bluuebunny/crossref_metadata_2025 huggingface dataset:
REGULAR STRUCTURES IN MEMBRANES
Authors: Knutton S. , Limbrick A. R. , Robertson J. D.
Published: May 2004

An "apical endocytic complex" in the ileal lining cells of suckling rats is described. The complex consists of a continuous network of membrane-limited tubules which originate as invaginations of the apical plasma membrane at the base of the microvilli, some associated vesicles, and a giant vacuole. The lumenal surface of this tubular network of membranes and associated vesicles is covered with a regular repeating particulate structure. The repeating unit is an ∼7.5-nm diameter particle which has a distinct subunit structure composed of possibly nine smaller particles each ∼3 nm in diameter. The ∼7.5-nm diameter particles are joined together with a center-to-center separation of ∼15 nm to form long rows. These linear aggregates, when arranged laterally, give rise to several square and oblique two-dimensional lattice arrangements of the particles which cover the surface of the membrane. Whether a square or oblique lattice is generated depends on the center-to-center separation of the rows and on the relative displacement of the particles in adjacent rows.

Four membrane faces are revealed by fracturing frozen membranes of the apical tubules and vesicles: two complementary inner membrane faces exposed by the fracturing process and the lumenal and cytoplasmic membrane surfaces revealed by etching. The outer membrane face reveals a distinct array of membrane particles. This array also sometimes can be seen on the outer (B) fracture face and is sometimes faintly visible on the inner (A) fracture face. Combined data from sectioned, negatively stained, and freeze-etched preparations indicate that this regular particulate structure is a specialization that is primarily localized in the outer half of the membrane mainly in the outer leaflet.
GROWTH CONTROL OF DIFFERENTIATED FETAL RAT HEPATOCYTES IN PRIMARY MONOLAYER CULTURE
Authors: Koch K. , Leffert H. L.
Published: May 2004

Serum-deficient ≤0.00003% vol/vol) conditioned medium (CM) obtained from primary cultures of fetal rat hepatocytes initiates DNA synthesis and mitosis in homologous quiescent cultures. CM similarly prepared from 3T3 fibroblast cultures is inactive.

At least two conditioning factors are involved in initiating DNA synthesis. The first of these, arginine, is obligatory, synthesized by the cells, and released into the culture medium. The second, a lipid or lipid-containing material, is stable to pH extremes (pH 2, pH 10) and chromatographs with an apparent R1 ∼0.5 on silica gel thin-layer plates using hexane-ether (4: 1) as the solvent system.

It is suggested that these cultured hepatocytes enter or leave the G0 or early G1 phase of the cell cycle as determined in part by their capacity to use available conditioning factor and nutrient components of the medium, in particular, arginine. Serum factors including serum fraction I (4), insulin, and possibly, lipid-like conditioning material appear to initiate DNA synthesis by controlling cellular processes involved with the enhanced utilization and synthesis of growth-limiting nutrients.
EPITHELIAL COLLAGENS AND GLYCOSAMINOGLYCANS IN THE EMBRYONIC CORNEA
Authors: Trelstad Robert L. , Hayashi Kimiko , Toole Bryan P.
Published: May 2004

The embryonic corneal epithelium synthesizes both collagen and chondroitin sulfate and excretes them across the basement membrane into the subepithelial space where they assemble into a spiraling orthogonal matrix of fibrils. The assembly of collagen into fibrils is first apparent at the outer face of the basement membrane in a region of ordered chondroitin sulfate molecules. Hyaluronate, another morphogenetically important corneal macromolecule, is produced at these early stages only by the inner endothelium. These correlated biosynthetic and ultrastructural data demonstrate discrete macromolecular products of the two corneal epithelia with differing morphogenetic properties and functions.
LYSOSOMAL PHYSIOLOGY IN TETRAHYMENA
Authors: Rothstein Thomas L. , Blum J. J.
Published: May 2004

The ingestion of 14C-labeled 9,10-dimethyl-1,2-benzanthracene particles, the extracellular release of acid phosphatase, ribonuclease, and α-glucosidase, and the egestion of preingested dimethylbenzanthracene particles by Tetrahymena taken from logarithmically growing cultures and resuspended in a dilute salt solution were followed in the presence of several pharmacologic agents. Serotonin, caffeine, and, to a lesser extent, dibutyryl cyclic AMP increased the rate of particle ingestion, but did not alter the rate of release of the three acid hydrolases studied. Added catecholamines did not affect either particle ingestion or acid hydrolase release, but particle ingestion was inhibited by the catecholamine antagonists, dichloroisoproterenol, desmethylimipramine, reserpine, and phenoxybenzamine. These drugs also increased the release of acid phosphatase and ribonuclease in 5-h incubations. Desmethylimipramine acted within 1 h to increase acid hydrolase release, but the effect of dichloroisoproterenol developed more slowly and was secondary to a change in cellular content of the hydrolases. Desmethylimipramine increased the energy of activation for the release of acid phosphatase, while dichloroisoproterenol did not. Both of these drugs enhanced the egestion of preingested dimethylbenzanthracene particles, supporting the view that acid hydrolase release occurs through a cytoproct egestion mechanism. Particle ingestion was also inhibited by colchicine, vinblastine, and cytochalasin B, but these agents had no effect on acid hydrolase release, thus further differentiating the properties of the ingestion mechanism from those of the egestion mechanism. It appears that both microtubules and microfilaments play a role in the ingestion process and that this process may be controlled in part by a cyclic AMP-mediated serotoninergic and adrenergic system.
MITOCHONDRIAL DNA REPLICATION IN SEA URCHIN OOCYTES
Authors: Matsumoto Lloyd , Kasamatsu Harumi , Pikó Lajos , Vinograd Jerome
Published: May 2004

Mitochondrial DNA (mtDNA) replicative intermediates from Strongylocentrotus purpuratus oocytes were isolated by ethidium bromide-CsCl density gradient centrifugation and examined by electron microscopy after formamide spreading. In some experiments, the mtDNA was radioactively labeled by exposing isolated oocytes to [3H]thymidine. Oocyte mtDNA replication appears to follow the displacement loop model outlined in mouse L cells. There are differences in detail. The frequency of D-loop DNA is much lower in oocytes, suggesting that the relative holding time at the D-loop stage is shorter. Duplex synthesis on the displaced strand occurs early and with multiple initiations. The frequency of totally duplex replicative forms, or Cairns' forms, is the highest reported for mtDNA. The differences may be related to the fact that oocyte mtDNA replication occurs in the absence of cell division and need not be coordinated with a cell cycle. Molecules with expanded D loops banded in the intermediate region between the lower and upper bands in an ethidium bromide-CsCl gradient, supporting the notion that displacement replication proceeds on a closed circular template which is subject to nicking-closing cycles. In mature sea urchin eggs, replicative forms are absent and virtually all the mtDNA is stored as clean circular duplexes. Some novel structural variants of superhelical circular DNA (molecules with denaturation loops and double branch-migrated replicative forms) are reported.
INFLUENCE OF GLUTARALDEHYDE AND/OR OSMIUM TETROXIDE ON CELL VOLUME, ION CONTENT, MECHANICAL STABILITY, AND MEMBRANE PERMEABILITY OF EHRLICH ASCITES TUMOR CELLS
Authors: Penttila Antti , Kalimo Hannu , Trump Benjamin F.
Published: May 2004

Effects of fixation with glutaraldehyde (GA), glutaraldehyde-osmium tetroxide (GA-OsO4), and osmium tetroxide (OsO4) on ion and ATP content, cell volume, vital dye staining, and stability to mechanical and thermal stress were studied in Ehrlich ascites tumor cells (EATC). Among variables investigated were fixation time, fixative concentration, temperature, osmolality of the fixative agent and buffer, total osmolality of the fixative solution, osmolality of the postfixation buffer, and time of postfixation treatment in buffer (Sutherland, R. M., et al. 1967. J. Cell Physiol. 69:185.).

Rapid loss of potassium, exchangeable magnesium, and ATP, and increase of vital dye uptake and electrical conductivity occurred with all fixatives studied. These changes were virtually immediate with GA-OsO4 or OsO4 but slower with GA (in the latter case they were dependent on fixative temperature and concentration) (Foot, N. C. 1950. In McClung's Handbook of Microscopical Technique. 3rd edition. 564.).

Total fixative osmolality had a marked effect on cell volume with OsO4 but little or no effect with GA or GA-OsO4. Osmolality of the buffer had a marked effect on cell volume with OsO4, whereas with GA or GA-OsO4 it was only significant at very hypotonic buffer osmolalities. Concentration of GA had no effect on cell volume. Osmolality of the postfixation buffer had little effect on cell volume, and duration of fixation or postfixation treatment had no effect with all fixatives.

Freezing and thawing or centrifugal stress (up to 100,000 g) had little or no effect on cell volume after all fixatives studied. Mechanical stress obtained by sonication showed that OsO4 alone produced poor stabilization and that GA fixation alone produced the greatest stabilization.

The results indicate that rapid membrane permeability changes of EATC follow fixative action. The results are consistent with known greater stabilizing effects of GA on model protein systems since cells were also rendered relatively stable to osmotic stress during fixation, an effect not noted with OsO4. After fixation with GA and/or OsO4 cells were stable to osmotic, thermal, or mechanical stress; this is inconsistent with several earlier reports that GA-fixed cells retain their osmotic properties.
IDENTIFICATION OF CHLOROPLAST COUPLING FACTOR BY FREEZE-ETCHING AND NEGATIVE-STAINING TECHNIQUES
Authors: Garber Melvin P. , Steponkus Peter L.
Published: May 2004

Identification of chloroplast coupling factor particles, by the freeze-etching and negative-staining techniques, was made utilizing chloroplast thylakoids isolated from spinach leaves. Complete removal of particles, comparable in diameter to purified coupling factor particles, from the outer surface of freeze-etched thylakoids was achieved by treatment with 0.8% silicotungstate. Reappearance of particles, comparable in diameter to purified coupling factor particles, on the outer surface of freeze-etched thylakoids was demonstrated by combining silicotungstate-treated thylakoids with purified chloroplast coupling factor. Negative-staining results were in agreement with the freeze-etch data. The results demonstrate that the chloroplast coupling factor particles are exposed on the outer surface.
BIOGENIC AMINES IN CULTURED NEUROBLASTOMA AND ASTROCYTOMA CELLS
Authors: Narotzky Robert , Bondareff William
Published: May 2004

The presence of biogenic amines in cultured cells of mouse neuroblastoma C-1300 (clone NB-2a) was suggested by fluorescence-microscope histochemistry. Incubation in media containing L-[14C]tyrosine and L-[14C]tryptophan for 24 h, followed by high-voltage electrophoresis, radiochromatogram scanning, and scintillation counting, confirmed the presence of [14C]dopamine, [14C]norepinephrine, [14C]epinephrine, [14C]serotonin, [14C]tyramine, and [14C]octopamine. Dopamine, norepinephrine, epinephrine, and serotonin were demonstrated spectrophotofluorometrically in concentrations, expressed as micrograms amine per milligram protein, of 1.19, 0.027, 0.038, and 0.148, respectively, for cells in a stationary growth phase.

Fluorescence-microscope histochemistry also suggested the presence of biogenic amines in cultured astrocytoma cells (cell line C6). Spectrophotofluorometric assay of cells in a stationary growth phase demonstrated intracellular dopamine, norepinephrine, epinephrine, and serotonin in concentrations significantly lower than those of neuroblastoma cells.
IN SITU LOCALIZATION OF GLOBIN MESSENGER RNA FORMATION
Authors: Harrison P. R. , Conkie D. , Affara N. , Paul J.
Published: May 2004

Globin mRNA levels in 11–15-day mouse fetal liver cells have been estimated by in situ hybridization of a highly labeled DNA copy (cDNA) of adult globin messenger RNAs (mRNAs) (globin cDNA) to fixed preparations of cells. Under the conditions employed, no significant in situ hybridization occurred to lymphoma cells (L 51787), mouse L cells, or hepatocytes; whereas reticulocytes from phenyl hydrazine-treated mice showed extensive in situ hybridization. The proportion of fetal liver cells showing predominantly cytoplasmic in situ hybridization increased from about 30% at the 11th day of development to 80–85% by days 13–15. Unlike more mature cells, proerythroblasts did not show in situ hybridization, except to a slight extent at later stages of development. These studies therefore indicate that globin mRNAs begin to accumulate during or shortly after the proerythroblastbasophilic erythroblast transition.

The fact that certain immature erythroid cells from 14-day fetal liver contain substantial amounts of globin mRNAs has been confirmed by comparing the hybridization in solution of globin cDNA to cytoplasmic RNA extracted from total fetal liver cells or from immature erythroid cells obtained by treatment of fetal liver cells with an antiserum raised against erythrocytes.
ULTRASTRUCTURAL AND BIOCHEMICAL STUDIES ON THE PRECURSOR RIBOSOMAL PARTICLES ISOLATED FROM RAT LIVER NUCLEOLI
Authors: Matsuura S. , Morimoto T. , Tashiro Y. , Higashinakagawa T. , Muramatsu M.
Published: May 2004

Sucrose density gradient analyses of pH 5.5 and pH 7.4 extracts from rat liver nucleoli revealed the presence of two broad peaks of approximately 60S and 80S, and 60S and 80–100S, respectively. Ribonucleoprotein (RNP) particles containing precursor ribosomal RNA in these peaks have been characterized by electron microscopy and RNA analyses. Spherical particles only were found in the 60S peak of the pH 5.5 extract, from which 28S RNA and smaller RNA (23S and 18S RNA) exclusively were extracted. In the broad 80S peak of the pH 5.5 extract, about 60% of the particles were spherical while 30% were rodlike. In the RNA species present there were 28S plus smaller RNA (80%) and 35S RNA (20%). The 60Speak of the pH 7.4 extract contained mainly spherical particles (84%), and the RNA species present was mostly 28S plus smaller RNA (89%). In addition to spherical particles (43%), a number of rodlike (31%) and filamentous molecules (26%) were observed in the heavier side of the 80–100S peak of the pH 7.4 extract, from which 45S (14%), 35S (26%), and 28S and smaller RNA (60%) were extracted. Thus the precursor ribosomal particles containing 45S RNA and 35S RNA appear to be filamentous and rodlike molecules, respectively. Folding of loose ribonucleoprotein filaments into compact, spherical, large subparticles may be part of the maturation process of ribosomal large subparticles, in addition to the so-called sequential cleavage of RNA.
NEURONAL PERIKARYA WITH DISPERSED, SINGLE RIBOSOMES IN THE VISUAL CORTEX OF MACACA MULATTA
Authors: Palay S. L. , Billings-Gagliardi S. , Chan-Palay V.
Published: May 2004

Numerous small and medium-sized neuronal perikarya in layers III and IV of the visual cortex display an unusual pattern of ribosomal distribution. Instead of being aggregated in clusters, spirals, rows, and other regular polysomal configurations, the ribosomes, whether free or attached to the endoplasmic reticulum, are randomly dispersed, with no discernible pattern. The endoplasmic reticulum in such cells is reduced to a few (perhaps only one) meandering, broad cisternae, which delimit broad fields of cytoplasmic matrix occupied almost solely by scattered, single ribosomes. The Golgi apparatus is elaborate. Mitochondria are either small and numerous or large and infrequent. The other organelles, including the nucleus and nucleolus, are not remarkable. Axonal terminals synapse in the normal fashion on the surfaces of these cells and their dendrites. Associated with these cells are more numerous intermediate cells in which a few to many polysomal clusters can be found. It is proposed that the neurons with dispersed, single ribosomes are inactive in protein synthesis and that the suspension of such an important metabolic activity is probably temporary. Thus, these cells are considered to be part of a population undergoing cyclic fluctuations in the intensity of protein synthesis that should be correlated with their specific neural behavior.
MOLECULAR COMPOSITION OF THE SUBMICROSOMAL MEMBRANE LIPID OF RAT BRAIN
Authors: Tamai Yoichi , Araki Shigeko Matsukawa , Katsuta Kimio , Satake Mei
Published: May 2004

Rough-surfaced and light and heavy smooth-surfaced microsomes were isolated from rat brain by means of discontinuous sucrose gradient centrifugation. Electron microscopically, the rough-surfaced microsomes were characterized by vesicles with ribosomes and the light and heavy smooth-surfaced microsomes by fairly homogeneous membrane features without ribosomes.

The rough-surfaced microsomal membranes were distinguished by the absence of glycolipids, such as ganglioside, cerebroside, and sulfatide. Cerebroside was exclusively recovered in the light smooth-surfaced microsomal membranes. Ganglioside and Na,K-ATPase were contained in larger amounts in the heavy smooth-surfaced microsomal membranes than in the light smooth-surfaced microsomal membranes in terms of protein content.

Among the three submicrosomal membranes, cholesterol and phospholipid were found in the largest amounts in the light smooth-surfaced microsomal membranes, where the molar ratio of cerebroside-cholesterol-phospholipid was about 1:10:10.

The membranes of rough- and smooth-surfaced microsomes were very similar in regards to the composition of phospholipid classes, although the fatty acid composition of the former contained a greater proportion of unsaturated fatty acids than that of the latter.

When the membrane proteins were analyzed by sodium dodecyl sulfate gel electrophoresis, some differences were observed between the light and heavy smooth-surfaced microsomal membranes.
INCREASED RATE OF ACETYLCHOLINESTERASE SYNTHESIS IN DIFFERENTIATING NEUROBLASTOMA CELLS
Authors: Lanks Karl W. , Dorwin Jan M. , Papirmeister Bruno
Published: May 2004

When neuroblastoma cells (N18) in vitro are maintained in the absence of serum, the specific activity of AChE begins to rise rapidly after an initial lag period of about 2–3 days, reaching a maximum level (10–20-fold increase) by 7 days after induction. In order to clarify the mechanism of induction, it was necessary to measure the rate of AChE synthesis and its sensitivity to metabolic inhibitors. Return of enzymatic activity after irreversible inhibition of AChE in "differentiated" cells was blocked by cycloheximide, but not by cordycepin or actinomycin D, suggesting that protein but not mRNA synthesis was required for replacement. By using the initial rate of this replacement as a measure of the rate of synthesis of the enzyme, it was shown that cells which had differentiated in the absence of serum synthesized AChE 50-fold faster on a specific activity basis than their undifferentiated counterparts. In contrast, cordycepin effectively blocked the increase in the rate of AChE synthesis that occurs as a result of serum deprivation, indicating that the induction process itself requires the synthesis of new mRNA. Axonation, another index of differentiation, was not completely blocked by inhibition of RNA or protein synthesis and presumably utilizes only pools of pre-existing structural proteins.
ULTRASTRUCTURAL ANALYSIS OF HEMATOPOIETIC COLONIES DERIVED FROM HUMAN PERIPHERAL BLOOD
Authors: Zucker-Franklin Dorothea , Grusky George
Published: May 2004

The ultrastructure of granulocyte colonies derived from normal human peripheral blood leukocytes cultured in semisolid media has been studied by a new method developed for this purpose. Fixation, dehydration, and embedding of the whole content of the Petri dish resulted in a block of Epon containing colonies made up of cells with the spatial orientation of those observed in living cultures. This permitted serial sectioning through entire colonies. Cell maturation in vitro appeared to parallel that of normal marrow. However, even the most mature cells retained cytoplasmic characteristics of more immature cells. This was particularly true for eosinophils which only rarely possessed granules with electron-dense crystalline "cores," a feature typical for mature eosinophils. In addition to the normal-appearing hematopoietic cells found within colonies, very large round or spindle-shaped cells were present between colonies and firmly attached to the bottom of the culture dish. Although the histochemical and functional characterization of these cells awaits further study, it is suggested that they are related to histiocytes or macrophages.

The technique described here should prove valuable in studies of the development, differentiation, and interaction of many types of cells.
THE PERMEABILITY OF GLOMERULAR CAPILLARIES TO GRADED DEXTRANS
Authors: Caulfield John P. , Farquhar Marilyn G.
Published: May 2004

Graded dextrans have been used as tracers to identify the primary permeability barrier(s) to macromolecules among the structural elements (endothelium, mesangium, basement membrane, epithelium) of the glomerular capillary wall. Three narrow-range fractions of specified molecular weights and Einstein-Stokes radii (ESR) were prepared by gel filtration: (a) 32,000 mol wt, ESR = 38 Å; (b) 62,000 mol wt, ESR = 55 Å; and (c) 125,000 mol wt, ESR = 78 Å. These fractions are known to be extensively filtered, filtered in only small amounts, and largely retained, respectively, by the glomerular capillaries. Tracer solutions were infused i.v. into Wistar-Furth rats, and the left kidney was fixed after 5 min to 4 h. The preparations behaved as predicted: initially, all three fractions appeared in the urinary spaces, with 32,000 > 62,000 » 125,000. The smallest fraction was totally cleared from the blood and urinary spaces by 2.5 h, whereas the intermediate and largest fractions were retained in the circulation at high concentrations up to 4 h. With all fractions, when particles occurred in high concentration in the capillary lumina, they were present in similarly high concentrations in the endothelial fenestrae and inner (subendothelial) portions of the basement membrane, but there was a sharp drop in their concentration at this level—i.e., between the inner, looser portions of the basement membrane and its outer, more compact portions. With the two largest fractions, accumulation of particles occurred against the basement membrane in the mesangial regions with time. No accumulation was seen with any of the fractions in the epithelial slits or against the slit membranes. Dextran was also seen in phagosomes in mesangial cells, and in absorption droplets in the glomerular and proximal tubule epithelium. It is concluded that the basement membrane is the main glomerular permeability barrier to dextrans, and (since their behavior is known to be similar) to proteins of comparable dimensions (40,000–200,000 mol wt). The findings are discussed in relation to previous work using electron-opaque tracers to localize the glomerular permeability barrier and in relation to models proposed for the functions of the various glomerular structural elements.
FOOD VACUOLE MEMBRANE GROWTH WITH MICROTUBULE-ASSOCIATED MEMBRANE TRANSPORT IN PARAMECIUM
Authors: Allen Richard D.
Published: May 2004

Evidence from a morphological study of the oral apparatus of Paramecium caudatum using electron microscope techniques have shown the existence of an elaborate structural system which is apparently designed to recycle digestive-vacuole membrane. Disk-shaped vesicles are filtered out of the cytoplasm by a group of microtubular ribbons. The vesicles, after being transported to the cytostome-cytopharynx region in association with these ribbons, accumulate next to the cytopharynx before they become fused with the cytopharyngeal membrane. This fusion allows the nascent food vacuole to grow and increase its membrane surface area. The morphology of this cytostome-cytopharynx region is described in detail and illustrated with a three-dimensional drawing of a portion of this region and a clay sculpture of the oral apparatus of Paramecium. Evidence from the literature for the transformation of food vacuole membrane into disk-shaped vesicles both from condensing food vacuoles in the endoplasm and from egested food vacuoles at the cytoproct is presented. This transformation would complete a system of digestive vacuole membrane recycling.
PINOCYTOSIS IN FIBROBLASTS
Authors: Steinman Ralph M. , Silver Jonathan M. , Cohn Zanvil A.
Published: May 2004

Horseradish peroxidase (HRP) was used as a marker to determine the rate of ongoing pinocytosis in several fibroblast cell lines. The enzyme was interiorized in the fluid phase without evidence of adsorption to the cell surface. Cytochemical reaction product was not found on the cell surface and was visualized only within intracellular vesicles and granules. Uptake was directly proportional to the administered concentration of HRP and to the duration of exposure. The rate of HRP uptake was 0.0032–0.0035% of the administered load per 106 cells per hour for all cells studied with one exception: L cells, after reaching confluence, progressively increased their pinocytic activity two- to fourfold. After uptake of HRP, L cells inactivated HRP with a half-life of 6–8 h. Certain metabolic requirements of pinocytosis were then studied in detail in L cells. Raising the environmental temperature increased pinocytosis over a range of 2–38°C. The Q10 was 2.7 and the activation energy, 17.6 kcal/mol. Studies on the levels of cellular ATP in the presence of various metabolic inhibitors (fluoride, 2-desoxyglycose, azide, and cyanide) showed that L cells synthesized ATP by both glycolytic and respiratory pathways. A combination of a glycolytic and a respiratory inhibitor was needed to depress cellular ATP levels as well as pinocytic activity to 10–20% of control values, whereas drugs administered individually had only partial effects. In spite of the availability of an accurate quantitative assay for fluid and solute uptake, the function of pinocytosis in tissue culture cells remains unknown.
PROPERTIES OF FLAGELLAR "RIGOR WAVES" FORMED BY ABRUPT REMOVAL OF ADENOSINE TRIPHOSPHATE FROM ACTIVELY SWIMMING SEA URCHIN SPERM
Authors: Gibbons Barbara H. , Gibbons I. R.
Published: May 2004

Sea urchin sperm were demembranated and reactivated with a solution containing 0.04% Triton X-100 and 0.03 mM ATP. The ATP concentration was then lowered abruptly by diluting the sperm suspension 50-fold into reactivating solution containing no ATP. The flagella of the sperm in the diluted suspension were not motile, but they were bent into a variety of stationary rigor wave forms closely resembling the wave forms occurring at different stages of the flagellar bending cycle during normal movement. The form of these rigor waves was unchanged upon storage for several hours in the presence of dithiothreitol and EDTA. Addition of 1 µM ATP induced slow relaxation of the waves, with most of the sperm becoming partially straightened over a period of about 30 min; somewhat higher concentrations gave a more rapid and complete relaxation. Concentrations of ATP above 10 µM induced resumption of normal beating movements. Addition of ITP, GTP, or GDP (up to 1 mM) produced no relaxation of the rigor waves. Digestion with trypsin to an extent sufficient to disrupt the radial spokes and the nexin links caused no change in the rigor wave forms, suggesting that these wave forms could be maintained by the dynein cross-bridges between the outer doublet tubules of the flagellar axoneme. Study of the effects of viscous shear on the rigor wave axonemes has shown that they are resistant to distortion by bending, although they can be twisted relatively easily.
ANALYSIS OF THE PINOCYTIC PROCESS IN RAT KIDNEY
Authors: Bode Folkert , Pockrandt-Hemstedt Hannelore , Baumann Karl , Kinne Rolf
Published: May 2004

Pinocytosis was induced in rat kidney by exposure to horseradish peroxidase (HRP). Pinocytic vesicle preparations were enriched after homogenization of kidney cortex by differential centrifugation and free-flow electrophoresis with HRP as an exogenous marker. Vesicles were identified by enzymatic analysis and by electron microscopy, including specific staining procedures. Typical brush-border enzymes such as alkaline phosphatase, aminopeptidase, 5'-nucleotidase, lysosomal acid phosphatase, and mitochondrial succinic dehydrogenase were reduced in the vesicular fraction, compared to the kidney cortex homogenate. Glucose-6-phosphatase and Na+-K+-ATPase were only slightly increased in the fraction. These results indicate that preparations of pinocytic vesicles from rat kidney cortex can be enriched. They have biochemical characteristics that differ from those of the cell organelles and membranes previously purified from renal tissue.
Quantitative studies of pinocytosis. II. Kinetics of protein uptake and digestion by rat yolk sac cultured in vitro.
Authors: Williams K E , Kidston E M , Beck F , Lloyd J B
Published: May 2004

Pinocytic uptake of 125I-labeled bovine serum albumin by 17.5-day rat visceral yolk sac cultured in vitro has been examined. Uptake was followed by intracellular digestion and, after an initial period, the content of radioactivity in the tissue itself remained constant during the incubation. Radiolabel was returned to the culture medium predominantly as (125I)iodotyrosine; exocytosis of undigested protein did not occur. The rate of uptake of labeled protein, which was constant within an experiment and reproducible between experiments, was much higher than that of a nondigestible macromolecule, 125I-labeled polyvinylpyrrolidone. The higher rate of uptake was a consequence of the protein entering the cells chiefly by adsorption to the plasma membrane being internalized; 125I-labeled albumin did not stimualte, nor did 125I-labeled polyvinylpyrrolidone inhibit pinocytosis. Different preparations of 125I-labeled albumin had characteristically different rates of uptake, probably reflecting differences in affinity for plasma membrane receptors. The physiological significance of the findings is discussed.
In vitro polymerization of microtubules into asters and spindles in homogenates of surf clam eggs.
Authors: Weisenberg R C , Rosenfeld A C
Published: May 2004

The eggs of the surf clam Spisula solidissima were artificially activated, homogenized at various times in cold 0.5 M MES buffer, 1mM EGTA at pH 6.5, and microtubule polymerization was induced by raising the temperature to 28 degrees C. In homogenates of unactivated eggs few microtubules form and no asters are observed. By 2.5 min after activation microtubules polymerize in association with a dense central cylinder, resulting in the formation of small asterlike structures. By 4.5 min after activation the asters formed in vitro contain a distinct centriole, and microtubules now radiate from a larger volume of granular material which surrounds the centriole. By 15 min (metaphase I) the granular material is more disperse and only loosely associated with the centriole. Microtubules are occasionally observed which appear to radiate directly from one end of the centriole. The organizing center can be partially isolated by centrifugation of homogenates of metaphase eggs and will induce aster formation if mixed with tubulin from either activated or unactivated eggs. Pretreatment of the eggs with colchicine does not prevent the formation of a functional organizing center. Complete spindles can also be obtained under polymerizing conditions by either homogenizing the eggs directly into warm buffer or by adding a warm high-speed supernate to spindles which have been isolated in a microtubule stabilizing medium. Extensive addition of new tubulin occurs onto the isolated spindles, resulting primarily in growth of astral fibers, although there occasionally appears to be growth of chromosomal fibers and of pole-to-pole fibers. Negatively stained aster microtubules have a strong tendency to associate side by side, and under some conditions distinct cross bridges can be observed. However, under other conditions large numbers of 300-400-A particles surround the microtubules; the presence of stain between particles can give the appearance of cross bridges.
A fine structure study of the anthocodium in Renilla mülleri. Evidence for the existence of a bioluminescent organelle, the luminelle.
Authors: Spurlock B O , Cormier M J
Published: May 2004

A fine structure study of the anthocodium of the sea pansy, Renilla mülleri, was undertaken. The anthocodium, a known site of bioluminescence, was selected in order to determine whether a structural entity could be found which would satisfy the biochemical and physiological features associated with the known sites of bioluminescence in this animal. These sites, termed lumisomes, have previously been shown to be small (0.1-0.2 mum), membrane-enclosed vesicles which contain all the proteins necessary for bioluminescence and its immediate control. One of the lumisomal proteins is an intensely green fluorescent protein and has been used as a probe for the detection of the cellular sites of bioluminescence. This green fluorescence was associated only with gastrodermal cells. We report the identification of a unique morphological entity, restricted to the cells of the gastrodermis, which satisfies the biochemical and physiological requirements for bioluminescence in Renilla. It is a large (4-6 mum), membrane-bounded subcellular organelle comparable in size to a subcellular structure whose green fluorescence is typically associated with the in vivo bioluminescence. Furthermore, it is filled with smaller membrane-bounded vesicles which have the same size and shape as the lumisomes. We suggest that the organelle identified in this study be termed a luminelle.
Meiotic DNA synthesis during mouse spermatogenesis.
Authors: Meistrich M L , Reid B O , Barcellona W J
Published: May 2004

The incorporation of radioactivity into various cells in the sequence of spermatogenesis was measured by preparing highly purified spermatozoan nuclei from the cauda epididymidis of mice at daily intervals after injection of (3H)thymidine. The stages of differentiation of these sperm at the time of thymidine administration were calculated from the kinetics of spermatogenesis. The procedure for purification of sperm nuclei included sonication, mechanical shearing, and treatment with trypsin, DNase, Triton X-100, 2M NaC1, and sodium dodecyl sulfate. DNA was isolated from these nuclei by treatment with dithiothreitol and pronase, followed by phenol extraction and ethanol precipitation. The levels of radioactivity in the epididymal sperm head preparations were low (less than 13 dpm/mouse) for 27 days after injection, and then rose dramatically to over 4 times 104 dpm/mouse. Further experiments demonstrated that the 11 dpm of 3H radioactivity contained in sperm heads at 21 or 26 days after injection of (3H)TdR was significantly above background and contamination levels from other cells or other sources. Most of the radioactivity was in the sperm DNA and represented incorporation of tritium from (3H)TdR into the nuclear DNA of meiotic cells at 0.002 percent of the rate of incorporation into S-phase cells. Little, if any, (3H)TdR was incorporation into the DNA of spermatids. The levels of DNA synthesis during the meiotic prophase in the mouse appear to be much lower than those reported for other organisms.
Actin associated with membranes from 3T3 mouse fibroblast and HeLa cells.
Authors: Gruenstein E , Rich A , Weihing R R
Published: May 2004

A protein component of membranes isolated from 3T3 mouse fibroblasts and HeLa cells has been identified as actin by peptide mapping. Extensive but apparently not total coincidence was found between the peptide maps of these two nonmuscle membrane-associated actins compared to chick skeletal muscle actin. Between 2 and 4 percent of the total membrane protein appears in the actin band on sodium dodecyl sulfate polyacrylamide gels of 3T3 membranes while about 4 percent of the membrane protein appears as the actin band from HeLa membranes. These values represent approximately the same proportion of actin to total protein found in the cell homogenates. Treatment of intact cells with levels of cytochalasin B sufficient to cause pronounced morphological changes did not change the amount of actin associated with the membrane in either 3T3 or HeLa cells. However, incubation of isolated membranes under conditions favoring conversion of actin from filamentous to monomeric form resulted in dissociation of approximately 80 and 60 percent of the actin from 3T3 and HeLa membranes, respectively. Thus, approximately 20 percent of 3T3 membrane actin and 40 percent of HeLa membrane actin remained associated with the membrane even under actin depolymerizing conditions.
Correlation of rhodopsin biogenesis with ultrastructural morphogenesis in the chick retina.
Authors: Mason W T , Bighouse K J
Published: May 2004

The developing chick retina from stages 39-45 has been examined by biochemical and electron microscope techniques. The levels of rhodopsin contained in the maturing chick retina were evaluated by detergent extraction and correlated with rod outer segment formation. It was found that the appearance of rhodopsin in significant levels preceded outer segment formation by at least 2 days, thus implying that rhodopsin is synthesized in the receptor cell inner segment and translocated to the outer limb when disk membrane biogenesis occurs. The level of rhodopsin continues to rise as the rod outer segment develops. Development of both rods and cones originates and proceeds most rapidly in the fundus or central region and proceeds toward the periphery. In general, rod outer segments were noted to develop far more rapidly than cone outer segments.
Molecular organization of rat prolactin granules: in vitro stability of intact and "membraneless" granules
Authors: Giannattasio G. , Zanini A , Meldolesi J
Published: May 2004

Studies carried out on a number of secretory cell systems suggest that the specific cytoplasmic granules in which the secretion products are stored before their release are complex organelles which can possess a distinct molecular organization. For instance, it has been reported that in some granules the segregated secretion products are organized into crystalline structures (1-3) or large intermolecular aggregates (4-8). It is likely that all phenomena of this type are favorable to the economy of the cell, in the sense that they reduce the energy required for storage of the secretion products. The prolactin (LTH) granules of the rat pituitary possess a number of morphological features which strongly suggest that the molecules(s) of their content might be arranged in a relatively stable structure. Thus, these granules are remarkably polymorphic in shape, and their membrane is usually separated from their content by a clear space. Furthermore, identifiable LTH granules devoid of their membrane are often seen in the pericapillary space, suggesting that upon discharge by exocytosis they are dissolved only slowly (9). However, no studies specifically concerned with the mechanisms of LTH storage have been reported so far. In order to obtain some information on this question, we have studied the behavior of isolated granule fractions incubated in vitro under a variety of carefully controlled experimental conditions.
Locations of 18S and 28S ribosomal genes on the chromosomes of the Indian muntjac.
Authors: Pardue M L , Hsu T C
Published: May 2004

The locations of genes coding for 18S and 28S ribosomal RNA have been mapped on metaphase chromosomes of the Indian muntjac M. muntjak by in situ hybridization with (3H)rRNA from the toad X. laevis. The results show that, in the muntjac, rDNA clusters are associated with the prominent secondary constrictions on the X and the Y1 chromos. In addition a cluster of rDNA is found near the tip of one arm on the longest pair of autosomes. The autosomal cluster of rDNAs usually does not express as a secondary constriction at metaphase.
Cell cycle and growth stage-dependent changes in the transport of nucleosides, hypoxanthine, choline, and deoxyglucose in cultured Novikoff rat hepatoma cells.
Authors: Plagemenn P G , Richey D P , Zylka J M , Erbe J
Published: May 2004

Populations of Novikoff rat hepatoma cells (subline N1S1-67) were monitored for the rates of transport of various substrates and for their incorporation into acid-insoluble material as a function of the age of cultures of randomly growing cells in suspension as well as during traverse of the cells through the cell cycle. Populations of cells were synchronized by a double hydroxyurea block or by successive treatment with hydroxyurea and Colcemid. Kinetic analyses showed that changes in transport rates related to the age of cultures or the cell cycle stage reflecte alterations in the V max of the transport processes, whereas the Km remained constant, indicating that changes in transport rates reflect alterations in the number of functional transport sites. The transport sites for uridine and 2-deoxy-D-glucose increased continuously during traverse of the cells through the cell cycle, whereas those for choline and hypoxanthine were formed early in the cell cycle. Increases in thymidine transport sites were confined to the S phase. Synchronized cells deprived of serum failed to exhibit normal increases in transport sites, although the cells divided normally at the end of the cell cycle. Arrest of the cells in mitosis by treatment with Colcemid prevented any further increases in transport rates. The formation of functional transport sites was also dependent on de novo synthesis of RNA and protein. Inhibition of DNA synthesis in early S phase inhibited the increase in thymidine transport rates which normally occurs during the S phase, but had no effect on the formation of the other transport systems. Transport rates also fluctuated markedly with the age of the cultures of randomly growing cells, reaching maximum levels in the mid-exponential phase of growth. The transport systems for thymidine and uridine were rapidly lost upon inhibition of protein and RNA synthesis, and thus seem to be metabolically unstable, whereas the transport systems for choline and 2-deoxy-D-glucose were stable under the same conditions.
Electron probe analysis of calcium transport by small intestine.
Authors: Warner R R , Coleman J R
Published: May 2004

Calcium transport in small intestine of rat and chick has been studied at the cellular level using the electron probe X-ray microanalyzer. Tissues were examined directly after removal as well as after incubation in a calcium solution. In both preparations, discrete calcium localizations were found associated with intracellular and extracellular goblet cell mucus. The in vitro preparations showed calcium in transit across the absorptive epithelium in discrete localizations. Although the primary path of transport was along lateral cell borders, some localizations were found in the cytoplasm in a supranuclear position. The effect of vitamin D depletion and repletion was to decrease and increase, respectively, the number of calcium localizations in transit across the epithelium. These results suggest that calcium is transported while in a sequestered form and indicate that goblet cell mucus plays a role in this transport process.
New immunolatex spheres: visual markers of antigens on lymphocytes for scanning electron microscopy.
Authors: Molday R S , Dreyer W J , Rembaum A , Yen S P
Published: May 2004

New immunochemical reagents consisting of antibodies bound to small latex spheres were used as visual markers for the detection and localization of cell surface antigens by scanning electron microscopy. Cross-linked latex spheres of various sizes from 300 to 3,4000 A in diameter were synthesized by aqueous emulsion copolymerization of methacrylate derivatives containing hydroxyl and carboxyl functional groups. Proteins and other molecules containing primary amino groups were covalently bonded to the acrylic spheres under a variety of mild conditions by the aqueous carbodiimide, cyanogen bromide, and glutaraldehyde methods. For use in the indirect immunochemical-labeling technique, goat antibodies directed against rabbit immunoglobulins were bonded to the spheres. These immunolatex reagents were shown to bind only to cells (red blood and lymphocytes) which had previously been sensitized with rabbit antibodies against cell surface antigens. Mouse spleen lymphocytes with exposed immunoglobulins on their surface (B cells) were labeled with these spheres and distinguished from unlabeled or T lymphocytes by scanning electron microscopy. The distribution of Ig receptors on lymphocytes was also studied using the spheres as visual markers. When lymphocytes were fixed with glutaraldehyde and subsequently labeled with the immunolatex reagents, a random distribution was observed by scanning electron microscopy; a patchy distribution was observed when unfixed lymphocytes were used. These results are consistent with studies using ferritin-labeled antibodies (S. De Petris and M. Raff. 1973. Nature [Lond.]. 241:257.) and support the view that Ig receptors on lymphocytes undergo translational diffusion. In addition to serving as visual markers for scanning electron microscopy, these latex spheres tagged with fluorescent or radioactive molecules have applications as highly sensitive markers for fluorescent microscopy and as reagents for quantitative studies of cell surface antigens and other receptors.
Messenger RNA metabolism of animal cells: possible involvement of untranslated sequences and mRNA-associated proteins
Authors: JR Greenberg
Published: May 2004

The past several years have seen a virtual revolution in the study of eukaryotic mRNA. Among the notable recent achievements are the positive identification of mRNA precursors in HnRNA, the enumeration of the DNA sequences from which mRNA is transcribed, and the finding that mRNA in cultured cells is much more stable than was previously believed. One of most far-reaching discoveries has been the finding that mRNA in eukaryotes contains poly A. This discovery, aside from providing a powerful tool for mRNA isolation, has generated a large body of research into the properties and metabolism of poly A itself. In addition, the finding of a poly A-associated protein has given a renewed stimulus to the study of proteins associated with mRNA. This review is devoted to a discussion of these and related achievements, and some of their implications
Hapten-sandwich labeling. II. Immunospecific attachment of cell surface markers suitable for scanning electron microscopy.
Authors: Nemanic M K , Carter D P , Pitelka D R , Wofsy L
Published: May 2004

A hapten-sandwich procedure has been used for immunospecific labeling of cell surface antigens with markers visible by scanning electron microscopy. Antihapten antibody was used to link hapten-modified tobacco mosaic virus, bushy stunt virus, or hemocyanin to hapten-modified human erythrocytes. The antihapten antibody bridge was also used to link the hapten-virus marker to hapten-modified antibodies against mammary tumor virus on mouse mammary tumor cells, or against immunoglobulin receptors on mouse splenic lymphocytes. In all cases, labeling was highly specific. With this technique, it is possible to (a) compare morphological features of cells bearing differing cell surface antigens, and (b) examine the arrangement of specific antigenic sites on a cell surface or their distribution relative to membrane structures such as microvilli.