bluuebunny/crossref_metadata_2025 huggingface dataset:
Synchronous exocytosis in Paramecium cells. VI. ultrastructural analysis of membrane resealing and retrieval
Authors: Plattner H. , Pape R. , Haacke B. , Olbricht K. , Westphal C. , Kersken H.
Published: April 2021

After the synchronous induction of exocytosis of secretory organelles (trichocysts) in Paramecium tetraurelia cells the process of membrane resealing and retrieval could be followed under synchronous conditions. The characteristic aggregates of membrane intercalated particles (MIPs) contained within the freeze-fractured cell membrane (rings and rosettes) and trichocyst membranes (annulus MIPs), in addition to collar striations on the top of trichocyst membranes, served as endogenous ultrastructural markers. This allowed us to follow the re-arrangement of membrane constituents during and after exocytosis with high temporal and spatial precision. Membrane specificity is maintained to a considerable extent (∼ 99·5%), as judged from the rare occurrence of aberrant resealing (according to freeze-fracture data) and from the rather minute shift of glycocalyx components (according to electron staining experiments) during normal membrane resealing. Coated pits are not involved in membrane retrieval (155 ghosts analysed); since the membrane regions involved in exocytotic fusion are backed by apposed materials, probably proteins, this may restrain membrane constituents from intermixing. Another factor for maintaining membrane specificity is the fact that resealing of the exocytotic opening occurs much more rapidly than in most other systems. The retrieval operates with a half-life of 3 (strain 7S) to 9 min (K401), the involvement of cortical microtubules in the retrieval can be largely excluded, since only two microtubules (of unidentified origin) were seen to approach ghost structures in 4074 cases analysed during this period of intense ghost retrieval. Phalloidin microinjected at a dose that blocked all cytoplasmic streaming (before synchronous exocytosis was induced) did not abolish membrane resealing and retrieval, which, therefore, may be passive processes.
Light and scanning electron microscopy of the same human metaphase chromosomes
Authors: Harrison Christine J. , Jack Elspeth M. , Allen Terence D. , Harris Rodney
Published: April 2021

A technique has been developed to examine the same G-banded human metaphase chromosomes, first in the light microscope and then in the scanning electron microscope (SEM). A structural involvement in chromosome banding was confirmed by a positional correlation between the G-positive bands observed in the light microscope and the circumferential grooves between the quaternary coils of the metaphase chromosomes, observed in the SEM. In further support of this the regions between the grooves showed a positional relationship with the G-negative or reverse (R) bands.

The examination of slightly extended metaphase chromosomes in the light microscope demonstrated that the G-banding pattern corresponded to that described by the Paris nomenclature for metaphase chromosomes. The arrangement of the circumferential grooves of the same chromosomes, observed in the SEM, was shown to relate to that described by the Paris nomenclature for prometaphase chromosomes. Therefore, using the SEM it is possible to demonstrate the details of prometaphase banding in metaphase chromosomes.
The cell cycle of symbiotic Chlorella I. the relationship between host feeding and algal cell growth and division
Authors: McAuley P. J.
Published: April 2021

When green hydra were starved, cell division of the symbiotic algae within their digestive cells was inhibited, but algal cell growth, measured as increase in either mean volume or protein content per cell, was not. Therefore, control of algal division by the host digestive cells must be effected by direct inhibition of algal mitosis rather than by controlling algal cell growth. The number of algae per digestive cell increased slightly during starvation, eventually reaching a new stable level.

A number of experiments demonstrated that although there was a relationship between host cell and algal mitosis, this was not causal: the apparent entrainment of algal mitosis to that of the host cells could be disrupted. Thus, there was a delay in algal but not host cell mitosis when hydra were fed after prolonged starvation, and algae repopulated starved hydra with lower than normal numbers of algae (reinfected aposymbionts or hydra transferred to light after growth in continuous darkness). Two experiments demonstrated a direct stimulation of algal cell division by host feeding. Relationships of algal and host cell mitosis to numbers of Artemia digested per hydra were different, and in hydra fed extracted Artemia algal, but not host cell, mitosis was reduced in comparison to that in control hydra fed live shrimp.

It is proposed that algal division may be dependent on a division factor, derived from host digestion of prey, whose supply is controlled by the host cells. Numbers of algae per cell would be regulated by competition for division factor, except at host cell mitosis, when the algae may have temporarily uncontrolled access to host pools of division factor. The identity of the division factor is not known, but presumably is a metabolite needed by both host cells and algae.
Changes in nucleoli and nucleolar fibrillar centres of chromaffin cells in rat adrenal medulla over a 24-hour period: An ultrastructural and stereological analysis
Authors: Robaglia Andrée , Seïte Raymond
Published: April 2021

A stereological and ultrastructural study was performed on the nucleoli of the adrenal medulla chromaffin cells of rats exposed to a standardized 12 h light/12 h dark cycle with free access to food and water. The animals were killed three at a time, every 4h during the 24-h span and fixed by intracardiac perfusion.

In these reticulated nucleoli, the stereological analysis over a 24-h period showed a variation dependent on the time of killing for the two parameters investigated, the mean nucleolar volume, , and the mean volume of the fibrillar centres, . The minimal value occurred at 0300 h (dark span) and the maximal one at 0700 h (at the onset of the light span). Between these two extreme values, increased 1’8-fold and 5’3-fold.

These data are compared with a previous description from our laboratory of circadian rhythm in nucleoli of sympathetic neurons of superior cervical ganglion in the same animals. Analogies and differences are pointed out, but apart from these considerations the present study provides a new example of temporal organization at the cellular level in the organelle involved in ribosomal RNA synthesis and ribosome assembly.
Rebinding of Tetrahymena 13 s and 21 s dynein atpases to extracted doublet microtubules the inner row and outer row dynein arms
Authors: Warner Fred D. , Perreault John G. , Mcilvain Jane H.
Published: April 2021

Ciliary axonemes from Tetrahymena contain a second salt-extractable ATPase distinguishable from outer arm 21 S dynein by sedimentation velocity (≃ 13 S), electrophoretic mobility and substrate specificity. As characterized by turbidimetric assay, gel electrophoresis in the presence of sodium dodecyl sulphate, ATPase activity and electron microscopy, the 13 S dynein ATPase rebinds to extracted doublet microtubules. Compared to structural-side (ATP-insensitive) 21 S dynein binding, which is moderately specific for the 24 nm outer row arm position, rebinding of 13 S dynein is highly specific but for the inner row arm position. However, 13 S dynein rebinds to the A subfibre with a spacing that coincides with the triplet spacing of the radial spokes (24–32–40 nm periods; 96 nm repeat). All of the major protein components present in the 13 S or 21 S fractions rebind to extracted doublets under conditions that both restore and activate dynein ATPase activity. Unlike active-side (ATP-sensitive) rebound 21 S dynein, rebound 13 S dynein is completely insensitive to dissociation by ATP-vanadate and does not independently decorate the B subfibre. The saturation profile for rebinding of 13 S dynein exhibits a lack of cooperativity between binding events (h = 1·0) similar to structural-side rebinding of 21 S dynein. At low 21 S/doublet stoichiometry there is no measureable competition between the 13 S and 21 S dyneins for binding sites on the A subfibre lattice, although at saturating concentrations of 21 S dynein, rebinding of 13 S dynein is blocked completely.
Surface features of human natural killer cells and antibody-dependent cytotoxic cells
Authors: Payne Claire M. , Linde Alec , Kibler Ruth , Poulos Bonnie , Glasser Lewis , Fiederlein Roger
Published: April 2021

The purpose of the present study was to examine the surface features of purified large granular lymphocytes (LGLs) (natural killer (NK) cells, antibody-dependent cytotoxic lymphoid (ADCL) cells, K-cells, Fcγ (+) third population (non-T, non-B) lymphoid cells, Tγ cells) by scanning electron microscopy (SEM) and to compare their surface features with granulocytes, monocytes and Fcγ (—) lymphoid cells that were all fixed for SEM under identical conditions. We have determined that 72–80% of LGLs enriched by rosette formation with sensitized erythrocytes or using Percoli gradients, have a complex microvillous surface (CMS) pattern identical to that of lymphocytes. The LGL fraction appears by SEM to represent a morphologically homogeneous population of cells. Monocytes prepared for SEM under identical conditions had distinct surface folds and granulocytes displayed numerous broad-based ridge-like profiles. The majority of lymphoid cells in an unfrac- tioned population have a CMS pattern when incubated at room temperature (25 °C) before fixation, and a sparse microvillous surface (SMS) pattern when incubated at body temperature (37°C). Ficoll-Hypaque (FH) also had a direct effect on the cell surface pattern. Over half of the unfractionated lymphoid cells displayed a CMS pattern after cells were washed free of FH and incubated at 37 °C before fixation. The CMS pattern is therefore not unique to LGLs but can be produced by the surface alteration of non-LGLs found in unfractionated buffy coat and mononuclear fractions. The interactions between LGLs and sensitized erythrocytes in an antibody-dependent cytotoxic assay system, and LGLs and K562 target cells in an NK assay system, were also examined. This is the first report that describes the surface features of human LGLs interacting with K562 target cells in an NK assay system. The LGL populations studied by SEM were determined to have a high percentage ofLeu-ll (+) and Leu-7 (+) cells. These same populations were also shown to have high antibody-dependent cytotoxicity and NK activity using the 51Cr release assay.
Changes in the transport of mitochondrial Ca2+ during the culture growth cycle of Tetrahymena pyriformis
Authors: Kim Yuri V. , Kudzina Ludmila Yu. , Zinchenko Valeri P. , Evtodienko Yuri V.
Published: April 2021

The properties of the Ca2+ transport system of mitochondria, isolated in various phases of growth of static cultures of Tetrahymena pyriformis, were studied. A large increase in the endogenous energy-dependent Ca2+ content of mitochondria was observed as cultures of T. pyriformis passed through the exponential and stationary phases of growth (approx. 0·25 and 50 nmol Ca2+ per mg mitochondrial protein, respectively). Simultaneously, the mitochondria dramatically lost their ability to withstand large concentrations of Ca2+ and ADP. However, in the latter case they were able to phosphorylate a large amount of ADP if the strong Ca2+chelator, ethylene glycol bis-β-aminoethyl ether)-N,N,N’,N’-tetraacetic acid, was initially present in the incubation medium.

Furthermore, all the changes observed in mitochondria from the stationary phase cells were completely reversed when cell proliferation was re-activated after the lag phase, either by reseeding the stationery cells in fresh growth medium or by oxygenation of the old medium. In aerobic conditions even a small addition of Ca2+ was able to induce rapid release of Ca2+ from mitochondria isolated during the stationary phase of growth. It is suggested that the redistribution of Ca2+between the mitochondria and the cytoplasm at the onset of the lag phase may serve as the main trigger for the subsequent biochemical and morphological changes observed in T. pyriformis.
Density-dependent survival of ehrlich ascites tumour cells in the presence of various substrates for energy metabolism
Authors: Zaporowska-Siwiak E. , Michalik M. , Kajstura J. , Korohoda W.
Published: April 2021

We have found that Ehrlich ascites tumour (EAT) cells, deprived of any carbon source, and suspended at a density of 2 ×105 cells/cm3, begin to die only after 12h of starvation, though it is known that under these conditions they lose over 80% of their ATP within 30 min. Moreover, we have found that the viability of the cells incubated in the absence of any substrate for energy metabolism is strongly dependent on the density of the cell suspension, and can be significantly improved simply by increasing the suspension density. This prompted us to investigate the density dependence of the maintenance of EAT cell viability in the presence of various substrates for energy metabolism and metabolic intermediates. It was found that:

(1) Glucose ensures 48 h viability of EAT cells irrespective of suspension density.

(2) Fatty acids and pyruvate as sole carbon source do not improve EAT cell survival.

(3) In the presence of glutamine as sole carbon source the EAT cell survival shows dependence on cell-suspension density. At densities of 1·6 ×106 to 3·2 × 106 cells/cm3 the cell viability is maintained at least as well as in the presence of glucose, but at low cell-suspension densities glutamine does not support cell viability.

(4) In the presence of glutamine, addition of 1 mM-inosine and 1 mM-uridine ensures high cell survival irrespective of the cell-suspension density.

(5) In the presence of inosine or uridine (10 mid) as sole carbon source, the EAT cell survival is the same as in the presence of glucose and does not depend upon cell-suspension density. Guanosine is less effective, whereas adenosine has no effect at all on the maintenance of EAT cell viability for 48 h.

(6)There is no correlation at all between EAT cell survival and the rate of lactic acid production. At a cell-suspension density of 1·6 × 106 cells/cm3 the cell survival is of the same order in the presence of glutamine as in the presence of glucose, in spite of the fact that in the first case the rate of lactic acid production is more than 20 times lower.

(7) There is no correlation between the capacity of particular nucleosides to support EAT cell survival and their effects on glycolysis and oxygen consumption.
The cystoskeleton of unstimulated blood platelets: Structure and composition of the isolated marginal microtubular band
Authors: Kenney Dianne M. , Linck Richard W.
Published: April 2021

Detergent-insoluble, marginal microtubular band (MB) cytoskeletons were isolated from unstimulated blood platelets after pretreatment with glycerol or with Taxol. MB cytoskeletons retained the shape of intact platelets and behaved in suspension as coherent structural units. The major structural component was a continuous coil of long microtubule(s), often with granular/amorphous material present in the centre; few typical actin filaments were observed. The coiled microtubules often had an amorphous surface coating, but no discrete inter-microtubule bridges were seen. Tubulin and actin (identified by immunochemical staining) were major polypeptides. None of the minor (> 10) polypeptide components comigrated with high molecular weight microtubule-associated proteins in brain tubulin. A novel polypeptide, resolved by two-dimensional electrophoresis and designated IEF-51K, was present in MB cytoskeletons in amounts approximately equivalent to each of the tubulin polypeptides. Evidence suggests that IEF-51K is a distinct, previously undescribed component of the platelet cytoskeletal system.
High density mammalian cell growth in leibovitz bicarbonate-free medium: Effects of fructose and galactose on culture biochemistry
Authors: Barngrover D. , Thomas J. , Thilly W. G.
Published: April 2021

The most commonly used buffering system for mammalian cell cultures is a bicarbonate/CO2 system, which requires CO2 regulators and incubators to supply a constant level of CO2. As a replacement, Leibovitz developed a bicarbonate-free medium, L15, with relatively high levels of certain amino acids in the free base form. We found that a modified form of L15, containing 10 mM-fructose instead of galactose, supported high density growth of Vero and MDCK cells, with maintenance of a stable pH and lactate/pyruvate ratio. We report here investigations of Vero and MDCK cell growth and culture biochemistry at different concentrations of the two carbohydrates. The initial fructose concentration in the medium affected the eventual pH of the medium, the rate of production of lactic acid and ammonia, and the fructose utilization rate. The initial galactose concentration affected the growth rate but did not affect eventual culture pH, the rates of lactate and ammonia production, or the rate of its own utilization. Thus, Leibovitz’ formula, modified to contain 10mM-fructose, appears to yield satisfactory stability of culture pH and the lactate/pyruvate ratio. At all concentrations of galactose tested, the lactate/pyruvate ratio drifted out of the physiological range.
Cell junctions and locomotion of the blastoderm edge in gastrulating chick and quail embryos
Authors: Andries L. , Harrisson F. , Hertsens R. , Vakaet L.
Published: April 2021

The blastoderm edge migrates by the active locomotion of a multilayer of epithelial cells, the so-called margin of overgrowth (MO), that uses the vitelline membrane as its substratum. The structural unity formed by the margin of overgrowth cells and their rapid migration suggest coordination of locomotion between individual cells. Using transmission electron microscopy of thin sections and freeze-fracture, we attempted to determine if the pattern of junctions of the migrating margin of overgrowth is related to the suggested cell-cell cooperation between individual cells in this region. In the leading edge there are large areas of closely apposed cell membranes. Incipient desmosomes and small gap junctions were observed. Tight junctions consisted of isolated strands or isolated networks of tight-junctional strands. In the proximal part of the margin of overgrowth the size of the gap junctions increased and the desmosomes were fully developed. Tight-junctional strands were either isolated or arranged into an isolated network. A broad belt of tight junctions was observed at the transition between margin of overgrowth and non-marginal cells. The distribution of the junctional elements in the MO suggests that junctions contribute to the maintenance of the structural and functional organization of the margin of overgrowth. Furthermore, the spatial distribution of the junctions might give information about the mechanism of locomotion of the margin of overgrowth.
Correlation of Na+, K+-ATPase content and plasma membrane surface area in adapted and de-adapted salt glands of ducklings
Authors: Merchant Juanita L. , Papermaster David S. , Barrnett Russell J.
Published: April 2021

During salt-water adaptation, an increase occurs in Na+, K+-ATPase content and surface area of the basolateral plasma membrane of the principal cell of the duck salt gland. To determine the degree to which these changes are correlated, accepted morphometric methods were used to determine numerical cell densities and plasma membrane surface densities of peripheral and principal cells. After adaptation, the plasma membrane surface area per principal cell was five times greater than in controls. Following de-adaptation, the plasma membrane content in principal cells returned to L9 times control levels. Two other cell constituents, mitochondria and lipid droplets, displayed similar quantitative changes. Na+, K+-ATPase content increased about fourfold with adaptation and decreased to near control levels with de-adaptation. Thus, changes in Na+, K+—ATPase content and basolateral plasma membrane surface area in adapting and de-adapting secretory epithelia of the salt gland occur nearly in parallel. These quantitative data enable Na+, K+-ATPase synthesis and degradation to be investigated in relation to membrane biogenesis.
Involvement of sperm sulphatases in early sperm–zona interactions in the hamster
Authors: Ahuja Kamal K. , Gilburt David J.
Published: April 2021

Sulphatase preparations from Abalone entrails, the limpet Patella vulgata and ox liver, as well as artificial substrates for these enzymes, were used in the hamster in -vitro fertilization system to study the possible roles of sperm sulphatases in sperm-zona pellucida interactions, p-nitrophenyl sulphate, p-nitrocatechol sulphate, ascorbate 2-sulphate, as well as D-glucopyranose and D-galactopyranose, both sulphated at the 3, 4 or 6 position but not the 2 position, inhibited fertilization in a dose-dependent manner. Sperm-egg fusion was not inhibited by the substrates used and eggs pre-treated with sulphates could readily be fertilized. Sperm motility and therefore viability was unaffected by inhibitory concentrations of substrates as determined by rhodamine 123 labelling of motile spermatozoa. Acrosomal integrity of rhodamine-labelled spermatozoa was assessed and found to be unaffected by inhibitory levels of substrates. Fertilization was inhibited by high concentrations of the two molluscan sulphatases but not by purified ox liver sulphatase. Pre-treatment of eggs with these enzymes did not prevent fertilization. Long-term exposure of hamster oocytes to N-acetylhexosaminidase or limpet sulphatase caused thinning and distension of the zona pellucida but these changes were not observed with the ox liver sulphatase. The results suggest that a glycosulphatase is probably released from hamster spermatozoa during sperm-egg adhesion and, or, penetration. If sperm-egg adhesion molecules are sulphated, the commercially available sulphatases would be unsuitable for their characterization.
Protein secretion in Tetrahymena thermophila: Characterization of the secretory mutant strain sb281
Authors: Maihle Nita J. , Satir Birgit H.
Published: April 2021

The ciliated protozoon Tetrahymena thermophila contains membrane-bounded secretory organelles termed mucocysts, the release of which has previously been characterized ultrastructurally as a model system for the events occurring during membrane fusion and protein secretion. Recently, a series of secretory mutant strains of Tetrahymena has been isolated following mutagenesis of a parental wild-type strain designated SB210. In this study, the correlates of non-release in one unique mutant strain of this series, designated SB281, are described. SB281 appears to express a diminished (undetectable) level of the major 34000 Mr proteinaceous secretory product of Tetrahymena, as determined by Western immunoblot analysis and indirect immunofluorescence labelling. Thin-section electron-microscopic studies of these cells reveal that they possess no docked or free mature mucocysts. In addition, freeze-fracture electron microscopy demonstrates that an intramembrane particle array termed the rosette, present in the plasma membrane of wild-type cells above sites of docked mucocysts, is absent in the plasma membrane of mutant SB281 cells. A morphometric analysis of intramembrane particles in the plasma membrane of both wild-type and mutant cells indicates that both strains have a similar intramembrane particle density in both leaflets of the the plasma membrane. Although assembled rosettes are missing in the plasma membrane of mutant cells, a 15 nm intramembrane particle size class does exist in the plasma membrane of the mutant, but this size class is significantly reduced in number relative to wild-type.
Effect of cytochalasin b on somatic cell hybrids between normal and transformed cells
Authors: Hickey Ivor , Mcconville Carmel , Mcmenamin Mary , Neill Rosemary
Published: April 2021

Cytochalasin B (CB) prevents cytokinesis in animal cells. In normal cells nuclear division and DNA synthesis are also blocked and the cells, held in the G1 phase of the cell cycle, remain either mononucleate or binucleate. In transformed cell lines DNA synthesis and nuclear division continue and the cells become multinucleate. We have examined the response to CB in two sets of somatic cell hybrids made between cells that display multinucleation after CB treatment and cells that do not. In a cross between transformed mouse LMTK cells and normal rat embryo lung cells, very little multinucleation was observed after treatment with CB for 7 days. The ability of the LMTK cells to form clones in soft agar was also significantly reduced in these hybrids. Segregant sub-clones that re-expressed both of these transformation phenotypes were isolated. These had reduced chromosome numbers.

A second cross was made between two variants of the BHK cell line, one of which displayed a high level of multinucleation in CB while the other did not. Again the hybrids showed a response similar to that of the non-multinucleating parent. From the results obtained with these two hybrids we conclude that the multinucleation induced in transformed cells by CB behaves as a recessive character in crosses with normal cells.
The kinetic polarities of spindle microtubules in vivo, in crane-fly spermatocytes. i. kinetochore microtubules that re-form after treatment with colcemid
Authors: Czaban B. Barbara , Forer Arthur
Published: April 2021

In newly formed chromosomal spindle fibres we determined the kinetic polarities of the microtubules, that is, the ends to which tubulin monomers add. Spindles disappeared after cells were continuously immersed in colcemid; then portions of the cells were continuously irradiated with a microbeam of near-ultraviolet light to reverse locally the effect of the colcemid. From the following lines of evidence we conclude: (a) that microtubules are organized by the chromosomes; and (b) that tubulin monomers add to the chromosomal spindle fibres at the kinetochore.

When chromosomes were irradiated chromosomal spindle fibres grew in different directions, not necessarily focussed to a common pole; this would not occur if the chromosomal spindle fibres were organized by poles. Chromosomal spindle fibres were sometimes associated with only some of the chromosomes; this would not occur if the fibres were organized by the poles. Thus, chromosomal spindle fibres are organized solely by chromosomes; these spindle fibres are functional since the associated chromosomes moved in anaphase.

When chromosomes were irradiated the re-formed spindle fibres grew up to 10 μm past the edges of the irradiating spot. Experimentally, free tubulin did not diffuse more than 4—5 μm from the irradiated spot. Thus we conclude that the tubulin monomers add at the kinetochores and not at the distal ends of the fibres.
The extracellular matrix of human amniotic epithelium: Ultrastructure, composition and deposition
Authors: Aplin J. D. , Campbell S. , Allen T. D.
Published: April 2021

Ultrastructural comparisons have been made between human amnion extracellular matrix in tissue and cell culture. Immunochemical analysis of matrix deposited by monolayers of cultured amnion epithelial cells has also been undertaken. The basal cell surfaces are highly invaginated with an associated basal lamina that is more electron dense at the distal tips of basal cell processes where hemidesmosomes are frequent. Immediately below the lamina densa is a zone rich in collagen bundles. In the underlying stroma two types of fibril predominate, one striated of 50 nm diameter and one of 18 nm diameter. The observations suggest that at gestational term the epithelial cells are still active in the production of matrix. Secretion appears to occur into invaginations in the basal cell surface where a loosely organized mixture of stromal-type and basal laminal-type aggregates is formed. In culture on plastic, cells also deposit a mixture of basal laminal (type IV collagen+ laminin) and stromal (collagens type I + III) components as well as fibronectin. However, segregation into a true basal lamina with underlying stroma does not occur in vitro, suggesting the need for an organized subcellular template to complete matrix morphogenesis. The in vitro and in vivo evidence suggest that the epithelium contributes to the subjacent dense collagenous zone as well as to the basal lamina.
Water ordering during the cell cycle: Nuclear magnetic resonance studies of the sea-urchin egg
Authors: Zimmerman S. , Zimmerman A. M. , Fullerton G.D. , Luduena R. F. , Cameron I. L.
Published: April 2021

Nuclear magnetic resonance was used to measure spin-lattice water proton relaxation times (T1) during the first cell cycle in sea-urchin zygotes of packed Strongylocentrotus purpuratus. Following insemination there was a 90% increase in the T1 value. The increase in T1 at fertilization could be accounted for by the accumulation of extracellular fluid between the egg surface and the fertilization envelope. The T1 value then remained without change during the first cell cycle, except at metaphase when there was a significant 13% decrease. The lowered T1 values measured at metaphase were not related to a change in the water content of the packed cells, which remained fairly constant throughout the cell cycle. High hydrostatic pressure, low temperature and colchicine (agents that depolymerize mitotic apparatus microtubules) did not affect the T1 values in fertilized eggs. Treatment in vitro of a microtubule protein preparation with low temperature and colchicine resulted in an increased T1, which accompanied the depolymerization of microtubule protein. Since depolymerization of the microtubules associated with the mitotic apparatus by high pressure, colchicine or low temperature does not alter the T1 of water protons in the cell, it is proposed that the increased state of ordered water molecules at metaphase is maintained by non-microtubular factor(s) of the metaphase egg.
Characterization of rat mammary cell types in primary culture: Lectins and antisera to basement membrane and intermediate filament proteins as indicators of cellular heterogeneity
Authors: Warburton Michael J. , Ferns Sharon A. , Hughes Christine M. , Rudland Philip S.
Published: April 2021

Three morphologically distinct major cell types were observed in primary cultures obtained from the mammary parenchyma of glands from virgin rats. These cell types consisted of small cuboidal epithelial cells, larger epithelioid cells and elongated cells. We have investigated the distribution of the basement membrane proteins laminin and type IV collagen, and the intermediate filament proteins vimentin and prekeratin, in these three cell types using immunofluorescence techniques. Antisera to the basement membrane proteins stain the large epithelioid cells and the elongated cells, but do not stain the small cuboidal cells. Polyclonal antiserum to keratin stains all the small cuboidal and large epithelioid cells, but only a small subpopulation of the elongated cells. However, a monoclonal antibody to keratin, LP34, stains only the large cuboidal and a proportion of the elongated cells. Vimentin antiserum fails to stain the small cuboidal cells but stains all the large epithelioid and elongated cells. In addition, peanut lectin, which binds only to ductal lining epithelial cells in the virgin rat mammary gland in vivo after their treatment with neuraminidase, binds to the small cuboidal cells after neuraminidase treatment but not to the other cell types. However, Griffonia simplicifolia agglutinin I, which specifically stains myoepithelial cells in vivo, binds to the large epithelioid and elongated cells but not to the small cuboidal cells. These results suggest that the small cuboidal cells are related to mammary ductal epithelial cells whereas the large epithelial and elongated cells have some characteristics of myoepithelial cells.
The behaviour in vitro of epidermal cells from normal and Pupoid foetus mutant mouse embryos
Authors: Anderson S. , Ede D. A.
Published: April 2021

Pupoid foetus (pf/pf) is a recessive lethal mutation of the mouse causing epidermal hypertrophy and disorganization of peripheral sensory nerves and mesoderm.

The comparative behaviour of epidermal cells from normal and pupoid foetus mutant mouse embryos was studied in vitro. Epidermal explants from the snout region of 12-5-to 13-day embryos were grown in culture for periods of up to 2 weeks. Cultures from both phenotypes were filmed using time-lapse cinemicrography for up to 3 days following explantation.

Paths of individual cells-were traced as they migrated from the explant, and their rate of locomotion and directional persistence were calculated. Differences in these parameters between the two phenotypes were tested statistically. The overall morphology of the cultures, and the tendency of the cells to detach from the periphery of the cell mass were also compared.

The results show that, between 25 and 60 h after explantation, epidermal cells frompupoidfoetus embryos move consistently more slowly than normal cells, and follow a more erratic path. This situation is reversed, however, between 16 and 25 h.

This suggests that the pf mutation causes an alteration in epidermal cells that affects their locomotion, and which is maintained for a minimum of 3 days in the absence of other influencing factors.
The ultrastructure of synchronized hela cells
Authors: Erlandson R. A. , Harven E. DE
Published: April 2021

Synchronous populations of mitotic HeLa cells were obtained by the controlled agitation method, and a detailed morphological study of the cells in all phases of the cell cycle was under-taken to correlate variations in cell structures to known coexisting biochemical events. Auto-radiographic techniques using tritiated thymidine (3H-TdR) were used to detect S1 cells, and colcemid was added to some G2 samples to prevent the cells from going into the next cycle, thus preventing contamination with G1 cells.

The approximate duration (in hours) of the 4 phases were as follows: M = 1·4, G1= 8–9, S = 7, G2 = 4, and the generation time (T) = 21 ± 2 h. Randomization of the cell populations became apparent in the G2 phase (contaminated with S and M cells) and was most likely a result of the genetic make-up of the individual (mixoploid) HeLa cells, nutritional factors (serum lots used), temperature shock, and handling effects. Polyribosomes shifted to monomeric ribosomes during late prophase, at which time nucleoli also break down. These changes are correlated with the drop in protein and RNA synthesis reported for mitotic mammalian cells. The Golgi apparatus persisted in a modified form throughout mitosis. The mid-body forms from the anaphase stem-body and may interfere with the separation of daughter cells. Our studies suggest that the mid-body goes to one of the daughter cells where remnants of this structure were seen in early G1 cells. Large numbers of polyribosomes and the presence of well-developed nucleoli (many attached to the nuclear envelope) characterized G1. These structures, which play a major role in protein and RNA synthesis, persist with slight variations throughout inter-phase. Dense fibrillar nuclear bodies were prominent in late G1 cells. Centrioles separate during G1, and replicate by orthogonal budding during the S phase. Reproducible labelling patterns which reflect the asynchronous multireplicon nature of DNA synthesis in mammalian cells were characteristic of the various stages of the 51 phase. Granular nuclear bodies, which were prominent in S and G2 cells, may correspond to the larger species of heterogeneous nuclear RNA found in HeLa cells. G2 cells were similar in appearance to S cells. As heterochromatin areas increased in late G2 and prophase, perichromatin granules (of unknown significance) became prominent. Mitochondria behaved as independent cell organelles throughout the cell cycle.

Hypertrophied RER, SER, and annulate lamellae, characterized the cytoplasm of colcemid-treated cells. The above changes are indicative of increased metabolic activity, and these structures may function in the production of colcemid-detoxifying enzymes in a manner analogous to that of drug-treated hepatocytes, such as those treated with phenobarbital.
An investigation of the molecular components of desmosomes in epithelial cells of five vertebrates
Authors: Suhrbier Andreas , Garrod David
Published: April 2021

We have shown previously, by fluorescent antibody staining, that desmosomal antigens are widely distributed in the tissues of vertebrate animals. Furthermore, we have demonsuated mutual desmosome formation between cells derived from man, cow, dog, chicken and frog. In this paper we have studied the components of desmosomes in a tissue or a cell line from each of these animals by immunoblotting with antibodies raised against the desmosomal components isolated from bovine nasal epithelium. Blotting was carried out on bovine nasal epithelial desmosomal cores, desmosome-enriched fractions derived from chicken and frog epidermis, nuclear matrixintermediate filament scaffolds derived from Madin-Darby bovine and canine cells (MDBK and MDCK), and unextracted cultured human foreskin keratinocytes.

The results show that desmosomes from all these sources contain high molecular weight proteins (desmoplakins) of similar or identical molecular weights (250 000 and 215 000). Antibodies against the two lower molecular weight desmosomal proteins (83 000 and 75 000) always recognized one or two bands in very similar molecular weight regions of the gels. The desmosomal glycoproteins were found to be much more variable than the proteins: they vary between sources in molecular weight, heterogeneity and antibody cross-reactivity. For instance, antibody specific for a group of glycoprotein bands of 175 000, 169 000 and 164 000 (Mr;) in bovine nasal epithelium recognizes three bands of 245 000, 230 000 and 210 000 in MDCK cells but only a single band of 190 000 in keratinocytes. In mammals, the 175 000-164 000 glycoproteins and the desmosomal adhesion molecules, the desmocollins (Mr 130 000 and 115 000 in cow’s nose), are immunologically distinct. In chicken and frog, however, there are glycoproteins that react with both anti-175 000—164 000 and anti-desmocollin antibodies, but there are also distinct desmocollin bands. The significance of these results is discussed in relation to conservation of desmosomal components and adhesion mechanisms. It is suggested that adhesion may be performed by a well-conserved protein domain and that the variation between desmosomal glycoproteins from different sources may be due to differences in their carbohydrate composition.
Structure of landmark loop ribonucleoprotein matrices in Pleurodeles waltlii lampbrush chromosomes visualized by scanning electron microscopy
Authors: Bonnanfant-JaïS M. L. , N’DA E. , Penrad-Mobayed M. , Angelier N.
Published: April 2021

Landmark loop ribonucleoprotein (RNP) matrices of Pleurodeles waltlii lampbrush chromosomes were systematically examined by scanning electron microscopy (SEM). The results, which corroborated similar studies by electron microscopy (EM), showed that RNP transcripts in normal loops, and RNP matrices in granular, globular and dense loops, are composed of one basic structure: an RNP particle with a diameter of 30 nm.

SEM observations also clarified the spatial arrangement of this particle in the RNP matrices of all the loop types examined.

The specific morphology of normal, granular, globular and dense loop RNP matrices depended on the degree of compaction of the transcription products; this compaction resulted both from the packaging of RNP transcripts and the progressive coiling of the loop axis.
Facultative heterochromatin in Locusta migratoria spermatogenesis: correlation between electron opacity and heteropycnosis
Authors: Friedländer Michael , Hauschteck-Jungen Elisabeth
Published: April 2021

The unpaired X chromosome of Locusta migratoria males is facultatively heterochromatic in the spermatocytes and early spermatids, showing an alternating cycle of positive-negative-positive heteropycnosis. Discrimination between facultative heterochromatin and euchromatin was made using a modification of the Feulgen reaction. It was found that the differential stainability of the DNA of the X chromosome is correlated with the electron opacity of its chromatin. The corresponding differential stainability and electron opacity should reflect differences in protein content or in spatial rearrangement since the amount of DNA in the X chromosome that is transmitted by the spermatozoa to the next generation should remain constant.
Optimization of environment for high density vero cell culture: effect of dissolved oxygen and nutrient supply on cell growth and changes in metabolites
Authors: Nahapetian Ara T. , Thomas James N. , Thilly William G.
Published: April 2021

This study was initiated for optimization of the environment of a technologically useful mammalian cell line for high density production. Cultures of Vero cells on microcarriers were perfused with 100%, 50%, 25% and 12-5% modified L15 media (galactose was replaced with 10 mM-fructose, with 4 HIM-glutamine and 5% foetal bovine serum) in phosphate-buffered saline at either 4 or 8 vol. day−1. Cell growth, pH, dissolved oxygen, and changes in the metabolites, lactate to pyruvate and lactate to ammonia indices, demonstrated that under the conditions used in the present study, perfusion of cultures with 50% L15 medium in PBS at 8 vol. day−1’ provided the optimum microenvironment for Vero cell growth. The highest cell density in the perfused cultures was 3×107 cells ml−1, which at these conditions was ten times higher than the maximum cell density (3×106 cells ml−1’) obtained in a batch culture. Nutrient supply and conditioning factors were the most probable growth-limiting factors in cultures that were perfused with 12·5% and 25% L15 media, while multilayering, limitation of available oxygen, and accumulation of metabolic endproducts in the cellular microenvironment were the most probable causes of a densitydependent inhibition of cell growth observed under the optimized and overfed (supply of 100% L15 medium at the rate of 8 vol. day−1’) culture conditions. Under the optimized environmental condition, the major source of energy was probably glutamine during the first week. However, significant utilization of fructose became evident at higher cell densities during the second week, when lactate production dramatically declined and reached an almost undetectable level, while respiration progressively assumed the predominant role in energy production. It is postulated that ‘available’ oxygen in the multicell-layered microenvironment of the optimized cultures was higher than in the overfed culture due to the greater utilization rate of oxygen for oxidation of excess nutrients in the overfed culture.
Organization of chromosomes in the mouse nucleus: Analysis by In situ hybridization
Authors: Katsumata Makoto , Lo Cecilia W.
Published: April 2021

We used in situ hybridization with various biotinylated DNA probes to characterize the distribution of mouse chromosomes in the nucleus. This entailed an examination of plasmid-marked chromosomes from two strains of transgenic mice and also an examination of the distribution of centromeres and total mouse genomic DNA. Our results show that, regardless of the hybridization probes used or the tissue types examined, with sections of mouse tissues embedded in paraffin all chromosomal DNA appeared to be localized to the nuclear periphery. This was observed in tissues fixed with either crosslinking or non-crosslinking fixatives, and submitted to a variety of prehybridization treatments. Further experiments with non-embedded nuclei fractionated from mouse liver revealed a similar distribution of DNA at the telomeric end of chromosome 3, but for centromeric DNA and total mouse genomic DNA, hybridization signals were observed both in the interior and at the periphery of the nucleus. These observations preclude the exclusive localization of chromosomes to the nuclear periphery. However, they indicate that a subset of centromeres are likely to be associated with the nuclear membrane, and that there is such an association at the telomeric end of chromosome 3. Overall, these results are compatible with previous observations of other investigators showing regions of contact between chromosomes and the nuclear membrane.
Characterization of a novel nuclear envelope protein restricted to certain cell types
Authors: Lord Janet M. , Thick Jane A. , Bunce Christopher M. , Taylor Alexander M. R. , Gallimore Phillip H. , Mills Deryck , Brown Geoffrey
Published: April 2021

The monoclonal antibody AGF2.3 identifies a nuclear envelope protein that is restricted to certain cell types. In particular, this antigen shows a reduced level of expression during haemopoietic cell maturation. In this study, we have examined the relationship of this protein to known nuclear envelope proteins that have a similar molecular mass. Antigen extraction and immunoelectron microscope studies revealed that the AGF2.3 protein is an integral membrane protein present at both the inner and outer aspects of the nuclear envelope. The protein is not associated with nuclear pores and therefore is distinct from pore complex proteins. The AGF2.3 protein does not have ATPase activity. Therefore, this protein is also distinct from a myosin heavy chain-like ATPase that is associated with the nuclear envelope. The AGF2.3 antibody identifies a novel nuclear envelope protein. Further studies of the biochemical nature of the AGF2.3 protein should provide insight into novel cellular processes at the nuclear envelope relating to the lineage or maturation status of cells.
Platelets contain a 21 ok microtubule-associated protein related to a similar protein in hela cells
Authors: Tablin Fern , Reeber Miriam J. , Nachmias Vivianne T.
Published: April 2021

We have demonstrated the presence of a 210K (K= 103Mr) microtubule-associated protein (MAP) in blood platelets and have studied its relationship to tubulin and to the cytoskeleton, using a well-characterized polyclonal antibody for the analysis. When platelet lysates were enriched for tubulin by an assembly cycle at 37°C, the 210K MAP was also enriched, as detected by Western blotting, while the antigen was not detected in pellets from cold-treated samples that lacked stabilized tubulin. Immunofluorescence of resting platelets showed that the 210K antigen colocalized with the microtubule coil in ring-like structures. On the other hand, in preparations of platelet cytoskeletons, the 210K antigen was present in samples from platelets in which the coil was disassembled (cold-treated without taxol pretreatment) as well as from platelets in which the coil was preserved (at 37°C without taxol, or 4 °C with taxol pretreatment). In chilled platelets with disassembled microtubule coils, indirect immunofluorescence using antibodies to 210K or tubulin gave a diffuse signal throughout the platelet cytoplasm. However, immunofluorescence of the 210K antigen in both resting and cold-treated platelets displayed discrete or patchy staining as compared to the continuous staining with antitubulin. We conclude that 210K MAP is present in platelets, that it copurifies with tubulin and that it is localized along the microtubule coil. Our results also suggest that the 210K MAP may interact with some other element(s) of the cytoskeleton, and hence that it might serve as a linking protein.