bluuebunny/crossref_metadata_2025 huggingface dataset:
Role of calcium in triggering rapid ultrastructural damage in muscle: a study with chemically skinned fibres
Authors: Duncan C. J.
Published: April 2021

Agents (A23187, caffeine) believed to raise [Ca]i in vertebrate cardiac and skeletal muscles cause rapid and characteristic subcellular damage in vitro and in vivo. By using saponin-skinned amphibian pectoris cutaneous muscle and Ca-EGTA-buffered solutions it is shown that low [Ca] consistently triggers the same rapid (2–20 min), ultrastructural damage.

Electron micrographs reveal a close similarity between the damaged intact and skinned preparations, namely loss of myofilament organization, specific Z-line damage, dissolution and hypercontraction bands, characteristic mitochondrial swelling and division. Where both actin and myosin filaments were lost, an underlying cytoskeletal network frequently remained, still attached to the Z-line framework.

Ca was effective in skinned preparations from 5×10−7M to 8×10−6M, within the concentration range experienced by a contracting muscle. Damage was [Ca]- and time-dependent and it is suggested that it is probably the active movement of Ca ions across key membrane sites that is critical in triggering damage of the myofilament apparatus. Strontium can substitute for Ca at higher concentrations. The action of saponin suggests that the chemically skinned cell is partially activated. Ca-triggering can be bypassed experimentally by membrane-active agents or by sulphydryl agents.

Ruthenium Red and trifluoperazine indirectly cause damage in the intact cell by raising [Ca] i. Studies with saponin-skinned cells and protease inhibitors show that changes in pHi, loss of ATP, Ca-activated neutral protease, or release of lysosomal enzymes (cathepsins B, D, L or H), are not involved in characteristic rapid myofilament damage.
Regulation of G
2 by cell size contributes to maintaining cell size variability within certain limits in higher plants
Authors: Navarrete Matilde H. , Cuadrado Antonio , Escalera Mariano , Cánovas Josél
Published: April 2021

The variability of (1) surface area projection (size) at which cells terminate DNA replication, (2) the area at which they initiate mitosis, (3) the area at which they divide, (4) the duration of G2, and (5) the duration of G2 plus mitosis (in fact, prophase + metaphase + anaphase) has been estimated in steady-state cell populations of Allium cepa root meristems. The coefficient of variation of cell area at termination of DNA synthesis was found to be 14% while the coefficient of variation of cell area at mitosis initiation was 13%. As there is also a substantial variability of G2 (the coefficient of variation was estimated to be 38%), the combination of these data indicates that cell size regulation of G2 contributes to maintaining cell size variability (and therefore DNA concentration) within certain limits. Mitosis also varies but less than G2 (the coefficient of variation of G2 + mitosis was found to be 31%). As the coefficient of variation of cell area at division (14%) is hardly larger than the coefficient of variation of cell area at initiation of mitosis, it can be suggested that coordination between cell size and mitosis duration helps to avoid a significant increase in the variability of cell size at the end of the division cycle.
Inhibition of cell adhesion by a synthetic polymer adsorbed to glass shown under defined hydrodynamic stress
Authors: Owens Norman F. , Gingell David , Rutter Paul R.
Published: April 2021

A co-polymer with hydrophobic and hydrophilic segments was allowed to adsorb from aqueous solution onto glass previously made hydrophobic by derivatization with octadecyl dimethylchlorosilane. The polymer is thought to adsorb via its hydrophobic segments, leaving the hydrophilic segments free to extend into the water. After allowing cells to settle on the treated surface, the shear stress at the chamber wall required to remove red blood cells, Dictyosteliiun discoideum amoebae and Escherichia coli was determined in a calibrated laminar flow chamber. On octadecyl glass a shear stress of 2·3 N m−2 evicts 50% of adherent red cells and E. coli. No D. discoideum amoebae could be removed at 5Nm−2. In striking contrast, the lowest experimentally obtainable shear stress of 0·03 N m−2 removes 97·0–99·5% of cells of all three types from the polymer-treated surface, even after a cell residence time of 1 h without flow in the absence of free polymer. The minimum shear stress of 0·03 Nm−2 corresponds to only ≈20 times the force of gravity on a red cell. The mechanism of action of the polymer and the implications of the results are discussed.
Spike-shaped oscillations in the absence of measurable changes in cyclic AMP concentration in a mutant of Dictyostelium discoideum*
Authors: Wurster Bernd , Mohn Renate
Published: April 2021

Periodic activities of Dictyostelium discoideum cells involve two types of oscillations, spikeshaped and sinusoidal. Spike-shaped oscillations are accompanied by the periodic synthesis and release of cyclic AMP, and cyclic AMP-activated cyclic AMP synthesis is believed to control these oscillations. Experiments described here call into question the importance of cyclic AMP in spikeshaped oscillations. Cell suspensions of strain agip43, an aggregation-deficient mutant of D. discoideum, displayed spike-shaped oscillations in light scattering with period lengths about 1-5 times larger than those of the parent strain. These oscillations were not accompanied by measurable oscillations of cyclic AMP and cyclic GMP. Applied cyclic AMP pulses elicited increases of two- to threefold in the cyclic AMP level and increases of seven-to ninefold in the cyclic GMP concentration. Cyclic AMP additions caused phase shifts in the oscillations of agip43 cells, suggesting that cyclic AMP receptors at the cell surface communicate with the oscillator. We interpret these results in terms of an oscillator not based on cyclic AMP. This oscillator should be coupled to the reaction system involving cyclic AMP synthesis and release. The latter can operate in an oscillatory manner in the parent strain Ax2 but not in mutant agip43.
Characterization of a heat shock-induced insoluble complex in the nuclei of cells
Authors: Littlewood T. D. , Hancock D. C. , Evan G. I.
Published: April 2021

The formation of an insoluble complex in isolated nuclei incubated at physiological temperature (37°C) is demonstrated. A similar complex is shown to form in the nuclei of intact cells subjected to temperatures that induce the classical heat-shock response. The formation of this complex occurs rapidly in response to hyperthermia and is induced by small increases in temperature both in vitro and in vivo. We have characterized the formation of the complex in isolated nuclei and the nuclei of intact cells. A small number of the subset of nuclear proteins involved in the complex have been identified. The significance of the loss of solubility of these proteins in the nucleus following hyperthermia is discussed.
Isolation of cytoskeletons from synchronized plant cells: the interphase microtubule array utilizes multiple tubulin isotypes
Authors: Hussey Patrick J. , Traas Jan A. , Gull Keith , Lloyd Clive W.
Published: April 2021

Biochemical analysis of the plant cytoskeleton has been hampered by an inability to isolate anything more than cytoskeletal fragments. Methods for isolating entire detergent-resistant cytoskeletons from carrot suspension cells are now reported. This enables the expression of tubulin isotypes to be studied in functional microtubular arrays, freed of the soluble pool by detergent extraction.

When osmotically cushioned with 0·4M-sorbitol, microtubule-stabilizing buffer and dimethylsulphoxide, carrot protoplasts can be extracted by mild detergent, without fragmenting. Cytoskeletons isolated by sucrose density centrifugation are shown by electron and fluorescence microscopy to contain a complex meshwork of three major fibrous sytems: F-actin, microtubules and bundles of 7 nm fibrils.

Plant cells can assemble tubulin into one of four microtubule arrays depending upon the phase of the cell cycle. Previous work had established that interphase cells contained multiple tubulin isotypes. However, the tubulins had been isolated by taxol assembly in vitro, which need not reflect patterns of usage by the interphase microtubule array in vivo. To address this problem, cells blocked in interphase were converted to cytoskeletons and their usage of tubulin isotypes determined by immunoblotting two-dimensional gels. This confirmed that all of the alpha and beta isotypes that can be identified on two-dimensional gels of carrot suspension cells are utilized by the interphase microtubule array.
intermediate filament formation after transfection with modified hamster vimentin and desmin genes
Authors: van den Heuvel Richard M. M. , van EYS Guillaume J. J. M. , Ramaekers Frans C. S. , Quax Wim J. , Vree Egberts Wilma T. M. , Schaart Gert , Cuypers H. Theo M. , Bloemendal Hans
Published: April 2021

Previously we cloned and characterized the hamster intermediate filament genes coding for vimentin and desmin. It was demonstrated that the cloned desmin gene was expressed after gene transfer and that the newly synthesized protein assembles into intermediate filaments. Here we present data on the transfection of modified vimentin and desmin genes onto simian virus 40-transformed hamster lens cells and HeLa cells. Modifications included: (1) removal of exons encoding the desmin COOH-terminal domain; (2) exchange of exons encoding the COOH-terminal domain of vimentin and desmin; and (3) deletion of part of exon I of desmin, coding for the NH2-terminal amino acids 4–148.

In transient transfection assays it was shown that the modifications in the COOH region had no detectable effects on the filament forming potential of the encoded proteins as demonstrated with desmin antibodies in the indirect immunofluorescence test. On the other hand, deletion of a considerable part of the first exon of the desmin gene results in a lack of bona fide intermediate filament formation. Immunoblotting with desmin antibodies of cell populations enriched for the transfected modified genes showed that the presence of the modified genes results in the synthesis of the corresponding proteins with the expected molecular weights. From our results we conclude that in vivo: (1) the presence of the COOH terminus is not essential for filament formation; (2) that an exchange of COOH-terminal parts of vimentin and desmin does not prevent assembly into intermediate filaments; and (3) that removal of the NH2 terminus of desmin affects intermediate filament formation.
Growth of endosymbiotic algae in the green hydra, Hydra Viridissima
Authors: Dunn Kenneth
Published: April 2021

Stable endosymbiosis depends upon balanced growth of the symbionts. In green hydra, coincident patterns of host and algal mitotic index suggest that coordinated reproduction provides for balanced growth. However, when hydra shrink during food shortage, the population of endosymbiotic algae in a green hydra must likewise decline in size. Thus far, no mechanism of reducing the size of the endosymbiont population has been described. Algal mitosis was found here to be stimulated by host feeding and clumped in its distribution among host cells, supporting the notion of some degree of control of algal mitosis exercised at the level of the host cell. However, comparisons of the rates of algal mitosis with the realized rates of algal population growth show that substantial numbers of algae disappear from hydra, in numbers in excess even of those necessary to accommodate host shrinkage. Only a small proportion of these lost algae was found to be expelled by hydra. Microscopic observations of the cells of macerated hydra show evidence of algal disintegration in nearly 50% of the digestive epithelial cells of regularly fed hydra. Coincidence of remnants of algal cells and food-derived materials within the same vacuoles suggests that algae are digested by host cells.
Development of macrociliary cells in Beroë I. Actin bundles and centriole migration
Authors: Tamm Signhild , Tamm Sidney L.
Published: April 2021

Differentiation of macrociliary cells on regener-ating lips of the ctenophore Beroë was studied by transmission electron microscopy. In this study of early development, we found that basal bodies for macrocilia arise by an acentriolar pathway near the nucleus and Golgi apparatus, in close association with plaques of dense fibrogranular bodies. Procentrioles are often aligned side-by-side in double layers with the cartwheel ends facing outward toward the surrounding plaques of dense granules. Newly formed basal bodies then dis-band from groups and develop a long striated rootlet at one end. At the same time, an array of microfilaments arises in the basal cytoplasm. The microfilaments are arranged in parallel strands oriented toward the cell surface. The basal body-rootlet units are transported to the apical surface in close association with the assembling actin filament bundle. Microfilaments run parallel to and alongside the striated rootlets, to which they often appear attached. Basal body–rootlet units migrate at the heads of trails of microfilaments, as if they are pushed upwards by elongation of their attached actin filaments. Near the apical surface the actin bundle curves and runs below the cell membrane. Newly arrived basal body–rootlets tilt upwards out of the microfilament bundle to contact the cell membrane and initiate ciliogen-esis. The basal bodies tilt parallel to the flat sides of the rootlets, and away from the direction in which the basal feet point. The actin bundle continues to enlarge during ciliogenesis. These results suggest that basal body migration may be driven by the directed assembly of attached actin filaments.
Heterogeneity and cell cycle analyses from time-lapse studies of human keratinocytes in vitro
Authors: Dover R. , Potten C. S.
Published: April 2021

We have analysed the behaviour of cultured epidermal Keratinocytes using time-lapse video recordings. We have found evidence for heterogeneity in the behaviour of the cells. Some lines underwent extensive self-renewal, thus expanding the population, while others produced daughter cells that migrated suprabasally and are presumed to have undergone terminal differentiation. We also present kinetic data on the cell cycle times, mitotic durations and post-mitoticresidence times. The latter is the time between a cell’s birth and eventual suprabasal migration. The data suggest that the ‘decision’ to migrate is in some way ‘programmed’ but that the actual migration is a stochastic process. Time-lapse analysis is a very powerful technique for lineage and cell kinetic analysis.
The site of ferricyanide photo-reduction in the lamellae of isolated spinach chloroplasts: a cytochemical study
Authors: Hall D. O. , Edge H. , Kalina M.
Published: April 2021

Ferricyanide was used as a Hill oxidant to localize the site of photoreduction in chloroplast lamellae. The ferrocyanide formed on illumination was complexed with copper ions to form insoluble, electron-dense precipitates of Cu ferrocyanide, which can be easily seen in unstained preparations in the electron microscope. Control experiments showed no precipitates in the dark, at zero time, in the absence of ferricyanide, or on addition of sodium ascorbate. Discrete precipitates of Cu ferrocyanide were seen on both stroma and grana lamellae. It is concluded that both stroma and grana lamellae have photosystem II activity necessary for reduction of ferricyanide.
Freeze-etch studies on the innervation of mesenteric arteries and vas deferens
Authors: Devine C. E. , Simpson F. O. , Bertaud W. S.
Published: April 2021

The innervation of mesenteric arteries and vas deferens of guinea-pig and vas deferens of mouse was examined by freeze-etching. Axons in bundles at large distances from the smooth muscle cells, were invested by Schwann cells and contained mainly neurotubules, while axons close to the smooth muscle cells had varicosities up to 1·6 μm in diameter and 20 μm long containing mainly small (approximately 50 nm) and large (approximately 100 nm) synaptic vesicles. Vascular axons differed from those in the vas deferens in that the former were at the medial adventitial border with an observed closest neuromuscular distance of approximately 200 nm and the latter were between smooth muscle cells at distances of 20–50 nm. Depressions of the axonal surface were seen and particles up to 15 nm were found on the axonal membrane.
The haemoglobins of healthy and anaemic Xenopus laevis
Authors: Maclean N. , Jurd R. D.
Published: April 2021

The haemoglobins of Xenopus laevis have been studied by carboxymethyl-cellulose column chromatography and by polyacrylamide gel disk electrophoresis. In Xenopus tadpoles, 2 haemoglobins are found (Xenopuj-HbF1 and Aer/opiiS-HbF2). Both haemoglobins persist throughout tadpole life: Aenopur-HbF1 is the main tadpole haemoglobin; Xenopus-HbF2 is present in rather smaller amounts. The proportion of Xenopus-HbF 1 to Xenopus-HbF2 increases as the tadpoles age. Almost all the tadpole haemoglobin disappears soon after metamorphosis, although traces persist throughout adult life. Two adult haemoglobins occur in Xenopus: they first appear during the later tadpole stages. Xenoput-HbA1 comprises the majority of the adult haemoglobin. Between one-twentieth and one-tenth of the adult haemoglobin consists of Xenopus- HbA2, which is not a polymer of Xenopus-HbA1.

Immunoglobulins respectively specific to pooled -Xenopus-HbF1 and Xenopus-HbF2, and to pooled Aeno/wr-HbA1 and Aenopui-HbA2 were raised. Samples of these antibodies were conjugated with fluorescein isothiocyanate.

Adult Xenopus were made anaemic by bleeding or by injection of phenylhydrazine. Fourteen days after the induction of anaemia it was found, using both chromatographic and electrophoretic techniques, that most anaemic toads had started to resynthesize Aenopur-HbF1. In some animals up to 11 % of the haemoglobin present was found to be Xenopus-FLbF1 and precipitin lines were obtained when the pooled haemoglobins from these animals were tested with a specific anti-Xenopnr-HbF immunoglobulin. During recovery from anaemia greatly enhanced amounts of Aenopur-HbA2 (up to 58 % of the total haemoglobin) were present in the blood. The amounts of tadpole haemoglobins present in such recovering toads were not noticeably greater than in healthy animals. Between 42 and 120 days after the induction of anaemia the haemoglobin profile of the toads regained normality.

Blood cell smears were made from anaemic toads which were known to possess abnormal amounts of Xenopus-HbF1 14 days after the induction of anaemia. The smears were treated with specific anti--Xenopus-HbF immunoglobulin conjugated with fluorescein isothiocyanate. Although up to 11 % of the haemoglobin present in the animals was Xenopus -HbF1, the number of fluorescing cells was no greater than in similarly treated smears from healthy control adults possessing less than 1 % Xenopus -HbFl.

It is concluded that during anaemia Xenopus starts to resynthesize one of its tadpole haemoglobins. This resynthesis occurs in many or most of the circulating red cells, and is not confined to a small population of cells. During recovery from anaemia enhanced quantities of Xenopus- HbA2 are made.
Development and deployment of cilia, basal bodies, and other microtubular organelles in the cortex of the ciliate Nassula
Authors: Tucker J. B.
Published: April 2021

The basal bodies contain more internal structures than those described for other organisms. For example, they possess 2 different and separate cartwheel structures. In cilia the 2 central tubules terminate proximally in a different fashion. Three types of cilia have been distinguished. Each type possesses a different array of rootlet fibres. During binary fisión one type develops 1 h before all the others while the ciliary shafts, but not the basal bodies, of a second type are being resorbed. These are later reformed when a third variety of cilia develop. Some new basal bodies and cilia are formed at least 30 min before they can be detected using silver staining. Studies of basal body morphogenesis based on this technique are pertinent to the separation of closely adjacent mature basal bodies rather than to their development. The arrangement and spacing of new basal bodies may be partly defined by their rootlet fibres which interconnect basal bodies before they attain their final positions in the cortex. Overlapping microtubules linking basal bodies apparently slide against each other during the final stages of basal body deployment. Heat shocks often inhibit the attachment of new basal bodies to the pellicle but ciliary and rootlet fibres assemble and grow out from the usual sites on these abnormally positioned basal bodies.

The pellicle is highly impermeable to water and the fine structure of its innermost layer resembles that of the waterproofing epicuticle of some insects. A cuff of this pellicular material forms around the distal end of each new basal body and then seals it to the undersurface of the pellicle. The outer wall of the pellicular chamber so formed breaks down when the ciliary shaft is assembled. This ‘cytoplasm-lock ‘mechanism may ensure that there is no marked change in the permeability of the pellicle when it is penetrated by outgrowing cilia.

A new type of ciliary membranelie is described. Some of its modified cilia possess flanges attached to their outer fibres. These cilia may have a sensory role. Membranelle basal bodies are linked by fibres exhibiting a non-repeating polarized banding pattern which may be involved in defining the arrangement and spacing of these basal bodies.

A ring of microfilaments encircles the narrow duct joining the contractile vacuole to its outlet pore. This ring may act as a sphincter and be involved in the control of water discharge. During binary fission contractile vacuole pores migrate posteriorly. They are apparently pulled by strands of microtubules and microfilaments attached at one end to a pore and anchored at the the other to more posteriorly situated basal bodies.
Ultrastructure of pseudocilia in Tetraspora lubrica (roth) ag
Authors: Lembi Carole A. , Walne Patricia L.
Published: April 2021

The flagella-like, but immobile, pseudocilia of the unicellular green alga, Tetraspora lubrica, have been characterized ultrastructurally and compared with the true flagella of the motile green alga, Chlamydomonas. The basal body and transitional regions of the 2 organelle systems are basically similar. Most of the marked differences occur in the pseudocilium proper, and a few important dissimilarities are found in the cytoplasmic portions of the apparatus. The major differences are: (1)4 instead of 2 proximal striated fibres in the pseudociliary apparatus; (2) a proximal-to-distal progressive decrease in subfibre number from 9 doublet fibres to a solitary fibre in the pseudocilium proper; (3) loss of interconnexions among the subfibres in the pseudocilium proper and a concomitant disorientation of those subfibres; (4) absence of major arms (dynein) on the A subfibre of peripheral doublets; (5) absence of the central pair of fibres. These and other differences are discussed with regard to their possible roles in loss of motility. Additional ultrastructural details - interconnexions between cytoplasmic microtubules and basal body subfibres of pseudocilia and flagella - are described and discussed in terms of their possible roles in the regulation of flagellar movement in motile organisms.
Sialic acid and cell aggregation
Authors: Mcquiddy P. , Lilien J.
Published: April 2021

Chick embryo neural retina cells from 7-, 10- and 14-day embryos and muscle cells from 9-day embryos, dissociated mechanically or with trypsin, reaggregate at the same initial rate whether treated before and during aggregation with active or inactive N-acetyl neuraminidase. JV-acetyl neuraminidase treatment also has no effect on the size or number of aggregates formed over 24 or 48 h of aggregation. It was also found that the amount of trypsin used to dissociate the tissue into single cells alters the initial rate of aggregation but has no effect on the size or number of aggregates formed over a 24-h period.
Distribution of hyaluronate and hyaluronate receptors in the adult lung
Authors: Green Shawn J. , Tarone Guido , Underhill Charles B.
Published: April 2021

In the present study, we have examined the distribution of both hyaluronate receptors and hyaluronate in adult hamster lung. The receptor for hyaluronate is a transmembrane glycoprotein of Mr 85 000 that interacts with actin filaments and is thought to mediate many of the effects that hyaluronate has on cell behaviour, such as cell-to-cell adhesion and migration. It was localized histochemically with a monoclonal antibody, designated as K-3, which specifically binds to the receptor. Hyaluronate was detected by a biotinylated form of cartilage proteoglycan, which binds with high affinity and specificity to hyaluronate. At the light-microscopic level, both hyaluronate and its receptor were present on the basolateral surfaces of bronchial and bronchiolar epithelium, suggesting that these two components interact with each other. This interaction may be important for maintaining the attachment of the epithelium to the basement membrane. In addition, hyaluronate was present in the adventitial regions of large pulmonary blood vessels. Receptors for hyaluronate were also evident on the surfaces of pulmonary macrophages, as demonstrated by the fact that K-3 immunoreactive cells coincided with cells positive for non-specific esterase, a characteristic feature of macrophages. The receptors on pulmonary macrophages probably mediate the effects that hyaluronate has on these cells with respect to cell-to-cell adhesion and migration.
G-proteins in mammalian gametes: An immunocytochemical study
Authors: Garty Nira B. , Galiani Dalia , Aharonheim Ami , Ho Yee-Kin , Phillips David M. , Dekel Nava , Salomon Yoram
Published: April 2021

The presence of transductory GTP(G)-regulatory proteins in mammalian gametes has been examined by indirect fluorescence immunocytochemistry. Using rabbit antisera to bovine rod /β γ-transducin (RAβγT), bovine rod holotransducin (AS–1), bovine rod α-transducin (RAαT), synthetic bovine rod α-transducin C-terminal decapeptide (AS–6), bovine brain α39Gο (RAα39), and two mouse monoclonal antibodies raised against frog retinal trans-ducin (4A), and rat brain β-tubulin, we demonstrated the presence of corresponding immunoreactive material in both rat oocytes and bovine ejaculated sperm. Immunostaining in the oocyte was evenly distributed on the oolemma, excluding the cell cytoplasm and zona pellucida. Immunoreactive material was also present in the cumulus cells that encapsulate the oocyte. In contrast, the immunofluorescence corresponding to transductory G–proteins was confined in sperm to functionally defined regions in the head and tail, in a manner specific for each antibody. While RA/β γT, AS-1 and RAα39 all stained the entire acrosome, AS–6 and RAαT stained only the acrosomal tip. Monoclonal antibody 4A stained the midpiece exclusively and anti-rat β-tubulin (a structural G–protein) stained the full length of the sperm tail. The existence of several G-protein types in mammalian gametes suggests their possible involvement in the regulation of various effector systems, in a manner reminiscent of somatic cells. The unique situation in sperm, where different G–proteins show distinct and specific patterns of distribution, further suggests their association with various effector systems in discrete functional domains.
Dna repair and transcriptional activity in genes
Authors: Bohr Vilhelm A.
Published: April 2021

Recent findings on the genomic heterogeneity of mammalian DNA repair have disclosed new features about molecular aspects of the repair processes and have also raised new interesting questions to help direct our research. The studies on preferential DNA repair in active genes demonstrate that there are variations in the repair processes corresponding to different parts of the genome and we are now attempting to study how these processes are regulated or controlled. In this commentary, it seems pertinent to discuss an important aspect of the preferential DNA repair mechanisms, the potential relationship between the DNA repair efficiency, the local chromatin structure and the transcriptional activity for a specific mammalian gene.

Most of the available results on DNA repair in specific genomic sequences are based on a technique to measure damage and repair after ultraviolet (u.v.) irradiation using quantitative hybridization (Bohr et al. 1985; Bohr & Okumoto, 1988) and have been reviewed recently (Bohr et al. 1987). The initial findings were that the essential, active gene dihydrofolate reductase (DHFR) was preferentially repaired in hamster and human cells (Bohr et. al. 1985; Mellon et al. 1986), and some results have been outlined in Table 1. The fine structure of DNA repair in and around the DHFR gene in CHO cells has been examined, and it was found that the preferential DNA repair was confined to a genomic region of about 60–80 kilobases. This region has been called a DNA repair domain and its size corresponds well with described higher order structures or loops in chromatin (Bohr et al. 1986). However, further studies are needed to clarify whether DNA repair is regulated within such loops in chromatin.
Visualization of the cytoplasmic Ca2+ gradient in Fucus serratus rhizoids: Correlation with cell ultrastructure and polarity
Authors: Brownlee Colin , Pulsford Ann L.
Published: April 2021

Fura-2 has been used with digital image analysis to visualize and quantify the cytoplasmic Ca2+ gradient in rhizoid cells of Fucus serratus. Ca2+ was higher at the growing rhizoid tip in about 50-60% of cells studied to date. Considerable variation in the pattern of the Ca2+ gradient has been found. Verapamil reduced but did not abolish the Ca2+ gradient. Nifedipine had no effect on Ca2+ distribution during the experimental period. Removing Ca2+ from the growth medium permeabilized the plasmalemma and allowed fura-2 to efflux from the cytoplasmic compartment, permitting an assessment of the degree of sequestration of the dye into vacuoles and vesicles. Comparisons of ratio images with electron micrographs suggested that lower Ca2+ levels were associated with the nuclear region, but there was no direct correlation between Ca2+ levels and vesicle distribution in healthy cells. The role of localized differences in Ca2+ distribution in the control of polarity is discussed.
The prometaphase configuration and chromosome order in early mitosis
Authors: Chaly Nathalie , Brown David L.
Published: April 2021

We have examined early mitotic stages in HeLa cells, mouse 3T3 fibroblasts and mitogen-activated mouse lymphocytes by immunofluorescence labelling with anti-tubulin and anti-centromere. Chromatin organization was monitored with the DNA-specific fluorochrome Hoechst 33258. This approach has led us to identify a modified Rabi array of chromosomes and spindle microtubules early in mitosis that is distinct from that at metaphase, and which we have called ‘the prometaphase configuration’. In the configuration, chromosomes are oriented so that telomeres are clustered at the outer surface, whereas centromeres are clustered inside the configuration, at the surface of a hollow spindle. Observations on cells earlier in mitosis indicate that the configuration is presaged by the spatial relationship between chromosomes and cytoplasmic microtubules in prophase and early prometaphase. We propose a model in which the prometaphase configuration represents an important step linking prophase and metaphase, serving to translate interphase spatial and intragenomic order into order at the metaphase plate.
Incorporation of tubulin from an evolutionarily diverse source, Physarum polycephalum, into the microtubules of a mammalian cell
Authors: Prescott A. R. , Foster K. E. , Warn R. M. , Gull K.
Published: April 2021

Physarum myxamoebal tubulin was injected into PtK2 cells to determine whether tubulin from this eukaryotic microbe could act as a reporter for microtubule growth and dynamics in a mammalian cell. The distribution of Physarum tubulin was determined by the use of a monoclonal antibody specific for Physarum tubulin and unable to detect mammalian tubulin. Physarum tubulin was incorporated into the microtubules of both interphase arrays and the mitotic spindle. Measurements of microtubule turnover kinetics were found to be similar to those of other studies in which chemically modified brain tubulin has been used. Results using this heterologous system demonstrate that tubulin from an evolutionarily diverse organism can be used as a marker for microtubule growth in mammalian cells. Furthermore, the Physarum tubulin was able to endow the injected cells with novel properties. Resistance to colchicine-induced microtubule disassembly, a characteristic of Physarum tubulin, was conferred on the injected PtK2 cells. Use of this heterologous reporter tubulin system has also revealed features of variation in microtubule dynamics both within individual cells and between cells.
Intracellular transport of the glycoprotein of VSV is inhibited by CCCP at a late stage of post-translational processing
Authors: Burkhardt Janis K. , Argon Yair
Published: April 2021

The appearance of newly synthesized glycoprotein (G) of vesicular stomatitis virus at the surface of infected BHK cells is inhibited reversibly by treatment with carbonylcyanide m-chlorophenylhydrazone (CCCP). Under the conditions used, CCCP treatment depleted the cellular ATP levels by 40-60%, consistent with inhibition of transport at energy-requiring stages. The G protein that ac-cumulates in cells treated with CCCP is heterogeneous. Most of it is larger than the newly synthesized G protein, is acylated with palmitic acid, and is resistant to endoglycosidase H (Endo H). Most of the arrested G protein is also sensitive to digestion with neuraminidase, indicating that it has undergone at least partial sialylation. A minority of G protein accumulates under these conditions in a less-mature form, suggesting its inability to reach the mid-Golgi compartment. The oligosaccharides of this G protein are Endo-H-sensitive and seem to be partly trimmed. Whereas sialylated G protein was arrested intracellularly, fucose-labelled G protein was able to complete its transport to the cell surface, indicating that a late CCCP-sensitive step separates sialylation from fucosylation. These posttranslational modifications indicate that G protein can be transported as far as the trans-Golgi in the presence of CCCP and is not merely arrested in the endoplasmic reticulum.
Effects of vitamin a on growth and differentiation of human tracheobronchial epithelial cell cultures in serum-free medium
Authors: Chopra D. P. , Klinger M. M. , Sullivan J. K.
Published: April 2021

Differentiating epithelial cell cultures from human tracheobronchial epithelium have been propagated in serum-free medium. The major objective of this study was to examine the trophic effects of vitamin A on cell multiplication and morphology of the tracheal cell cultures. The cellular responses were analyzed in terms of growth kinetics, morphological and ultrastructural alterations and secretion of glycoconjugates. Cell cultures in control medium exhibited characteristics of epithelial cells including microvilli on cell surfaces, desmosomes between cells, and numerous secretory vesicles in the cytoplasm. Vitamin A at 10−6 M and 10−7 M inhibited cell replication and enhanced the secretion of [3H]glucosamine-labeled glycoconjugates. Further, vitamin A increased the production of plasma membrane vesicles and acquisition by the cells of a highly secretory ultrastructure. This in vitro model of human epithelial cells will be important in the investigation of various aspects of growth and differentiation.
Saccharide expression on wounded endothelial cell monolayers in vitro
Authors: Ball R. Y. , Stoddart R. W. , Jones C. J. P. , Mitchinson M. J.
Published: April 2021

Monolayer cultures of porcine aortic endothelial cells were used as a model of the endothelium of large arteries. Linear wounds were produced in such cultures by scraping and the subsequent sequence of events in nearby cells was analysed. The earliest detectable event was cellular spreading at the margins of the wounds (2h) followed by cell migration (starting at 6–8 h) and cell proliferation in regions adjacent to the wound (16 h and later). Cell spreading was associated with the appearance of saccharides selectively at the spreading margins of the cells, which bound the lectins, ConA, LCA and PSA, and were sensitive to a-mannosidase. Terminal a-mannosyl residues were therefore present.

The appearance of these saccharides suggests a mechanism by which monocytes might adhere to and/or migrate through the endothelium of vessels at sites of cellular response to injury.
Morphometric analysis of isolated Zea mays sperm
Authors: Wagner Vincent T. , Dumas Christian , Mogensen H. Lloyd
Published: April 2021

Recently developed techniques for the isolation of sex cells of flowering plants represent an important first step toward eventual detailed cytological and biochemical analyses and experimental manipulations of higher plant gametes that was not previously possible. In the present investigation we have analysed a population of isolated viable male gametes from the standpoint of qualitative and quantitative ultrastructure in order to elucidate the process of double fertilization in angiosperms. The vast majority of the Zea mays sperm cells were found to be structurally intact spherical cells. Morphometric analysis of 400 isolated sperm cells indicates that the average cell is 7·66 μm in diameter, has volume and surface areas of 235·3 μm3 and 184·3 μm2, respectively, and contains a variously shaped, often curved nucleus, which occupies 38·5% of the cell volume. Other cell constituents and their relative volumes (expressed as percent ages of the protoplasm) included in the analysis are: vacuoles, 9·3%; endoplasmic lamellae, 5·9%; mitochondria, 3·5%; osmiophilic bodies, 1·2%; Golgi complex, 0·6%; and hyaloplasm (background cytoplasm), 41·1%. No plastids or microtubules were observed in the hybrid line used.

Two types of nuclei were found, i.e. heterochromatic and non-heterochromatic. Although statistical analysis of cell components shows some significant differences between cells of the two nuclear types, we do not believe that they represent the two sperms of a pair within pollen grains, since they do not occur in a 1:1 ratio. It is more likely that the variability within the sperm population is the result of slightly different developmental states at the time of anther dehiscence.
Expression of a cell surface antigen in Dictyostelium discoideum in relation to the cell cycle
Authors: Krefft Marianne , Weijer Corneeis J.
Published: April 2021

We have previously shown binding of a monoclonal antibody MUD 9 to the cell surface of Dictyostelium discoideum amoebae and slug cells. In the slug stage the prestalk region was predominantly labelled, while vegetative amoebae showed a great heterogeneity in binding. In the present paper it is shown that the heterogeneous label of vegetative amoebae is due to differences in MUD 9 binding by cells in different cell cycle phases. Cells were synchronized by dilution from stationary phase and the level of MUD 9 binding was determined. Synchrony was determined by investigating increase in cell number and changes in the volume distribution of the cells, and by estimating the number of cells in S phase by monitoring bromodeoxyuridine (BUdR) incorporation. Simultaneously the amount of MUD 9 binding was determined by quantitative microscopy and flow cytometry. The amount of MUD 9 label varies during the cell cycle. The highest amount of label is found on cells early in the cell cycle, i.e. S-phase. These results support the finding that the developmental fate of Dictyostelium discoideum cells depends among other things on the cell cycle position of the cells at the moment of starvation.
Identification of a spindle-associated protein in ciliate micronuclei
Authors: Keryer Guy , Loubresse Nicole Garreau De , Bordes Nicole , Bornens Michel
Published: April 2021

Ciliated protozoa display a nuclear dualism, with germinal micronuclei and a somatic macronucleus. During mitosis, which proceeds without disruption of the nuclear envelope, a spindle is organized within the micronucleus from, presumably, intranuclear microtubule-organizing centres (MTOCs). In order to characterize these MTOCs, monoclonal antibodies generated against human centrosomes were screened on several ciliates and particularly on Paramecium tetraurelia. In this ciliate, the monoclonal antibody CTR 532, which decorates centrosomal and spindle-associated components in mammalian cells, specifically labelled the micronuclei during interphase. At the electron-microscope level, it stained a fibrous material surrounding microtubules localized on the inner face of the nuclear envelope. During mitosis this decoration extended all over the metaphase spindle. At all stages of the cell cycle, the decoration remained specific to the micronucleus and was absent not only from all of the various cytoplasmic and cortical microtubule arrays but also from the macronuclei, even at early stages of their development from the zygotic nucleus. CTR 532 recognizes a single 170xl(rMr polypeptide in the cytoskeletal fraction that contains micronuclei and this polypeptide is absent in the cytoskeletal fraction of amicro-nucleate cells.
Digital image processing of optical density wave propagation in Dictyostelium discoideum and analysis of the effects of caffeine and ammonia
Authors: Siegert Florian , Weijer Cornelis
Published: April 2021

Waves of chemotactic movement during the early phase of aggregation in Dictyostelium discoideum were analyzed by digital image processing in a manner that immediately shows the following parameters: wave propagation velocity, period length, wave amplitude und wave shape. We have characterized the aggregation of AX-2 and the streamer F mutant NP 377 in terms of these parameters and investigated the influence of caffeine and ammonia. It was found that during normal aggregation oscillation frequency increases while at the same time wave propagation velocity decreases. Caffeine, a known inhibitor of cyclic AMP relay, reduces oscillation frequency and wave propagation velocity in a dose-dependent manner but most notably leads to the appearance of bimodal (harmonic) oscillations. These bimodal waves are also found in streamer F mutants without caffeine during early aggregation.

The effect of caffeine is interpreted as an increase in the average chemotactic deadaptation time due to elevated cyclic GMP levels after a cyclic AMP stimulus. This increased deadaptation time results in some cells responding to every chemotactic signal, while others respond only to every second signal, leading to mixed population behavior and hence biphasic optical density waves. Ammonia has no significant influence on oscillation frequency and wave propagation velocity but shows a clear increase in the amplitude of the optical density waves. This may indicate a more vigorous chemotactic response by individual cells or a better synchronization of the responding cell populations due to shortened chemotactic deadaptation times.
The disruption of dictyosomal integrity by caffeine in the green algal flagellate, Gloeomonas Kupfferi
Authors: Domozych David S.
Published: April 2021

The endomembrane system of the green algal flagellate, Gloeomonas kupfferi, is a highly ordered complex of endoplasmic reticulum sheets, 14–18 dictyosomes, vesicles and 8–12 peripheral vacuoles centered around a large nucleus. Each dictyosome is a highly polar cisternal stack with distinct cis and trans faces and an assortment of different-sized vesicles. The methylxanthine, caffeine, in concentrations of 1–2 mM, has been shown to be an effective disrupting agent of dictyosome integrity. For short treatments (1 h), caffeine causes an inhibition of typical trans-face vesiculation. Dictyosomes expand laterally and trans-face cisternal edges fuse. The resultant morphological products includeexaggerated circular profiles and multidictyosomal masses. During longer treatment times (3–4 h), cisface cistemae swell and ultimately disintegrate into large vesicles. Trans-face cistemae also vesiculate but do so into masses of tiny vesicles. The caffeine effects are reversible for up to 12 h, after which time cytological changes result in cell death. Phosphatase cytochemistry was also employed in this study to help reveal localized effects of caffeine. A putative explanation of the action of caffeine is presented.
Diacylglycerols and PMA induce actin polymerization and distinct shape changes in lymphocytes: relation to fluid pinocytosis and locomotion
Authors: Keller H. U. , Niggli V. , Zimmermann A.
Published: April 2021

Shape changes have been determined in human blood lymphocytes stimulated with OAG, diC8, PMA, colchicine or the hexapeptide fNLPNTL in short-term assays (30 min). Distinct types of shapechange responses were observed. Colchicine was active in generating a relatively small proportion of polarized lymphocytes (front-tail polarity). OAG, diC8 and PMA produced different types of shape change (non-polar cells with surface projections), and these were closely associated with an increase in actin polymerization and a shift of F-actin into the projections at the cell periphery. The diacylglycerols OAG and diC8 produced biphasic dose-response curves leading to rounding up of cells at very high stimulant concentrations. PMA produced no comparable biphasic response when tested over a much wider concentration range. Though the nonpolar cells with surface projections generated by OAG, diC8 or PMA showed vigorous shape changes, they lacked significant locomotor activity. α-Phorbol, 4α-PDD, lumicolchicine or fNLPNTL were inactive. Small blood lymphocytes stimulated by OAG, diC8 or PMA showed a very small increase in the net uptake of FITC-dextran by fluid pinocytosis. Unlike neutrophils, which show a high net uptake, lymphocytes did not concentrate FITC-dextran in large granules, indicating that they do not develop a ‘storage’ compartment in the form of large vesicles. However, small fluorescent spots were consistently found in at least a fraction of blood lymphocytes. The results indicate that stimulated surface movement may be instrumental in fluid pinocytosis. Diacylglycerols may act as second messengers to induce pinocytosis, shape changes and altered actin polymerization in lymphocytes.