bluuebunny/crossref_metadata_2025 huggingface dataset:
Contraction and organization of collagen gels by cells cultured from periodontal ligament, gingiva and bone suggest functional differences between cell types
Authors: Bellows C. G. , Melcher A. H. , Aubin J. E.
Published: April 2021

Monkey periodontal ligament fibroblasta (MPLF cells), human gingival fibroblasts (HGF cells), rat embryonic calvaria cells (REC cells), porcine periodontal ligament epithelial cells (PPLE cells) and rat osteosarcoma 17/2 cells (ROS cells) were incorporated into 3-dimensional collagen gels plated in 60 mm Petri dishes in order: first, to measure the capacity of these cell types to contract; second, to investigate cell-collagen and intercellular relationships during contraction; and third, to define the cellular contribution to tissue contraction in an in vitro system. Measurements at times up to 72 h on 3 ml gels containing 5×105 cells and with a collagen concentration of 1·20 mg/ml showed that MPLF cells contracted the gels at a significantly greater rate (P < 0·001) than did the other cell types. In addition, contraction started sooner and was of greater extent than with the other cells. HGF cells contracted the gels more rapidly than REC and PPLE cells, while ROS cells caused no contraction. Several stages of gel compaction could be defined: (1) the attachment of cells to collagen; (2) cellular spreading within the collagen fibre matrix; (3) organization and alignment of collagen fibres by cell processes; (4) cell migration; (5) establishment of intercellular contacts; and (6) the development of a cellular reticular arrangement within the gel and the extension of this arrangement into a 3-dimensional, tissue-like, honeycomb network. Electron microscopic observations on 0·1 ml gels containing MPLF cells showed that, in the early contractile phase, numerous cell processes attached to or enclosed collagen fibrils. These processes contained micro-filamentous material and few organelles. In compacted gels, the cells contained an increased amount of distended rough endoplasmic reticulum and Golgi membranes. Since MPLF cells have the capacity for vigorous contraction of the collagen gels and since they develop a reticular, 3-dimensional structure in compacted gels that is reminiscent of the relationship of periodontal ligament fibroblasts to collagen fibres in vivo, it is suggested that they could provide the major force necessary for tooth eruption in vivo. This system also provides a well-defined in vitro model to study the sequential stages that occur during contraction processes.
Effect of hyaluronic acid on neutrophil adhesion
Authors: Forrester John V. , Lackie J. M.
Published: April 2021

The effects of hyaluronate on rabbit neutrophil adhesion were studied using a variety of techniques. Exogenous hyaluronate inhibited neutrophil aggregation under conditions of both turbulent flow and constant shear rate. Hyaluronate also inhibited neutrophil adhesion to glass. Inhibition was dose-dependent above 100 μg ml−1 and a minimum molecular weight for hyaluronate of 1 × 104 was required. These effects were not simply the result of increased bulk viscosity of the hyaluronate-containing medium, nor did they appear to be mediated by putative cell-surface receptor mechanisms. Instead, physical factors such as steric hindrance and/or changes in the interfacial free-energy exchange at the cell surface due to the unusual hydrodynamic properties of the hyaluronate molecule were considered to be more important.

Since neutrophil migration in vivo occurs through hyaluronate-rich connective tissue matrices, the relevance of these findings for processes such as inflammation and wound healing is clear.
Variability in individual cell cycles of Saccharomyces cerevisiae
Authors: Lord P. G. , Wheals A. E.
Published: April 2021

The kinetics of cell proliferation of Saccharomyces cerevisiae were studied at 4 growth rates using time-lapse cinephotomicrography. Cells were grown on media with a high refractive index to reveal greater intracellular detail under the phase-contrast microscope. The morphological cell-cycle events scored were: bud emergence, nuclear migration, nuclear division, onset of cytokinesis and cell separation. Cell size was measured at cell separation and at bud emergence. The daughter-cycle time was always longer than the parent-cycle time mainly due to the large difference in the lengths of the unbudded phases. Parent cells had a shorter budded period than daughter cells. The large variance in daughter-cycle times was accounted for by the large variance in the lengths of the unbudded phase of daughter cells. The duration and variability of the periods in the cycle from nuclear migration onwards were equivalent for parent and daughter cells. Daughter cells were always smaller than parent cells at division. There was wide variation in cell size at both division and bud emergence. The results indicated that a modified deterministic model could best explain cell proliferation kinetics in yeast. The data were used to evaluate 2 different models. The * sloppy size control’ model of Wheals (1981a) was more consistent with the data than the ‘tandem’ model of Shilo, Shilo & Simchen (1976). The distribution of unbudded periods of daughter cells suggested that there was an additional incompressible period not present in parent cells.
Mitochondrial morphology in the spermatozoa of the mexican axolotl, Ambystoma mexicanum
Authors: Keyhani Ezzatollah , Lemanski Larry F.
Published: April 2021

Thin-section and freeze-fracture electron microscopy of immature and mature spermatozoa of the Mexican axolotl, Ambystoma mexicanum, revealed numerous small spherical mitochondria with diameters ranging from 0·15 to 0·22 μm. Both the spherical form and the small size of these mitochondria were confirmed by serial thin-section studies. In mature spermatozoa, the mitochondria are located in the midpiece region, in tight contact with each other, exhibiting an almost crystalline arrangement. They do not surround the midpiece, but form a semicircular sheet over the sustained filament. The portion of the midpiece on the side of the undulating membrane and the flagellum is devoid of mitochondria. The plasma membrane in the midpiece region is tightly apposed to the mitochondria, so that in freeze-fracture or scanning electron microscopy the mitochondria seem to protrude through the plasma membrane. We suggest that the unusual organization of mitochondria in axolotl sperm facilitates the oxidative processes and increases the efficiency of ATP production and/or distribution within the cell.
Bs i-b4 isolectin as a probe for an investigation of membrane alterations and transformation phenotypes of mouse l cells
Authors: Stanley Wayne S. , Chu E. H. Y.
Published: April 2021

BS I-B4, an α-D-galactopyranosyl-binding isolectin from Bandeiraea simplicifolia seeds, was found to interact differently with transformed mouse L cells and non-transformed mouse 3T3 cells. The lectin induces detachment of 3T3 cells but increases adhesiveness and clustering of L cells. However, the induced cell aggregation does not lead to cell fusion.

A variant clone of L cells, resistant to BS I-B4, which had lost the capacity for agglutination in the presence of the lectin, was isolated. Fluorescence binding studies of this variant suggest a lesion involving a and β-D-galactopyranosyl units on its cell-surface structures. Although the variant cells form colonies in a methylcellulose medium, they do not produce tumours, as do the parental cells, when transplanted in athymic nude mice. The results demonstrate that alterations in cell membrane glyco-conjugates play an important role in tumourigenesis of animal cells, but anchorage-independent growth in vitro, as one of the transformation phenotypes, cannot be correlated absolutely with tumourigenicity in vivo.
Nuclear bodies in the maturing egg cell of a fern, Pteridium aquilinum
Authors: Bell P. R.
Published: April 2021

Nuclear bodies about 250 nm in diameter, and with a strong affinity for uranium and acriflavine, appear in the nuclei of maturing egg cells of Pteridium. Many enter well-defined evaginations of the nucleus. The nuclear bodies are almost wholly digested by Pronase, but are resistant to ribonuclease and deoxyribonuclease. Radioactive labelling gives no evidence of the presence of nucleic acids, but X-ray microprobe analysis indicates phosphorus. It is concluded that the bodies consist entirely of acidic protein, possibly phosphorylated. This protein may be a structural component of the nucleus, temporarily displaced and aggregated as a consequence of the fine dispersal of the chromatin.
Immunocytochemistry of the acellular slime mould Physarum polycephalum: I. Preparation, morphology, and reliability of results concerning cytoplasmic actomyosin patterns in sandwiched plasmodia
Authors: Naib-Majani Wallid , Osborn Mary , Weber Klaus , Wohlfarth-Bottermann Karl-Ernst , Hinssen Horst , Stockem Wilhelm
Published: April 2021

Small phaneroplasmodia of Physarum polycephalum migrate, under sandwich conditions between two agar sheets and a membrane of cellophane, as thin protoplasmic sheets. This method suitably simulates the situation in the natural habitat of acellular slime moulds; i.e. the narrow clefts of the forest soil. The highly differentiated system of cytoplasmic fibrils displayed under these conditions survives both long-term extraction with glycerol and fixation with methanol, procedures that remove the strong inherent autofluorescence, thus allowing the use of immunocytochemical studies. The complicated fibrillar system of sandwiched plasmodia consists of: (1) a membrane-associated cortical filament layer in the anterior region; (2) a more or less regular polygonal fibrillar network in the intermediate region; and (3) a helically twisted fibrillar system encircling endoplasmic pathways as well as isolated strands in the posterior region. So far, three different cytoskeletal proteins have been identified immunocytochemically as constituents of the fibrillar structures: actin, myosin and AM-protein (fragmin). No positive identification of α-actinin, filamin and tropomyosin was obtained using antibodies against vertebrate proteins. Electron microscopy of glycerol-extracted specimens treated with antibodies against actin and myosin revealed that the 6 nm filaments consist of actin, whereas the electron-dense material between single actin filaments appears to be myosin. The AM-protein modulating the polymer status of actin is located in all fibrillar structures.
Bacillus thuringiensis var israelensis crystal δ-endotoxin: effects on insect and mammalian cells in vitro and in vivo
Authors: Thomas Wendy E. , Ellar David J.
Published: April 2021

Bacillus thuringiensis var israelensis parasporal crystal ô-endotoxin was purified by ultracentrifugation on a discontinuous sucrose gradient. Native δ-endotoxin crystals showed no detectable toxicity in the in vitro and in vivo systems that are described. By contrast alkali-solubilized crystal δ-endotoxin caused rapid cytological and cytopathological changes in Aedes albopictus, Choris-toneura fumiferana 63 CF1, Spodoptera frugiperda and Trichoplusia ni cell lines as observed by phase-contrast microscopy and vital staining.

Mouse fibroblasts, primary pig lymphocytes and three mouse epithelial carcinoma cell types showed a similar response to the alkali-soluble crystal δ-endotoxin. In addition the soluble crystal δ -endotoxin protein caused haemolysis of rat, mouse, sheep, horse and human erythrocytes.

Intravenous administration of the alkali-soluble crystal δ-endotoxin to Balb.c mice at a dose rate of 15-30μg of protein per gram body weight resulted in rapid paralysis followed by death within 12h. Subcutaneous inoculation of 15–30μg of protein per gram body weight resulted in death of suckling mice in 2-3 h. The alkali-solubilized crystal δ-endotoxin was not toxic however, when administered per os.

A comparison is made with a similar alkali-soluble fraction from the parasporal crystal δ-en-dotoxin of B. thuringiensis var kurstaki. With the exception of the Lepidopteran cell line, Choris-toneura fumiferana 63 CF1, this soluble crystal δ-endotoxin protein showed no in vitro or in vivo toxicity, and no haemolytic activity.
X-ray microanalysis of hela s3 cells I. Instrumental calibration and analysis of randomly growing cultures
Authors: Warley Alice , Stephen J. , Hockaday A. , Appleton T. C.
Published: April 2021

Cryo-ultramicrotomy and X-ray microanalysis were used to study the elemental composition of HeLa S3 cells. Quantitation was achieved by reference to elemental standards of known concentration made up in 25% gelatin. Analysis of standards showed linear calibration for each of the elements studied: Na, P, S, Cl, K. Standardization was validated by comparing flame-photometric analysis of gelatin containing sodium potassium tartrate with that of X-ray microanalysis. Freeze-dried sections of cells showed good morphology and analysis of whole sections of the cells showed that K/Na varied in individual cells. Low K/Na could not be ascribed to cell damage or to the sequestering of Na in any particular subcompartment of the cells. Treatment with ouabain caused changes in levels of all the elements studied and resulted in a low K/Na ratio in all cells.
Modification of flagellar waveform and adenosine triphosphatase activity in reactivated sea-urchin sperm treated with N-ethylmaleimide
Authors: Cosson Marie Paule , Tang Wen-Jing Y. , Gibbons I. R.
Published: April 2021

Treatment of demembranated aea-urchin sperm for 1-2 min with 10μMW-cthylmaleimide (Mall-NEt) at pH80 prior to reactivation with 1 mM-ATP causes the asymmetry of the flagellar waveform to become desensitized to the presence or absence of Ca2+ in the reactivating medium. In such sperm, changes in concentration of free Ca2+ between 10−7M and 10−3M have no effect on the asymmetry of the waveforms as measured by the turning rate of the sperm in radians per beat cycle, while the beat frequency and the propulsive efficiency of the waves remain unchanged from the values observed in control preparations not treated with MalNEt. A somewhat more prolonged treatment with MalNEt causes a progressive decrease in the bend angles of the flagellar waves, while the beat frequency and the wavelength still remain largely unchanged. Further extension of the treatment with MalNEt causes complete loss of motility.

Little ATP-induced sliding of the doublet tubules is observed upon treatment with trypsin of sperm flagella that have been rendered non-motile with MalNEt. However, the preparations of solubilized dynein 1 obtained by 0·6M-NaCl extraction of axonemes treated with MalNEt appear almost identical to those obtained from untreated axonemes, both in terms of the amount solubilized and in the specific ATPase activities of their latent and Triton-activated forms. These preparations also appear capable of restoring much of the beat frequency of dynein-1-depleted flagella. These results suggest that the observed desensitization to Ca2+ and decrease in bend angle result from the reaction of MalNEt with axonemal polypeptides that are not part of the dynein 1 particle extracted with 0·6M-NaCl.

The rate of ATP hydrolysis by demembranated sperm rendered non-motile with MalNEt remains relatively high, and it decreases about 50% when the flagella are broken by brief homogenization. This ‘homogenizer-sensitive’ ATPase activity appears to be derived from some flagellar regulatory mechanism, which controls the ATPase activity of intact non-motile axonemes.
Identification of the cohesion molecule, contact sites B, of Dictyostelium discoideum
Authors: Chadwick C. M. , Garrod D. R.
Published: April 2021

Polyspecific antibodies were raised against vegetative cells of Dictyostelium discoideum, strain Ax2. Monovalent (Fab′) fragments of antibodies CMC 1, 5, 7 and 12 blocked completely the cohesion of vegetative cells. Antibody CMC 1 was studied in detail. The Fab′ of this blocked the cohesion of aggregation-competent cells by 40%. It also caused some loss of cell contact in aggregation streams. In so doing the contacts that remained were mostly at the ends of the cells. Immunofluorescence showed that CMC 1 Fab′ bound to both the cytoplasm and the surface of fixed cells. It also bound to the surface of live cells. A control (N Fab′) also bound to the cell surface but did not block vegetative cell cohesion.

An extract of vegetative cells was obtained using the detergent Triton X-100. D. discoideum proteins were immunoprecipitated from this extract using protein A-Sepharose and CMC 1 immunoglobulin G (IgG). These immobilized proteins absorbed the cohesion-blocking activity of CMC 1 Fab′.

About 30 proteins were obtained when the Triton-soluble fraction was immunoprecipitated with IgG of CMC 1, 5, 7 and 12. Five of these were found to be cell surface proteins by the technique of lactoperoxidase-catalysed radio-iodination. These proteins had molecular weights of 178 000, 166 000, 126 000 and 64 000. CMC 12 IgG immunoprecipitated an additional cell surface protein of 46000 molecular weight.

Slices of polyacrylamide gel containing each of the five proteins identified as possible contact sites were fixed, washed and incubated with CMC 1 Fab′. Gel that contained protein of 178 000, 166 000 and 64 000 molecular weight had no effect on the activity of CMC 1 Fab′. However, Fab′ that had been incubated with gel containing protein of 126000 molecular weight no longer blocked cell cohesion.
Behaviour and structure of the leading lamella in moving fibroblasts: I. Occurrence and centripetal movement of arcshaped microfilament bundles beneath the dorsal cell surface
Authors: Heath Julian P.
Published: April 2021

Arc-shaped bundles of microfilaments are frequently found beneath the dorsal surface of the leading lamella of chick embryo fibroblasts. These structures, called arcs, form parallel to, and 2 – 10μm from the leading edge of the lamella and they then move centripetally through the lamella and disappear in front of the cell nucleus. Arcs move centripetally at a mean speed of 1 · 33 (±0 · 08 S.E.) μm min − 1 relative to the substratum.

Arcs are specifically associated with cells that are actively protrusive. They are common in fanshaped fibroblasts and in those cells that are newly spreading on a substratum. Arcs are absent from fibroblasts that have only small lamellae or that are polygonal. The correlation between arc formation and protrusive activity is also clearly shown by the locomotory behaviour of fan-shaped cells. When protrusion of the lamella is increased by tail detachment, fibroblasts often develop numerous arcs, which chase each other backwards through the lamella. Conversely, arc formation ceases during contact inhibition of locomotion.

In cells with large convex-edged lamellae there is a dorsal submembraneous sheath of microfilaments. All of the filaments comprising the sheath are oriented parallel to the margin of the lamella. Arcs are regions of the sheath where the microfilaments are more densely packed and hence visible by phase-contrast microscopy. There is no obvious relationship between the dorsally situated arcs and microfilament sheath, and the stress fibres that are associated with the ventral cell surface.

The similarities between the movement and behaviour of arcs and the centripetal transport of particles on the surface of the lamella suggest a role for the microfilament sheath in the movement of particles.
Nucleoside diphosphokinase and cell cycle control in the fission yeast Schizosaccharomyces pombe
Authors: Dickinson J. Richard
Published: April 2021

Centrifugal elutriation was used to prepare synchronous cultures of Schizosaccharomycespombe. Nucleoside diphosphokinase activity was measured throughout the cell cycle. In the wild-type strain (972) nucleoside diphosphokinase activity doubled in a stepwise fashion. The midpoint of the rise in enzyme activity was at 0 ·65 of a cycle, 0·29 of a cycle before the next S phase. Synchronous cultures of the mutant wee l ·6 were also prepared. In this strain S phase is delayed, occurring about 0·3 cycle later than in the wild-type. In wee 1 ·6 the midpoint of the stepwise doubling in nucleoside diphosphokinase activity occurred at 0 · 084; showing that the rise in enzyme activity is also delayed.

Addition of cycloheximide to an exponentially growing culture caused an immediate inhibition of protein synthesis, yet nucleoside diphosphokinase activity continued to increase exponentially for a further 300 min. This indicates that the stepwise doubling of nucleoside diphosphokinase activity during the cell cycle is not achieved by a simple control on protein synthesis.

Two temperature-sensitive cdc− mutants were also used: cdc2-33, a mutant whose single genetic lesion results in the twin defects of a loss of mitotic control and a loss of commitment to the cell cycle; and cdc10-129, which has a defect in DNA replication. In both mutants a temperature shift-up of an asynchronously growing culture from the permissive (25 °C) to the restrictive temperature (36·5°C) results in a rapid inhibition of DNA replication. In both mutants nucleoside diphosphokinase continues to increase exponentially. Therefore, although nucleoside diphosphokinase is required for DNA replication, apparently DNA replication is not required for an increase in nucleoside diphosphokinase activity.
Microspectrophotometric and autoradiographic study of the timing and duration of pre-meiotic DNA synthesis in Paramecium caudatum
Authors: Fujishima Masahiro
Published: April 2021

Timing and duration of pre-meiotic G1, S and G2 phases in Paramecium caudatum have been clarified by microspectrophotometry of DNA content and autoradiography with [H]thymidine ([H]dThd) and [3H]deoxyuridinemonophosphate ([3H]dUMP). Microspectrophotometric measurement of DNA content showed that cells in stationary phase have a G| micronucleus, and that pre-meiotic DNA synthesis begins with the micronuclear swelling and ends immediately before the nucleus enters meiotic prophase. The results indicate that the micronucleus is in G1 during the first T5 h after onset of mating reaction at 25 °C and in pre-meiotic S phase during the following 1 h, and that Gl phase is very short or lacking. Therefore, in P. caudatum, the transition point from pre-meiotic interphase to meiotic prophase is at or very close to the end of pre-meiotic DNA synthesis.

Autoradiographic study showed that although [3H]dThd and [3H]dUMP were well utilized in pre-mitotic DNA synthesis, only [3H]dUMP was well utilized in pre-meiotic DNA synthesis. [3H]dThd was utilized very little in pre-meiotic DNA synthesis even if the precursor was taken into the cell, suggesting that the activity of thymidine kinase is low in these cells.
Interaction between vascular endothelial cells and vascular intimal spindle-shaped cells in vitro
Authors: Bono David De , Green Catherine
Published: April 2021

The interactions between human or bovine vascular endothelial cells and fibroblast-like vascular intimal spindle-shaped cells have been studied in vitro, using species-specific antibodies to identify the different components in mixed cultures. Pure cultures of endothelial cells grow as uniform, nonoverlapping monolayers, but this growth pattern is lost after the addition of spindle cells, probably because the extracellular matrix secreted by the latter causes the endothelial cells to modify the way they are attached to the substrate. The result is a network of tubular aggregates of endothelial cells in a three-dimensional ‘polylayer’ of spindle-shaped cells. On the other hand, endothelial cells added to growth-inhibited cultures of spindle-shaped cells will grow in sheets over the surface of the culture. Human endothelial cells grown in contact with spindle-shaped cells have a reduced requirement for a brain-derived endothelial growth factor. The interactions of endothelial cells and other connective tissue cells in vitro may be relevant to the mechanisms of endothelial growth and blood vessel formation in vivo, and emphasize the potential importance of extracellular matrix in controlling endothelial cell behaviour.
Effects of ethidium bromide on cells of the cockroach central nervous system
Authors: Leech C. A.
Published: April 2021

The effects of ethidium bromide were tested on isolated cockroach interganglionic connectives, either with the perineurium intact or desheathed. Intact connectives showed small, reversible depolarizations in response to 25 mM-ethidium. Desheathed connectives showed much larger, concentration-dependent depolarizations and the membrane potentials repolarized only slowly after washing out ethidium. The ethidium-induced depolarization could be blocked by raising the K concentration in the salines and was also shown to be reduced by rubidium, caesium, ammonium and ouabain. These treatments also reduced the degree of staining of nerve cords by ethidium, which was demonstrated to enter the cells.
Reduction in the mitochondrial membrane potential of Toxoplasma gondii after invasion of host cells
Authors: Tanabe Kazuyuki , Murakami Kenji
Published: April 2021

The membrane potential of Toxoplasma gondii, an obligatory intracellular protozoan parasite, was monitored with the cationic permeant fluorescent dye rhodamine 123 (R123). Fluorescence microscopy revealed R123 to be partitioned predominantly in a restricted part of the parasite, which consisted of twisted or branched tubules, or of granular bodies. These structures were frequently connected to each other. The dye retention by these structures was markedly reduced by treating R123-labelled parasites with the proton ionophore, carbonylcyanide m-chlorophenylhydrazone, the potassium ionophore, valinomycin and the inhibitor of electron transport, antimycin A. Thus, these structures are regarded as the parasite mitochondria. Another cationic fluorescent dye, rhodamine 6G, stained the parasite mitochondria, whereas a negatively charged fluorescent dye, fluorescein, and the neutral compounds, rhodamine 110 and rhodamine B, did not. This fact indicates that R123 monitored the parasite mitochondrial membrane potential. T. gondii-infected 3T3 cells were also stained with R123. In contrast to the mitochondria of extracellular parasites, those of intracellular parasites failed to take up the dye. The absence of fluorescence in intracellular parasites persisted until the infected host cells ruptured and liberated daughter parasites 1 day after infection. Parasites, liberated from the host cells, either spontaneously or artificially by passing the infected cells through a 27G needle, regained the ability to take up the dye. After direct microinjection of R123 into the vacuole in which the parasite grows and multiplies, the dye appeared in the host-cell mitochondria but not in the parasite’s mitochondria. Thus, we conclude that the mitochondrial membrane potential of T. gondii was reduced after invasion of host cells by the parasite.
Repetitive titin epitopes with a 42 nm spacing coincide in relative position with known a band striations also identified by major myosin-associated proteins an immunoelectron-microscoplcal study on myofibrils
Authors: Fürst Dieter O. , Nave Rüdiger , Osborn Mary , Weber Klaus
Published: April 2021

A direct titin-thick filament interaction in certain regions of the A band is suggested by results using four new monoclonal antibodies specific for titin in immunoelectron microscopy. Antibodies T30, T31 and T32 identify quasi-repeats in the titin molecule characterized by a 42-43 nm repeat spacing. These stripes seem to coincide with striations established by others on negatively stained cryosections of the A band. Antibodies T30 and T32 recognize epitopes matching five or two of the seven striations per half sacromere known to harbor both the myosin-associated C-protein and an 86K (K = 103 Mr) protein. Antibody T31 labels two stripes in the P zone, which correspond to the two positions where decoration is seen with 86K protein, but not with C-protein. The single titin epitope defined by antibody T33 is located 55 nm prior to the center of the M band. This position seems to coincide with the M7 striation defined by others on negatively stained A bands.

The T33 epitope position proves that the titin molecule, which is known to be anchored at the Z line, also penetrates into the complex architecture of the M band. The titin epitopes described here enable us to begin to correlate known ultrastructural aspects of the interior part of the A band with the disposition of the titin molecule in the sarcomere. They raise the question of whether there is a regular interaction pattern between titin and the thick filaments. Because of the limit of resolution of immunoelectron microscopy, it remains to be seen whether the repetitive titin epitopes of the C and P zones coincide exactly with the position of the myosin-associated proteins or lie on a similar lattice, out of register with these proteins.
Autoradiographic analysis of formylpeptide chemoattractant binding, uptake and intracellular processing by neutrophils
Authors: Janeczek Amelia H. , Marasco Wayne A. , Alten Pierson J. Van , Walter Robert J.
Published: April 2021

Formylpeptide chemoattractant binds specifically to leukocyte cell surface receptors and during chemotactic activation, is internalized and processed by these cells. Using electron microscope autoradiographic techniques, we have examined the binding, uptake and disposition of fNle-Leu-Phe-Nle-[125I]Tyr-Lys by rabbit peritoneal neutrophils. Cells were incubated with ligand for 15 min at 4°C, rinsed and then further incubated in buffer at 4°C, 15°C, 24°C, or 37°C for 0–40min. For all cells incubated at 4°C, grains were predominantly localized on the plasma membrane. Initially, this formylpeptide was seen in small clusters or microaggregates that were not associated with coated pits. Upon further incubation at 37°C for 1 min, formylpeptide clustering on the plasma membrane increased and extensive internalization of formylpeptide was observed. Endocytosis of formylpeptide-receptor complexes clearly involved uncoated membrane pits and vesicles but did not appear to involve coated pits or coated vesicles. In the following 3 min, peptide proceeded in waves through compartments composed of small uncoated endocytic vesicles, then large vesicles, and then into dense granules. After 4 min at 37°C, the most active phase of intracellular processing subsided. The percentage of grains in the cytoplasm, granule and small vesicle compartments very gradually increased during the remainder of the 40 min incubation. Formylpeptide neither bound at 4 °C nor accumulated at 37 °C on the cell surface in proximity to the underlying Golgi/centrosome region of the cell. At 24°C, processing was slowed but formylpeptide-receptor complexes proceeded through a similar series of compartments. The q for formylpeptide uptake at 37 °C was 15-20 s, whereas at 24°C the q was approximately 10min. No uptake was observed at 15°C. The distinctive characteristics of formylpeptide binding and receptor-complex uptake seen here may be essential in initiating and maintaining continued chemotactic responsiveness.
The polarization of fibroblasts in early primary cultures is independent of microtubule integrity
Authors: Middleton C. A. , Brown A. F. , Brown R. M. , Karavanova I. D , Roberts D. J. H. , Vasiliev J. M.
Published: April 2021

Fibroblasts in culture apparently require an intact system of microtubules in order to adopt and maintain a polarized morphology. In contrast, the polarization of a number of epithelial cell types has been shown to be microtubule-independent. Reconciliation of these apparently contradictory data is difficult, however, because the epithelial cells were studied in short-term primary cultures while the fibroblasts were studied in secondary or longer-term cultures. To clarify the situation we have examined the effects of the microtubule-disrupting drugs, colcemid and nocodazole, on the polarization of a single cell type, the chick heart fibroblast (HF), maintained in both primary (1°) and secondary (2°) cultures.

Immunofluorescence observations of both types of culture showed that in control medium the cells contained abundant microtubules, which were absent if the cells were cultured in medium containing either colcemid or nocodazole. The effects of microtubule-disrupting drugs on the polarization of the cells were quantified using two measures of cell shape, elongation and dispersion, both of which increase with increasing polarization. The results show that microtubule-disrupting drugs do not have a significant effect on the polarization of HF spreading in 1 ° culture but significantly reduce the polarization of HF spreading in 2° cultures. The effects of microtubule disruption on HF that had been maintained in 1° culture for 6h, 24 h or 48 h were also quantified. These measurements demonstrate that the polarization of HF in 1° culture becomes increasingly sensitive to microtubule disruption over this period until, after 48 h, they are as sensitive as HF in 2° cultures to microtubule disruption. The results show that the polarization of HF, cultured immediately after their isolation from the parent tissue is, like that of some epithelial cells, microtubule-independent but that during the first 48 h in culture these fibroblasts change in such a way that their polarization becomes microtubuledependent.
Ultrastructure and gold-immunolabelling of cell-substratum adhesions (podosomes) in rsv-transformed bhk cells
Authors: Gavazzi Isabella , Nermut Milan V. , Marchisio Pier Carlo
Published: April 2021

Rous sarcoma virus-transformed BHK (RSV/B4-BHK) cells develop peculiar dot-like adhesions, that have been named podosomes, which, in the presence of serum, aggregate into ring- or crescentshaped adhesion sites, the rosettes of podosomes. We have used the lysis-squirting technique and gold-immunolabelling to study the 3D- organisation of podosomes and the location of vinculin, gelsolin, phosphotyrosine-containing proteins and pp60src at an ultrastructural level. Podosomes appear to be conical bodies, 0 ·l-0 ·5 μm high, made by a dense aggregation of actin oligomers and several associated proteins, connected in the rosette by actin filaments. Gelsolin and some phosphotyrosine-containing proteins are found within the podosomes, often associated with the actin filaments, while vinculin is found predominantly at the podosome periphery, associated with microfilaments, and pp60arc is located on the adjacent plasma membrane.
Three-dimensional localisation of DNA in the nucleolus of Spirogyra by correlated optical tomography and serial ultra-thin sectioning
Authors: Jordan E. Gwyn , Rawlins David J.
Published: April 2021

Spirogyra nucleoli were shown by three-dimensional optical microscopy of DAPI fluorescence to contain DNA with a pattern and distribution matching those of the fibrillar centres. This was confirmed using different species with nucleoli showing different sizes of fibrillar centre. Much lower levels of fluorescence were seen corresponding to the dense fibrillar component. Nearly all the DAPI fluorescence arises from the fibrillar centres or from regions very close to their surface, indicating that this is the site of nucleolar transcription.
Appearance and origin of snRNP antigens in chick erythrocyte nuclei reactivated in heterokaryons
Authors: Bergman Mathias , Nyman Ulf , Ringertz Nils , Pettersson Ingvar
Published: April 2021

Fusion of terminally differentiated chick erythrocytes (CE) with transcriptionally active rat myoblasts results in heterokaryons in which the CE nuclei undergo reactivation of RNA synthesis and splicing. In order to analyze the transport and assembly of small nuclear ribonucleoprotein (snRNP) particles and larger molecular complexes engaged in RNA processing, we have examined CE nuclei in heterokaryons for the presence of four U snRNP-related nuclear antigens (Sm, 70000Mr, F78 and M3G-cap) and for one antigen (La), associated with RNA polymerase III transcripts. Inactive erythrocyte nuclei showed low levels of Sm and F78 antigens, but the other antigens were undetectable. Immediately after fusion, the fluorescence of the pre-existing chicken Sm antigen was detected in the CEn, and then the intensity of the signal increased rapidly during reactivation. The other antigens appeared more slowly, reaching full intensity at different time points after fusion. Blocking of chick transcription did not block the appearance of Sm, 70000Mr, cap and La antigens but did effectively inhibit the appearance of the F78 antigen. It has previously been demonstrated that the structure recognized by this monoclonal antibody is physically associated with functional splicing complexes. Blocking of translation in heterokaryons abolished uptake of snRNPs into the chicken nuclei. Taken together, the results indicate that rat snRNP complexes were imported into the chick nuclei after assembly in the cytoplasm. For all the studied antigens, except F78, this translocation was independent of chick RNA synthesis. The appearance of the F78 antigen was totally dependent on expression of chicken genes.
A 116000 M
r nucleolar antigen specific for the dense fibrillar component of the nucleoli
Authors: Masson C. , Andre C. , Arnoult J. , Geraud G. , Hernandez-Verdun D.
Published: April 2021

In ATT, a human autoimmune serum, we found anti-nucleolar antibodies that recognized nucleolar antigens confined to a single nucleolar compartment, the dense fibrillar component (DFC). We localized these antigens by immunoelectron microscopy in DFC of HeLa cell nucleoli both on Lowicryl sections and cryoultrathin sections without embedding. The antigens were solubilized by incubation with 2 M NaCl but not by RNase or DNase treatment. The ATT serum crossreacted with rat liver nucleoli and PtK1 cell nucleoli in which immunofluorescence labelling displayed a clumpy pattern. During mitosis, the antigens dispersed in the cytoplasm until late telophase, when they gathered in the prenucleolar bodies. In human peripheral lymphocytes, or HeLa cells treated with actinomycin D, the antigens were still present but the fluorescence intensity decreased. By immunoblotting using human nuclear extracts, the ATT serum bound to a 116 000 Mr protein at dilutions up to 1:2000. The reactivity of this band diminished with actinomycin D-treated nuclear extracts. Two minor bands were also observed at 97 and 70K (K=103Mr). Immunopurification by competition or elution demonstrated that the 116K antigens were at the origin of the nucleolar labelling. This DFC marker appeared to be different from the NORsilver-stained proteins, which in our preparations exhibited apparent molecular weights of 105,80 and 38 –40K. In addition, these 116K antigens did not exhibit the characteristics described for DNA topoisomerase I, fibrillarin or nucleolin. We propose the 116K antigen as a new marker of the DFC of the nucleoli.
Prompt heat-shock and heat-shifted proteins associated with the nuclear matrix-intermediate filament scaffold in Drosophila melanogaster cells
Authors: Ornelles David A. , Penman Sheldon
Published: April 2021

Elevated temperatures induced the synthesis of several new proteins in Drosophila melanogaster cells. Besides the conventional heat shock (HS) proteins, another set of temperature-induced proteins has been found. These latter resemble the prompt HS proteins of mammalian cells. The prompt HS proteins of Drosophila differ from the well-known conventional HS proteins in the following properties: (1) synthesis of the prompt HS proteins is insensitive to the transcription inhibitor actinomycin D, which blocks the appearance of conventional HS proteins; (2) induction of the prompt HS proteins requires a significantly higher temperature than conventional HS proteins; (3) prompt HS proteins associate strictly with the nuclear matrix-intermediate filament complex (NM-IF), while the conventional HS proteins are found in all subcellular fractions; (4) prompt HS proteins of Drosophila are induced by high temperature alone while the conventional HS proteins are also produced by a variety of stress conditions.

Resinless-section electron micrographs show an altered nuclear matrix morphology in heat-shocked cells. The nuclear matrix fibers are altered in spatial distribution and have much additional electron-dense material. This added material probably reflects the soluble proteins shifted into the nuclear matrix at high temperature. The prompt HS proteins can be distinguished clearly from heat-shifted proteins by several criteria. Also, the prompt HS proteins are distinct from the heat-insensitive viral proteins of a persistent virus (HPS-1).
Nucleation of microtubules from mitotic centrosomes is modulated by a phosphorylated epitope
Authors: Centonze Victoria E. , Borisy Gary G.
Published: April 2021

At the onset of mitosis a class of proteins appears that possess a phosphorylated epitope recognized by the monoclonal antibody MPM-2. Immunofluorescence staining shows that a subset of these proteins is associated with the centrosomes of the mitotic apparatus. The appearance of these proteins coincides with the increased microtubule nucleating capacity of the centrosomes. We have tested whether growth of microtubules from mitotic centrosomes in a lysed cell model is dependent on the availability of the phosphorylated epitope by blocking the epitope with a specific antibody or by modifying it by removal of the phosphate. Centrosomes incubated with purified tubulin nucleate microtubule asters. However, preincubating the centrosomes with MPM-2 blocks all microtubule nucleation. Pretreating mitotic centrosomes with alkaline phosphatase also inhibits nucleation. These data suggest that the phosphorylated epitope recognized by MPM-2 is important for microtubule nucleation.
Substratum-induced stress fiber assembly in vascular endothelial cells during spreading in vitro
Authors: Yost Jeffrey C. , Herman Ira M.
Published: April 2021

We tested whether aortic endothelial cell (EC)-synthesized substrata, which modulate smooth muscle cell proliferation and EC motility following injury, could influence EC actin cytoskeleton and spreading in vitro. A partial characterization of the substrata indicates that the substratum prepared by deoxycholic acid extraction (DOC-derived substratum) is enriched with fibronectin and type IV collagen. Substratum prepared by removal of the intact monolayer with 20 mM EGTA in PBS (EGTA-derived substratum) contains fibronectin and heparan sulfate proteoglycan, but no type IV collagen. Morphometric analyses were performed on fixed and cytoskeletal antibody treated EC in order to quantitate the extent of spreading and stress fiber (SF) assembly. Compared to plastic, the DOC-derived substratum, a collagenase-treated DOC-derived substratum (CT-DOC-derived substratum) and the EGTA-derived substratum promote EC spreading 2·3-, 2·9- and 1·7-fold, respectively. In addition, there are 4·2-, 4·1- and 2·0-fold more SF on DOC-, CT-DOC- and EGTA-derived substrata, respectively, when compared to plastic. Subcellular fractionation and immunoprecipitation of cytoskeletal proteins from metabolically labeled EC were performed prior to electrophoresis and fluorography. The DOC-derived substratum increases immunoprecipitable actin and myosin 3-to 4.5-fold in both fractions compared to the EGTA-derived substratum and plastic. Collagenase treatment of the DOC-derived substratum partially inhibits this increase. Cycloheximide treatment prevents the rise in soluble actin and myosin as well as causing a reduction in SF number by 1/2 on the DOC-derived substratum and 2/3 on CT-DOC-derived substratum. We propose that fibronectin-collagen interactions are, in part, responsible for inducing endothelial synthesis of cytoskeletal proteins required for SF assembly. This substratum-induced actin-cytoskeletal reorganization facilitates EC spreading in vitro.
Ultrastructure of chromatin II. Three-dimensional reconstruction of Isolated fibers
Authors: Woodcock Christopher L. , McEwen Bruce F. , Frank Joachim
Published: April 2021

Electron-microscope tomography has been used to reconstruct isolated, negatively stained chromatin fibers from Necturus maculosus erythrocytes. Tilt series micrographs from +70° to —70° at 5° intervals were obtained, allowing a reconstruction resolution of 3.3 nm for fibers lying parallel to the tilt axis. The fibers were found to be flattened in the plane of the carbon support, and also stained differentially according to the distance from the carbon. A number of methods of presenting the three-dimensional information were explored. Especially useful was an automatic peak search method for locating putative nucleosome positions coupled with the production of a computer-generated model. Other valuable techniques included the generation of projection stereograms and construction of solid models. A peripheral location of nucleosomes in the chromatin fiber was indicated, and helical arrangements of nucleosomes were observed over short regions. However, no long-range ordering of nucleosomes was apparent. The extent to which this lack of order may be the result of events occurring during the preparation of chromatin for electron microscopy is discussed.
Monoclonal antibodies to Leishmania mexicana promastigote antigens I. Secreted acid phosphatase and other proteins share epitopes with lipophosphoglycan
Authors: Ilg Thomas , Menz Beatrice , Winter Gerhard , Russell David G. , Etges Robert , Schell Dietmar , Overath Peter
Published: April 2021

The abundant surface glycolipid, lipophosphoglycan (LPG), of Leishmania promastigotes is composed of phosphosaccharide repeating units linked via a phosphosaccharide core to a conserved lyso alkyl-phosphatidylinositol membrane anchor. It is shown in this paper that monoclonal antibodies (mAbs) directed against LPG also react with an acid phosphatase secreted by L. mexicana promastigotes. Acid phosphatase purified by column chromatography (apparent Mr=100000) reacts in immunoblots with the anti-LPG mAb AP3 and another mAb, L3.13, which does not recognize LPG. mAb L3.13 was used to purify the enzyme by affinity chromatography. The resulting glycoprotein has the same molecular weight and binds AP3 on immunoblots. The secreted phosphatase is non-covalently associated with a high molecular weight, galactose-containing glycan or proteoglycan that is recognized by both AP3 and L3.13. In addition to acid phosphatase, other parasite proteins appear to be modified by LPG epitopes.
Cycling of intracellular free calcium and intracellular pH in Xenopus embryos: a possible role in the control of the cell cycle
Authors: Grandin Nathalie , Charbonneau Michel
Published: April 2021

The scope of this commentary is to propose the incorporation of recent data, obtained from Xenopus eggs and embryos, into the models of cell cycle regulation by MPF, a universal M-phase Promoting Factor operating in most, if not all, mitotic cells, from yeast to human. These new data are: (1) the cycling activity of MPF in Xenopus eggs is temporally and functionally related to the cycling activity of intracellular pH (pHi) (Grandin and Charbonneau, 1990a); (2) cell division in Xenopus embryos is accompanied by oscillations of the intracellular free calcium activity ([Ca2+]1) (Grandin and Charbonneau, 1991). There is now definitive evidence that one of the molecular components of the cell cycle, the ‘master oscillator’ (or cytoplasmic clock), is represented by MPF (Masui and Markert, 1971) and its correlated cdc2 kinase activity and cyclin level (see, for instance, Draetta and Beach, 1989; Murray, 1989; Murray and Kirschner, 1989). In addition, a variety of different systems have revealed a direct implication of [Ca2+]1 variations in mitotic events (reviewed by Berridge and Irvine, 1989; Hepler, 1989). Although Xenopus embryonic cells have become, in addition to yeast, one of the most important systems for studying the molecular biology of the cell division cycle, almost no attention has been paid to the possible involvement of ionic messengers, particularly Ca2+, in the control of mitosis in Xenopus embryos. This lack of attention to the possible role of [Ca2+]1 variations in the cell division cycle of Xenopus embryos was due to their repeatedly noted absence. However, the recent demonstration of Ca2+ oscillations occurring with a periodicity equal to that of the cell division cycle in Xenopus embryos (Grandin and Charbonneau, 1991) now offers an opportunity of re-evaluating the already proposed models of cell division.