bluuebunny/crossref_metadata_2025 huggingface dataset:
Effect of calcium on oxytocin-induced contraction of mammary gland myoepithelium as visualized by nbd-phallicidin
Authors: Moore David M. , Vogl A. Wayne , Baimbridge Kenneth , Emerman Joanne T.
Published: April 2021

The effect of calcium on oxytocin-induced contraction of myoepithelial cells was visualized with NBD-phallacidin, a fluorescent stain for filamentous actin. In the absence of oxytocin, the cells appeared relaxed; long, branching processes radiated from the cell bodies. In the presence of 50nM-oxytocin, myoepithelial cells contracted into smaller spoke-shaped bodies in which the arms were shorter and thicker. Electron microscopy confirmed the morphological differences between oxytocin-treated and untreated myoepithelium. To determine a role for extracellular calcium, tissue was incubated in EGTA, then exposed to oxytocin, with or without added calcium. Contraction occurred in the presence of oxytocin plus additional calcium but not in the absence of calcium. When the tissue was incubated with the calmodulin antagonist trifluoperazine (TFP) in calcium-containing medium, oxytocin did not induce myoepithelial cell contraction. These data support previous results obtained with a myosin light-chain phosphorylation assay implicating calcium and calmodulin in oxytocin-induced contraction. Furthermore, NBD-phallicidin visualization of myoepithelial cells demonstrates that the effect of calcium on contraction is physiologically significant.
Cell spreading on serum is not identical to spreading on fibronectin
Authors: Mcinnes Colin , Knox Peter , Winterbourne David J.
Published: April 2021

Adhesion and spreading of cell lines on dishes coated with serum-derived proteins were studied after removal of cell-surface proteoglycans. A mixture of glycosaminoglycans lyases from heparin-induced Flavobacterium heparinum removed 80% of the [35S] sulphate-labelled glycosaminoglycans from the surface of attached cells within 30 min, but this had little effect on cell morphology. The rate of cell attachment to dishes coated with serum was unaffected by prior treatment of cells with this mixture of glycosaminogly can lyases. While a heparan sulphate lyase preparation abolished cell spreading in response to fibronectin there was no effect of the enzyme on the spreading mediated by vitronectin. These results suggest that, although heparan sulphate is required for spreading on purified fibronectin, the spreading stimulated by serum under routine culture conditions requires neither cellular heparan sulphate nor serum-derived fibronectin.
Functional monopolar spindles caused by mutation in MGR, a cell division gene of Drosophila melanogaster
Authors: González Cayetano , Casal José , Ripoll Pedro
Published: April 2021

Mutation in the gene merry-go-round (mgr) of Drosophila causes a variety of phenotypic traits in somatic and germinal tissues, such as poly-ploid cells, metaphasic arrest, postmeiotic cysts with 16 nuclei, and spermatids with four times the normal chromosome content. The most characteristic phenotype is the appearance of mitotic and meiotic figures where all chromosomes are arranged in a circle. Treatment with anti-mitotic drugs and the phenotype of double mutants mgr asp (asp being a mutation altering the spindle) show that these circular figures need a functional spindle for their formation. These abnormal figures are caused by monopolar spindles similar to those observed after different treatments in several organisms. All mutant traits indicate that mgr performs a function necessary for the correct behaviour of centrosomes, thus opening this or-ganelle to genetic analysis.
Structure of myofibrils at extra-junctional membrane attachment sites in cultured cardiac muscle cells
Authors: Atherton Blair T. , Behnke Monica M.
Published: April 2021

The composition and organization of myofibrils at extra-junctional membrane attachment sites in cultured neonatal rat cardiac muscle cells were analysed by immunofluorescence and electron microscopy. When myofibril terminals attached to the cell membrane via focal contacts at regions of the sarcolemma that lacked intercalated discs, they appeared to be non-striated and resembled thick actin cables. Although the non-striated terminals contained actin, myosin and alpha-actinin, the proteins were not organized into recognizable sarcomeres at the light microscopic level. Analysis of the structure of the terminals in the electron microscope confirmed that the usual sarcomeric organization and attachments to the sarcolemma were markedly modified. The non-striated myofibril terminals differed in structure from both stress fibres in non-muscle cells and stress fibre-like structures present in embryonic heart cells in culture. Non-striated myofibril terminals attached to the cell membrane by lateral contact with extra-junctional electron-dense membrane plaques rather than by insertion by their ends into the fascia adherens. It is proposed that the structure and composition of membrane-attachment points for myofibrils may have an influence on the structure, organization or stability of contractile elements in cardiac muscle.
DNA replication and repair of Tilapia cells II. Effects of temperature on DNA replication and ultraviolet repair in Tilapia ovary cells
Authors: Chen J. Don , Yew F. H.
Published: April 2021

TO-2 is a fish cell line derived from the Tilapia ovary. It grows over a wide range of temperature (15–34°C). While most fish cells lack DNA excision repair and are hypersensitive to ultraviolet light (u.v.), Tilapia cells are more u.v.-resistant than mammalian cells. In this paper we report the effects of temperature on DNA replication and u.v. repair in TO-2 cells.

When the cells were moved from 31 °C to the sublethal high temperature of 37°C, the rate of DNA synthesis first decreased to 60%, then speedy recovery soon set in, and after 8h at 37°C the rate of DNA synthesis overshot the 31 °C control level by 180%. When moved to low temperature (18°C) Tilapia cells also showed an initial suppression of DNA synthesis before settling at 30% of the control level. u.v. reduced but could not block DNA synthesis completely. The inhibition was overcome in 3 h at 37, 31 and 25°C, but not at 18°C. Initiation of nascent DNA synthesis was blocked at 4 Jm−2 in TO-2 cells compared with ≤ljm−2 in mammalian cells. After 9Jm−2 u.v. irradiation, low molecular weight DNA replication intermediates started to accumulate, and they could be chased into high molecular weight DNA with little delay.

TO-2 cells showed low levels of u.v.-induced excision repair; but this was prominent compared with other fish cells. The u.v.-induced incision rate has been measured at various temperatures, and the activation energy of incision estimated to be 13 kcal mol−1 (1 cal ≈ 4·I84 J).
Response of aggregating chick corneal cells to modifiers of n-linked oligosaccharides, endoglycosidase h and deoxymannojirimycin
Authors: Overton Jane
Published: April 2021

Chick corneal epithelium takes on its mature conformation between 11 and 16 days of incubation. Earlier work has shown that desmosome frequency increases during this period, reaching its highest rate at 154 days. In the present report aggregation rates of cells from embryos of 11 days and those of 154 days are compared. Younger cells, which form fewer desmosomes, aggregate at a more moderate rate than older cells. In addition, younger cells bind less concanavalin A (ConA) than older cells. To determine if increase in ConA binding could be related to these cellular responses, aggregating cells were exposed to endoglycosidase H (EndoH) and to deoxymannojirimycin. This treatment should permit comparison of the response of cells that have a normal complement of N-linked oligosaccharides with those that have reduced high-mannose or complex type sugars. The effectiveness of EndoH under the conditions used was confirmed by failure of treated glycoprotein after separation by SDS-PAGE and electroblotting to bind ConA. Aggregation rates of both older and younger cells were unaffected, as measured by disappearance of single cells, though older cells formed somewhat smaller aggregates at the highest dosage used. Desmosome formation was markedly reduced in the presence of the enzyme, even in the absence of other changes in the fine structure. At the highest dose of the enzyme the fine structure of older but not younger cells showed indications of blockage of transport. Deoxymannojirimycin appears to cause a build-up of high-mannose groups, since treated cells showed increased incorporation of [3H]mannose. In addition, SDS-PAGE of an NP40-extracted cell fraction showed a similar distribution of proteins, but incorporation of [3H] mannose differed between inhibitor-treated cells and controls. There was no effect on cell fine structure and junctional frequency was normal. Thus N-linked sugars appear to be important in this system to permit re-formation of normal cell contacts, but there is no evidence that processing is required.
Fibronectin observed in the nuclear matrix of hela tumour cells
Authors: Zerlauth Gerold , Wesierska-Gadek Józefa , Sauermann Georg
Published: April 2021

We have investigated the intracellular distribution of insoluble fibronectin in HeLa tumour cells. By indirect immunofluorescence microscopy fibronectin was detected in the nuclear region, but not in the region of the cell surface. Isolated nuclei and isolated nuclear matrices also stained for fibronectin. By quantitative enzyme-linked immunosorbent assay (ELISA), fibronectin was found almost exclusively in the subcellular fraction of isolated nuclear matrices. Using immunoblotting techniques fibronectin was detected in nuclear matrices isolated from cells grown in standard and in fibronectin-depleted medium. The data demonstrate that fibronectin in HeLa tumour cells is preferentially associated with the nuclear matrix.
Intracellular diaphragmed fenestrae in cultured capillary endothelial cells
Authors: Montesano Roberto , Orci Lelio
Published: April 2021

The endothelium of visceral capillaries is characterized by the occurrence of numerous fenestrae, which are usually bridged by a thin, singlelayered diaphragm. Both in vivo and in vitro, diaphragmed fenestrae perforate the endothelial cell cytoplasm in the most attenuated regions of the cell. We report here that in capillary endothelial cells grown under experimental conditions promoting the development of intracellular lumina (for example, suspension within a threedimensional collagen matrix), diaphragmed fenestrae can form in a unique, previously undescribed intracellular location - that is, within thin cytoplasmic septa separating contiguous luminal compartments.
Flexible-substratum technique for viewing cells from the side: Some in vivo properties of primary (9+0) cilia in cultured kidney epithelia
Authors: Roth Karen E. , Rieder Conly L. , Bowser Samuel S.
Published: April 2021

Cells cultured on thin plastic (e.g. Formvar, Teflon, polycarbonate) membranes can be clearly imaged from the side in vivo by video microscopy. We have used this flexible-substratum technique to examine the behaviour and properties of primary cilia in confluent cultures of the kidney epithelial cell lines PtK1 PtK2, LLC-PK1, MDCK and BSC-40. In these cells primary cilia appear as rigid rods, up to 55 um long, which project at various angles from the dorsal cell surface. The length distribution of primary cilia in confluent cultures is a distinct characteristic of each established kidney cell line examined, with LLC-PKi exhibiting three distinct length populations. Primary cilia of kidney cell lines bend passively in response to flow but do not display propagated bending or vortical motions. Up to 26% of the cilia in the cell types examined possess one or more conspicuous swellings along the ciliary shaft. Treatment with 0·05% trypsin, which is sufficient to cause cell rounding, does not induce the resorption or shedding of the cilium. These direct observations demonstrate that kidney epithelial-cell primary cilia are non-motile and longer than previously thought, and suggest that their length represents a phenotypic marker for each cell line.
Tight-junctional strands first appear in regions where three cells meet in differentiating olfactory epithelium: A freeze-fracture study
Authors: Menco Bert Ph. M.
Published: April 2021

Tight junctions of the olfactory epithelium of rat embryos were studied at the 14th day of gestation and during their subsequent development. Two different epithelial morphologies could be distinguished at the 14th gestational day. In one group of embryos the epithelial surface appeared undifferentiated, with tight-junctional strands found exclusively in regions where three cells met. The main orientation of these strands is in a direction parallel to the longitudinal orientation of the epithelial cells. These junctions resemble tight junctions that interconnect three cells, i.e. tricellular tight junctions, in that respect. However, unlike these the junctions mainly have single strands of particles, whereas tricellular junctions usually consist of paired strands of particles. Tight-junctional strands were completely absent in areas where two cells met. These areas, i.e. those of incipient bicellular tight junctions, had gap-junction-like aggregates of intra-membranous particles. Another group of 14-day-old embryos displayed a differentiating olfactory epithelial surface with bicellular as well as tricellular tight-junctional strands. The latter ones were paired. Here too the tight-junctional belts displayed some gap-junction-like aggregates of particles, but there were considerably fewer of these than earlier. As one or the other tight-junctional appearance was always seen in a single freeze-fracture replica, it is reasonable to assume that the two tight-junctional appearances reflect a sequential pattern of differentiation peculiar to the whole surface of the olfactory epithelium, i.e. to surfaces of receptor cells as well as to surfaces of supporting cells. It would appear that, at the onset of olfactory epithelial differentiation, tight junctions first interconnect cells in regions where three cells meet and that tricellular strand formation precedes the formation of bicellular strands. When strands were present at the 14th day of embryonic development, their numbers were lower than those found later. However, strand packing, expressed as the density per micrometre of strands parallel to the epithelial surface, increased beginning at the 16th day of embryonic development.
Induction of fibroblast spreading by Mn2+: A possible role for unusual binding sites for divalent cations in receptors for proteins containing arg-gly-asp
Authors: Edwards J. G. , Hameed H. , Campbell G.
Published: April 2021

Mn2+ at low (μM) concentrations fulfils the divalent cation requirement for spreading of BHK21 cells on fibronectin. At much higher concentrations, Mn2+ (and to a small extent also Mg2+) induces spreading on haemoglobin, not normally an adhesive protein. Since high Mn2+ also induces spreading of BHK variants unresponsive to exogenous fibronectin, it is unlikely to be acting as a cofactor for secreted cellular fibronectin or by stimulating its secretion. High Ca2+, but not Mg2+, inhibits the induction of spreading by Mn2+ on haemoglobin.

Pre-treatment of cells with high concentrations of trypsin decreases the rate of spreading induced by Mg2+ on fibronectin, and by Mn2+ on haemoglobin, to similar extents.

High and low Mn2+ could induce spreading, either by different mechanisms or through a common pathway. In the second case, at both concentrations, Mn2+ could act by binding to Ca2+/Mg2+ sites in one or more receptors for adhesion proteins. This would require binding of Mn2+ or Mg2+ to these sites to activate the receptors in the absence of adhesion proteins, and the effect of such proteins to be to increase the affinity of the sites for metal ions.

The sites in question may be formed by sequences homologous to those found in the extracellular domains of the vitronectin receptor and platelet membrane glycoprotein lib. Although very similar to the Ca2+-binding loop in the EF hand of calmodulin, these sequences more closely resemble bacterial galactose-binding protein in lacking one of the conserved co-ordinating sidechains.
Membrane-cytoskeleton interactions in the flagellum: A 240000 mr surface-exposed glycoprotein is tightly associated with the axoneme in chlamydomonas moewusii
Authors: Reinhart Frederick D. , Bloodgood Robert A.
Published: April 2021

The flagellar surface of Chlamydomonas moewusii is a dynamic structure involved in several adhesive and motile events. In this report, we describe for the first time the flagellar membrane components of vegetative C. moewusii. A glycoprotein (or pair of glycoproteins) with an apparent molecular weight of 240 ×103 is the dominant flagellar protein (other than the tubulins) in this species of Chlamydomonas. Both a rabbit polyclonal antibody (designated P-19) and the lectin concanavalin A recognize this 240K (K= 103Mr) glycoprotein on nitrocellulose transblots of flagellar proteins. Fluorescence microscopic studies using these same two probes suggest that the 240K glycoprotein is exposed at the flagellar surface. Direct evidence that the 240K glycoprotein is exposed at the flagellar surface is provided by vectorial labelling with a N-hydroxysuccinamide-derivitized biotin reagent (NHS-LC-biotin). Nonionic detergent extraction of isolated flagella fails to solubilize most of the 240K glycoprotein, although it completely removes the flagellar membranes as demonstrated by transmission electron microscopy. Furthermore, immunofluorescence microscopy of isolated axonemes demonstrates that both P-19-defined epitopes and surface-biotinylated proteins continue to be associated with the axoneme structure after detergent treatment. These observations demonstrate that the 240K flagellar protein is a glycoprotein that is both exposed at the flagellar surface and tightly coupled to the underlying cytoskeleton (axoneme). Because of its cell surface orientation and axonemal linkage, it is likely that the 240K glycoprotein plays an important role in the adhesive and/or motile phenomena exhibited by the C. moewusii flagellar surface.
Cell biology of the stigma ofBrassica campestrisin relation to Co2 effects on self-pollination
Authors: O’neill Patricia , Singh Mohan B. , Knox R. B.
Published: April 2021

The effect of carbon dioxide in blocking the sporophytic self-incompatibility system in Brassica campestris occurs within the first 2h of pollination at the pollen-stigma interface. Percentage germination of self pollen on the stigma was found to be similar in air and in the presence of 5% CO2. The CO2 effect therefore must occur after pollen germination, modifying the interaction between pollen tubes and stigma cells. Lectin binding studies showed the presence of fucosyl but not galactosyl residues on the stigma surface. Gel electrophoresis of plant extracts showed that stigma esterase activity is marked in comparison to other plant tissue. This activity is shown histo-chemically to be localized on the stigma cell surface and in the nucleus. Carbonic anhydrase has been detected on the stigma surface by two different histochemical methods and its possible relationship to the CO2 effect is discussed.
T-cell-mediated cytolysis: Analysis of killer and target cell deformability and deformation during conjugate formation
Authors: Foa Colette , Mège Jean-Louis , Capo Christian , Benoliel Anne-Marie , Galindo Jean-Rémy , Bongrand Pierre
Published: April 2021

T-cell-mediated cytolysis is initiated by the formation of strong adhesions between killer and target cells. The present work was aimed at determining whether T lymphocytes might exert some mechanical stress on targets during the binding process.

Target S194 myeloma cells were thus conjugated to cytotoxic T lymphocytes (CTLs) raised by mixed lymphocyte culture or a cloned lymphoid line that was no longer cytolytic (TG2OUA2). After incubation periods of various lengths, conjugates were processed for electron microscopy and micrographs were digitized for computerized analysis: the cell surface curvature (at the micrometre level) and rugosity (at the submicrometre level) were quantified in free and adhesion-involved regions. Also, the size of cell interaction areas and the distribution of intermembrane distances were measured. Finally, TG2OUA2 and target cells were aspirated into glass micropipettes with calibrated pressure in order to assay their resistance to deformation by mechanical forces. The following conclusions were suggested.

(1) Formation of extensive contact zones (with a linear size of several micrometres) with tight intermembrane adhesion (more than 30% of the membrane contours in adhesive zones were separated by an apparent distance lower than 500 Å) was essentially completed within less than one minute.

(2) CTLs or TG2OUA2 cells were more villous than their targets, and they seemed to deform in adhesive zones in order to adapt to the target contour, rather than imposing some deformation on the target. This may be a general feature of actively adherent cells.

(3) Some CTLs, but no TG2OUA2 cells, exhibited conspicuous protrusions extending towards the bound target. In this case, the target cell but not the CTL displayed markedly increased roughness in the adhesion area.

(4) TG2OUA2 cells were more resistant to mechanical deformation than S194 target cells, in accordance with previous reports suggesting that the membrane of CTLs was more resistant than that of target tumour cells.

Hence, CTLs might transiently impose mechanical stress on the target membrane during the course of lethal-hit delivery.
A biochemical investigation into the inhibitory effects of glucosamine and N-acetylglucosamine on the aggregation in vitro of embryonic chick muscle cells
Authors: Lloyd C. W. , Kemp R. B.
Published: April 2021

The effect of glucosamine and N-acetylglucosamine on aggregation and energy metabolism was investigated over an 8-h period in cells dissociated by 0·25 % (w/v) trypsin from the skeletal muscle of 9-day-old chick embryos. At 8 h, 0·023 M glucosamine and N-acetylglucosamine inhibited the aggregation of cells suspended in Eagle’s minimal essential medium by 18·9 % and 16·4% respectively, as judged on a basis of aggregate size.

Glucosamine and N-acetylglucosamine reduced the cellular ATP level by a mean of 36 % and 27 % respectively; values reflected in the 32% and 19% loss of total adenine nucleotides caused by these sugars. The adenine nucleotide balance was also changed from a mean control ATP/AMP ratio of I3·7 to 8·75 by N-acetylglucosamine and to 8·0 by glucosamine.

Intracellular lactate/pyruvate ratios were similarly disrupted in cells incubated with 0·023 M hexosamine. Although the hourly values fluctuated, it was seen that the amount of lactic acid relative to pyruvic acid, considered as an average for the 8-h period, was raised from 6:1 in controls to 8:1 in N-acetylglucosamine-treated and to 10:1 in glucosamine-treated cell preparations. Compared to controls at 8 h, glucosamine enhanced the production of lactate into the suspension medium by 99%. The N-acetyl analogue caused cells to produce more lactic acid than did controls for 4–5 h only, for by 8 h 25 % less of this metabolite was assayed in the culture medium.

The incorporation of D-[U-14C]glucose into glycogen paralleled the results of extracellular lactic acid assays. N-acetylglucosamine inhibited the incorporation by 30 % at 4 h, although by 6 h, and for the remainder of the experimental period, there was more 14C-labelled glycogen in these cells than in controls. By contrast, glucosamine inhibited the incorporation of radioactive glucose into glycogen by 42% at 4 h and, unlike N-acetylglucosamine, consistently thereafter.

Glucosamine also enhanced cellular oxygen uptake throughout the experimental period, to the extent of 59 % at 8 h. The oxygen uptake of N-acetylglucosamine-treated cells was similar to controls until about the 5th hour, when there was a subsequent inhibition which had accumulated to 13 % by the end of the experiment. The release of 14COs by cells was inhibited by glucosamine. This hexosamine depressed production by 19% at 12 h whereas N-acetylglucosamine inhibited this evolution by 9 % at this time.

The metabolic effects of these hexosamines on chick muscle cells in vitro are mainly attributed to a central alteration of the adenine nucleotide balance although certain other documented effects of glucosamine are considered to be involved. An inhibition of cell aggregation by glucosamine and N-acetylglucosamine is discussed in terms of a depressed cellular metabolic economy.
Developmental changes occurring in isolated intact etioplasts
Authors: Wellburn Florence A. M. , Wellburn A. R.
Published: April 2021

When dark-grown plants are illuminated, the characteristic morphology of the etioplast changes. The tubules of the paracrystalline prolamellar body disperse as the membranes reorganize and ‘grow out’ as ‘perforated’ thylakoids. Similar changes are expected during the greening of the isolated etioplasts of Avena. Under certain preparative conditions the morphological changes vary from those previously reported for intact tissue.

Following low illumination at a low temperature the process of outgrowth is slowed down, resulting in the outgrowth of regularly spaced tubules from the prolamellar body into the stroma. These tubules appear to develop slowly into normal thylakoids by a process of tubule fusion to form perforated thylakoids. A diagram is included to show how this is believed to take place. The outgrowing tubules have an outer diameter of 25–28 nm with a lumen of 7·0–7·6 nm diameter.

Two types of protein body are observed, the ‘stromacentre ’ which is a regular feature of A vena plastids and a ‘crystallite’ which is only observed under certain conditions. The ‘stromacentre’ is a fibrillar spherulite with fibrils (diameter 9 nm) in bundles up to 200 nm long which in turn are aggregated to form the characteristic spherulite of the stromacentre. The crystallite is composed of fibrils 7·5–8·5 nm in diameter lying parallel to each other with a regular spacing of 12·5 nm. The crystallite appears to develop when the etioplast is damaged and has started to vesiculate.

Particles which are larger than either plastid or cytoplasmic ribosomes have also been observed. These vary in diameter from 18 to 24·5 nm and are arranged in rows adjacent to the thylakoids.
Particles within membranes: a freeze-etch view
Authors: Flower N. E.
Published: April 2021

Particles are commonly present on the membrane faces revealed by freeze-etching. The number, distribution and size of these particles vary considerably both between different membranes and, in many cases, between the 2 fracture faces found in individual membranes. Many of the larger particles appear to be too large to fit totally within smooth-surfaced membranes, so raising the question of how particles, especially the larger ones, are contained within membranes. This could be accomplished by a local reorganization of the membrane’s internal structure such that small particles would be totally enclosed within smooth-surfaced membranes, while large particles would protrude from the membrane surface. Alternatively, all sizes of particles could be contained within membranes by a bulging of the 2 component lamellae such that protuberances, having a larger diameter than the underlying particles, would arise on the membrane surface.

Evidence is presented to show that in the case of specialized particles, which are located in rows around the base of flagella in the mollusc Cominella maculosa, protuberances are present on the membrane surface. However, it is possible that particles could be accommodated within membranes from other tissues by a different mechanism, and only further work will decide whether or not the present findings can be applied to these other membrane surfaces.
Mitosis in the fission yeast Schizosaccharomyces pombe: a comparative study with light and electron microscopy
Authors: McCully E. Kathleen , Robinow C. F.
Published: April 2021

Mitosis in Schisosaccharomyces pombe has been followed in living cells by phase-contrast microscopy and studied in fixed and suitably stained preparations by light microscopy. Successful preservation of nuclear fine structure in this yeast, not previously achieved, has allowed us to confirm and extend the observations made with light microscopy.

Without first arranging themselves on a metaphase plate, mitotic chromosomes become grouped in 2 clusters radiating, finger-like, from 2 points of attachment at opposite poles of an elongating nucleus. At these 2 sites electron microscopy reveals the presence of disk-shaped electron-dense organelles which we have called kinetochore equivalents (KCE). At mitosis the KCEs are connected across the nucleus by a narrow bundle of parallel microtubules which we refer to as the spindle.

Integration of our observations has led us to propose that at mitosis the separation of the KCEs and their attached chromosomes is initiated by a differential expansion of the nuclear envelope restricted to the region between recently divided KCEs and that expansion of the nuclear envelope later becomes general, resulting in a marked elongation of the nucleus. Displacement of the nuclear contents to the ends of the elongated nucleus gives it the shape of a dumbbell.

The elongation of the microtubule bundle keeps in step with the elongation of the nucleus but does not appear to be the cause of it. It may have the function of keeping the separated KCEs rigidly apart.

During mitosis the nucleolus persists and stretches out within the unbroken envelope of the nucleus as it elongates. Towards the end of division equal amounts of nucleolar material are found in the rounded ends of the dumbbell-shaped nucleus.

The break up of the dumbbell shape into daughter nuclei seems to involve the breaking of its tenuous middle part and a pivoting of its 2 ends in opposite directions.

In the course of our work on mitosis we have become aware of features in the cytoplasm of growing S. pombe cells which are described here for the first time. The cells invariably contain several prominent vacuoles containing an extremely electron-dense material which stains metachromatically with toluidine blue and may be polyphosphate. The mitochondria are of special interest for 2 reasons. First, because they have unique mesosome-like membrane invaginations and secondly, because a mitochondrion is regularly associated with the single KCE by the side of the interphase nucleus, as well as with each one of the 2 KCEs that occupy opposite ends of the intranuclear spindle during mitosis.
Longitudinal growth of striated muscle fibres
Authors: Williams Pamela E. , Goldspink G.
Published: April 2021

The direct determination, by counting, of the number of sarcomeres in series along the length of single teased muscle fibres taken from mice of different ages showed that the increase in fibre length during normal growth is accompanied by a large increase in sarcomere number The greatest increase occurs during the 3 weeks after birth. By counting the number of muscle fibre nuclei in single teased fibres it was shown that the number of nuclei per fibre increases with age, and that this increase continues beyond the point at which the fibres have ceased to grow in length. It is suggested that post-natal increase in nuclear number is associated with both increase in length and increase in girth of the muscle fibres.

By injecting tritiated adenosine into young mice, an attempt was made to label newly formed actin filaments and ribosomes and thus to determine the region where new sarcomeres are laid down during increase in fibre length. Using autoradiography and scintillation counting it was shown that the radioactive label was incorporated more into the ends than into the middle regions of the muscles. The implication of these findings is that new sarcomeres are added on serially at the ends of the muscle fibres. An investigation, at the ultrastructural level, of muscle fibres taken from foetal and newborn mice indicates that the end of the fibre is a region of active development. This area is characterized by numerous ribosome formations and by myofilaments which are not organized into myofibrils. Cells which can occasionally be seen fusing with the end regions of young muscle fibres indicate a possible way in which nuclei are added to the growing fibre.

Immobilization experiments have shown that it is possible to alter both the rate and the extent of the post-natal increase in sarcomere number. Immobilization of limb joints, by means of plaster casts, so that the muscle is held in either the extended or the shortened position results in the number of sarcomeres along the fibres falling far short of that in the fibres from control muscles. Removal of the restriction is followed by a rapid increase in the number of sarcomeres in series and a return to the normal level within a period of about 4 weeks. These experiments indicate that for normal growth to occur, it is important for a muscle to be able to contract isotonically.
The nucleoskeleton: Artefact, passive framework or active site?
Authors: Cook P. R.
Published: April 2021

“As seen in the living cell the nucleus most commonly appears as a clear, rounded, sac-like body bounded by a delicate membrane and often showing no visible structure save for the presence within it of one or more smaller rounded bodies, the nucleoli. After coagulation by fixing agents, the nucleus offers a much more complicated appearance containing… a net-like framework….”

“Very often no trace of the framework is seen before coagulation sets in; and this has led to a skeptical attitude concerning it on the part of some observers.”
The nuclear matrix: Structure and composition
Authors: Verheijen Ron , Venrooij Walther Van , Ramaekers Frans
Published: April 2021

The term nuclear matrix was first introduced by Berezney & Coffey (1974) to denote a highly structured residual framework obtained from rat liver nuclei by sequential salt extractions, detergent and nuclease treatments. The isolated three-dimensional structure consisted almost entirely of protein. Subsequent studies showed that when protease inhibitors were included in all isolation steps and ribonuclease (RNase) was omitted, the isolated nuclear matrix contained RNA as the second most abundant component (Herman et a/. 1978; Miller et. al. 1978b ; Shaper et al. 1979; Berezney, 1980; van Eekelen & van Venrooij, 1981; Mariman et al. 1982a; Fey et al. 1986a,b). In this review the term nuclear matrix is defined as the biochemical entity that can be isolated after sequential extraction of cells with non-ionic detergents, nucleases and high-salt buffers (Shaper et al. 1979). With respect to the nucleases, it should be stated here that several authors use only deoxyribonucleases (DNases), while others use DNases in combination with RNases. Nuclear matrices have been isolated from a wide variety of mammalian and non-mammalian cell types (reviewed by Shaper et al. 1979; Barrack & Coffey, 1982).
The initial stages in the action of an insecticidal d-endotoxin of Bacillus thuringiensis var. Israelensis on the epithelial cells of the malpighian tubules of the insect, Rhodnius prolixus
Authors: Maddrell S. H. P. , Lane N. J. , Harrison J. B. , Overton J. A. , Moreton R. B.
Published: April 2021

The effects of the 27×103Mr insecticidal δ-endotoxin from Bacillus thuringiensis var. israelensis have been studied using, as a model system, isolated insect Malpighian tubules. At all concentrations of the toxin higher than 1 μg ml−1 (4×10−8 moll−1) applied to the outer surface of the tubules, fluid secretion failed within about 30 min. Except at very high concentrations, where failure always takes at least 30 s, there was an inverse relationship between the concentration of toxin and the time of failure of toxin-treated tubules. During exposure to toxin, the tubules were initially unaffected for a relatively long period and then rapid failure occurred. If the tubules were removed into toxin-free saline just before failure would have occurred, fluid secretion remained normal for at least 2h, but on return to the original toxin-containing saline failure was almost immediate. The toxin was found not to bind to the basement membrane.

Ultrastructural changes became evident as tubule failure occurred. These initially involvedmodifications to the basal side of the cells, but later also to the luminal microvilli. Intercellular junctions became disassociated and cytoplasmic vacuolization occurred. The population of intra-membranous particles in the basal membranes became reduced with time.

Our findings suggest the following hypothesis for the initial stages in the interaction of the toxin with the tubules. Toxin molecules attach to the accessible cell membranes progressively and irreversibly. They do not readily associate by diffusing laterally in the membrane, so that toxic effects develop only when sufficiently large numbers of them attach close together. The molecules may then associate in some way as a complex, perhaps forming a pore in the membrane. Relatively few such pores lead rapidly to cell failure and death.
Release of lysosomal enzymes in Tetrahymena: A Ca2+-Dependent secretory process
Authors: Tiedtke A. , Rasmussen L. , Florin-Christensen J. , Florin-Christensen M.
Published: April 2021

The ciliate Tetrahymena thermophila releases lysosomal enzymes into nutrient and starvation media. We show here that this process occurs selectively, i.e. without leakage of cytoplasmic components, as indicated by lack of release of isocitrate dehydrogenase, a cytosolic enzyme with high activity in Tetrahymena. The role of intracellular Ca2+ in the process was also investigated. The Ca2+ ionophore A23187 has strong stimulatory effects on this release. Ionophore stimulation is maximal in the presence of extracellular Caz+ but can occur also in its absence. Quin 2 fluorescence measurements indicate that intracellular Ca2+ increases in both cases. Mg2+ completely prevents the stimulatory effects of A23187. lonomycin, another Ca2+ ionophore, also stimulates lysosomal enzyme release with a maximal response in the presence of extracellular Ca2+. Measurements of extracellular isocitrate dehydrogenase showed that ionophore-stimulated lysosomal enzyme release can take place without leakage of cytoplasmic components. The observations that divalent cation ionophores stimulate selective lysosomal enzyme release and that this effect is strongest in the presence of external Caz+ indicate that this cation plays a crucial role in the control of this process in Tetrahymena. Together with other observations they support the view that a subpopulation of Tetrahymena lysosomes has properties like those of secretory vesicles.
Conjugation of Paramecium tetraurelia cells: selective wheat germ agglutinin binding, reversible local trichocyst detachment and secretory function repair*
Authors: Pape Regina , Haacke-Bell Birgit , Lüthe Norbert , Plattner Helmut
Published: April 2021

Conjugation of ciliated protozoa involves precisely defined stages. In Paramecium there occur: (1) the formation of ciliary contacts; (2) local loss of cilia and formation of narrow contacts between anterioventral somatic cell membranes; followed by (3) formation of small cytoplasmic bridges (which allow for the exchange of molecular components only); before (4) their enlargement enables the cells to exchange micronuclei. This work concentrates on stages (2) and (3) in P. tetraurelia cells. We analysed lectin binding during conjugation, using mainly secretory mutants nd6 and tl (occasionally nd9-28°C and 7S (wildtype) cells); (tl is a trichocyst-free mutant, nd strains are non-discharge mutations). Our most essential findings are: already early contact sites (stage (2)) can be labelled with wheat germ agglutinin-fluorescein isothiocyanate; only early stages (2) can be reversed with an excess of WGA; no selective labelling was found with concanavalin A-fluorescein isothiocyanate (commonly observed with other ciliate species); WGA binding sites most probably involve GlcNAc residues. As known before, trichocysts disappear from contact sites in stages (2)/(3). We then analysed this process in more detail. Trichocysts are locally detached from the cell membrane (without transfer to the other conjugant) and re-inserted after conjugation. From our data obtained with nd6μl con jugants one can conclude that trichocysts (in particular secretory lectins or glycoproteins contained in them) cannot play a regulatory role in the conjugation process, since conjugation precedes secretory function repair. We also present evidence for a secretory function repair in nd mutations by tl cells (though tl cells are devoid of any recognizable ‘exocytotic apparatus’). nd6 cells are more difficult to ‘cure’ than nd9-28°C. The ‘curing effect’ seems to emanate from the conjugation zone. This might indicate the occurrence of diffusible ‘curing factors’ exchanged mainly in stage (3). Finally, we show that secretory function repair in nd cells induces the assembly of ‘fusion rosettes’, i.e. the formation of the wild-type ultrastructure of the trichocyst release sites, to about the same extent as nd cells are functionally repaired (visualized by the release of trichocysts in response to two different trigger agents).
Ubiquitin gene expression in Dictyostelium is induced by heat and cold shock, cadmium, and inhibitors of protein synthesis
Authors: Müller-Taubenberger A. , Hagmann J. , Noegel A. , Gerisch G.
Published: April 2021

Ubiquitin is a highly conserved, multifunctional protein, which is implicated in the heat-shock response of eukaryotes. The differential expression of the multiple ubiquitin genes in Dictyostelium discoideum was investigated under various stress conditions. Growing D. discoideum cells express four major ubiquitin transcripts of sizes varying from 0·6 to 1·9 kb. Upon heat shock three additional ubiquitin mRNAs of 0·9, 1·2 and 1·4 kb accumulate within 30 min. The same three transcripts are expressed in response to cold shock or cadmium treatment. Inhibition of protein synthesis by cycloheximide leads to a particularly strong accumulation of the larger ubiquitin transcripts, which code for polyubiquitins. Possible mechanisms regulating the expression of ubiquitin transcripts upon heat shock and other stresses are discussed.
Formation of plasma membrane domains in rat hepatocytes and hepatoma cell lines in culture
Authors: Maurice M. , Rogier E. , Cassio D. , Feldmann G.
Published: April 2021

In vivo, proteins of the hepatocyte plasma membrane are asymmetrically distributed, making it possible to distinguish a sinusoidal, a lateral and a canalicular domain. The conditions that determine hepatocyte plasma membrane polarity have been investigated in vitro, using three monoclonal antibodies directed against integral membrane proteins, which were characteristic of each domain. The localization of the three antigens was studied by immunolabelling of hepatocytes isolated from adult rat liver, primary monolayer cultures and rat hepatoma cell lines. When hepatocytes were isolated, the three antigens spread over the entire cell surface. The lateral antigen redistributed at lateral sites as soon as cell-cell contacts were established, 4h after the beginning of primary culture. The sinusoidal and canalicular antigens became asymmetrically distributed after 48 h of primary culture, after the formation of bile canaliculus-like structures. In most of the hepatoma lines studied, the three antigens were expressed, except that the canalicular antigen was fully expressed in differentiated clones only. The lateral antigen was always distributed on thecontiguous membranes of clustered hepatoma cells, whereas the sinusoidal and canalicular antigens were localized on the entire plasma membrane. However, in a few cells of some clones in which bile canaliculus-like structures were observed, the canalicular membranes were strongly labelled only with the canalicular antibody. In the absence of bile canalicular formations, in both primary culture and cell lines, the canalicular antigen and, to a lesser extent, the sinusoidal antigen accumulated in the Golgi apparatus, suggesting that their transport to the cell surface was altered in the absence of a bile pole. These results show that in hepatic cells, polarization of the plasma membrane is determined by: (1) the existence of cell-cell contacts, which is correlated with the domain-specific localization of the lateral antigen; and (2) the formation of bile canaliculi, which would trigger the development of an asymmetrical distribution of the sinusoidal and canalicular antigens.
Effect of enzymic (collagenase) harvesting on the intracellular Na+/k+ Ratio of swiss/3t3 cells as revealed by x-ray microanalysis
Authors: Szállási Z. , Szállási Á. , Boján F. , Zs.-Nagy I.
Published: April 2021

Swiss/3T3 cell cultures were harvested with 0·05% collagenase and after centrifugation the pellet was prepared by the freeze-fracture/freeze-drying (FFFD) method for bulk-specimen X-ray microanalysis. Time-dependent variations in the intracellular monovalent elemental concentrations (Na+, K+ and Cl-) as well as of the Na+/K+ ratio were followed for 120 min subsequent to harvesting. The quantitative measurements revealed a very considerable increase in the intracellular Na+ and Cl- accompanied by a decrease in the K+ concentration as soon as 5 min after harvesting. The Na+/K+ ratio had increased by this time to about 1-5 on average. These changes indicate a sustained depolarization of the cell membrane. During the first 60 min this depolarization tended to normalize as demonstrated by an exponential decrease in the intracellular Na+ and Cl- and an increase in the K+ content involving a decrease in the Na+/K+ ratio. The total intracellular monovalent ion concentration remained almost constant during this post-harvesting period. These results suggest that harvesting represents a serious depolarizing stimulus to the cells, the consequences of which are restored only after 1·2 h. These alterations should be taken into consideration during various experimental designs when using anchorage-dependent cell cultures.
The role of transport signals and retention signals in constitutive export from animal cells
Authors: Cutler D. F.
Published: April 2021

The question: ‘Do proteins using the constitutive secretory pathway need signals to be retained or signals to be transported?’, has been a fertile source of speculation over the last decade or so. This is at least partly because little direct evidence bearing on the problem has been available until recently. The speculators have fallen into two groups, whose positions can be oversimplified as follows: (1) The secretory pathway is like a conveyer-belt to which a protein gains access through possession of a signal that couples translation to translocation into the endoplasmic reticulum (the signal sequence). Once in the ER, the protein will be carried passively to the cell surface. Membrane proteins will end up in the plasma membrane, soluble proteins will be secreted. To have an intracellular final destination, a signal of some kind that retains the protein within the cell must be present on the transported protein (a ‘retention signal’). (2) The secretory pathway is made up of a number of discrete steps. To move from one organelle to the next along this pathway, a positive signal of some kind must be present on the protein. This signal interacts with a ‘receptor’ of some kind, which mediates its transport at each step (a ‘transport signal’).