bluuebunny/crossref_metadata_2025 huggingface dataset:
THE PERSISTENCE OF HEMOPOIETIC STEM CELLS IN VITRO
Authors: Meints Russell , Goldwasser Eugene
Published: May 2004

Cells capable of forming colonies in spleens of irradiated mice (CFU) are lost temporarily when bone marrow cells from rats or mice are maintained in culture. Rat marrow CFU go through a minimum at about 3 days after which there is a slow increase in the number of CFU in culture, reaching a maximum at 9 days. Mouse marrow CFU reach a minimum at 3 days and a maximum at 7 days. Some rat marrow CFU persist in culture for as long as 28 days.
RAPID DEGENERATION OF AMPULLARY ELECTRORECEPTOR ORGANS AFTER DENERVATION
Authors: Szamier R. Bruce , Bennett Michael V. L.
Published: May 2004

Electroreceptors (ampullary organs) of the transparent catfish (Kryptopterus bicirrhus) lie in the epidermis, and contain spherical receptor cells that receive purely afferent innervation from the lateral line nerve. Section of this nerve causes rapid degenerative changes to occur in the receptors. Fine structural alterations occur in the receptor cell synapses and nerve fiber 6–12 h postoperatively. Disruption of the receptor cells begins by 18 h and most are lost by 48 h. By 72 h supporting cells and secretory cells also show marked degeneration, and by 96 h they may be totally lost. The rapid degeneration of the electroreceptor organs of Kryptopterus should make them a useful preparation for analysis of neurotrophic functions.
FINE STRUCTURE OF NERVE FIBERS AND GROWTH CONES OF ISOLATED SYMPATHETIC NEURONS IN CULTURE
Authors: Bunge Mary Bartlett
Published: May 2004

The leading tips of elongating nerve fibers are enlarged into "growth cones" which are seen in tissue culture to continually undergo changes in conformation and to foster numerous transitory slender extensions (filopodia) and/or a veillike ruffling sheet. After explantation of 1-day-old rat superior cervical ganglia (as pieces or as individual neurons), nerve fibers and tips were photographed during growth and through the initial stages of aldehyde fixation and then relocated after embedding in plastic. Electron microscopy of serially sectioned tips revealed the following. The moving parts of the cone, the peripheral flange and filopodia, contained a distinctive apparently filamentous feltwork from which all organelles except membranous structures were excluded; microtubules were notably absent from these areas. The cone interior contained varied forms of agranular endoplasmic reticulum, vacuoles, vesicles, coated vesicles, mitochondria, microtubules, and occasional neurofilaments and polysomes. Dense-cored vesicles and lysosomal structures were also present and appeared to be formed locally, at least in part from reticulum. The possible roles of the various forms of agranular membranous components are discussed and it is suggested that structures involved in both the assembly and degradation of membrane are present in the cone. The content of these growing tips resembles that in sensory neuron growth cones studied by others.
APPEARANCE AND DISTRIBUTION OF FERRITIN IN MOUSE PERITONEAL MACROPHAGES IN VITRO AFTER UPTAKE OF HETEROLOGOUS ERYTHROCYTES
Authors: Fedorko Martha E. , Cross Nicholas L. , Hirsch James G.
Published: May 2004

Mouse peritoneal macrophages have been studied in vitro after ingestion of treated rat, rabbit, or sheep erythrocytes. Under light microscopy, phagocytic vacuoles persist up to 24 h. Macrophages lose benzidine reactivity about 5 h after red cell ingestion, and they become prussian blue positive at 2 days. Ultrastructural studies show little or no ferritin in control macrophages not fed erythrocytes. In contrast, after red cell ingestion, ferritin is widely distributed in the cytoplasmic matrix and in some cytoplasmic granules by 48 h. The Golgi complex, pinocytic vacuoles, endoplasmic reticulum, nuclei, and mitochondria do not contain ferritin. Between 2 and 4 days, ferritin in cytoplasmic granules increases, concomitant with decrease in the ferritin in the cytoplasmic matrix. Evidence is presented suggesting that ferritin in the cytoplasmic matrix is translocated into cytoplasmic granules by autophagy. Polyacrylamide gel studies on macrophages after uptake of red blood cells labeled with radioiron confirm that macrophages produce radiolabeled ferritin by 4 days.
PEROXISOMES IN INNER ADRENOCORTICAL CELLS OF FETAL AND ADULT GUINEA PIGS
Authors: Black Virginia H. , Bogart Bruce I.
Published: May 2004

Abundant membrane-bounded granules, 0.1–0.45 µm in diameter, occur among the elements of the smooth-surfaced endoplasmic reticulum in zona fasciculata and zona reticularis adrenocortical cells of guinea pigs. Acid phosphatase cannot be cytochemically demonstrated in them, and they are therefore distinct from lysosomes. Incubation in medium containing 3,3'-diaminobenzidine results in dense staining of the granules, identifying them as peroxisomes. These small peroxisomes increase in number as fetal adrenocortical cells differentiate, and they appear to arise from dilated regions of endoplasmic reticulum. They maintain interconnections with the smooth endoplasmic reticulum and with one another.
ANIONIC SITES OF HUMAN ERYTHROCYTE MEMBRANES
Authors: Nicolson Garth L.
Published: May 2004

The effects of pH, trypsin, and phospholipase C on the topographic distribution of acidic anionic residues on human erythrocytes was investigated using colloidal iron hydroxide labeling of mounted, fixed ghost membranes. After glutaraldehyde fixation at pH 6.5–7.5, the positively charged colloidal particles were bound to the membranes in small randomly distributed clusters. The clusters of anionic sites were reversibly aggregated by incubation at pH 5.5 before fixation at the same pH. These results correlate with the distribution of intramembranous particles found by Pinto da Silva (J. Cell Biol. 53:777), with the exception that the distribution of anionic sites on a majority of the fixed ghosts at pH 4.5 was aggregated instead of dispersed. The randomly distributed clusters could be nonreversibly aggregated by trypsin or phospholipase C treatment of intact ghosts before glutaraldehyde fixation. Previous glutaraldehyde fixation prevented trypsin and pH induced aggregation of the colloidal iron sites. Evidence that N-acetylneuraminic acid groups are the principal acidic residues binding colloidal iron was the elimination of greater than 85% of the colloidal iron labeling to neuraminidase-treated cell membranes. Colloidal iron binding N-acetylneuraminic acid residues may reside on membrane molecules such as glycophorin, a sialoglycoprotein which contains the majority of the N-acetylneuraminic acid found on the human erythrocyte membrane.
PERMEABILITY OF MUSCLE CAPILLARIES TO EXOGENOUS MYOGLOBIN
Authors: Simionescu Nicolae , Simionescu Maia , Palade George E.
Published: May 2004

Whale skeletal muscle myoglobin (mol wt 17,800; molecular dimensions 25 x 34 x 42 Å) was used as a probe molecule for the pore systems of muscle capillaries. Diaphragms of Wistar-Furth rats were fixed in situ at intervals up to 4 h after the intravenous injection of the tracer, and myoglobin was localized in the tissue by a peroxidase reaction. Gel filtration of plasma samples proved that myoglobin molecules remained in circulation in native monomeric form. At 30–35 s postinjection, the tracer marked ∼75% of the plasmalemmal vesicles on the blood front of the endothelium, 15% of those located inside and none of those on the tissue front. At 45 s, the labeling of vesicles in the inner group reached 60% but remained nil for those on the tissue front. Marked vesicles appeared on the latter past 45 s and their frequency increased to ∼80% by 60–75 s, concomitantly with the appearance of myoglobin in the pericapillary spaces. Significant regional heterogeneity in initial labeling was found in the different segments of the endothelium (i.e., perinuclear cytoplasm, organelle region, cell periphery, and parajunctional zone). Up to 60 s, the intercellular junctions and spaces of the endothelium were free of myoglobin reaction product; thereafter, the latter was detected in the distal part of the intercellular spaces in concentration generally equal to or lower than that prevailing in the adjacent pericapillary space. The findings indicate that myoglobin molecules cross the endothelium of muscle capillaries primarily via plasmalemmal vesicles. Since a molecule of this size is supposed to exit through both pore systems, our results confirm the earlier conclusion that the plasmalemmal vesicles represent the large pore system; in addition, they suggest that the same structures are, at least in part, the structural equivalent of the small pore system of this type of capillaries.
BIOCHEMICAL CYTOLOGY OF TRICHOMONAD FLAGELLATES
Authors: Müller Miklós
Published: May 2004

To determine the localization of several enzymes in Tritrichomonas foetus, the axenic KV-1 strain was grown in Diamond's medium with bovine serum, homogenized in 0.25 M sucrose, and subjected to analytical differential and isopycnic centrifugation. The fractions were assayed for their enzymatic composition and examined electron microscopically. NADH and NADPH dehydrogenases, about 90% of the catalase, and two hydrolases, α-galactosidase and manganese-activated ß-galactosidase I are in the nonsedimentable part of the cytoplasm. α-Glycerophosphate and malate dehydrogenases are associated with a large particle, whose equilibrium density in sucrose gradients is 1.24. This particle corresponds to that population of the paracostal and paraxostylar granules which, having a uniform granular matrix surrounded by a single membrane, resemble microbodies from other organisms. The small sedimentable portion of catalase (about 10% of the total activity) is not associated with these granules and equilibrates at density 1.22. The nature of the subcellular entity carrying catalase could not be ascertained. Hydrolases with a pH optimum around 6–6.5 (protease, ß-N-acetylglucosaminidase, ß-N-acetylgalactosaminidase, and cation-independent ß-galactosidase II), as well as a large part of acid phosphatase, are associated with a population of large particles which equilibrate at densities from 1.15 to 1.20. The hydrolases in these granules lose their structure-bound latency easily after freezing and thawing. These particles correspond to another population of the paracostal and paraxostylar granules which have varied shape and inhomogeneous content with frequent myelin figures, indicating a digestive function. The rest of the phosphatase and most of the acid ß-glucuronidase activity are in a smaller granule fraction with an equilibrium density around 1.18. The latency of these enzymes is quite resistant to freezing and thawing. This particle population consists of smaller, very often flattened vesicles and granules, many of which are clearly fragments of the prominent Golgi apparatus of the cell.
RNA SYNTHESIS BY EXOGENOUS RNA POLYMERASE ON CYTOLOGICAL PREPARATIONS OF CHROMOSOMES
Authors: Sederoff R. , Clynes R. , Poncz M. , Hachtel S.
Published: May 2004

Cytological preparations of Drosophila polytene chromosomes serve as templates for RNA synthesis carried out by exogenous RNA polymerase (Escherichia coli). Incorporation of labeled ribonucleoside triphosphates into RNA may be observed directly by autoradiography. Because of the effects of rifampicin, actinomycin D, ribonuclease, high salt, and the requirement for all four nucleoside triphosphates, we conclude that the labeling observed over chromosomes is due to DNA-dependent RNA polymerase activity. Using this method, one can observe RNA synthesis in vitro on specific chromosome regions due to the activity of exogenous RNA polymerase. We find that much of the RNA synthesis in this system occurs on DNA sequences which appear to be in a nondenatured state.
LYSOSOMAL PHYSIOLOGY IN TETRAHYMENA
Authors: Rothstein Thomas L. , Blum J. J.
Published: May 2004

Log-phase Tetrahymena were washed and resuspended in a dilute salt solution supplemented with glucose, acetate, pyruvate, or carmine, as desired, and then incubated for 5 h. Intra- and extracellular activities of acid phosphatase, α-glucosidase, and ribonuclease were assayed. Extracellular activities were corrected for proteolytic degradation. The three nutritive substrates affected both the amount and pattern of extracellular enzyme release, but carmine had no effect. Intracellular activities declined early in the starvation period, but partially recovered with time, particularly α-glucosidase activity. Acetate reduced the decline in acid phosphatase activity; acetate and glucose enhanced the recovery of α-glucosidase activity; carmine had no effect on intracellular enzyme activities. Protein content changed little and was unaffected by the addition of substrates. Glycogen content increased during incubation; acetate and glucose enhanced the increase.
VITAMIN B12 AND THE MACROMOLECULAR COMPOSITION OF EUGLENA
Authors: Johnston Pamela Leban , Carell Edgar F.
Published: May 2004

When vitamin B12 is added to B12-deficient cultures of Euglena gracilis, the cells undergo two relatively synchronous cell divisions within a shorter than usual period of time, apparently as a result of a transitory shortening of the cell cycle. The first cell division pulse, occurring 4.5 h after addition of B12, is preceded by the completion of DNA duplication, but appears to involve no net synthesis of RNA or protein. Before the second round of cell division at about 11 h, a significant amount of DNA synthesis is observed. This time it is accompanied by a minor increase in the RNA and protein content of the culture. The cellular contents of RNA and protein were observed to decrease steadily after the resumption of cell division in B12-depleted cultures receiving the vitamin. Ultimately all three macromolecules returned to their nondeficient, plateau stage levels; by this time, cell division had ceased.
THE HISTONES ASSOCIATED WITH CONDENSED AND EXTENDED CHROMATIN OF MOUSE LIVER
Authors: Johmann Carol , Eckhardt Ronald A. , Gorovsky Martin A.
Published: May 2004

Histones were extracted from isolated mouse liver nuclei, and from mouse liver condensed and extended chromatin. Mouse liver histones were found to be very similar to those of calf thymus in their solubility properties, relative electrophoretic mobilities, and molecular weights as determined on SDS-polyacrylamide gels. Quantitative analysis by high-resolution gel electrophoresis demonstrated a remarkable similarity between the histones of condensed chromatin and those of extended chromatin. However, minor differences were found. A unique subspecies was found only in condensed chromatin histone and the relative amounts of fractions F2A1 and F2A2 differed in the two types of chromatin. The ratio of the parental to the acetylated form of F2A1 was identical in the two chromatin samples. Since DNA extracted from the condensed chromatin fraction consisted of approximately 50% satellite DNA, the general similarities between the histones of condensed and extended chromatin make it likely that even this simple, highly repetitive DNA is complexed with a number of histone subfractions.
SELECTIVE RELEASE OF CONTENT FROM MICROSOMAL VESICLES WITHOUT MEMBRANE DISASSEMBLY
Authors: Kreibich Gert , Debey Pascale , Sabatini David D.
Published: May 2004

Rat liver rough microsomes treated with a series of desoxycholate (DOC) concentrations from 0.003 to 0.4% were analyzed by isopycnic sucrose density gradient centrifugation in media containing high or low salt concentrations. Tritium-labeled precursors administered in vivo were used as markers for ribosomes (orotic acid, 40 h), phospholipids (choline, 4 h), membrane proteins (leucine, 3 days), and completed secretory proteins of the vesicular cavity (leucine, 30 min). Within a narrow range of DOC concentrations (0.025–0.05%), the vesicular polypeptides were selectively released from the microsomes, while ribosomes, nascent polypeptides, and microsomal enzymes of the electron transport systems were unaffected. The detergent concentration which led to leakage of content was a function of the ionic strength and of the microsome concentration. At the lowest effective DOC concentration the microsomal membranes became reversibly permeable to macromoles as shown by changes in the density of the vesicles in Dextran gradients and by the extent of proteolysis by added proteases. Incubation of rough microsomes with proteases in the presence of 0.025% DOC also led to digestion of proteins from both faces of the microsomal membranes and to a lighter isopycnic density of the membrane vesicles.
AUTORADIOGRAPHIC EVIDENCE FOR MANY SEGREGATING DNA MOLECULES IN THE CHLOROPLAST OF OCHROMONAS DANICA
Authors: Gibbs Sarah P. , Poole Ronald J.
Published: May 2004

Light-grown cells of Ochromonas danica, which contain a single chloroplast per cell, were labeled with [methyl-3H]thymidine for 3 h (0.36 generations) and the distribution of labeled DNA among the progeny chloroplasts was followed during exponential growth in unlabeled medium for a further 3.3 generations using light microscope autoradiography of serial sections of entire chloroplasts. Thymidine was specifically incorporated into DNA in both nuclei and chloroplasts. Essentially all the chloroplasts incorporated label in the 3-h labeling period, indicating that chloroplast DNA is synthesized throughout the cell cycle. Nuclear DNA has a more limited S period. Both chloroplast DNA and nuclear DNA are conserved during 3.3 generations. After 3.3 generations in unlabeled medium, grains per chloroplast followed a Poisson distribution indicating essentially equal labeling of all progeny chloroplasts. It is concluded that the average chloroplast in cells of Ochromonas growing exponentially in the light contains at least 10 segregating DNA molecules.
OSMOTIC REGULATION OF α-AMYLASE SYNTHESIS AND POLYRIBOSOME FORMATION IN ALEURONE CELLS OF BARLEY
Authors: Armstrong Judith E. , Jones Russell L.
Published: May 2004

Water stress inhibits the gibberellic acid (GA3)-induced synthesis of α-amylase in aleurone layers of barley (Hordeum vulgare L.). Electron microscope evidence indicates that the effect of water stress induced by 0.6 M solutions of polyethylene glycol (PEG) is to reduce the binding of ribosomes to the endoplasmic reticulum. This was confirmed by sucrose density gradient centrifugation of polyribosome preparations from stressed cells. The reduction in polyribosome formation does not result from reduced ribosome activity as measured by [3H]peptidylpuromycin formation. Thus, calculation of percent active ribosomes shows that osmoticum has little effect on the ability of ribosomes to incorporate puromycin into nascent protein. Water stress does not cause a marked decrease in the total RNA level of aleurone cells. Estimates of total RNA in postmitochondrial supernatant fractions from stressed cells show only a reduction of 8–9% relative to the control. Membrane synthesis measured by [14C]choline incorporation is depressed by 15% in cells stressed with 0.6 M PEG for 2.5 hours.
COMPARISON AND CHARACTERIZATION OF NUCLEAR ISOLATION PROCEDURES AS APPLIED TO CHICK OVIDUCT
Authors: Knowler John T. , Moses Harold L. , Spelsberg Thomas C.
Published: May 2004

A number of methods for the preparation of chick oviduct nuclei have been compared. Nuclei have been isolated in hypertonic sucrose and citric acid and the product has been characterized with respect to cleanliness, ultrastructure, RNA polymerase activity, RNA integrity, and chromatin composition. The study demonstrates that the choice of oviduct nuclear isolation procedure will depend markedly on the purpose for which the nuclei are required. Thus, nuclei prepared entirely in high-molarity sucrose retain the highest levels of RNA polymerase. Those prepared rapidly in the presence of citric acid retain nuclear RNA in an essentially undegraded state. Finally, a bulk preparation is described which, because of its adaptability and high yield of morphologically intact nuclei using large amounts of tissue, is ideal for use in preparing chromatin. Conditions are described by which isolated nuclei can be stored for up to 6 months and retain their morphology, chemical characteristics, and RNA polymerase activity.
PARENCHYMAL CELLS FROM ADULT RAT LIVER IN NONPROLIFERATING MONOLAYER CULTURE
Authors: Chapman Geoffrey S. , Jones Albert L. , Meyer Urs A. , Montgomery Bissell D.
Published: May 2004

Hepatic parenchymal cells from adult rats, established in vitro as a monolayer, have been evaluated by electron microscopy. Within 24 h after the initial seeding, the incubated cells were polygonal and in close apposition with three to six neighboring cells. The ultrastructure of the monolayer cells was examined at this time and after 3 and 10 days of incubation. With the exception of a few enlarged mitochondria, organelles in both the 1- and 3-day monolayer cells were indistinguishable quantitatively and morphologically from those found in the intact liver. After 10 days of incubation, however, the rough-surfaced endoplasmic reticulum (RER) had become dilated and vesiculated. In all cells studied, portions of RER were found in a close spatial relationship to mitochondria. From its frequency, this association appeared to be more than fortuitous, and the organelle complex may represent a functional unit necessary for new membrane formation, as suggested previously. The Golgi complexes of 1- and 3-day cells contained very low density lipoprotein-sized particles, which suggests that the monolayer cells synthesize lipoproteins. These electron microscope observations demonstrate that adult hepatic parenchymal cells in monolayer retain for several days the subcellular structural elements characteristic of normally functioning hepatocytes.
Spermiogenesis in Lumbricus herculeus
Authors: Gatenby J. Brontë , Dalton A. J.
Published: May 2004

Small pieces of the sperm sacs of Lumbricus herculeus were fixed for 4 hours in chrome-osmium, embedded in methacrylate, sectioned with a Porter-Blum microtome, and studied with a R.C.A. EMU-2C electron microscope.

Each spermatid of a group developing synchronously is attached by a cytoplasmic strand to a common nutrient protoplasmic mass. This mass contains mitochondria and yolk bodies but is anucleate.

The proximal centriole, that is, the centriole nearer the nucleus, is at first associated with a small peg which becomes firmly attached to the nuclear membrane. Later these two bodies become separated during the development of the middle-piece which is differentiated in the usual manner from a nebenkern formed by the fusion of 6 or 7 mitochondria.

The acrosome develops in relation to the dictyosome (Golgi body), itself composed of 8 or more individual flattened sacs and situated in the cytoplasm opposite the point of attachment of the spermatid to the nutrient mass. Soon after its formation, the acrosome becomes incorporated into a cytoplasmic appendage or acrosome carrier. The carrier moves from its original position, along the lateral border of the elongating nucleus, to the distal margin of the nucleus where the acrosome is deposited. No evidence was found of a centriole located at the point of junction between nucleus and acrosome as suggested by earlier workers.
Dynamics of Spindle Formation and Its Inhibition by Chemicals
Authors: Taylor Edwin W.
Published: May 2004

The formation of the mitotic spindle of the newt cell in tissue culture has been studied, using polarized light. The rate of formation was measured and it was shown that the spindle increased in length at a constant rate until the maximum was attained. During metaphase the spindle shortened to about 50 to 60 per cent of its original length, reaching a minimum just before anaphase. No birefringence was detected in late anaphase in the spindle region after the chromosome masses had separated.

The effects of certain compounds which are believed to inhibit protein synthesis were investigated. Chloramphenicol added in early prophase prevented the formation of a spindle of normal length. The possible relation of chloramphenicol to the synthesis of spindle proteins is discussed.
Differents aspects du fer dans l'organisme
Authors: Bessis Marcel , Breton-Gorius Janine
Published: May 2004

Des cellules réticulaires remplies de molécules de ferritine et d'hémosidérine se trouvent au centre "d'ilots érythroblastiques."

Dans la zone de contact entre la cellule réticulaire chargée de ferritine et les érythroblastes, on voit des invaginations et des petites vacuoles au bord desquelles adhérent des molécules de ferritine. Il est postulé que la ferritine passe de la cellule réticulaire centrale dans les érythroblastes par ce mécanisme, apparenté à la pinocytose (rhophéocytose).

Dans tous les érythroblastes normaux, il existe du fer sous forme de ferritine. Celle-ci peut se trouver à l'état dispersé ou agglomérée en amas. Lorsque ces amas sont assez gros, ils sont visibles au microscope optique: ce sont les granules des sidéroblastes.

On trouve du fer dans les mitochondries, mais rarement à l'état normal. Il est soit sous forme de granules ferritiniques soit sous forme de micelles ferrugineuses.

Dans les thalassémies et d'autres maladies s'accompagnant d'un trouble de l'hémoglobinogénèse, on trouve, en grande quantité, du fer visible au microscope électronique, dans les érythroblastes. Il s'y trouve sous forme de ferritine, en amas ou dispersé. Il existe parfois en grande quantité dans les mitochondries soit sous forme de ferritine, soit sous forme de micelles ferrugineuses.

Il semble que soit objectivé ainsi le trouble de la synthèse de l'hémoglobine: le fer inutilisé s'accumule dans des érythroblastes hypochromes.

Il est probable qu'à l'etat normal, le fer est métabolisé dans les mitochondries. Dans le thalassémies et les anémies hypochromes hypersidérémiques, il semble souvent bloqué dans ces organites.
Fine Structure of Cellular Inclusions in Measles Virus Infections
Authors: Kallman Frances , Adams John M. , Williams Robley C. , Imagawa David T.
Published: May 2004

Cells which are infected with measles virus have been known for some time to contain inclusion material that is distinguishable from normal cellular components by application of traditional staining methods and observation in the light microscope. The fine structure of the inclusion material contained in HeLa cells infected with Edmonston strain of measles virus has been examined in the electron microscope. Two steps have been found necessary in this study: (1) the recognition by phase-contrast microscopy of the living cell of bodies that are defined as inclusion material when the cells are classically stained; and (2) the recognition in the electron microscope of inclusion-body material that had previously been identified in the living cell.

The fine structure of the nuclear and cytoplasmic inclusion material in osmium-treated cells was found to consist mainly of randomly arrayed filaments of low electron density. Dense, highly ordered arrays of filaments were found near the center of the nuclear inclusions, sometimes as a two-dimensional, nearly orthogonal arrangement. If the size of the measles virus is taken to be around 100 mµ in diameter, the strands seen in the inclusions cannot be fully formed virus.
Electron Microscopy of Neurospora crassa Mycelia
Authors: Shatkin A. J. , Tatum E. L.
Published: May 2004

The fine structure of vegetative mycelia of the filamentous Ascomycete, Neurospora crassa, has been investigated by the standard techniques of electron microscopy. Addition of uranyl nitrate to the methacrylate-embedding medium minimized disruption of the specimens—an accident often observed in the preparation of microbial material.

This report describes the presence of a chitinous polysaccharide wall containing fine fibrils embedded in a homogeneous matrix. A sinuate plasma membrane lies adjacent to the inner wall surface. This membrane is often closely associated with the endoplasmic reticulum of the cytoplasm. Numerous mitochondria of the classical type, and dense particles of 10 mµ diameter occur throughout the cytoplasm. The nuclear region is surrounded by a double membrane with pore openings. Associated with the nuclear envelope is a dense area, the nucleolus.

The significance of these observations and their relationship to other forms is discussed.
A BIOCHEMICAL AND CYTOCHEMICAL STUDY OF ADENOSINE TRIPHOSPHATASE ACTIVITY IN THE PHLOEM OF NICOTIAN A TABACUM
Authors: Gilder Jamison , Cronshaw James
Published: May 2004

A biochemical and cytochemical study has been made of the distribution of ATPase in mature and differentiating phloem cells of Nicotiana tabacum and of the substrate specificity and effects of fixation on enzyme activity. Homogenates of unfixed leaf midveins and midveins fixed in formaldehyde-glutaraldehyde were assayed for enzyme activity by determining the amount of Pi, liberated per milligram of protein from various substrates in a 30 min period at pH 7.2. In fresh homogenates, hydrolysis of ATP was not significantly different from that of ITP, CTP, and UTP. Hydrolysis of GTP was slightly higher than that of ATP. ATP hydrolysis by fresh homogenates was 17% more extensive than that of ADP, 76% more extensive than that of 5'-AMP, and was inhibited by fluoride and p-chloromercuribenzoate (PCMB). There was little or no hydrolysis of the competitive inhibitors 2'- and 3'-AMP nor with the alternate substrates p-nitrophenylphosphate (PNP) or ß-glycerophosphate (ß-GP). In homogenates of material fixed in formaldehyde-glutaraldehyde for 1¼ h, ATPase activity was 13% preserved. Hydrolysis of ATP by fixed homogenates was not significantly different from that of ADP, 5'-AMP, ITP, CTP, and GTP. Hydrolysis of UTP was lower. Fluoride and PCMB inhibited fixed ATPase activity. The results of cytochemical localization experiments using a lead phosphate precipitation technique were in agreement with the biochemical results. Similar localization patterns were obtained with the nucleoside triphosphates ATP, CTP, GTP, ITP, and UTP. Activity was also localized with ADP and 5'-AMP but not with the competitive inhibitors 2'- and 3'-AMP, nor with PNP or ß-GP. Little or no reaction product was deposited in other controls incubated without substrate or with substrate plus fluoride, PCMB, or N-ethylmaleimide. ATPase activity was demonstrated chiefly at the plasma membrane of mature and differentiating phloem cells and was associated with the P-protein of mature sieve elements. It is suggested that the phloem transport system derives its energy from the demonstrated nucleoside triphosphatase activity.
INHIBITION OF CELL GROWTH IN THE G1 PHASE BY ADENOSINE 3',5'-CYCLIC MONOPHOSPHATE
Authors: Froehlich Jeffrey E. , Rachmeler Martin
Published: May 2004

Incorporation of tritiated thymidine into acid-precipitable material was used to measure the rate of DNA synthesis in secondary cultures of human diploid fibroblasts. Confluent cultures of human diploid fibroblasts, which are synchronized in the G1 phase due to contact inhibition, were released from growth inhibition either by the addition of fresh medium to the cultures or by trypsinization and replating at nonconfluent densities. Either treatment resulted in a synchronous wave of DNA synthesis beginning 10–15 h after treatment and peaking at 20–25 h. In confluent cultures stimulated by fresh medium, either the addition of 0.25 mM N6, O2-dibutyryl-adenosine 3',5'-cyclic monophosphate (db-cAMP) to the medium in the interval 4–8 h after stimulation or the replacement of the fresh medium in that same 4 h interval with the depleted medium present on the cells for the 2 day period before stimulation delayed the synchronous onset of DNA synthesis in the cultures by about 4 h. In nonconfluent cultures freshly seeded from trypsinized confluent cultures, this same depleted medium obtained after a 2 day incubation of fresh medium on confluent cultures is shown to support the progress of the cells into S phase; however, the addition of 0.25 mM db-cAMP to the medium 3½ h after replating still partially prevented the initiation of DNA synthesis in the cultures. The results are discussed in terms of the role of serum and cAMP in the control of cell growth in fibroblast cultures.
STIMULATION BY INSULIN OF RNA SYNTHESIS IN CHICK FIBROBLASTS
Authors: Baseman Joel B. , Paolini Domenic , Amos Harold
Published: May 2004

After the addition of insulin to monolayers of chick fibroblasts previously incubated in serum-free medium, the rates of protein and RNA synthesis increase continuously during the first 8–10 h. Little stimulation of DNA synthesis or mitosis results with the addition of insulin alone in contrast to the addition of fresh serum which stimulates both markedly. The stimulation in RNA synthesis does not result from expansion of the nucleotide pool but is correlated with increases in RNA polymerase activity. All major classes of RNA are stimulated; processing of preribosomal RNA to 28S and 18S and the association of this mature RNA with ribosomes appear to occur normally. The kinetics of stimulation of 5S RNA differ from those of the synthesis of 4S and of ribosomal RNA. Insulin and serum appear to affect the synthesis or stability of certain transcripts differentially.
NEW MEMBRANE FORMATION DURING CYTOKINESIS IN NORMAL AND CYTOCHALASIN B-TREATED EGGS OF XENOPUS LAEVIS
Authors: de Laat Siegfried W. , Bluemink John G.
Published: May 2004

The electrical membrane potential (Em) and electrical membrane resistance (Rm) were measured continuously during the first cleavage of Xenopus eggs, using intracellular microelectrodes. A sharp hyperpolarization of Em and decrease in Rm can be observed from 6 to 8 min after the onset of cleavage. This moment coincides with the onset of the insertion of new membrane (Bluemink and de Laat, 1973) leading to the formation of the interblastomeric membrane during normal cleavage. Removal of the vitelline membrane or exposure to cytochalasin B (CCB) leads to exposure of the entire surface area of the membrane newly formed during cleavage. These conditions allow for a direct measurement of the permeability properties of the new membrane. It was found that under these conditions Em reaches values about 3 times more negative and Rm reaches values about 1.5–3 times smaller than during normal cleavage. The extent of reduction of Rm can be correlated with the surface area of the newly formed membrane. We conclude that the new membrane has different ionic permeability properties than the pre-existing membrane (most probably a relatively high permeability for K+ ions). Its mean specific resistance is 1–2 kΩ·cm2, as against 74 kΩ·cm2 for the pre-existing membrane. No influence of CCB on the permeability properties of the pre-existing or new membrane could be detected.
THE MYOCARDIAL INTERSTITIUM: ITS STRUCTURE AND ITS ROLE IN IONIC EXCHANGE
Authors: Frank J. S. , Langer G. A.
Published: May 2004

The structures present in the rabbit myocardial interstitium have been defined and quantified. Stereological methods were used for the quantification. The extracellular space contains abundant ground substance (23%) distributed in a homogeneous mat throughout the space and within the T tubules. The remainder of the space contains 59% blood vessels, 6% "empty" space, 4.0% collagen, and 7.0% connective tissue cells. The arrangement of the interstitium in relation to the myocardial cells and the capillaries has been described. In addition, the extracellular space was measured using extracellular markers: 14C sucrose (neutrally charged), 35SO4 (negatively charged), and 140La (positively charged). The La+++ space differed markedly from the other two (P << 0.001), indicating extensive binding of La+++ to polyanionic extracellular structures. Cetylpyridinium chloride, a cationic detergent specific for polysaccharides, caused precipitation of the ground substance and marked decrease in the La+++ space. This study indicates the considerable structural complexity of the interstitium. The effects of an abundant negatively charged protein-polysaccharide within the interstitium has been discussed in terms of cation exchange in arterially perfused tissue.
LOCALIZATION OF TRANSPORT ADENOSINE TRIPHOSPHATASE IN RAT CORNEA
Authors: Leuenberger Peter M. , Novikoff Alex B.
Published: May 2004

Increasingly, physiological and biochemical data on the mammalian cornea have emphasized the importance of the endothelial rather than the epithelial layer in regulating the state of hydration of the cornea, via a transport ATPase. This view is supported by the observations reported here. With the Ernst method, the first cytochemical procedure to be readily responsive to ions and ouabain, the major sites of enzyme reaction product are the intercellular spaces of the endothelial layer. This localization was not found by us with the so-called ATPase method of Wachstein and Meisel.
BRIDGES BETWEEN MICROTUBULES
Authors: McIntosh J. R.
Published: May 2004

Bridges between microtubules have been studied with the electron microscope in the axostyle of Saccinobaculus and in various tubule systems of chicken testis, including the helix of tubules surrounding the elongating spermatid nucleus and the flagellum of the sperm tail. In addition to the previously described periodic bridges, evidence is presented that nonperiodic bridges exist between certain tubules. An analysis of axial spacing between adjacent nonperiodic bridges suggests that these structures are attached to periodic binding sites on the microtubule wall, but that not all the binding sites are filled. The bridges appear nonperiodic as a result of random occupancy of some fraction of the periodic sites. The distribution of these binding sites is related to the substructure of the microtubule wall as seen with negative staining and optical diffraction.
ANALYTICAL STUDY OF MICROSOMES AND ISOLATED SUBCELLULAR MEMBRANES FROM RAT LIVER
Authors: Beaufay Henri , Amar-Costesec Alain , Feytmans Ernest , Thinès-Sempoux Denise , Wibo Maurice , Robbi Mariette , Berthet Jacques
Published: May 2004

The series introduced by this paper reports the results of a detailed analysis of the microsomal fraction from rat liver by density gradient centrifugation. The biochemical methods used throughout this work for the determination of monoamine oxidase, NADH cytochrome c reductase, NADPH cytochrome c reductase, cytochrome oxidase, catalase, aminopyrine demethylase, cytochromes b5 and P 450, glucuronyltransferase, galactosyltransferase, esterase, alkaline and acid phosphatases, 5'-nucleotidase, glucose 6-phosphatase, alkaline phosphodiesterase I, N-acetyl-ß-glucosaminidase, ß-glucuronidase, nucleoside diphosphatase, aldolase, fumarase, glutamine synthetase, protein, phospholipid, cholesterol, and RNA are described and justified when necessary.
CHARACTERIZATION OF A UNIQUE MUSCLE CELL LINE
Authors: Schubert David , Harris A. John , Devine Carrick E. , Heinemann Stephen
Published: May 2004

A clonal cell line derived from a mouse neoplasm is described which shares many properties with smooth muscle. The cells have electrically excitable membranes capable of generating overshooting action potentials, and they contract both spontaneously and with electrical stimulation. They respond to the iontophoretic application of acetylcholine with a depolarizing response, and to norepinephrine with a hyperpolarizing response. Electron microscopy reveals that the cells have a morphology similar in many, but not all, respects to that of smooth muscle cells in vivo. The cells secrete soluble collagen-like molecules in addition to several proteins of undefined function. Finally, there is an increase in the specific activities of creatine phosphokinase and myokinase associated with increased cell density and the cessation of cell division.
FORMATION OF BONE TISSUE IN CULTURE FROM ISOLATED BONE CELLS
Authors: Binderman Itzhak , Duksin Dan , Harell Arieh , Katzir (Katchalski) Ephraim , Sachs Leo
Published: May 2004

A system is described for the formation of bone tissue in culture from isolated rat bone cells. The isolated bone cells were obtained from embryonic rat calvarium and periosteum or from traumatized, lifted periosteum of young rats. The cells were cultured for a period of up to 8 wk, during which time the morphological, biochemical, and functional properties of the cultures were studied. Formation of bone tissue by these isolated bone cells was shown, in that the cells demonstrated osteoblastic morphology in light and electron microscopy, the collagen formed was similar to bone collagen, there was mineralization specific for bone, and the cells reacted to the hormone calcitonin by increased calcium ion uptake. Calcification of the fine structure of the cells and the matrix is described. Three stages in the calcification process were observed by electron microscopy. It is concluded that these bone cells growing in vitro are able to function in a way similar to such cells in vivo. This tissue culture system starting from isolated bone cells is therefore suitable for studies on the structure and function of bone.
ECDYSONE-MEDIATED STIMULATION OF DOPA DECARBOXYLASE ACTIVITY AND ITS RELATIONSHIP TO OVARIAN DEVELOPMENT IN AEDES AEGYPTI
Authors: Schlaeger Dorothy A. , Fuchs Morton S. , Kang Suk-Hee
Published: May 2004

Very little dopa decarboxylase activity is detectable in adult female mosquitoes Aedes aegypti which have not been allowed to engorge blood. However, when such females are injected with the molting hormone ß-ecdysone a marked stimulation of this enzyme's activity is observable. No stimulation is observed in males similarly injected, nor in females injected with cholesterol or a juvenile hormone mimic. In addition, ecdysone injection initiates ovarian development in these anautogenous non-blood-fed mosquitoes. The extent of stimulation in both cases is dependent upon the amount of ß-ecdysone administered. These results suggested that ecdysone may play a role in ovarian development in Aedes and led us to hypothesize that a normal blood meal may trigger the synthesis, activation, or release of this hormone endogenously. Using the radioimmune assay for ecdysone developed by Borst and O'Connor (Science [Wash. D. C.] 178:4–18.), we found that the titer of an antigenic-positive material, presumably ecdysone or a closely related analogue, substantially increased 24 h after blood feeding, thereby supporting our postulation.
CHANGES IN THE RIBOSOME CONTENT, PRINCIPAL MICROSOMAL PROTEIN COMPOSITION, AND SECRETORY CHARACTER OF MAMMARY EPITHELIAL ROUGH ENDOPLASMIC RETICULUM DURING DIFFERENTIATION
Authors: Slaby Frank , Brown Carolyn
Published: May 2004

The equilibrium density distribution, protein composition, and secretory character of mouse mammary epithelial rough microsomes have been determined during differentiation. The density range exhibited by the rough microsomes broadens during mammary development; rough microsomes within the 1.25–1.29 g/ml density range appear soon after conception and then within the 1.30–1.34 range after the onset of lactation. The appearance of these denser microsomes represents the progressive increase of the average ribosome content of the rough endoplasmic reticulum (ER) during gestation and lactation. Fractionation of rough microsomal proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis reveals that two proteins, having molecular weights of 57,000 and 76,000, occur to a significant extent only during lactation and are then most prominent in the very dense rough microsomes of the 1.30–1.34 range. Nascent polypeptide chains discharged (by incubation with puromycin) from 17-days lactation rough microsomes in either the 1.21–1.29 or 1.30–1.34 density range are distributed equally between the intra- and extravesicular compartments. Whereas 36% of the chains are discharged intravesicularly from 1-day lactation rough microsomes in the 1.30–1.34 range, only 25% are so discharged from those in the 1.21–1.29 range. The results indicate (a) that there is no correlation between the relative levels in lactation rough microsomes of the two microsomal proteins which become prominent during lactation and the extent of secretory activity and (b) that for a short period after parturition the rough ER elements bearing high surface densities of ribosomes have a greater proportion of ribosomes synthesizing milk proteins than the rough ER elements with moderate ribosome densities.
KERATIN SYNTHESIS DURING DEVELOPMENT OF THE EMBRYONIC CHICK FEATHER
Authors: Kemp D. J. , Dyer P. Y. , Rogers G. E.
Published: May 2004

The synthesis of keratin proteins during development of the embryonic chick feather was studied by quantitative gel electrophoresis of the reduced and carboxymethylated proteins. The results demonstrated a coordinated synthesis of the major keratin proteins, during and after the onset of keratin synthesis. The results from gel electrophoresis correlated well with electron microscope visualization or keratin fibrils in the developing feathers. Autoradiography at the electron microscope level indicated that the feather cells lose the ability to synthesize DNA before keratin synthesis begins, but retain the ability to synthesize RNA after keratin synthesis begins.
GROWTH AND LABILITY OF CHAETOPTERUS OOCYTE MITOTIC SPINDLES ISOLATED IN THE PRESENCE OF PORCINE BRAIN TUBULIN
Authors: Inoué Shinya , Borisy Gary G. , Kiehart Daniel P.
Published: May 2004

Purified tubulin solutions stabilized and augmented the birefringence (BR) of isolated Chaetopterus spindles. Tubulin was extracted from pig brain tissue in cold PEG buffer (0.1 M piperazine-N-N'-bis[2-ethane sulfonic acid], 1 mM ethylene bis-[oxyethylenenitrilo]tetraacetate, [EGTA], 2.5 mM guanosine triphosphate, [GTP], pH 6.94, at 25°C), and purified by two cycles of a reversible, temperature-dependent assembly-disassembly procedure. The spindle BR of the meiotic metaphase-arrested oocytes of Chaetopterus decreased linearly at a rate of 1.5 nm/min when perfused with PEG buffer without tubulin. In this hypotonic, calcium-chelating solution, the cell lysed within 1.5 min, and after a brief, transient rise, the BR disappeared in ca. 4 min from the time of buffer application. Cells perfused with tubulin in PEG buffer also showed BR decay at the same rate until cell lysis. Immediately upon cell lysis the spindle BR increased, initially at ca. 2.3 nm/min and then more slowly until the BR attained or exceeded intact cell values. Spindle and asters grew considerably larger than those in intact cells. From the kinetics of the transient BR increase after lysis, we infer that, initially, Chaetopterus cytoplasmic tubulin contributes to increased BR; further augmentation required added pig brain tubulin and most probably reflects the addition and incorporation of heterologous porcine tubulin into the spindle and asters. Isolated, augmented spindles depolymerized rapidly at 6°C. Upon return to 23°C, spindle BR returned slowly in tubulin-PEG. The BR of the isolates also decayed in solutions containing calcium ions 2.5 mM in excess of the EGTA. However, the isolates did not respond, or responded very slowly, to 1 mM colchicine or Colcemid and to dilution of tubulin with PEG solution. Microinjection into Chaetopterus oocytes of tubulin-PEG, but not PEG alone, enhanced spindle and aster BR which reversibly disappeared upon chilling the cell.
UNDER-REPLICATION OF RIBOSOMAL CISTRONS IN POLYTENE CHROMOSOMES OF RHYNCHOSCIARA
Authors: Gambarini A. G. , Lara F. J. S.
Published: May 2004

DNA preparations obtained from several tissues of Rhynchosciara americana and two related species, R. milleri and R. papaveroi, were hybridized to R. americana rRNA. The percentage of hybridization was found to be higher in tissues with low polyteny than in tissues with high polyteny, suggesting a relationship between the amount of rDNA and the tissue polyteny. This could be explained by under-replication of ribosomal cistrons in polytene cells, such as those from the salivary gland. Only slight tissue-dependent changes in the percentages of hybridization can be observed in heterologous hybridization using Xenopus laevis rRNA. The possibility that these experiments could not detect differences in the amount of ribosomal cistrons among tissues is discussed. The female:male ratio for the percentages of hybridization in the salivary gland of R. americana agrees with the results obtained by in situ hybridization experiments (16, 17) which have shown that the rRNA cistrons are distributed among chromosomes other than chromosome X.