 cells pulse labeled with (3H)thymidine. Initiation events and subsequent chain growth occurring over short stretches (up to three replication units in length) of chromosomal DNA were analyzed. The results show that adjacent units usually initiate replication synchronously and that this synchrony is related to the proximity of initiation sites. In addition, adjacent units are of similar size and the rates of replication fork progression within units and on adjacent units are similar. The rate of fork progression increases with increasing replication unit size. Finally, no evidence for fixed termination sites for the units has been found. These observations suggest that despite large variations in size of replication units, timing of initiation events, and rates of fork progression found in chromosomal DNA as a whole, these processes are closely regulated within subchromosomal clusters of active replication units.
Actin filaments in the acrosomal reaction of Limulus sperm. Motion generated by alterations in the packing of the filaments.
Authors: Tilney L G
Published: May 2004

When Limulus sperm are induced to undergo the acrosomal reaction, a process, 50 mum in length, is generated in a few seconds. This process rotates as it elongates; thus the acrosomal process literally screws through the jelly of the egg. Within the process is a bundle of filaments which before induction are coiled up inside the sperm. The filament bundle exists in three stable states in the sperm. One of the states can be isolated in pure form. It is composed of only three proteins whose molecular weights (mol wt) are 43,000, 55,000, and 95,000. The 43,000 mol wt protein is actin, based on its molecular weight, net charge, morphology, G-F transformation, and heavy meromyosin (HMM) binding. The 55,000 mol wt protein is in equimolar ratio to actin and is not tubulin, binds tenaciously to actin, and inhibits HMM binding. Evidence is presented that both the 55,000 mol wt protein and the 95,000 mol wt protein (possibly alpha-actinin) are also present in Limulus muscle. Presumably these proteins function in the sperm in holding the actin filaments together. Before the acrosomal reaction, the actin filaments are twisted over one another in a supercoil; when the reaction is completed, the filaments lie parallel to each other and form an actin paracrystal. This change in their packing appears to give rise to the motion of the acrosomal process and is under the control of the 55,000 mol wt protein and the 95,000 mol wt protein.
Calcium-containing structures in vertebrate glial cells. Ultrastructural and microprobe analysis.
Authors: Gambetti P , Erulkar S E , Somlyo A P , Gonatas N K
Published: May 2004

Electron probe microanalysis has revealed that vesicular or cisternal structures containing electron-dense material in frog ependymal glial cells contain deposits of calcium and phosphorus. The so-called "osmiophilic particles" in human astrocytes also contain calcium. It is suggested that these organelles are storage sites of calcium.
Two improved methods for preparing ferritin-protein conjugates for electron microscopy.
Authors: Kishida Y , Olsen B R , Berg R A , Prockop D J
Published: May 2004

Two improved procedures were developed for activating ferritin so that the ferritin could be covalently linked to antibodies. One procedure involved use of a water-soluble carbodiimide and N-hydroxysuccinimide to prepare ferritin-containing activated esters. The other involved activation of the ferritin with excess glutaraldehyde. The ferritin-antibody conjugates prepared with the two procedures were shown to have a number of properties which made them suitable for locating antigenic components in cells.
Further characterization of embryonic tendon fibroblasts and the use of immunoferritin techniques to study collagen biosynthesis.
Authors: Olsen B R , Berg R A , Kishida Y , Prockop D J
Published: May 2004

Morphological studies were carried out on fibroblasts from chick embryo tendons, cells which have been used in a number of recent studies on collagen biosynthesis. The cells were relatively rich in endoplasmic reticulum and contained a well-developed Golgi complex comprised of small vesicles, stacked membranes, and large vacuoles. Techniques were then devised for preparing cell fragments which were penetrated by ferritin-antibody conjuates but which retained the essential morphological features of the cells. Finally, the new procedures were employed to develop further information as to how collagen is synthesized. As reported elsewhere, preliminary studies with ferritin-labeled antibodies showed that prolyl hydroxylase was found in the endoplasmic reticulum of freshly isolated fibroblasts and that procollagen is found in both the cisternae of the endoplasmic reticulum and the large Golgi vacuoles. In the experiments described here, the cells were manipulated so that amino acids continued to be incorporated into polypeptide chains but assembly of the molecule was not completed because hydroxylation of prolyl and lysyl residues was prevented. The results indicated that these manipulations produced no change in the distribution of prolyl hydroxylase. Examination of the cells with ferritin conjugated to antibodies which reacted with protocollagen, the unhydroxylated form of procollagen, demonstrated that protocollagen was retained in the cisternae of the endoplasmic reticulum during inhibition of the prolyl and lysyl hydroxylases. Assays for prolyl hydroxylase with an immunologic technique demonstrated that although the enzyme is found within the endoplasmic reticulum, it is not secreted along with procollagen. The observations provided further evidence for a special role for prolyl hydroxylase in the control of collagen biosynthesis.
Protein migration into nuclei. II. Frog oocyte nuclei accumulate a class of microinjected oocyte nuclear proteins and exclude a class of microinjected oocyte cytoplasmic proteins.
Authors: Bonner W M
Published: May 2004

Nuclear contents or cytoplasm from Xenopus oocytes labeled with (35-S)methionine or (3-H)proline (donor oocytes) were reinjected into unlabeled oocytes (recipient oocytes). The radioactivity injected as nuclear contents was found to enter and accumulate in the recipient oocyte nucleus. In contrast, the radioactivity injected as cytoplasm was found to enter but not to accumulate in the recipient oocyte nucleus. Sodium dodecyl sulfate (SDS) gel electrophoresis of the nucleus and cytoplasm of donor oocytes revealed the existence of three classes of labeled proteins in these oocytes: those proteins found predominantly in the nucleus (N proteins), those found predominantly in the cytoplasm (C proteins), and those found in both the nucleus and cytoplasm at similar concentrations (B proteins). SDS gel electrophoresis of the nucleus and cytoplasm of recipient oocytes showed that N proteins entered and accumulated in the nucleus but that B proteins partitioned about equally between the nucleus and cytoplasm. A similar analysis of oocytes injected with labeled cytoplasm showed that C proteins did not enter the nucleus but again B proteins partitioned about equally between the nucleus and cytoplasm.
Evidence for firm linkages between microtubules and membrane-bounded vesicles.
Authors: Allen R D
Published: May 2004

Direct evidence is presented in support of the widely held idea that membrane-bounded vesicles can bind firmly to microtubules. This is shown in P. caudatum which contains ribbons of straight microtubules located in open cytoplasm and uniquely associated with the disk-shaped vesicles. These vesicles frequently lie flat against the face of the ribbons at a constant distance of 30-40 nm. Under certain conditions the ribbons are compressed into zigzag pattern, but the vesicles continue to maintain their 30-40 nm spacing with the tubules and The author's interpretation of this phenomena is that the vesicles and the microtubules are strongly bound together. This interaction appears to be via a filamentous material rather than bridges.
The site of cellulose synthesis. Hormone treatment alters the intracellular location of alkali-insoluble beta-1,4-glucan (cellulose) synthetase activities.
Authors: Shore G , Maclachlan G A
Published: May 2004

Membrane preparations from growing regions of 8-day old Pisum sativum epicotyls contain multiple beta-1,4-glucan (cellulose) synthetase activities (UDP- or GDP-glucose: beta-1,4-glucan-glucosyl transferase), and the levels of some of these are influenced by treatments with the growth hormone, indoleacetic acid (IAA). When membranes from control epicotyl segments (zero time) are fractionated by isopycnic sedimentation in sucrose density gradients, all of the synthetase activities are associated mainly with Golgi membrane (density 1.55 g/cm3). After decapitation and treatment of epicotyls with IAA, synthetases also appear in a smooth vesicle fraction (density 1.11 g/cm3) which is rich in endoplasmic reticulum (ER) marker enzyme. Major fractions of these synthetases are not recovered in association with plasma membrane or washed cell walls. When [14-C]sucrose is supplied in vivo to segments +/- IAA, radioactive cellulose is deposited only in the wall. Cellulose or cellodextrin precursors do not accumulate in those membranes in which synthetase activities are recovered in vitro. In experiments where tissue slices containing intact cells are supplied with [14C]sugar nucleotide in vitro, alkali-insoluble beta-1,4-glucan is synthesized (presumably outside the protoplast) at rates which greatly exceeded (20-30 times) those obtained using isolated membrane preparations. Progressive disruption of cell structure results in increasing losses of this high activity. These results are consistent with the interpretation that Golgi and ER-associated synthetases are not themselves loci for cellulose synthesis in vivo, but represent enzymes in transit to sites of action at the wall:protoplast omterface. There they operate only if integrity of cellular organization is maintained.
Permeability of muscle capillaries to small heme-peptides. Evidence for the existence of patent transendothelial channels.
Authors: Simionescu N , Siminoescu M , Palade G E
Published: May 2004

Two heme-peptides (HP) of about 20-A diameter (heme-undecapeptide [H11P], mol wt approximately 1900 and heme-octapeptide [H8P], mol wt approximately 1550), obtained by enzymic hydrolysis of cytochrome c, were sued as probe molecules in muscle capillaries (rat diaphragm). They were localized in situ by a perixidase reaction, enhanced by the addition of imidazole to the incubation medium. Chromatography of plasma samples showed that HPs circulate predominantly as monomers for the duration of the experiments and are bound by aldehyde fixatives to plasma proteins to the extent of approximately 50% (H8P) to approximately 95% (H11P). Both tracers cross the endothelium primarily via plasmalemmal vesicles which become progressively labeled (by reaction product) from the blood front to the tissue front of the endothelium, in three successive resolvable phases. By the end of each phase the extent of labeling reaches greater than 90% of the corresponding vesicle population. Labeled vesicles appear as either isolated units or chains which form patent channels across the endothelium. The patency of these channels was checked by specimen tilting and graphic analysis of their images. No evidence was found for early or preferential marking of the intercellular junctions and spaces by reaction product. It is concluded that the channels are the most likely candidate for structural equivalents of the small pores of the capillary wall since they are continuous, water-filled passages, and are provided with one or more strictures of less than 100 A. Their frequency remains to be established by future work.
A morphometric study of cellular autophagy including diurnal variations in kidney tubules of normal rats.
Authors: Pfeifer U , Scheller H
Published: May 2004

Cellular autophagy in convoluted tubules of kidney was studied in 24 rats, killed in pairs at constant time intervals during one diurnal cycle, by (a) morphometric evaluation of tubular cells by the point-counting method in randomly sampled micrographs, and (b) selective search for autophagic vacuoles (AV) directly on the electron microscopy screen. The total area of tubular cells recorded in the electron microscopy sections was 93 X 10(-4) mum2. Since the distal convoluted tubules, covering about 12% of the whole tubulocellular area, contained only 3-4% of all AV, they were omitted from the main calculations. The number of AV per area unit and the total amount of segregated material showed a distinct diurnal rhythm, synchronous for the different types of AV which were distinguished from each other according to their contents. The minimum was found during the night, the maximum during the day. This rhythm appears similar to that described elsewhere in liver cells. The mean segregated fractions were calculated from the relation of segregated to nonsegregated material in proximal convoluted tubular cells. The segregated fraction of the mitochondria was 4.4 X 10(-4). This value could account for the degradation of all mitochondria in a cell within 15 days, i.e., the upper limit of the lifetime of mitochondrial DNA in the cortex of the kidney, if one assumes that a mitochondrion is destroyed within 10 min after being segregated. The degregated fraction of microbodies was 11.7 X 10(-4). This suggests a shorter lifetime of these organelles. It is concluded that cellular autophagy plays a significant role in the turnover of cytoplasmic constituents, including the membranes of the endoplasmic reticulum.
Microendocytosis in eosinophilic leukocytes.
Authors: Komiyama A , Spicer S S
Published: May 2004

Rat peritoneal eosinophils were examined after intraperitoneal infusion either of a mixture of phosphate-buffered saline (PBS) and colloidal gold or of fetal calf serum. These cells characteristically contained vesiculotubular structures, cuplike structures, and small granules during centrifugation. The cup-shaped structures and elaborate labyrinths of vacuole-like spaces increased markedly after injection of the PBS-colloidal gold mixture, presumably as features of heightened microendocytic activity. The vesiculotubular structures increased greatly after infusion of fetal calf serum. A few cyrstalloid granules exhibited fine-structural changes after the PBS-colloidal gold injection, and more numerous crystalloid granules appeared altered after fetal calf serum. Infrequent small granules contained a lucent, crystal-like silhouette after the fetal calf serum injection. Eosinophils evidenced microendocytic uptake of gold spherules into coated vesicles, the cup-shaped structures, and the small granules, but not into the vesiculotubular structures or crystalloid granules after intraperitoneal infusion of the PBS-gold mixture. Strong unmasked acid phosphatase activity in small granules contrasted with the general lack of activity in normal-appearing crystalloid granules and moderate activity in apparently altered crystalloid granules, presumably reflecting active and latent forms of enzyme in the different granules.