bluuebunny/crossref_metadata_2025 huggingface dataset:
The Striated Musculature of Blood Vessels
Authors: Karrer H. E.
Published: May 2004

The musculature of small lung veins, of the thoracic portion of the inferior vena cava, and of other thoracic veins of the mouse have been studied in the electron microscope. Tissues were fixed in 1 per cent osmium tetroxide buffered with veronal, to which either sodium chloride or sucrose had been added. Methacrylate or araldite served as embedding matrices. Phosphotungstic acid or uranyl acetate was used to stain some of the preparations. Thin sections were examined in a Siemens and Halske Elmiskop Ib electron microscope.

The entire musculature of the veins examined was of the striated type. It represents a variety of cardiac muscle, characterized by centrally located nuclei, typical mitochondria, and narrow I bands. Many I bands cannot be recognized at all. H and M bands are likewise indistinct. There is a double array of primary and secondary myofilaments. Mitochondria are large and numerous and contain many cristae. The endoplasmic reticulum consists of longitudinal tubules which run through the whole sarcomeres and bypass Z bands, and of transverse tubules which accompany Z bands. Some "triads," located at Z levels, consist of flattened vacuoles flanked by such transverse tubules. Small vesicles located at Z bands, close to the nucleus, and beneath the plasma membrane may represent still other portions of the reticulum.
A Diazo Coupling Method for the Electron Microscopic Localization of Cholinesterase
Authors: Lehrer Gerard M. , Ornstein Leonard
Published: May 2004

The presence of cholinesterase at the myoneural junction of intercostal muscle has been demonstrated in both light and electron microscopic preparations. A new simultaneous diazo coupling technique using α-naphthyl acetate as substrate and "hexazonium pararosanilin" as coupler has been applied to cold formalin-fixed tissues. After postfixation in buffered osmium tetroxide the sites of esterase activity are faithfully demonstrated at a high level of resolution. The details of cholin-esterase distribution and some technical aspects of the procedure are discussed.
Electron Microscopic Observations of Rabbit Antibodies
Authors: Hall C. E. , Nisonoff A. , Slayter H. S.
Published: May 2004

Electron micrographs were obtained showing the individual, shadow-cast macromolecules from solutions of purified anti-p-azobenzoate rabbit antibody and of normal γ-globulin. The two materials look alike and consist mainly of asymmetrical rod-like particles about 30 to 40 A in diameter. Lengths are not constant but the weight average is about 250 A for the antibodies and about 200 A for the γ-globulin. The average observed dimensions are reasonably consistent with values deduced from physical-chemical methods, although the shape is more nearly that of a cylindrical rod rather than the ellipsoid employed in hydrodynamical theory. Mixtures of antibody and specific dihaptenic dye were examined in attempts to establish the mode of the specific aggregation. At the high dilutions necessary for electron microscopy (0.1 mg./ml.), the effect of the dye was small and tended to be masked by non-specific aggregation on drying. The evidence suggests that under these conditions the specific reaction involves an end-to-end aggregation of the elementary particles to produce a weight average length about twice that of the pure antibody.
Observations on the Microanatomy of the Spermatozoid of the Bracken Fern (Pteridium aquilinum)
Authors: Manton Irene
Published: May 2004

Salient features of structure of the spermatozoid of a fern (Pteridium aquilinum) have been determined by a combination of visual and ultraviolet microscopy, with electron microscopy of shadow-cast whole mounts and thin sections, using magnifications up to but not exceeding 50,000 diameters. Attention has been concentrated on the arrangement rather than on the internal details of the various parts. The most important component, apart from the spirally wound nucleus, numerous (about 40) cilia, and mitochondria, is a sheet of parallel fibres spirally wound near the surface of a cone of cytoplasm to which all the other major components are in various ways attached. The diameter of the individual fibres is of the order of 200 A. A few details are given of other minor cell constituents including additional mechanical materials, starch-containing leucoplasts, and the smaller cytoplasmic inclusions.
Studies on the Mitotic Apparatus of the Sea Urchin by Means of Antigen-Antibody Reactions in Agar
Authors: Went Hans A.
Published: May 2004

The primary purpose of the experiments reported in this paper was to gain information on the molecular origin of the mitotic apparatus. Antisera were prepared against unfertilized sea urchin (Strongylocentrotus purpuratus) egg antigens and mitotic apparatus antigens. These were permitted to react with various antigen solutions in Ouchterlony agar gel diffusion plates, and the resultant precipitation patterns analysed. The results revealed that the mitotic apparatus contains probably no more than two antigens (precursor-1 component and precursor-2 component) and that these are shared by the unfertilized egg. Absorption and fractionation techniques indicated that in the unfertilized egg the precursor-1 component is present both as a "soluble" protein and as an insoluble form tenaciously associated with intracellular structural elements. A survey of dividing and non-dividing tissues for the precursor-1 component revealed that it was restricted to tissues in which mitotic activity could be detected microscopically.

No immunochemical relationship could be detected between the mitotic apparatus and proteins extracted, by various methods, from the lantern muscle.
Chemical and Morphological Studies of Bacterial Spore Formation
Authors: Young I. Elizabeth , Fitz-James Philip C.
Published: May 2004

The development of both the spore and parasporal protein crystal of Bacillus cereus var. alesti was followed using chemical and cytological techniques. The changes which led to the formation of the fore-spore were similar to those already described for Bacillus cereus. However, adjacent to the developing fore-spore a small inclusion became discernible in phase contrast. This protein inclusion during its growth was differentiated from the chromatin and lipid-containing inclusions by sequential staining techniques.

During spore and crystal formation no net synthesis of either nucleic acid was detected. Tracer studies with radioactive phosphorus confirmed that the spore chromatin was derived from that in the vegetative cell. These same studies also indicated that a turnover of ribonucleic acid occurred during the sporulation process.

During their formation both the spore and crystal incorporated methionine-35S from the medium and from cellular material into a bound form. Sequential extractions with alkali and with alkaline-thioglycollate reagent revealed that the solubility characteristics of the mature crystal were possibly related to the presence of intermolecular disulphide bonds which developed after the major synthesis of the crystal was complete.

The synthetic nature of sporogenesis and crystal formation is discussed with reference to the concept of "endotrophic" sporulation.
MICROTUBULE SURFACE LATTICE AND SUBUNIT STRUCTURE AND OBSERVATIONS ON REASSEMBLY
Authors: Erickson Harold P.
Published: May 2004

Neuronal microtubules have been reassembled from brain tissue homogenates and purified. In reassembly from purified preparations, one of the first structures formed was a flat sheet, consisting of up to 13 longitudinal filaments, which was identified as an incomplete microtubule wall. Electron micrographs of these flat sheets and intact microtubules were analyzed by optical diffraction, and the surface lattice on which the subunits are arranged was determined to be a 13 filament, 3-start helix. A similar, and probably identical, lattice was found for outer-doublet microtubules. Finally, a 2-D image of the structure and arrangement of the microtubule subunits was obtained by processing selected images with a computer filtering and averaging system. The 40 x 50 Å morphological subunit, which has previously been seen only as a globular particle and identified as the 55,000-dalton tubulin monomer, is seen in this higher resolution reconstructed image to be elongated, and split symmetrically by a longitudinal cleft into two lobes.
REGULATION OF PHOSPHOENOLPYRUVATE CARBOXYKINASE AND TYROSINE TRANSAMINASE IN HEPATOMA CELL CULTURES
Authors: van Rijn Han , Bevers Marinus M. , van Wijk Roeland , Wicks Wesley D.
Published: May 2004

The ability of N6, O2'-dibutyryl cyclic AMP (DBcAMP) to regulate a number of metabolic events in four lines of cultured rat hepatomas has been examined. Although dexamethasone induces tyrosine transaminase in all four lines, DBcAMP induces this enzyme normally only in H35 cells. A slight increase in transaminase activity was seen with MH1C1 cells and HTC cells, but no effect was detectable in RLC cells. In contrast, phosphoenolpyruvate carboxykinase activity is increased by both agents in H35 and MH1C1 cells, but neither had any effect in HTC or RLC cells.

DBcAMP caused a rapid inhibition of the growth rate and DNA synthesis and an increase in protein content in both H35 and MH1C1 cells but not in HTC or RLC cells. The effect of DBcAMP on DNA synthesis in MH1C1 cells could be reversed by deoxycytidine as is also the case with H35 cells.

The resistance of HTC and RLC cells to DBcAMP was not due to reduced uptake or deacylation as judged by studies with [3H]DBcAMP. The cyclic nucleotide appears to enter the cells by passive diffusion as the intracellular concentration approaches that in the medium within 30–60 min. Possible explanations for the differential responses observed are discussed.
CRYOPROTECTANT-INDUCED REDISTRIBUTION OF INTRAMEMBRANOUS PARTICLES IN MOUSE LYMPHOCYTES
Authors: McIntyre John A. , Gilula Norton B. , Karnovsky Morris J.
Published: May 2004

This study demonstrates, by freeze fracture, clustering of intramembranous particles caused by cryoprotectant treatment of intact unfixed mouse lymphoid cells. Both T and B cells react in a similar fashion, while similar clustering of particles is not observed in some other cell types. The intramembranous particles can be aggregated by incubating unfixed cells in glycerol or dimethylsulfoxide (DMSO) before freezing. The aggregation phenomenon is dependent on the length of time of exposure and the concentration of the cryoprotectants. Further, the cells remain viable and the cryoprotectant-induced clustering is completely reversible. Prefixation of glycerol-treated cells with glutaraldehyde prevents the formation of these particle clusters, and unfixed nonglycerinated cells show no clusters. Thin sections of cells exposed to glycerol or DMSO without previous fixation exhibit bizarre membrane alterations and numerous other degenerative changes. These observations stress the importance of prefixation of lymphoid cells before exposure to glycerol or DMSO, as well as indicate that the membrane characteristics of certain cell types may be probed by glycerol treatment of unfixed cells.
A FINE STRUCTURAL INVESTIGATION OF SURFACE SPECIALIZATIONS AND THE CORTICAL REACTION IN EGGS OF THE CNIDARIAN BUNODOSOMA CAVERNATA
Authors: Dewel William C. , Clark Wallis H.
Published: May 2004

Developing oocytes of the cnidarian Bunodosoma cavernata are located within the mesoglea of the mesenteries of the gastrovascular cavity. The cortex of the more mature vitellogenic oocytes contains numerous, electron-dense, membrane-bound, cortical granules. The surface of these oocytes possesses prominent radially projecting structures termed cytospines. Each cytospine has a core of microfilaments, 50–70 Å in diameter, that extends basally as a rootlet through the cortical layer. During spawning, ova lacking any extraneous investments are released from the enclosing gastrodermis. As a consequence of fertilization or events associated with the earliest stages of development the ova undergo a massive cortical reaction. This reaction, which occurs during or just after release of the ova, involves extensive reorganization of the cortical layer. The cortical granule membranes and egg surface membrane fuse and vesiculate resulting in the massive discharge of granule contents. This event is accompanied by the loss of vesicular remnants of cortical ooplasm and the disruption of cytospine organization. Light and electron microscope comparisons of unreacted and reacted eggs show that the reaction results in a significant decrease in egg diameter with the oolemma of the reacted egg reorganizing in a position centripetal to its original location.
THE EFFECTS OF PARATHYROID HORMONE, COLCHICINE, AND CALCITONIN ON THE ULTRASTRUCTURE AND THE ACTIVITY OF OSTEOCLASTS IN ORGAN CULTURE
Authors: Holtrop Marijke E. , Raisz Lawrence G. , Simmons Hollis A.
Published: May 2004

The ultrastructure of osteoclasts was examined in fetal rat bones after stimulation or inhibition of resorption in culture. A central ruffled border area completely encircled by a clear zone was considered to represent the resorbing system of the cell. The proportion of ruffled border and clear zone in osteoclast cross sections was compared with changes in bone resorption as measured by the release of previously incorporated radioactive calcium (45Ca). In control cultures 55% of the osteoclast cross sections showed an area closely apposed to bone and this consisted mainly of clear zone; only 11% showed ruffled borders. Treatment with parathyroid hormone (PTH) increased 45Ca release, increased the frequency of finding areas closely apposed to bone (79%), and markedly increased the frequency of the ruffled border area (64%).

Colchicine given concurrently with PTH decreased the number of osteoclasts. Colchicine or calcitonin treatment after PTH stimulation decreased the proportion of ruffled border area significantly by 1 h; this was followed by a decrease in 45Ca release. These inhibited osteoclasts resembled osteoclasts from control, unstimulated cultures, suggesting that the cells had returned to their inactive state.

Colchicine-treated osteoclasts also showed a loss of microtubules and a massive accumulation of 100 Å filaments, suggesting that synthesis of microtubular subunits had increased.
CELL CYCLE-SPECIFIC CHANGES IN HISTONE PHOSPHORYLATION ASSOCIATED WITH CELL PROLIFERATION AND CHROMOSOME CONDENSATION
Authors: Gurley Lawrence R. , Walters Ronald A. , Tobey Robert A.
Published: May 2004

Preparative polyacrylamide gel electrophoresis was used to examine histone phosphorylation in synchronized Chinese hamster cells (line CHO). Results showed that histone f1 phosphorylation, absent in G1-arrested and early G1-traversing cells, commences 2 h before entry of traversing cells into the S phase. It is concluded that f1 phosphorylation is one of the earliest biochemical events associated with conversion of nonproliferating cells to proliferating cells occurring on old f1 before synthesis of new f1 during the S phase. Results also showed that f3 and a subfraction of f1 were rapidly phosphorylated only during the time when cells were crossing the G2/M boundary and traversing prophase. Since these phosphorylation events do not occur in G1, S, or G2 and are reduced greatly in metaphase, it is concluded that these two specific phosphorylation events are involved with condensation of interphase chromatin into mitotic chromosomes. This conclusion is supported by loss of prelabeled 32PO4 from those specific histone fractions during transition of metaphase cells into interphase G1 cells. A model of the relationship of histone phosphorylation to the cell cycle is presented which suggests involvement of f1 phosphorylation in chromatin structural changes associated with a continuous interphase "chromosome cycle" which culminates at mitosis with an f3 and f1 phosphorylation-mediated chromosome condensation.
SYNAPTIC VESICLE DEPLETION AND RECOVERY IN CAT SYMPATHETIC GANGLIA ELECTRICALLY STIMULATED IN VIVO
Authors: Pysh Joseph J. , Wiley Ronald G.
Published: May 2004

This study examined the ultrastructure of presynaptic terminals after short periods of vigorous acetylcholine (ACh) secretion in the cat superior cervical ganglion in vivo. Experimental trunks of cats anesthetized with chloralose-urethane were stimulated supra-maximally for periods of 15–30 min and at several frequencies including the upper physiological range (5–10 Hz). Stimulated and contralateral control ganglia from each animal were fixed by intra-arterial aldehyde perfusion, processed simultaneously, and compared by electron microscopy. Stimulation produced an absolute decrease in the number of synaptic vesicles, an enlargement of axonal surface membrane, and distinct alterations in the shape of presynaptic terminals. Virtually complete recovery occurred within 1 h after stimulation at 10 Hz for 30 min. These results support the hypothesis that ACh release at mammalian axodendritic synapses occurs by exocytosis of synaptic vesicles resulting in the incorporation of vesicle membrane into the presynaptic membrane and that synaptic vesicles subsequently are reformed from plasma membrane.
ISOLATION OF CILIATED OR UNCILIATED BASAL BODIES FROM THE RABBIT OVIDUCT
Authors: Anderson Richard G. W.
Published: May 2004

Techniques have been developed for the isolation of basal bodies with cilia attached or for the isolation of only basal bodies from the rabbit oviduct. Oviducts are removed, cut open, and placed in an extraction medium composed of 0.25 M sucrose, 0.001 M EDTA, 0.025 M KCl, 0.02 M Hepes buffer pH 7.5, and 0.05% Triton X-100. After the oviduct is agitated in this medium on a Vortex mixer for ½ h, the lumenal cortex of each ciliated cell, containing 200–300 basal bodies with cilia attached, is released as a unit. The cortices and the intact nuclei, which are also released from the disrupted cells, form a pellet when the extraction medium is centrifuged at 600 g for 10 min. When cortices which contain only basal bodies are to be isolated, the oviduct is subjected to conditions which remove the cilia prior to being processed as above. The cilia are removed when the oviduct is placed in a medium of 0.25 M sucrose, 0.01 M CaCl2, 0.02 M Pipes buffer pH 5.5, and 0.05% Triton X-100 and continuously agitated for 15 min on a Vortex mixer. The low pH and Ca++ solubilize the transition region of the cilium and also prevent the cell from being disrupted. The cortices can be partially purified if the 600-g pellet is resuspended in 2.2 M sucrose pH 6.5 and centrifuged at 40,000 g for 2 h. Under these conditions, 85% of the nuclei form a pellet and the cortices float to the surface of the sucrose. In addition to the basal bodies or basal bodies with cilia, the cortices contain some adherent cytoplasm, a few fibers, and a few vesicles which may be remnants of mitochondria or endoplasmic reticulum. The structure of the cilia and the basal bodies isolated with either procedure is normal.
POROUS SUBSTRUCTURE OF THE GLOMERULAR SLIT DIAPHRAGM IN THE RAT AND MOUSE
Authors: Rodewald Richard , Karnovsky Morris J.
Published: May 2004

The highly ordered, isoporous substructure of the glomerular slit diaphragm was revealed in rat and mouse kidneys fixed by perfusion with tannic acid and glutaraldehyde. The slit diaphragm was similar in both animal species and appeared as a continuous junctional band, 300–450 Å wide, consistently present within all slits formed by the epithelial foot processes. The diaphragm exhibited a zipper-like substructure with alternating, periodic cross bridges extending from the podocyte plasma membranes to a central filament which ran parallel to and equidistant from the cell membranes. The dimensions and spacing of the cross bridges defined a uniform population of rectangular pores approximately 40 by 140 Å in cross section and 70 Å in length. The total area of the pores was calculated to be about 2–3% of the total surface area of the glomerular capillaries. Physiological data indicate that the glomerular filter functions as if it were an isoporous membrane which excludes proteins larger than serum albumin. The similarity between the dimensions of the pores in the slit diaphragm and estimates for the size and shape of serum albumin supports the conclusion from tracer experiments that the slit diaphragm may serve as the principal filtration barrier to plasma proteins in the kidney.
CYCLIC CHANGES IN THE CELL SURFACE
Authors: Everhart Leighton P. , Rubin Robert W.
Published: May 2004

Cytochalasin B (CB) shows a marked concentration-dependent inhibition of the incorporation of [3H]thymidine into Chinese hamster ovary cells. This inhibition was shown to result from an inhibition of thymidine uptake, not from an inhibition of DNA synthesis. Cells normally acquire the capacity to transport thymidine as they move from the G1 stage of the cell cycle into the S phase. If CB is added to cells while they are in G1, they do not acquire the ability to transport thymidine as they enter S. However, the addition of CB to cells that are already in S has no effect on their ability to transport thymidine. These results are discussed in terms of a model in which elements involved in thymidine transport enter the cell surface membrane as the cells move from G1 to S. It is proposed that CB prevents this structural transition by binding to the cell surface.
CYCLIC CHANGES IN THE CELL SURFACE
Authors: Everhart Leighton P. , Rubin Robert W.
Published: May 2004

The surface morphology of Chinese hamster ovary cells treated with cytochalasin B (CB) has been examined using the scanning electron microscope. The cells respond to treatment with CB by retracting peripheral processes, rounding up, and assuming a smooth or gently convoluted surface. This response occurs within minutes. Cells in different stages of the cell cycle all respond in a similar manner. When CB is removed from treated cells by washing with conditioned medium, the cells regain their normal surface conformation within minutes. The surface topography of these released cells is characteristic of their stage in the cell cycle. Because CB causes an alteration in the morphology of the cell surface and because of the speed of the response and recovery, it is proposed that the primary site of action of CB is the cell surface.
MICROFILAMENTS IN EPIDERMAL CANCER CELLS
Authors: Malech Harry L. , Lentz Thomas L.
Published: May 2004

The occurrence and structure of microfilaments in epidermal cancers induced in mice by treatment with 3,4-benzpyrene were investigated with the electron microscope. With malignant change, pleomorphic, undifferentiated cells with a cortical zone of microfilaments became increasingly abundant. The microfilaments were 40 Å in diameter and occupied the cortex of the cells beneath the plasma membrane, extended into cell processes, and were situated in the cores of microvilli. At high magnification, the filamentous areas were formed by an interconnected meshwork of filaments which in favorable planes had a polygonal arrangement. When exposed to high concentrations of cytochalasin B, the microfilaments became clumped and moderately disrupted. At the same time, the processes and microvilli of the cells were blunted. The structure of these filaments and their sensitivity to cytochalasin B place them in a class of microfilaments believed to be related to cell motility. Their presence in malignant cells may be correlated with the motile, invasive properties of these cells.
CYTOCHEMICAL LOCALIZATION OF MALATE SYNTHASE IN GLYOXYSOMES
Authors: Trelease Richard N. , Becker Wayne M. , Burke John J.
Published: May 2004

Cytochemical staining techniques for microbodies (peroxisomes) are limited at present to the enzymes catalase and α-hydroxy acid oxidase, and neither technique can distinguish glyoxysomes from other microbodies. Described here is a procedure using ferricyanide for the cytochemical demonstration by light and electron microscopy of malate synthase activity in glyoxysomes of cotyledons from fat-storing cucumber and sunflower seedlings. Malate synthase, a key enzyme of the glyoxylate cycle, catalyzes the condensation of acetyl CoA with glyoxylate to form malate and release free coenzyme A. Localization of the enzyme activity is based on the reduction by free CoA of ferricyanide to ferrocyanide, and the visualization of the latter as an insoluble, electron-opaque deposit of copper ferrocyanide (Hatchett's brown). The conditions and optimal concentrations for the cytochemical reaction mixture were determined in preliminary studies using a colorimetric assay developed to measure disappearance of ferricyanide at 420 nm. Ultrastructural observation of treated tissue reveals electron-opaque material deposited uniformly throughout the matrix portion of the glyoxysomes, with little background deposition elsewhere in the cell. The reaction product is easily visualized in plastic sections by phase microscopy without poststaining. Although the method has been applied thus far only to cotyledons of fat-storing seedlings, it is anticipated that the technique will be useful in localizing and studying glyoxylate cycle activity in a variety of tissues from both plants and animals.
THE NATURE OF HYPOTHALAMO-NEUROHYPOPHYSEAL NEUROSECRETION IN THE RAT
Authors: Kent Christine , Williams M. A.
Published: May 2004

The nature of hypothalamo-neurohypophyseal neurosecretion was examined in the rat by means of intraventricular injections of tritiated amino acids. Quantitation of autoradiographs was used at the light microscope level to study the sites of synthesis of proteins and their time of arrival in the neural lobe. Electron microscope autoradiographs were used to study the labeling of neural lobe tissue. It was concluded that the great majority of the labeled material was translocated inside dense-cored granules and was probably composed mostly of neurophysins.

The effect of ether anesthesia was also examined. It was found to remove the dense cores from about 20% of the granules in the neural lobe tissue, a process accompanied by the loss of most of their labeled material. The mechanism of the ether effect is discussed and compared to the normal secretion process.
SPHEROIDAL BODIES IN THE JUNCTIONAL SARCOPLASMIC RETICULUM OF LIZARD MYOCARDIAL CELLS
Authors: Forbes M. S. , Sperelakis N.
Published: May 2004

The sarcoplasmic reticulum (SR) of lizard (Anolis carolinensis) myocardial cells has been examined, with particular attention being paid to the structural details of the peripheral couplings (junctional SR). Spheroidal bodies are present within the opaque core of junctional SR; these can be seen both in sections made en face and in sections cut to show the apposition of the junctional SR with the sarcolemma. Opaque junctional processes extend between the sarcolemma and the peripheral junctional SR. The myocardial cells in addition contain some SR cisternae deep within the cells which also possess opaque cores composed of spheroids. Although the significance of the junctional SR spheroidal bodies is unknown, it is thought that they could act as a matrix on which enzymes such as calcium-specific ATPase may be located.
MITOCHONDRIAL AND CYTOPLASMIC RIBOSOMES FROM TETRAHYMENA PYRIFORMIS
Authors: Curgy Jean-Jacques , Ledoigt Gérard , Stevens Barbara J. , André Jean
Published: May 2004

Mitochondrial and cytoplasmic ribosomes from Tetrahymena pyriformis have been isolated and studied by the techniques of polyacrylamide gel electrophoresis and electron microscopy used in conjunction. Although the two ribosome types show the same coefficient of sedimentation (80S) in sucrose gradients, they can be distinguished by gel electrophoresis: mitoribosomes migrate in a single band, considerably slower than the cytoribosome band. Electron microscope observations of negatively stained cytoribosomes show typical rounded or triangular profiles, about 275 x 230 Å; mitoribosome profiles are much larger and clearly elongate, about 370 x 240 Å. An electron-opaque spot delimits two nearly equal size subunits. In mixtures of mito- and cytoribosomes, each type can be recognized by its characteristic electrophoretic mobility and by its distinctive fine structure. Cytoribosomal 60S and 40S subunits each produce a distinct electrophoretic band. On the contrary, neither electrophoretic analysis, using a variety of conditions, nor electron microscopy is able to discern two different subunit types in the single 55S mitoribosomal subunit peak. Electrophoretic analysis of RNA shows that both ribosomal RNA species are present in the mitoribosomal subunit fraction. These results establish that mitoribosomes from T. pyriformis dissociate into two subunits endowed with the same sedimentation coefficient, the same electrophoretic mobility, and a similar morphology.
MEMBRANE JUNCTIONS IN THE INTERMEMBRANE SPACE OF MITOCHONDRIA FROM MAMMALIAN TISSUES
Authors: Saito Akitsugu , Smigel Murray , Fleischer Sidney
Published: May 2004

There have been several reports describing paracrystalline arrays in the intermembrane space of mitochondria. On closer inspection these structures appear to be junctions of two adjoining membranes. There are two types. They can be formed between the outer and inner mitochondrial membranes (designated outer-inner membrane junctions) or between two cristal membranes (intercristal membrane junctions). In rat heart, adjoining membranes appeared associated via a central dense midline approximately 30 Å wide. In rat kidney, the junction had a ladder-like appearance with electron-dense "bridges" approximately 80 Å wide, spaced 130 Å apart, connecting the adjoining membranes. We have investigated the conditions which favor the visualization of such structures in mitochondria. Heart mitochondria isolated rapidly from fresh tissue (within 30 min of death) contain membrane junctions in approximately 10–15% of the cross sections. This would indicate that the percentage of membrane junctions in the entire mitochondrion is far greater. Mitochondria isolated from heart tissue which was stored for 1 h at 0°–4°C showed an increased number of membrane junctions, so that 80% of the mitochondrial cross sections show membrane junctions. No membrane junctions are observed in mitochondria in rapidly fixed fresh tissue or in mitochondria isolated from tissue disrupted in fixative. Thus, the visualization of junctions in the intermembrane space of mitochondria appears to be dependent upon the storage of tissue after death. Membrane junctions can also be observed in mitochondria from other stored tissues such as skeletal muscle, kidney, and interstitial cells from large and small intestine. In each case, no such junctions are observed in these tissues when they are fixed immediately after removal from the animal. It would appear that most studies in the literature in which isolated mitochondria from tissues such as heart or kidney were used were carried out on mitochondria which contained membrane junctions. The presence of such structures does not significantly affect normal mitochondrial function in terms of respiratory control and oxidative phosphorylation.
HUMAN VASCULAR ENDOTHELIAL CELLS IN CULTURE
Authors: Gimbrone Michael A. , Cotran Ramzi S. , Folkman Judah
Published: May 2004

Human endothelial cells, obtained by collagenase treatment of term umbilical cord veins, were cultured using Medium 199 supplemented with 20% fetal calf serum. Small clusters of cells initially spread on plastic or glass, coalesced and grew to form confluent monolayers of polygonal cells by 7 days. Cells in primary and subcultures were identified as endothelium by the presence of Weibel-Palade bodies by electron microscopy. A morphologically distinct subpopulation of cells contaminating some primary endothelial cultures was selectively subcultured, and identified by ultrastructural criteria as vascular smooth muscle. Autoradiography of endothelial cells after exposure to [3H]thymidine showed progressive increases in labeling in growing cultures beginning at 24 h. In recently confluent cultures, labeling indices were 2.4% in central closely packed regions, and 53.2% in peripheral growing regions. 3 days after confluence, labeling was uniform, being 3.5 and 3.9% in central and peripheral areas, respectively. When small areas of confluent cultures were experimentally "denuded," there were localized increases in [3H]thymidine labeling and eventual reconstitution of the monolayer. Liquid scintillation measurements of [3H]thymidine incorporation in primary and secondary endothelial cultures in microwell trays showed a similar correlation of DNA synthesis with cell density. These data indicate that endothelial cell cultures may provide a useful in vitro model for studying pathophysiologic factors in endothelial regeneration.
AN UNUSUAL MITOTIC MECHANISM IN THE PARASITIC PROTOZOAN SYNDINIUM SP
Authors: Ris Hans , Kubai Donna F.
Published: May 2004

Syndinium and related organisms which parasitize a number of invertebrates have been classified with dinoflagellates on the basis of the morphology of their zoospores. We demonstrate here that with respect to chromosome structure and chemistry as well as nuclear division, they differ fundamentally from free-living dinoflagellates. Alkaline fast green staining indicates the presence of basic proteins in Syndinium chromosomes. Chromatin fibers are about 30 Å thick and do not show the arrangement characteristic of dinoflagellate chromosomes. The four V-shaped chromosomes are permanently attached at their apexes to a specific area of the nuclear membrane through a kinetochore-like trilaminar disk inserted into an opening of the membrane. Microtubules connect the outer dense layer of each kinetochore to the bases of the two centrioles located in a pocket-shaped invagination of the nuclear envelope. During division kinetochores duplicate, and each sister kinetochore becomes attached to a different centriole. As the centrioles move apart, apparently pushed by a bundle of elongating microtubules (central spindle), the daughter chromosomes are passively pulled apart. During the process of elongation of the central spindle, the cytoplasmic groove on the nuclear surface which contains the central spindle sinks into the nuclear space and is transformed into a cylindrical cytoplasmic channel. A constriction in the persisting nuclear envelope leads to the formation of two daughter nuclei.
THE LYSOSOME PERIPHERY: BIOCHEMICAL AND ELECTROKINETIC PROPERTIES OF THE TRITOSOME SURFACE
Authors: Gersten Douglas M. , Kimmerer Thomas W. , Bosmann H. Bruce
Published: May 2004

Normal rat liver lysosomes were isolated by the technique of loading with Triton WR-1339. Purity of the preparation was monitored with marker enzymes; a high enrichment in acid hydrolases was obtained in the tritosome fraction. In 0.0145 M NaCl, 4.5% sorbitol, 0.6 mM NaHCO3, pH 7.2 at 25°C the tritosomes had an electrophoretic mobility of -1.77 ± 0.02 µm/s/V/cm, a zeta potential of 23.2 mV, a surface charge of 1970 esu/cm2, and 33,000 electrons per particle surface assuming a tritosome diameter of 5 x 10-7 m. Treatment of the tritosomes with 50 µg neuraminidase/mg tritosome protein lowered the electrophoretic mobility of the tritosome to -1.23 ± 0.02 µm/s/V/cm under the same conditions and caused the release of 2.01 µg sialic acid/mg tritosome protein. Treatment of the tritosomes with hyaluronidase did not affect their electrophoretic mobility, while trypsin treatment elevated the net negative electrophoretic mobility of the tritosomes. Tritosome electrophoretic mobilities indicated a homogeneous tritosome population and varied greatly with ionic strength of the suspending media. pH vs. electrophoretic mobility curves indicated the tritosome periphery to contain an acid-dissociable group which likely represents the carboxyl group of N-acetylneuraminic acid; this was not conclusively proven, however, since the tritosomes lysed below a pH of 4 in the present system. Total tritosome carbohydrate (anthrone-positive material as glucose equivalents) was 0.19 mg/mg tritosome protein while total sialic acid was 3.8 µg (11.4 nmol)/mg tritosome protein.

A tritosome "membrane" fraction was prepared by osmotic shock, homogenization, and sedimentation. Approximately 25% of the total tritosome protein was present in this fraction. Analysis by gas-liquid chromatography and amino acid analyzer showed the following carbohydrate composition of the tritosome membrane fraction (in microgram per milligram tritosome membrane protein): N-acetylneuraminic acid, 14.8 ± 3; glucosamine, 24 ± 3; galactosamine, 10 ± 2; glucose, 21 ± 2; galactose, 26 ± 2; mannose, 31 ± 5; fucose, 7 ± 1; xylose, 0; and arabinose, 0. The results indicate that the tritosome periphery is characterized by external terminal sialic acid residues and an extensive complement of glycoconjugates. Essentially all the tritosome N-acetylneuraminic acid is located in the membrane and about 53% of it is neuraminidase susceptible.
EXCITABLE MEMBRANE ULTRASTRUCTURE
Authors: Peracchia Camillo
Published: May 2004

Cross-sectioned and cross-fractured crayfish axons display regions in which axon and Schwann cell surface membranes are regularly curved and project into the axoplasm. At these regions (projections) the two membranes run precisely parallel, separated by a gap of 130–140 Å. Longitudinal fractures through the axons expose the inner fractured surface of either the internal (face A) or the external (face B) leaflet of axon and adjacent Schwann cell surface membranes. On both membranes the projections appear as elongated structures oriented with the long axis parallel to the long axis of the nerve fiber. On face A of the axon surface membrane they are seen as elongated indentations 0.5–1.2-µm long, 0.12–0.15-µm wide. The indentations contain parallel chains of globules. The chains repeat every 120–125 Å and are oriented obliquely in such a way that if one looks at the axon surface from the extracellular space, the axis of the chains is skewed counterclockwise to the long axis of the indentations by an acute angle (most often 55–60°). The globules repeat along the chain every 80–85 Å. Globules of adjacent chains are in register in such a way that the axis on which globules of neighboring chains are aligned forms an angle of 75–85° with the axis of the chains. The complex structure can be defined as a globular array with a rhomboidal unit cell of 80–85 x 120–125 Å. On face B of the axon surface membrane the complementary image of these structures is seen. The projections of the Schwann cell surface membrane also contain groupings of globules; however, these differ from those in the axonal projections in size, pattern of aggregation, and fracture properties. Several possible interpretations of the meaning of these membrane specializations could be proposed. They could be: (a) structures involved in the mechanism of excitation, (b) regions of presumed metabolic couplings, and (c) areas of cell-to-cell adhesion.
A ZONAL ROTOR METHOD FOR THE PREPARATION OF MICROPEROXISOMES FROM EPITHELIAL CELLS OF GUINEA PIG SMALL INTESTINE
Authors: Connock M. J. , Kirk P. R. , Sturdee A. P.
Published: May 2004

A method is described for the preparation of catalase particles from homogenates made from suspensions of epithelial cells of the small intestine of the guinea pig. Electron microscope examination of the preparations revealed the presence of small diaminobenzidine-positive particles measuring 0.1–0.3 nm in diameter and resembling the microperoxisomes observed by Novikoff and Novikoff (1972. J. Cell Biol. 53:532.). Analytical data upon which the method is based are presented. The technique consisted of a rate zonal separation of microperoxisomes from large particles followed by an isopycnic separation from less dense organelles. Application of the method yielded microperoxisomes purified between 20- and 30-fold.
HISTONE F1 OF TETRAHYMENA MACRONUCLEI
Authors: Gorovsky Martin A. , Keevert Josephine Bowen , Pleger Gloria Lorick
Published: May 2004

Histone fraction F1 has been isolated and purified from macronuclei of the ciliated protozoan, Tetrahymena pyriformis. In many respects, Tetrahymena F1 is similar to that of other organisms. It is the only Tetrahymena histone soluble in 5% perchloric acid or 5% trichloroacetic acid, has a higher molecular weight than any other Tetrahymena histone, is the histone most easily dissociated from Tetrahymena chromatin, and is susceptible to specific proteolytic cleavage. However, unlike F1 in all other organisms, Tetrahymena F1 is not the slowest-migrating histone fraction when analyzed by polyacrylamide gel electrophoresis at low pH. Tetrahymena F1 also exhibits unusual behavior in sodium dodecyl sulfate-containing polyacrylamide gels, migrating faster than calf thymus F1 at pH 10, and slower than calf thymus F1 at pH 7.6. Tetrahymena F1 was found to be highly phosphorylated in rapidly growing cells, suggesting that the relationship between cell replication and F1 phosphorylation previously observed in mammalian cells may extend to all eukaryotes. The observation that extensive F1 phosphorylation occurs in macronuclei, which divide amitotically, argues against a unique role for F1 phosphorylation in the process of chromosome condensation at mitosis.
EVIDENCE FOR DISCONTINUOUS REPLICATION OF CIRCULAR MITOCHONDRIAL DNA MOLECULES FROM NOVIKOFF RAT ASCITES HEPATOMA CELLS
Authors: Koike Katsuro , Wolstenholme David R.
Published: May 2004

Double-forked circular molecules of mitochondrial DNA (mtDNA) from rat tissues, indicated by their form and size to be replicative intermediates, are of two structurally distinct classes. Molecules of the first class are totally double stranded. Molecules of the second class are defined by one daughter segment being totally or partially single stranded. Length histograms of daughter segments measuring between 2% and 44% of the total 5-µm molecular contour were constructed from samples of both classes of replicating molecules derived from mtDNA or Novikoff rat ascites hepatoma cells. For single strand-containing molecules, the lengths fell into eight distinct, reproducible groups with mean values separated by 4.1–7.6% of the circular contour length. For totally double stranded molecules, the lengths fell into seven groups, corresponding to seven of the groups found for single strand-containing molecules. These results suggest that along at least 44% of the contour of mtDNA molecules there exist discrete points at which DNA synthesis tends to be arrested. This may indicate that there are pauses in normal mtDNA synthesis. However, as the DNA used in these experiments was isolated from mitochondrial fractions, the findings may indicate that continuation of synthesis beyond specific points on the nucleotide strands requires a factor which is not available after cell disruption.
CALCIUM-CONTAINING ELECTRON-DENSE STRUCTURES IN THE AXONS OF THE SQUID GIANT SYNAPSE
Authors: Hillman D. E. , Llins R.
Published: May 2004

Following the Oschman and Wall technique, electron-dense structures (EDS) were found on unstained, unosmicated membranes of squid giant synapse axons. These densities contain high concentrations of calcium and phosphorus as identified by energy dispersive X-ray analysis. Based on the signal strength, the quantity is significantly greater than that of other regions of the membrane or tissue spaces. The calcium EDS occur as plaques or globules along the axonic membrane, and small globules are found between sheath cell processes. EDS also occur at the synaptic site. These densities were correlated with the opacity change seen in giant axons. It is proposed that these structures represent sites where the calcium-binding protein found by other investigators has become nearly saturated with calcium.
ASSOCIATION OF CALCIUM WITH MEMBRANES OF SQUID GIANT AXON
Authors: Oschman J. L. , Hall T. A. , Peters P. D. , Wall B. J.
Published: May 2004

Giant axons from the squid, Loligo pealei, were fixed in glutaraldehyde and postfixed in osmium tetroxide. Calcium chloride (5 mM/liter) was added to all aqueous solutions used for tissue processing. Electron-opaque deposits were found along the axonal plasma membranes, within mitochondria, and along the basal plasma membranes of Schwann cells. X-ray microprobe analysis (EMMA-4) yielded signals for calcium and phosphorus when deposits were probed, whereas these elements were not detected in the axoplasm.
ANALYTICAL STUDY OF MICROSOMES AND ISOLATED SUBCELLULAR MEMBRANES FROM RAT LIVER
Authors: Beaufay Henri , Amar-Costesec Alain , Thinès-Sempoux Denise , Wibo Maurice , Robbi Mariette , Berthet Jacques
Published: May 2004

Rat liver microsomal fractions have been equilibrated in various types of linear density gradients. 15 fractions were collected and assayed for 27 constituents. As a result of this analysis microsomal constituents have been classified, in the order of increasing median density, into four groups labeled a, b, c, and d. Group a includes: monoamine oxidase, galactosyltransferase, 5'-nucleotidase, alkaline phosphodiesterase I, alkaline phosphatase, and cholesterol; group b: NADH cytochrome c reductase, NADPH cytochrome c reductase, aminopyrine demethylase, cytochrome b5, and cytochrome P 450; group c: glucose 6-phosphatase, nucleoside diphosphatase, esterase, ß-glucuronidase, and glucuronyltransferase; group d: RNA, membrane-bound ribosomes, and some enzymes probably adsorbed on ribosomes: fumarase, aldolase, and glutamine synthetase. Analysis of the microsomal fraction by differential centrifugation in density gradient has further dissociated group a into constituents which sediment more slowly (monoamine oxidase and galactosyltransferase) than those of groups b and c, and 5'-nucleotidase, alkaline phosphodiesterase I, alkaline phosphatase, and the bulk of cholesterol which sediment more rapidly (group a2). The microsomal monoamine oxidase is attributed, at least partially, to detached fragments of external mitochondrial membrane. Galactosyltransferase belongs to the Golgi complex. Group a2 constituents are related to plasma membranes. Constituents of groups b and c and RNA belong to microsomal vesicles derived from the endoplasmic reticulum. These latter exhibit a noticeable biochemical heterogeneity and represent at the most 80% of microsomal protein, the rest being accounted for by particles bearing the constituents of groups a and some contaminating mitochondria, lysosomes, and peroxisomes. Attention is called to the operational meaning of microsomal subfractions and to their cytological complexity.
GENETIC CONTROL OF MITOCHONDRIAL THYMIDINE KINASE IN HUMAN-MOUSE AND MONKEY-MOUSE SOMATIC CELL HYBRIDS
Authors: Kit Saul , Leung Wai-Choi
Published: May 2004

Distinctive thymidine (dT) kinase molecular forms are present in mouse, human, and monkey mitochondria. Disk polyacrylamide gel electrophoresis (disk PAGE) analyses have shown that the mitochondrial-specific dT kinases differ from cytosol dT kinases in relative electrophoretic mobilities (Rm). Furthermore, the mouse mitochondrial dT kinase differs in Rm value from primate mitochondrial dT kinases. The mouse and primate cytosol dT kinases can also be distinguished.

Disk PAGE analyses have been carried out on the cytosol and mitochondrial dT kinases of human-mouse (WIL-8) and monkey-mouse (mK·CVIII) somatic cell hybrids in order to learn whether the mitochondria of the hybrid cells contained murine mitochondrial-specific, primate mitochondrial-specific, or both dT kinases. WIL-8 cells were derived from cytosol dT kinase-negative, mitochondrial dT kinase-positive mouse fibro blasts and from cytosol dT kinase-positive, mitochondrial dT kinase-positive human embryonic lung cells; they contained mostly mouse chromosomes and a few human chromosomes, including the determinant for human cytosol dT kinase. The mK·CVIII cells were derived from cytosol dT kinase-negative, mitochondrial dT kinase-positive mouse kidney cells and from cytosol dT kinase-positive, mitochondrial dT kinase-positive monkey kidney cells; they contained mostly mouse chromosomes and a few monkey chromosomes, including the determinant for monkey cytosol dT kinase. Disk PAGE analyses demonstrated that the mitochondria of human-mouse and monkey-mouse somatic cell hybrids contained the mouse-specific mitochondrial dT kinase but not the human- or monkey-specific mitochondrial dT kinase. These findings suggest that primate cytosol and mitochondrial thymidine kinase genes are coded on different chromosomes.