bluuebunny/crossref_metadata_2025 huggingface dataset:
Masking of extracellular collagen and the co-distribution of collagen and fibronectin during matrix formation by cultured embryonic fibroblasts
Authors: Little C.D. , Chen W.T.
Published: April 2021

Embryonic chicken heart fibroblasts (CHF) were examined by double immunofluorescence in order to study the interactions of collagen and fibronectin. Double-labelling studies of permeabilized whole mounts of CHF, cultured for 15 min to 2 days after plating, detected extracellular collagen at very early times (15 min to 1 h) but not at times later than 3 h. Abundant fibronectin was seen at all times. These observations suggested that the collagen/procollagen antigen was being “masked' by matrix components at the later times. This possibility was tested in two ways: (1) immunolabelling of frozen sections of multilayered 4-day CHF cultures, which showed extensive extracellular collagen superimposable with fibronectin. (2) Limited trypsinization of 4-day CHF cultures, which allowed collagen to be immunolabelled concomitantly with a partial decrease in the degree of fibronectin labelling. The results are discussed in terms of the secretion and assembly of collagen and fibronectin in cultures of embryonic fibroblasts.
Poly(A) RNA populations, polypeptide synthesis and macromolecule accumulation in the cell cycle of the eukaryote Chlorella
Authors: John P.C. , Lambe C.A. , McGookin R. , Orr B. , Rollins M.J.
Published: April 2021

Synchronous cultures of Chlorella, that were obtained with minimum metabolic perturbation by centrifugal selection, reveal that progress through the cell cycle requires no change in the poly(A)+ mRNA population, although changes do occur during nutritional adaptation. Of the abundant soluble proteins, 93% are synthesized continuously through the cell cycle and those that are discontinuous show similar patterns in control cells. The synthesis of proteins is compared with parallel studies of accumulation of enzyme activity and it is shown that there is no discrepancy in their pattern of accumulation when both are studied under the same culture conditions. The eukaryote cell cycle can allow stable relative rates of synthesis of most proteins and balanced rates of accumulation of most enzyme activities. Macromolecule classes differ in their rates of accumulation throughout the cell cycle: total RNA increases linearly, poly(A)+ RNA accumulation is restricted to G1 phase, but total protein accumulation accelerates smoothly through G1, S and mitosis phases, pausing at cytokinesis. There is no evidence that the cell cycle requires an extensive programme of differential enzyme synthesis. The cycle can therefore proceed with minimum disturbance of metabolism required for growth.
The motive force of the migrating pseudoplasmodium of Dictyostelium discoideum under dark and light conditions
Authors: Kitami Masanobu
Published: April 2021

As a contribution to the understanding of the mechanism of the collective motion of the cell mass in the migrating pseudoplasmodium its motive force has been determined. The measurements in the present paper were made by an original method using centrifugal force. The maximum value of the motive force of a migrating pseudoplasmodium in the dark was estimated as 0·6–1·6 dyn. This value is comparable to the motive force of protoplasmic streaming in Physarum polycephcdum. In addition, the motive force in phototactic migration, which has not previously been estimated, was measured. Illumination from the front increased the motive force, whereas illumination from the back produced the opposite results. That is to say, illumination could directly influence the motive force. Furthermore, some interesting results were obtained by measuring the changes in the velocity of the pseudoplasmodia placed under four conditions (combinations of illuminated or non-illuminated, and parallel or antiparallel to the direction of the external force). These results suggest the necessity of taking into consideration the direct effect of light upon the motility of the cells in the migrating pseudoplasmodium.
Spreading of blastomeres from eight-cell mouse embryos on lectin-coated beads
Authors: Kimber S. J. , Surani M. A. H.
Published: April 2021

Single blastomeres from 8-cell embryos adhere to lectin-coated agarose beads. The cells spread over the surface of peanut lectin (PNA), wheat germ lectin (WGA) and concanavalin A (Con A)-coated agarose beads, and this was prevented by sugars with high affinity for the appropriate lectin. Spreading was also prevented by cytochalasin D at a concentration (0·5-1·0 Ag/ml) known to inhibit compaction of whole 8-cell embryos. Colcemid (1–2 μg/ml) only depressed spreading, as measured by linear contact distance, to 70–80 % of that for the controls, while removal of calcium from the medium with EDTA had little effect on spreading. Fucose promoted spreading on WGA beads. Cells from 8-cell embryos will not adhere to negatively charged G-Sephadex and CM-Sephadex beads. They do adhere to positively charged DEAE-Sephadex beads but do not spread on them.

In an attempt to inhibit cell spreading at compaction in intact 8-cell embryos, these were disaggregated in the presence of various (34) saccharide-containing molecules. Neither any sugar-containing molecules alone, nor combinations of N-acetyl-glucosamine, galactose and mannose, fucose and galactose, and fucose and lactose were able to inhibit compaction at physiological concentrations. However, blastocyst formation was inhibited to various extents by high non-physiological concentrations (50 mm) of D-glucose, D-mannose, L-fucose, D-arabinose, N-acetyl-D-glucosamine, N-acetyl-D-galactosamine, α-lactose, 25 mM-α-meliblonic acid and by 2 mg/ml fucoidin, ovine mucin and desialylated ovine mucin. Our results indicate that interaction of PNA, WGA and Con A with molecules on the cell surface that bind these lectins can trigger the cells to spread. However, a simple lectin-sugar interaction alone may not account for the spreading of cells on one another at compaction in whole 8-cell embryos.
Movement and guidance of migrating mesodermal cells in Ambystoma maculatum gastrulae
Authors: Nakatsuji Norio , Gould Andrew C. , Johnson Kurt E.
Published: April 2021

A scanning electron microscopic study in early gastrulae of Ambystoma maculatum showed that migrating presumptive mesodermal cells were strongly oriented toward the animal pole. They had lamellipodia and filopodia at their leading edges, and rounded or tapering, tail-like, trailing edges. Of the cells whose polarization could be determined unequivocally, 81 % appeared to be directed in a quadrant toward the animal pole, and 93 % were directed to some extent away from the blastopore. This strong orientation suggests that specific mechanisms direct cell movement, in addition to the non-specific dispersive mechanism of the contact inhibition of cell movement. There is a network of fine extracellular fibrils that covers the inner surface of the ectodermal layer. Filopodia of the migrating cells frequently attach to and appear to follow the fibrils, suggesting that the fibrils serve as a guiding substratum for cell attachment and movement. There are areas where the fibrils are apparently aligned along the blastopore - animal pole axis, and a preliminary statistical analysis using micrographs at high magnification showed a significant alignment parallel to the blastopore - animal pole axis. This fibril alignment could cause the strong orientation of the mesodermal cells by means of contact guidance.
Ascospore development in the fission yeasts Schizosaccharomyces pombe and S. japonicus
Authors: Tanaka Kenji , Hirata Aiko
Published: April 2021

The fine structure of ascospore formation in the fission yeasts Schizosaccharomyces pombe and Schizosaccharomyces japonicus var. japonicus was studied by serial thin-sectioning and electron microscopy. The morphogenetic events were almost the same in both species. Ascospore development was initiated by the formation of the forespore membrane on the cytoplasmic side of the differentiated nucleus-associated organelle (NAO) in the interval between meiosis I and II in S. pombe, or during the post-meiotic nuclear division in S. japonicus, and the process proceeded almost synchronously through the two or four nuclei in the ascus. The forespore membrane developed by fusion of the cytoplasmic vesicles and this was clearly demonstrated in S. japonicus where the behaviour of vesicles involved in the forespore membrane development could be traced as they were marked by the presence of electron-dense granules. The staining technique, by phosphotungustic acid-chromic acid (PTA-CA) after treatment with periodic acid, was used to attempt to elucidate the origin and the nature of the forespore membrane. The method specific to plasmalemma-type membranes stained both ascus and ascospore plasmalemmas; the forespore membrane was not stained at first but developed the same affinity for stain as the plasma membrane in the course of ascospore development. The results suggest that the forespore membrane did not come directly from the ascus plasma membrane, but from another membrane system such as the endoplasmic reticulum. Spore wall material was deposited in the space between the inner and outer leaflets of the forespore membrane.
The detergent-resistant cytoskeleton of higher plant protoplasts contains nucleus-associated fibrillar bundles in addition to microtubules
Authors: Powell Andrew J. , Peace Geoffrey W. , R. Slabas Antoni , Lloyd Clive W.
Published: April 2021

Cytoskeletons prepared from carrot protoplasts often retain the nucleus, which is associated with bundles of 7 nm fibrils. These were also seen in cytoskeletons from oil palm, maize and Italian rye grass cells. By examining such cytoskeletons by immunofluorescence, negative staining and stereo scanning electron microscopy it appears that the bundles could be part of a more delicate system of fibrils distal to the nucleus. Optical diffraction of the bundles, staining with heavy meromyosin, drug treatments and phalloidin-staining all confirm that the bundles are not composed of actin. Bundles could not be isolated from such cytoskeletons in quantities sufficient for analysis, but by disrupting protoplasts prepared from stationary-phase cells it was possible to purify fibrillar bundles that appeared in the lysate. These were similar in size and properties to the nucleus-associated bundles. The former were shown by sodium dodecyl sulphate/polyacrylamide gel electrophoresis to be composed of several proteins and were sensitive to reducing agents. Since they are not composed of actin, the possibility was explored that these fibrils are related to the P-proteins of phloem sieve tube and accessory cells.
Migration of cultured vascular cells in response to plasma and platelet-derived factors
Authors: Bernstein Lori R. , Antoniades Harry , Zetter Bruce R.
Published: April 2021

Phagokinetic migration of cultured vascular cells was tested in response to human plateletrich serum (‘serum’) and human platelet-poor plasma serum (‘plasma’). The cell types tested included bovine aortic endothelial cells, human umbilical vein endothelial cells, human haemangiomal capillary endothelial cells, bovine adrenal microvascular pericytes, and bovine aortic smooth muscle cells. Human serum stimulated a significant increase in the rate of migration for all five cell types. Human plasma stimulated the endothelial cells to migrate but had no effect on the migration of pericytes or smooth muscle cells.

Highly purified platelet-derived growth factor (PDGF) stimulated dose-dependent migration of smooth muscle cells causing a 50 % increase in phagokinetic track area relative to controls. Neither pericyte nor endothelial cell migration was stimulated by PDGF. Rabbit antiserum to human PDGF completely blocked the smooth muscle cell migration induced by either 10% serum or 1 ng/ml pure PDGF. Purified platelet factor IV (PF4) stimulated migration of pericytes but not of smooth muscle cells nor endothelial cells. Sheep antiserum to human PF4 completely blocked the pericyte migration induced by either 10 % serum or 1 μg/ml pure PF4. These results indicate that PDGF is the primary factor in serum responsible for the migration of cultured aortic smooth muscle cells and that PF4 is a critical factor required to induce the migration of pericytes. Other factors present in both plasma and serum control the migration of vascular endothelial cells.
Effects of conditioned medium on nucleoside uptake, cell cycle progression and apparent DNA repair
Authors: Downes C. S. , Johnson R. T. , Yew F. F.
Published: April 2021

Cells in culture can condition the medium they grow in so as to inhibit cellular uptake of nucleosides. Some conditioned media specifically block the uptake of cytosine arabinoside and deoxycytidine, but not other deoxyribonucleosides; some also block thymidine uptake; some have no effect. Detransformation of cells by sodium butyrate can alter their medium-conditioning properties. The inhibition of uptake acts at the level of nucleoside phosphorylation; it is not affected by inhibitors of nucleoside degradation. Cell cycle progression is also blocked by conditioned medium. Inhibition of nucleoside uptake by conditioned medium can cause serious errors in estimations of the incision step of DNA repair, or of DNA replication.
Inhibition of Dictyostelium discoideum differentiation in monolayers in vitro by endogenous and exogenous lectins
Authors: Laroy Kathy , Weeks Gerald
Published: April 2021

Spore-cell differentiation in monolayers in vitro of two sporagenous mutants of Dictyostelium discoideum, HM18 and HM15, is markedly inhibited by relatively low concentrations of the exogenous lectins, Concanavalin A (ConA) and wheat germ agglutinin (WGA) and by somewhat higher concentrations of the endogenous lectin, discoidin. The selective inhibition of spore cell formation by ConA occurs toward the end of the differentiation process, as shown by studies involving the addition or removal of ConA at various times during the period of differentiation. In addition, ConA has no deleterious effect on the expression of the pre-spore cell specific enzyme, UDP-galactosyl transferase. It is likely, therefore, that the maturation of pre-spore cells into spore cells is the most sensitive locus of lectin inhibition.

At higher concentrations of WGA and ConA, both stalk- and spore-cell formation are inhibited, but increased concentrations of discoidin do not have this effect.
Conditioning of a culture substratum by the ectodermal layer promotes attachment and oriented locomotion by amphibian gastrula mesodermal cells
Authors: Nakatsuji Norio , Johnson Kurt E.
Published: April 2021

We have found that ectodermal fragments of Ambystoma maculatum gastrulae deposit immense numbers of 0-1 pm diameter extracellular fibrils on plastic coverslips. When migrating mesodermal cells from A. maculatum gastrulae are seeded on such conditioned plastic substrata, they attach and begin migrating after 15-30 min in vitro. We did a detailed analysis of the relationship between fibril orientation and cell migration using time-lapse cinemicrography, scanning electron microscopy, and a microcomputer with a graphics tablet and morphometric program. We found that cells move in directions closely related to the orientation of fibrils. Usually fibrils are oriented in dense arrays with a predominance of fibrils running parallel to the blastopore-animal pole axis of the explant, and cells move preferentially along lines parallel to the blastopore-animal pole axis. When fibrils are unaligned, cells move at random. We have also shown that cells move with a slightly stronger tendency towards the animal pole direction. These results are discussed concerning the mechanism of specific cell migration during amphibian gastrulation.
Osmium-impregnation patterns of the golgi complex in the epididymal epithelial cells of castrated and testosterone-injected mice
Authors: Yamaoka Ikuo , Yamamoto Kozo , Urabe Nobuko , Nagatani Yoshimi
Published: April 2021

After prolonged exposure of mouse epididymal epithelial cells to a solution of osmium tetroxide, reduced osmium compounds were detected in the Golgi cisternae and in the cytoplasm adjacent to the Golgi complex. Their appearance changed in time under certain conditions. Eight days after castration each cisterna of the regularly arranged Golgi lamellae fragmented into small vesicles, in which deposits of reduced osmium compound were in reduced amounts or completely absent, but no notable decrease of the fine reduced osmium particles in the cytoplasm adjacent to the Golgi complex occurred. The amount of deposit in the Golgi cisternae in castrated mice recovered to the normal level after the subcutaneous injection of testosterone for two weeks. On the other hand, the osmium particles observed in the cytoplasm adjacent to the Golgi complex during the recovery process of the lamellar structure increased in amount. This study showed that the reduced osmium compounds were of two kinds, and that the deposits contained in the Golgi cisternae were related to secretory products, and might be controlled by hormonal factors, but the fine reduced osmium particles that appeared in the cytoplasm adjacent to the Golgi complex might be regulated by other factors.
Characterization of proteins from the cytoskeleton of Giardia lamblia
Authors: Crossley Richard , Holberton David V.
Published: April 2021

Proteins from the axonemes and disc cytoskeleton of Giardia lamblia have been examined by sodium dodecyl sulphate/polyacrylamide gel electrophoresis. In addition to tubulin and the 30 × 103 molecular weight disc protein, at least 18 minor components copurify with the two major proteins in Triton-insoluble structures. The most prominent minor bands have the apparent molecular weights of 110 × 103, 95 × 103 and 81 × 103.

Protein of 30 × 103 molecular weight accounts for about 20% of organelle protein on gels. In continuous 25 mM-Tris-glycine buffer it migrates mostly as a close-spaced doublet of polypeptides, which are here given the name giardins.

Giardia tubulin and giardin have been purified by gel filtration chromatography in the presence of sodium dodecyl sulphate. Well-separated fractions were obtained that could be further characterized.

Both proteins are heterogeneous when examined by isoelectric focusing. Five tubulin chains were detected by PAGE Blue 83 dye-binding after focusing in a broad-range ampholyte gel. Giardin is slightly less acidic than tubulin. On gels it splits into four major and four minor chains with isoelectric points in the pl range from 5· 8 to 6’2.

The amino acid composition of the giardin fraction has been determined, and compared to Giardia tubulin and a rat brain tubulin standard. Giardins are rich in helix-forming residues, particularly leucine. They have a low content of proline and glycine; therefore they may have extensive a-helical regions and be rod-shaped.

As integral proteins of disc microribbons, giardins in vivo associate closely with tubulin. The properties of giardins indicate that in a number of respects -molecular size, charge, stoichiometry -their structural interaction with tubulin assemblies will be different from other tubulin-accessory protein copolymers studied in vitro.
Differential effects of K+ and Na+ on specific bands of isolated polytene chromosomes of Chironomus Tentans
Authors: Lezzi M. , Gilbert L. I.
Published: April 2021

Previous investigations on the effects of monovalent cations on the gene activities (formation of puffs) of polytene chromosomes utilized whole salivary glands or nuclei and suggested that the K+/Na+ ratio in the nuclear sap was of primary importance in influencing gene activities. To determine whether these effects of ions were direct or indirect, we have employed isolated polytene chromosomes incubated in salt solutions of varying ionic composition. Our observations reveal differential effects of K+, Na+, Mg2+, Ca2+ and combinations thereof on specific chromosome regions. The band at region I-18-C that is specifically affected by K+ is the same one that puffs when salivary gland nuclei are incubated in a K+-rich medium and this is believed to be an ecdysone-specific puff. Bands at region I-19-A are specifically affected by Na+ and are the same ones that form puffs when salivary gland nuclei are incubated in a Na+-rich medium. This region is believed to be juvenile hormone-specific. Effects on the chromosome are also described that are the result of incubation in solutions containing Mg2+ and Ca2+. Analogies are drawn between the observed effects of ions on isolated chromosomes and their effects on chromatin isolated from mammalian cells. For example, when chromatin is exposed to solutions of increasing ionic strength, hydration is followed by aggregation and histone dissociation. This may be analogous to what is observed in the isolated chromosome under similar conditions; namely, expansion, contraction and expansion.
The effects of some metabolic inhibitors on the ability of SV40 virus in transformed cells to be detected by cell fusion
Authors: Watkins J. F.
Published: April 2021

When mouse fibroblasts transformed by SV 40 virus are fused with African Green Monkey kidney cells about 5 % of the heterokaryons produce infective SV 40 virus. If the transformed mouse cells are treated with iododeoxyuridine before fusion, the percentage of heterokaryons producing SV40 virus is increased to between 50 and 80 %; if the transformed cells are treated with 8-azaguanine, the percentage of heterokaryons yielding virus is increased to between 59 % and 96 %. This shows that whether the SV40 virus can be detected by cell fusion depends on the state of the transformed cells beforehand. A new method for titrating SV 40 virus is described.
Studies on the ‘incisures’ of schmidt and lanterman
Authors: Hall Susan M. , Williams P. L.
Published: April 2021

Since its initial description over a century ago the ‘incisure’ of Schmidt-Lanterman has usually been regarded as a histological artifact, or a post-mortem change. With the establish ment of in vivo discrete zones in the myelin sheath, an ultrastructural study was undertaken and correlation attempted between their electron-microscopical appearances and their range of states seen in vivo and produced in vitro.

It was demonstrated that under conditions of hypotonicity the incisure dilates and further that it closes in hypertonic solutions. Such behaviour is reversible within certain limits. Ultrastructurally, the incisural region appears as a highly complex zone in which the repeat characteristics of the compact myelin sheath are drastically altered. At the incisure, the major dense line of the myelin splits to enclose a granular Schwann cytoplasm which frequently contains a single central helical microtubule, dense bodies and small vesicles. The intraperiod line also splits, dilatation of the incisure involving further widening of this intraperiod line gap beyond the normal confines of the incisure. Desmosomoid stacks are a common feature, particularly of the outer layers of incisures of both mature and immature fibres. Surface invaginations, vesicles, longitudinal microtubules, filaments, mitochondria and granular endoplasmic reticulum are typical constituents of peri-incisural Schwann cytoplasm. The incisure may be related to fibre growth, remodelling and degeneration; to dynamic changes of internodal contour—possibly peristaltic; or may operate as a fluid-satellite cell-neuron interchange pathway.
The three cortical membranes of the gregarines: I. Ultrastructural organization of Gregarina blaberae
Authors: Schrével J. , E. Caigneaux D. , Gros D. , Philippe M.
Published: April 2021

Gregarines, parasitic protozoa of invertebrates, possess a highly differentiated cell surface, with three cortical membranes and associated structures. Transmission electron microscopy and freezefracture reveal the presence of two cytomembranes lying uniformly under the plasma membrane. The density and the distribution of the intramembraneous particles (IMPs) in the plasma membrane of Gregarina blaberae are similar to those reported for other eukaryotic cells. The IMP density is lower in the cytomembranes than in the plasma membrane. The distribution of IMPs in the different fracture faces of the two cytomembranes suggests that they are in topological continuity, forming either side of a flattened vesicle or cisterna. The sizes of the cytomembrane IMPs show a high variability. The nature of the IMPs, both for the plasma membrane and the cytomembranes, is discussed with regard to the integral proteins and glycoproteins of the ghost.

The cell surface of G. blaberae exhibits numerous longitudinal folds with three types of cortical membrane-associated structures: 12 nm filaments, an internal lamina, and homogeneous structures described as ‘rippled dense structures’. The 12 nm filaments, running under the cytomembranes along the longitudinal axis of each fold, exhibit the properties of intermediate filaments. Their distribution in mature cells and during the growth process suggests a participation in cell surface morphogenesis. The internal lamina, also localized under the cytomembranes, would stabilize each fold and assure a scaffolding function between the numerous folds. The rippled dense structures, settled on the external cytomembrane, show a regular distribution at the top of each fold. The membrane-associated structures are discussed with regard to the gliding movement mechanism.
Cytasters induced within unfertilized sea-urchin eggs
Authors: Kuriyama Ryoko , Borisy Gary G.
Published: April 2021

Conditions that induce the formation of asters in unfertilized sea-urchin eggs have been investigated. Monasters were formed by treatment of eggs with acidic or basic sea-water, or procaine- or thymol-containing sea-water. A second treatment step, incubation with D2O-containing, ethanol-containing or hypertonic sea-water induced multiple cytasters. The number and size of cytasters varied according to the concentration of agents and duration of the first and second treatments, and also upon the species of eggs and the season in which the eggs were obtained. Generally, a longer second treatment or a higher concentration of the second medium resulted in a higher number of cytasters per egg.

Asters were isolated and then examined by light and electron microscopy. Isolated monasters apparently lacked centrioles, whereas cytasters obtained from eggs undergoing the two-step treatment contained one or more centrioles. Up to eight centrioles were seen in a single aster; the centrioles appeared to have been produced during the second incubation. Centrospheres prepared from isolated asters retained the capacity to nucleate the formation of microtubules in vitro as assayed by light and electron microscopy.

Many microtubules radiated from the centre of isolated asters, whether they contained centrioles or not. This observation is consistent with many other reports that microtubule-organizing centres need not contain centrioles.
Mutational disruption of the 9 + 2 structure of the axoneme of Chlamydomonas Flagella
Authors: Forest Charlene L.
Published: April 2021

A mutant of Chlamydomonas, gam-5, which has short but variable-length, paralysed flagella has been characterized using electron microscopy and genetics. The flagellar axoneme shows varying degrees of disorganization of its 9 + 2 structure, and the structures that attach to the microtubule pairs may be wholly or partly missing or, if present, abnormal. Flagellar length is inversely related to temperature, and gametes of this mutant are generally incapable of mating at higher temperatures. This pleiotropic mutation shows Mendelian (2:2) segregation and is unlinked to the mating-type locus. It is suggested thatgiwz-5 leads to the production of a thermal-sensitive flagellar component, which results in increased assembly and/or maintenance difficulties as temperature is increased.
Mitochondria of Tetrahymena pyriformis: Enumeration and sizing of isolated organelles using a coulter counter and pulse-height analyser
Authors: POOLE R. K.
Published: April 2021

An electronic particle counter (Coulter Counter ZBI) and pulse-height analyser (Channelyzer C1000) have been used to measure numbers and sizes of mitochondria isolated from the ciliated protozoon Tetrahymena pyriformis. Differential centrifugation of disrupted organisms, followed by single-step sub-fractionation of the mitochondrial fraction on sucrose gradients yielded a population of organelles extensively enriched in the activities of mitochondrial marker enzymes. Gradient-purified mitochondria (approx. 3 × 109 particles (mg protein)-1) were stable in electrolyte, exhibited unimodal volume distributions and were somewhat larger (0·93 ± 0·13(S.D.) μm3; 19 preparations) than organelles in a crude mitochondrial fraction. Glutaraldehyde fixation of mitochondria in sucrose gradients decreased the apparent volume to 0·6 ± 0·06 μm3 (6 preparations). Based on the recovery in the mitochondrial fraction of mitochondrial membrane-bound cytochromes from a suspension of intact cells, the number of mitochondria per cell was estimated to be approximately 1440, representing 15 % of the total cell volume. Isolated mitochondria were osmotically sensitive and exhibited an apparent marked contraction on adding Ca2+ (10 μM-10mM). Addition of chloramphenicol (500 μgml-1) to exponentially growing Tetrahymena cultures resulted in an almost immediate cessation of cell division and a dramatic decrease in cell volume. Mitochondria purified from such cells were much smaller than control mitochondria (0·21 ± 0·02 μm3; 7 measurements); their population density was approximately 900 per cell, equivalent to 11 % of total cell volume. The measurements of mitochondrial populations using the Coulter Counter and electron microscopy are in good agreement.
Effects Of Culture Conditions On The Proliferation, Morphology And Migration Of Bovine Aortic Endothelial Cells
Authors: Schor A. M. , Schor S. L. , Allen T. D.
Published: April 2021

Various culture conditions, such as the presence of ascorbic acid, initial plating density and the nature of the substratum (plastic, gelatin or native collagen gels), influenced the growth, morphology and migration of three cloned populations of adult bovine aorta endothelial cells.

Aorta endothelial cells showed two distinctive and reversible morphological phenotypes. Cells presenting a free apical surface were polygonal and formed sheets of overlapping or non-overlapping cells, depending on the culture conditions. When the cells were able to establish adhesive interactions over their entire cell surface they adopted an elongated shape and formed meshworks of interconnected ‘sprouting’ cells. The cells were capable of migrating into a collagen gel from both their basal and apical surfaces. Once in the gel, they formed characteristic, compact, threedimensional meshworks.
Meiosis and early cleavage in Drosophila melanogaster eggs: Effects of the claret-non-disjunctional mutation
Authors: Kimble Mary , Church Kathleen
Published: April 2021

The claret-non-disjunctional (cand) mutation is a female-specific mutation in Drosophila melanogaster, which causes a high frequency of mortality (89–90 %) in eggs laid by the homozygous females. Among the progeny that survive to the adult stage, greater than 50 % are aneuploid and/ or mosaic for the X and number 4 chromosomes. The genetic studies of cand indicate that it is homologous to the claret mutation (ca-simulans) in Drosophila simulons.

The cytological effect of the cand mutation on meiosis I and the early cleavage divisions has been observed at the light microscope level in stage 14 ovarian and fertilized uterine eggs, respectively. Four classes of metaphase I figures were observed. These include those with: (1) two or more spindles; (2) spindles that were abnormally wide and the bivalents widely separated; (3) unipolar spindles; and (4) apparently normal bipolar spindles. The three abnormal classes of metaphase I figures included 80 % of the eggs examined at this stage. Among the cleavage stage eggs examined 69 % showed highly abnormal mitotic figures, including multipolar spindles, and the nuclei in these eggs were found in clusters, rather than dispersed throughout the ooplasm.

In addition to the cytological abnormalities observed, 17–23 % of the eggs produced by the cand females showed morphological abnormalities. These abnormalities included eggs having three or four dorsal filaments, eggs that had a truncated shape, and abnormally small eggs. These abnormalities may not be an aspect of the cand syndrome, but they are due to recessive genes located on the third chromosome.

Although the cand and ca-simulans mutations both affect the formation of the spindle apparatus during meiosis and the early cleavage divisions, the effects of these two mutations differ considerably in detail. The effect of the ca-simulans mutation appears to be more severe than the effect of cand.

A model to explain the relationship between the effect of the cand mutation on meiosis I and the early cleavage divisions is presented, and evidence to support the model is discussed.
Selective Extraction With Sarkosyl And Repolymerization In Vitro Of Cytoskeleton Proteins From Giardia
Authors: Crossley Richard , Holberton David V.
Published: April 2021

Sarkosyl has been used to dissociate structures in cytoskeletons isolated from Giardia lambda. Results from sodium dodecyl sulphate/polyacrylamide gel electrophoresis and from electron microscopy of insoluble residues show that the solubilization of components is partly selective. At low concentrations of detergent (<0·3%), microribbons and microtubules of the disc cytoskeleton disappear, but doublet microtubules from axonemes resist extraction. Consequently, the microribbon protein giardin is extracted into solution more completely than tubulin.

Soluble proteins in 0·l % Sarkosyl have been fractionated by gel filtration chromatography in Bio-Gel P300. Giardin elutes in two positions: as a low molecular weight subunit, and in early fractions corresponding to a larger particle size in which subunits might be forming oligomers.

Supernatants prepared in 0·5 % Sarkosyl were diluted with 0·1 M-KC1 or 0· 1 mM-MgCl2 to bring about reaggregation of the cytoskeleton proteins. Reassembled structures seen in negatively stained preparations were polymorphic. Some tubulin ribbons of 5nm protofilaments were identifiable; also there were large fibres and some flat sheets of very thin filaments. Electron micrographs of sheets have been analysed by optical diffraction. The transforms show that the lateral separation of the fine filaments is about 2·5 nm. Axial periodicities from features spaced along filaments were weak. A 3·75 nm layer-line has been detected, corresponding to a similar periodicity found earlier in microribbons.
Role of the cellular matrix in haemopoiesis. I. Synthesis of glycosaminoglycans by mouse bone marrow cell cultures
Authors: Gallagher J.T. , Spooncer E. , Dexter T.M.
Published: April 2021

Haemopoietically active mouse bone marrow cultures, incubated for 48 h with [3H]glucosamine and Na2(35)SO4, synthesized radiolabelled hyaluronic acid, heparan sulphate and chondroitin sulphate. Heparan sulphate was enriched in a trypsin extract of the adherent cells whereas hyaluronic acid and chondroitin sulphate were distributed mainly to the culture medium. Analysis of nitrous acid scission products of heparan sulphate by gel chromatography demonstrated the close association of N- and O-sulphate groups along the polysaccharide chain. Chondroitinase AC degradation established the copolymeric nature of chondroitin sulphate in which about 38% of the hexuronic acid residues were in the form of GlcUA. Studies on non-haemopoietic cultures, derived from W/Wv mice or from normal marrow cells maintained in foetal calf serum instead of horse serum, indicated that adherent stromal cells were the major source of glycosaminoglycans.
Determination of patterning in the pollen wall of Lilium henryi
Authors: Sheldon Judith M. , Dickinson H. G.
Published: April 2021

Centrifugation of living pollen mother cells of Lilium henryi has permitted the modification of complex walls developed on the four daughter microspores. This experimental approach has not only provided an insight into the mechanism by which patterning is established, but has also identified the stages in cell development when wall determinants are present. These determinants can be first detected late in the meiotic prophase, but are not necessarily active at the cell surface at this early stage. The fact that treatment during cytokinesis may affect subsequent wall formation, combined with the obvious interference of the colpus with the spore patterning, indicates pattern determination to be a lengthy process, commencing during late prophase and reaching completion in the young tetrad. The study of walls formed on enucleate cell fragments suggests that neither the spindle nor the cytoskeleton is intimately involved in formation of the basic reticulate patterning. It is proposed that material is inserted into the mosaic of the plasma membrane and is reorganized biophysically to form contiguous ‘plates’, which then modify the properties of the membrane in such a way that the cytoplasmic protrusions that constitute the first stages of wall formation only develop in the interstices between them. Changes in the plasma membrane have been detected over this period and it seems that a population of coated vesicles is closely involved in these events. Examination of the cytoskeleton of the young spore suggests that microtubules play a critical rôle in the positioning of the colpus, and reveals ways by which only small changes in this skeleton could give rise to multicolporate grains.
Membrane recycling and the control of secretory activity in pollen tubes
Authors: Picton Jill M. , Steer Martin W.
Published: April 2021

Pollen tubes of Tradescantia growing on media containing 10−3M-Ca2+ and 10−2M-Ca2+ exhibit growth rates of 28 μm min−1 and 7 μm min−1, respectively. The rates of vesicle production by the dictyosomes in these tubes were determined from the rates at which vesicles accumulated in the cytoplasm after treatment with cytochalasin D. Although the vesicle requirements for these two growth rates are considerably different, it was found that vesicle production rates were the same. The results indicate that, in pollen tubes, membrane recycling is occurring and that dictyosome activity is not regulated according to the vesicle requirements for tube growth.
Kinetics of In vitro ageing of mouse embryo fibroblasts
Authors: Karatza Calliopi , Stein Wilfred D. , Shall Sydney
Published: April 2021

The kinetics of ageing of normal mouse embryo fibroblast cells in culture have been determined. The growth pattern during every passage was established. It was observed that the growth pattern was not exponential, but that the growth rate declined progressively both within and with every passage.

We also estimated the cell cycle parameters using the Fraction of Labelled Mitoses method at every passage. We found that the cell cycle duration was constant throughout the lifespan of this cell strain; the median value of the cell cycle duration was found to be 15·5 ± 0·5 h (S.D., n = 8).

From these two sets of observations we infer that the fraction of dividing cells declines smoothly from the beginning of the culture. Our data exclude quite positively any description of ageing of the fibroblast population in terms of a catastrophe or any abrupt change in the population. Our data are also inconsistent with a linear decline in growth fraction. On the contrary, we observed that there was a gradual and smooth decline in the growth rate of the strain, due to a smoothly declining growth fraction. This smooth change in the growth behaviour of this cell strain is accurately described by the mortalization theory of Shall & Stein in which the single parameter γ (gamma), describes the change in reproductive potential over the entire lifespan. The parameter γ describes the rate at which the doubling time of the culture increases. It is the number of generations at which half of the newborn cells are themselves reproductively sterile. Our present data provided an estimate of γ for this cell strain, which was consistent during the entire lifespan of the strain; the best estimate of γ for this cell strain was 20·3 ± 0·6 generations (S.D., n = 19).
Effects of monensin on the processing and intracellular transport of influenza virus haemagglutinin in infected mdck cells
Authors: Edwardson J. M.
Published: April 2021

The role of the Golgi complex in the intracellular transport of influenza virus haemagglutinin in infected MDCK cell monolayers has been investigated using monensin, a carboxylic ionophore known to disrupt the functioning of this organelle in other cell types. In untreated cells metabolically labelled 5 h post-infection with [35S]methionine haemagglutinin was first seen in core glycosylated form, which was sensitive to the enzyme endo-β-N-acctylglucosaminidase H (endo H). After approximately 20 min this form was converted into a terminally glycosylated, endo H-resistant form. In the presence of monensin core glycosylation of haemagglutinin was not affected, but terminal glycosylation was interrupted. Two new forms of haemagglutinin were observed, both of which were smaller than the core glycosylated form. Of these, the larger was endo H-sensitive while the smaller was endo H-resistant. These new (and uncharacterized) forms of haemagglutinin are likely to be intermediates in the normal process of terminal glycosylation, which are revealed as a result of the inhibition by monensin of the transport of haemagglutinin through the stack of Golgi cisternae.

In untreated cells 85 % of the pulse-labelled haemagglutinin had reached the plasma membrane after 90 min of chase, as revealed by its sensitivity to externally applied trypsin. In monensin-treated cells, on the other hand, only 55 % of the haemagglutinin had reached the plasma membrane after 90 min of chase, while 94% had arrived there after 180 min of chase.

At 5 h post-infection the density of envelope proteins detected at the apical surface of the monolayer by immunofluorescence microscopy was greatly reduced by monensin treatment. Budding of virions from the apical surface of the monolayer at 4 and 7 h post-infection was also reduced, and the normal Golgi complexes were replaced by distended vacuoles that appeared to contain poorly preserved virions.
Video digitizer analysis of birefringence along the lengths of single chromosomal spindle fibres: I. Description of the system and general results
Authors: Schaap Catherine J. , Forer Arthur
Published: April 2021

A new system, based on a video digitizer interfaced to a microcomputer, has been developed to quantify birefringence of individual chromosomal spindle fibres from videotaped images of spindles. (The system also can be used for any other purpose that requires the analysis of video intensities.) Retardations along the lengths of single chromosomal spindle fibres have been studied throughout metaphase and anaphase in cells kept at constant temperatures. The instrumental readings are accurate to within less than 0·06 nm retardation, but operationally the retardation values along a single chromosomal spindle fibre can vary by up to 0·15nm, primarily because of variation in operator definition of the spindle fibre. Retardations vary with position along the fibre. During anaphase the retardations along a given chromosomal spindle fibre do not move poleward, but rather change as if the oriented material is disorganized at the kinetochore. The retardation at the kinetochore of a chromosomal spindle fibre does not change during anaphase, except for non-predictable jumps of 20 –30 % that sometimes occur. Thus there is no ‘decay of birefringence’ during anaphase, such as has been described in other species. In this regard our data, that pertain only to single chromosomal spindle fibres, differ from those previously published; we argue that this is because the published data deal with mixtures of chromosomal and continuous spindle fibres, and because changes in birefringence can appear to occur, artef actually, when measurements of birefringence are made at a single spot in a spindle.
Inactivation of mitotic factors by ultraviolet irradiation of hela cells in mitosis
Authors: Adlakha Ramesh C. , Wang Yong C. , Wright David A. , Sahasrabuddhe Chintaman G. , Bigo Helene , Rao Potu N.
Published: April 2021

‘Extracts from mitotic HeLa cells, when injected into fully grownXenopus larois oocytes, exhibit maturation-promoting activity (MPA) indicated by germinal vesicle breakdown (GVBD) and chromosome condensation. Recently, we observed that the MPA of mitotic cell extracts is neutralized by the inhibitors of mitotic factors (IMF) in HeLa cells, which are activated at telophase and remain active throughout the G1 period. The activity of the IMF coincides with the process of chromosome decondensation, which begins at telophase and continues until the beginning of S phase, when chromatin reaches its most decondensed state. The objective of the present study was to investigate whether these two phenomena - chromosome decondensation and the activation of IMF -were related. The activity of IMF was measured in N2O-blocked mitotic HeLa cells, in which chromosome decondensation was induced by exposure to ultraviolet light, and subsequent incuba tion in medium containing inhibitors of DNA synthesis, hydroxyurea and arabinosylcytosine (araC). u.v. irradiation activated IMF in mitotic HeLa cells but not in cell extracts. In contrast, much less activation of IMF was seen even at very high doses of X-irradiarion. The IMF seemed to inactivate the mitotic factors directly by forming a complex that precipitated on heating at 60°C for 15 min. Mg2+ or polyamines (i.e. spermine, spermidine, and putrescine), agents known to promote chromatin condensation partially restored the MPA of the u.v.-irradiated mitotic cell extracts. These results tend to support the conclusion that the IMF play a role in the decondensation of chromosomes.
Activation of potential initiation sites of dna replication and induction of new initiation within the same cycle by puromycin in Drosophila polytene chromosomes
Authors: Mukherjee A. S. , Chatterjee Chandana
Published: April 2021

DNA replication in the polytene chromosomes of larval salivary glands of Drosophila hydei has been examined under normal physiological conditions, with or without puromycin, by an autoradiographic procedure using [3H]thymidine. It has been demonstrated that puromycin induces new labelling patterns that can be identified as initiation patterns. Such initiation patterns were found to have been induced during both the initial (interband labelling patterns) and the terminal (discontinuous) phases. Both induced patterns are sensitive to a-amanitin. These findings lead us to suggest that (a) the puff-interband-labelled patterns are indeed the initial patterns, and (b) the induction of new initiation, which is normally known to be prevented until the completion of the cycle, can be brought about by puromycin. The results also suggest the probable existence of a pool of protein factor(s) for the initiation of a replication cycle already present in the cell system.
Monocytes from circulating blood fuse in vitro with purified osteoclasts in primary culture
Authors: Zambonin Zallone A. , Teti A. , Primavera M.V.
Published: April 2021

The origin of osteoclasts from mononuclear phagocytes and the addition of new nuclei to already differentiated osteoclasts have already been documented by several authors, but the factors controlling these events have not yet been elucidated. With the aim of investigating this problem, monocytes, isolated from circulating blood of laying hens and cultured previously for 5 days, were added to osteoclasts isolated from the medullary bone of the same hen and cultured at low density. The cultures were either fixed and observed under phase contrast at 24-h intervals for 5 days or filmed by time-lapse cinemicrography. With both techniques the formation of extensive areas of membrane contacts, generally followed by fusion between some monocytes and osteoclasts, was observed. The absence of added resorbing factors and the possible mechanisms by which osteoclast precursors are recruited in vivo are discussed.