bluuebunny/crossref_metadata_2025 huggingface dataset:
Mechanism of enhanced fusion capacity of mouse red cells infected with Plasmodium berghei
Authors: Nakornchai Sunan , Sathitudsahakorn Chiraporn , Chongchirasiri Suntanee , Yuthavong Yongyuth
Published: April 2021

Plasmodium berghei-infected mouse red cells have enhanced fusion capacity as triggered by addition of poly(ethylene glycol) in the presence of Ca2+. The uptake of Ca2+ in P. berghei-infected cells is greater than in normal cells, and the difference in Ca2+ uptake was found to be enhanced in the presence of poly (ethylene glycol). Fusion of normal and P. berghei-infected red cells by poly (ethylene glycol) was significantly inhibited by N-tosyl-L-lysylchloromethyl ketone and phenyl-methylsulphonyl fluoride. In addition, ethyleneglycolbis(aminoethylether) tetra-acetate, N -ethyl-maleimide, iodoacetamide, cystamine and tetrathionate also prevented fusion in both systems. In contrast, N -tosyl-L-phenylalanylchloromethyl ketone and N-tosyl-L-arginine methyl ester did not inhibit cell fusion. The latter enhanced fusion of infected cells but was without effect on normal cells. These results indicate that a Ca2+-activated thiol proteinase may be involved in membrane fusion in malaria-infected as well as in normal red cells. However, differences in the effect of proteinase inhibitors and substrate on fusion and Ca2+ entry show that the processes leading to fusion may not be identical.
Independent expression of type i collagen and fibronectin by normal fibroblast-like cells
Authors: Connor Nigel S. , Aubin Jane E. , Sodek Jaro
Published: April 2021

A double-label immunofluorescence technique was used to determine whether all normal fibroblast-like cells cultured from porcine connective tissues simultaneously express both type I collagen and fibronectin (FN). Cells surrounding Explants in primary cultures from gingival and periodontal ligament (PL) tissue were heterogeneous in the expression of collagen and FN. Mass population cells at first and second subculture after primary explanting of gingival tissue were also heterogeneous in the expression of collagen and FN. When clonal populations were analysed, four distinct phenotypes were observed : Coll+/FN+; C0II+/FN–; Coll–/FN+; Coll–/FN–. Mass populations of PL cells were more homogeneous than gingival fibroblasts and when clones were analysed only two phenotypes were observed. These data suggest that normal fibroblast-like cells in vitro are capable of expressing collagen and FN independently and that FN-negative cells may utilize a FN-independent attachment mechanism in culture.
Distribution of desmosomal components in the tissues of vertebrates, studied by fluorescent antibody staining
Authors: Cowin P. , Mattey D. , Garrod D.
Published: April 2021

In previous work we used immunofluorescent staining with specific antibodies to study the distribution of five desmosomal antigens in the epithelia of different vertebrate animals. We showed that all five antigens were present in all epithelia studied in human, bovine, rat, guinea pig, chick and frog (Rana piptens) tissues. It was concluded that desmosomes are highly conserved structures. This paper extends those studies: (a) by including three other species, a lizard (Lacerta viridis), the axolotl (Ambystoma mexicanum) and the trout (Salmo trutta), and (b) by looking at several tissues in more detail.

The principal results are as follows. (1) The epidermis of all species down to the frog stain with equal intensity for all desmosomal antigens. (2) In the epidermis of axolotl and trout, staining for desmosomal plaque constituents is present, but staining for the desmosomal glycoproteins is greatly reduced or absent. (3) Within mammalian species as well as chick, lizard and frog, staining for the 115 × 103 and 100 × 103 molecular weight desmosomal glycoproteins is less intense in non-epidermal tissues than in the epidermis, while staining for desmosomal plaque constituents and for the 150 × 103 molecular weight glycoprotein is undiminished. It is possible, therefore, that slight differences exist between certain glycoproteins of epidermis and non-epidermal epithelia. (4) The hearts of lower vertebrates (lizard, frog, axolotl and trout) stain only for individual desmosomal plaque antigens. (5) The pillar cells of trout gill stain, adjacent to their collagenous columns, for one desmosomal plaque antigen. There is a fibrous cytoplasmic mat in this position but no desmosomes. Thus one of the desmosomal antigens may have a function outside the desmosome.
Arginine synthesis by hepatomas in vitro: II. Isolation and characterization of morris hepatoma variants unable to convert ornithine to arginine, and modulation of urea-cycle enzymes by dexamethasone and cyclic-amp
Authors: Goss S. J.
Published: April 2021

‘77orn’, a derivative of the Morris rat hepatoma 7777, stably expresses high levels of ornithine transcarbamoylase (OTC) and carbamoylphosphate synthetase I (CPS-I), and is able to grow indefinitely in ornithine-medium (medium with ornithine in place of arginine). Variants that have lost this ability are isolated from 77orn by a ‘suicide’ selective technique dependent on the cellular incorporation of [3H]ornithine. These variants, which have reduced levels of CPS-I, or of both CPS-I and OTC, are shown to have developed multiple hormonal requirements; their enzyme deficiencies can be reversed by use of an appropriately supplemented medium. In particular, CPS-I is inducible by dexamethasone and dibutyryl-cyclic-AMP in combination. Cholera toxin can be used instead of cyclic-AMP, but then butyrate is additionally required if the induction is to be maintained in the long term. The use of these agents in excess can depress OTC. Several other hepatomas, and also explanted foetal rat liver cells, have similar requirements for CPS-I expression. It is argued that multiple hormonal requirements for CPS-I production are normal in liver cells in vitro, and that hormone-independent hepatomas should be regarded as abnormal. The implications of this for the somatic cell genetic investigation of differentiation are briefly discussed.
Division of peribacteroid membranes in root nodules of white clover
Authors: Robertson John G. , Lyttleton Pamela
Published: April 2021

Division of peribacteroid membranes in the cytoplasm of root nodules of white clover was found, from a study of serial thin sections prepared for electron microscopy, to accompany division of the bacteroids. It was also observed that the peribacteroid membranes appeared to have adhered to various sites on the surface of the bacteroid envelope outer membranes. Wherever peribacteroid membranes were constricted as though undergoing division in the region of the cleft formed by partial division of the bacteroids, these constrictions could be related to the point of adhesion of the peribacteroid membranes to the surface of the bacteroids within the cleft. It was concluded that adhesion of the peribacteroid membranes to the bacteroid envelope outer membranes is likely to be a critical element in the process of division of the peribacteroid membranes. Differences in the degree of adhesion between peribacteroid membranes and the bacteroid envelope outer membranes may explain variations in the number of bacteroids enclosed by peribacteroid membranes in nodules of different legumes.
Lateral fusion of membranes in bacteroid-containing cells of leguminous root nodules
Authors: Gunning B. E. S.
Published: April 2021

Bacteroid-containing cells of leguminous root nodules (Pisum and Trifolium) provide an environment in which the membrane envelope surrounding each bacteroid can exhibit unusual properties. The bacteroids are crowded, and frequently neighbouring membrane envelopes come into close contact. In such cases the two envelopes can fuse laterally to give a symmetrical tripartite membrane, probably consisting of a composite inner layer sandwiched between the outer leaflets of the two parent membranes. The expanse of fused membrane forks at its periphery, i.e is confluent with both of the envelopes from which it is derived, and seems to be relatively stable.
The effects of irradiation with high energy electrons on the structure and reactivity of native and cross-linked collagen fibres
Authors: Grant R. A. , Cox R. W. , Kent C. M.
Published: April 2021

Native and cross-linked rat tail tendon collagen was irradiated with high energy electrons up to a maximum dose of 100 × 104 J kg − 1 (100 Mrd). Fibres irradiated dry showed greater damage when examined in the electron microscope using negative staining techniques than those irradiated wet. Cross-linking with glutaraldehyde prior to irradiation resulted in the band structure being preserved even at the highest dose but an unequal shrinkage of the bands was noted.

Irradiation altered the reactivity of both native and cross-linked collagen with collagenase and elastase. Wet-but not dry-irradiated native collagen became resistant to collagenase. Both wet- and dry-irradiated specimens were digested with elastase. Cross-linked collagen, normally resistant to both elastase and collagenase, became sensitive after varying doses of radiation; different results were obtained after irradiation in the wet and dry states.

Irradiated collagen reacted abnormally with various histological stains and tended to resemble elastin in tinctorial properties.

No marked changes were noted in the amino acid composition of the collagen after irradiation. Solubility in dilute alkali, acetic acid and hot water was decreased after irradiation of collagen in the wet state and increased after irradiation in the dry state.

The results were consistent with the hypothesis that electron irradiation of collagen in the dry state results in scission of the polypeptide chains and that, in the presence of water, this is accompanied by the formation of intermolecular bonds. It also appears that changes in the configuration of the polypeptide chains accompany both these processes.
Chloramphenicol-related changes in mitochondrial ultrastructure
Authors: Smith Una , Smith D. S. , Yunis Adel A.
Published: April 2021

An electron-microscopic examination of bone marrow mitochondria derived from 10 patients before and after chloramphenicol (CAP) therapy has shown that CAP induces an ultrastructural modification resulting in an increase in the density of the mitochondrial matrix. These changes resemble the condensed mitochondrial configurations which have previously been interpreted in terms of electron transfer and oxidative phosphorylation. However, in the concentrations employed in this study, CAP has no effect on mitochondrial respiration or on oxidative phosphorylation, but is a potent inhibitor of mitochondrial protein synthesis. Evidence relating this effect of the drug to its myelotoxicity has been supported by a comparative study demonstrating lack of effect of a drug such as tetracycline which is not known to be myelotoxic, and the excel lent correlation between the extensiveness of mitochondrial changes in the marrow and the serum level of free CAP. The results were confirmed in a parallel study on insect flight muscle mitochondria, in which the metabolic state under which the drug is administered can be controlled and where the regular disposition of the mitochondrial cristae provides an ideal model for the study of configurational changes.
Improvements in a chemically defined medium for the growth of mouse cells (strain LS) in suspension
Authors: Birch J. R. , Pirt S. J.
Published: April 2021

In the medium used in previous work, both growth rate and maximum cell population density were reduced in protein-free medium. This was due to iron limitation. In a medium supplemented with iron, the average minimum population doubling time was reduced from 78 to 37 h and the average maximum cell population density increased from 1·6×106 to 3·3 × 106/ml.

In the improved medium described, sodium pyruvate, α-ketoglutaric acid, methylcellulose and polyvinylpyrrolidone are no longer required although methylcellulose prevents an initial fall in population which occurs in polymer-free medium. The present study indicates that amino acids now limit maximum cell population density. By increasing the concentration of the relevant amino acids we have obtained maximum cell populations in excess of 5 × 106 cells/ml without medium change.

Finally we describe a method for measuring the growth of the LS cell in the denned medium by an opacity method.
Kinetics of cell spreading in the presence of different concentrations of serum or fibronectin-depleted serum
Authors: Knox Peter
Published: April 2021

A morphometric assay has been used to show that speed of cell spreading is sensitive to the level of serum in the surrounding growth medium. There was an inverse relationship between serum concentration and speed of spreading; as serum concentration was increased from 0·1% to 100% cells took progressively longer to become fully spread. The time taken to achieve complete spreading in serum concentrations of 0·1, l·0, 10 and 100% serum was 45, 60, 105 and 240 min, respectively. At serum concentrations of 3% and above, fibronectin depletion had no effect on rates of spreading. Only at concentrations below 3% was there an effect of fibronectin depletion and although cells became fully spread in the depleted serum they took longer to do so in comparison to native serum. It is suggested that plasma fibronectin plays no role in serum-stimulated spreading at serum concentrations of 3% and above since at these concentrations rates of spreading are identical whether fibronectin is present or not. At concentrations of 3% and above spreading is mediated by the 7OK spreading factor. At serum concentrations below 3% both fibronectin and 70K factor are effective. The reason for the lack of effect of fibronectin at higher serum concentrations is an inhibition by albumin and other plasma proteins. In contrast, 7OK factor is not affected by exogenous protein concentration. The effect of exogenous protein concentration on cell adhesion is discussed in terms of cell migration between compartments that have different extracellular protein concentrations.
Chick embryonic pigmented retina is one of the group of epithelioid tissues that lack cytokeratins and desmosomes and have intermediate filaments composed of vimentin
Authors: Docherty R. J. , Edwards J. G. , Garrod D. R. , Mattey D. L.
Published: April 2021

Using sodium dodecyl sulphate/polyacrylamide gels to analyse detergent-insoluble residues, and indirect immunofluorescence, we have found that the major protein of intermediate filaments in cultures and freshly explanted fragments of chick embryonic retinal pigment epithelium (RPE) is vimentin. Moreover, these cells also fail to stain with antibodies against cytokeratins and most components of true desmosomes (maculae adhaerentes). Staining with anti-vinculin antibody suggests that the principal intercellular junction is the zonula adherens. Thus although RPE is an epithelium according to all other criteria, it belongs to a group of tissues (including vascular endothelium, iris and lens-forming epithelium) that have intermediate filaments composed of vimentin and possess neither cytokeratins nor desmosomes. That a tissue can be fully epithelial by other criteria, whilst lacking these components, is in agreement with other work, which has shown a lack of effect of micro-injection of antibodies to cytokeratin, and of the suppression of desmosome formation, on epithelial organization in culture.

Although our observations were made solely on chick embryonic tissue, we suggest that published ultrastructural studies are consistent with the possibility that RPE of other species, including human, may lack true desmosomes.
The transcriptional activity of individual ribosomal dna gene clusters is modulated by serum concentration*
Authors: De Capoa A. , Marlekaj P. , Baldini A. , Archidiacono N. , Rocchi M.
Published: April 2021

The activity of ribosomal gene clusters has been studied by cytological methods in human cultured cells grown in different amounts of serum under controlled experimental conditions. It has been shown that increasing amounts of serum induce an increase in ribosomal RNA synthesis at the single cell level. Furthermore, the concomitant identification of individual rRNA gene clusters by fluorescent techniques allowed us to demonstrate: (1) that individual gene clusters have differential transcriptional activity and differential frequency of activation; (2) that ribosomal gene activity is closely associated with the amount of silver-positive gene product and; (3) that environmental variations modulate rRNA synthesis by repressing or derepressing specific gene clusters.
The effect of medium changes on the growth and metabolism of the human diploid cell, W1-38
Authors: Griffiths J. B.
Published: April 2021

It has been established that although an inhibitory interaction occurs when a culture of human diploid cells become crowded together (contact inhibition of growth), multiple-layered cell sheets are obtained by using a continuous medium perfusion culture. A similar effect is obtained when the culture medium is changed at frequent intervals, and this paper reports the effects of a medium change on cell growth and metabolism.

A direct relationship was found between cell yield and the number of medium changes given to a culture. This was an unexpected result because normally when a culture is prolonged by additional feeding the cell yield shows a diminishing return. The amino acid and glucose uptakes and growth yields (the ratio of the amount of cell dry weight produced to substrate used) were determined and they also showed that a unit amount of growth occurred per medium change, and that cessation of growth was accompanied by cessation of nutrient uptake and metabolism. Medium changes had a profound affect on cellular metabolism, especially on DNA and protein synthesis. As a culture approached confluency, DNA, RNA and protein synthesis were sequentially inhibited. After a medium change there was a sequential stimulation of DNA, RNA and protein synthesis in the same order as they were inhibited. The inhibitory mechanism that is affected by cell crowding is obviously reversed by a medium change.

The results presented in this paper suggest that contact inhibition of growth primarily affects DNA synthesis and that if the cell is able to take up a sufficient supply of nutrients in a crowded culture then this inhibition can be overcome.
Studies on the formation of ‘floating’ guard cell mother cells in Anemia
Authors: Galatis B. , Apostolakos P. , Palafoutas D.
Published: April 2021

The protodermal cells producing the ‘floating’ guard cell mother cells (GMCs) in three Anemia species undergo an extraordinary polarization and an unexpected shaping. During interphase an intercellular space is initiated at the internal proximal end of the cell, while the polar region bulges outwards. At this stage a microtubule girdle traverses the cortical cytoplasm underneath the rims of the external periclinal wall curvature. In addition, another system of microtubules converges on a cortical site adjoining the wall delimiting the intercellular space and, or, the neighbouring region of the internal periclinal wall (internal polar cortical site, IPCS). Vacuoles are found in all regions of the cell except for that between the centrally located nucleus and the intercellular space.

As the cell approaches mitosis, the growing vacuolar system retreats from the cytoplasmic region below the external periclinal wall curvature. In most cells the polarized cytoplasm forms an inclined truncated cone, the bases of which abut on the external periclinal wall curvature and the wall lining the IPCS. The organization of the cortical microtubule cytoskeleton does not change significantly during preprophase-prophase. A preprophase microtubule band (PMB) is localized in the cortex lining the rims of the external periclinal wall curvature, while some microtubules traverse the IPCS and the cytoplasm adjacent to the neighbouring wall regions. The mitotic spindle axis is diagonal, while the cell plate separating the GMCs exhibits an unusual mode of growth. It gradually encircles the proximal daughter nucleus, becoming funnel-shaped. One of its periclinal edges fuses with the external periclinal wall area lined by the PMB cortical zone and the other with the internal periclinal wall area adjoining the IPCS. The latter region seems to behave like the PMB cortical zone.

The results show that the morphogenetic mechanism underlying the formation of the conical GMCs includes a series of highly integrated processes, initiated or carried out during cell polarization.
Adhesion, growth and morphology of human mesothelial cells on extracellular matrix
Authors: Niedbala M. J. , Crickard K. , Bernacki R. J.
Published: April 2021

Human mesothelial cells (HMC) cover a variety of serosal surfaces and have been shown to rest upon an underlying subcellular basement membrane in vivo. Bovine corneal endothelial cells produce an extracellular matrix (ECM) in vitro that mimics HMC subcellular basement membrane and was found to modulate HMC adhesion, morphology and proliferation in vitro. Our results indicated that within minutes after plating, a high percentage (>80%) of HMC firmly attached to ECM. Active cellular migration and subsequent proliferation were observed leading to the formation of a well-organized closely apposed cell monolayer. However, when cells were plated on plastic, the rate of cell attachment was much lower and the proliferative rate of HMC grown on plastic also was strikingly lower (exponential doubling time 4·3 days) than that of cells grown on ECM (exponential doubling time 2·4 days). Cells upon reaching confluency on plastic were markedly enlarged as compared to confluent cells grown on ECM. These observations corroborated differences in final cell density where it was noted that HMC cultured on ECM demonstrated a 10fold greater final cell density as compared to cells grown on plastic. Results from these studies illustrate the fact that phenotypic expression as well as proliferative responsiveness of HMC can be modulated by adhesive interactions with preformed ECM.
Mutants of Polysphondylium pallidum showing delayed modifications of glycoproteins are altered in a regulatory signal for development
Authors: Toda K. , Francis D. , Gerisch G.
Published: April 2021

Binding of a monoclonal antibody, Ab293, to cell-surface glycoproteins of Polysphondylium pallidum is known to be blocked by L-fucose, and Fab of this antibody has been shown to inhibit intercellular adhesion of aggregation-competent cells. Mutants with delayed expression of the carbohydrate epitope, ep293, recognized by the antibody, have been shown to be retarded and altered in cell aggregation. The present study shows that ep293 is a modification of carbohydrate structure that is subject to regulation not only in mutant but also in wild-type cells; ep293 is expressed at an early stage of exponential growth in wild-type and only after 12 h of starvation in mutant PN6002. Proteins are already glycosylated before the epitope is expressed. The developmental regulation of pallidin, a lectin known to be an unglycosylated protein, was investigated in parallel with ep293 using a monoclonal antibody. Pallidin was expressed at about the same time as the carbohydrate epitope in cells of the wild-type as well as the mutant. These results indicate a regulatory signal to which various events are coupled. Induction of ep293 and expression of pallidin are two of these events, and mutants such as PN6002 are altered in the timing of the signal.
Junctional Complexes of Epithelial Cells
Authors: Skerrow C. J.
Published: April 2021

This book contains the proceedings of a Symposium held in London in February, 1986, and attended by almost all the leading workers in the field of junction research. The first three chapters are concerned with desmosomes: their general molecular organization, the more detailed structure of the plaque, and the effects of calcium on desmosome assembly and stability. Six shorter chapters on gap junctions cover their physical and molecular structure (with both sides of current controversies being represented) and also functional studies on junctional communication in adult and embryonic tissue. A chapter on the role of uvomorulin in tight junction formation is followed by a more general treatment of cell adhesion molecules and rules for their expression during development. The last two chapters deal with factors affecting epithelial cell interactions in vitro and the role of cell junctions in cancer.
Structure and molecular organization of higher plant coated vesicles
Authors: Coleman Julian , Evans David , Hawes Chris , Horsley David , Cole Louise
Published: April 2021

Suspension-cultured cells of carrot contain three populations of coated vesicles, associated with the plasma membrane (84–91 nm diameter), Golgi dictyosomes and the partially coated reticulum (61–73 nm diameter). These were observed by thin sectioning, dry-cleaving and rapid-freeze deep-etching of cells. Dissociation of clathrin coats with Tris, released triskelions that were morphologically identical with those from mammalian tissue. The triskelion arm length of carrot clathrin was greater (61 nm versus 44–50 nm), but packaging results in clathrin cages of pentagons and hexagons of similar size to those from mammalian cells.

SDS—PAGE of Tris-released triskelion preparations revealed a complex of three polypeptides of 190, 60 and 57(× −)Mr. The 190× 103Mr protein is the plant clathrin heavy chain, slightly larger than the mammalian heavy chain. The 60 and 57(× 103) Mr bands showed the same sensitivities to protease treatment as mammalian light chains. Triskelion preparations containing these three proteins reassembled into polyhedral cages.

These results are discussed in relation to the structural organization of coated vesicles and clathrin cages in other systems.
The relative significance of growth rate and interdivision time in the size control of cultured mammalian epithelial cells
Authors: Hola M. , Riley P. A.
Published: April 2021

Genealogies of a line of mammalian epithelial cells (GPK) have been constructed from timelapse film of monolayer cultures and measurements made of initial (post-divisional) cell size, final (pre-divisional) cell size and interdivision time (IDT). The mean initial cell volume was 2696 ±404 (S.D.) μm3, the mean final volume was 5247 ± 696 μm3 and the mean IDT was 985 ± 84 min.

Cell size regulation must be by modulation of either the growth rate or the length of the growth period. Increase in size was strongly correlated with the average rate of growth (increase in volume per unit time) (R = 0·94, P <4 0·001), whilst no correlation was found between increase in size and IDT.

Although a negative correlation was found between initial volume and IDT (P<0·02), this appeared to be due to differences in IDT between sister cells being correlated with differences in their initial volumes (P<0·02), as indicated by the lack of correlation between mean sister IDT and mean sister initial volume.

The regulatory effect of growth rate was demonstrated by a negative correlation between growth rate and the initial volume of the cell (P< 0·005), which is independent of differences between siblings. The mean growth rate of sibling cells was found to be negatively correlated with both the maternal growth rate (P<0·01) and the maternal volume increase (P<0·005). This implies that the growth rate of division products (which manifest similar growth rates) is influenced by the growth of the progenitor cell.

Key words: cell size regulation, growth rates, interdivision times.
Turnover of the carboxy-terminal tyrosine of α-tubulin and means of reaching elevated levels of detyrosination in living cells
Authors: Wehland Juergen , Weber Klaus
Published: April 2021

Monoclonal antibodies specific for either the tyrosinated (Tyr) or the detyrosinated (Glu) form of α-tubulin were elicited with synthetic peptides spanning the carboxy-terminal sequences of the two forms. While almost all microtubules (MTs) are usually of the Tyr-tubulin type (Tyr-rich MTs) some MTs containing noticeable amounts of Glu-tubulin (Glu-rich MTs) were found in many but not all cell lines studied. Glu-rich MTs seemed absent from proliferating CHO and N115 neuroblastoma cells. When differentiation of these cells was initiated by the addition of forskolin for CHO, or by serum deprivation for N115, elevated levels of microtubular Glu-tubulin were observed. In differentiated N115 cells Glu-tubulin was restricted to MT of elongated cell processes and was not found in growth cones and many MT of the cell body. Elevated levels of Glu-tubulin were also characteristic of other differentiated cell types, including neurones and myotubes but were not found in glial cells, astrocytes and fibroblasts in the same primary cultures.

Additional experiments suggested that the restricted distribution of Glu-tubulin is the result of MT subsets with different stabilities. Results with mitotic drugs indicated that detyrosination occurs on MTs rather than on soluble tubulin and that stabilization of MTs usually favours the detyrosination process. Evidence for a functional α-tubulin cycle involving an inherent carboxypeptidase and a recharging ligase was apparent in 3T3 cells from the preponderance of Glu-rich MTs induced by taxol treatment or the microinjection of certain antibodies either protecting the detyrosinated form (Glu-tubulin antibodies) or inhibiting retyrosination (ligase antibodies). As the same treatment of CHO cells resulted in comparable arrays of Glu-rich MTs only when forskolin was also present, different cell types may differ in the level of active carboxypeptidase.

The results are discussed in terms of possible functions of the tyrosination/detyrosination cycle of α-tubulin. While most results can be explained on the basis of‘older’ and, consequently, more detyrosinated MTs, others raise the possibility that cyclic-AMP-dependent events and certain environmental influences known to induce either a morphological transformation or a differentiation event may influence the carboxypeptidase inherent in the α-tubulin cycle.
Preliminary characterization of maturation-promoting factor from yeast Saccharomyces cerevisiae
*
Authors: Tachibana Kazunori , Yanagishima Naohiko , Kishimoto Takeo
Published: April 2021

It has been known for some time that maturation-promoting factor (MPF) appears in a wide variety of eukaryotic cells at M phase and exerts equal M-phase-promoting activity in both meiotic cells and mitotic cells in a non-specific manner. MPF was extracted from cdc20 mutant cells of the yeast Saccharomyces cerevisiae synchronized at M phase by incubation at the restrictive temperature. When injected into immature oocytes of Xenopus laevis, yeast MPF caused meiosis re-initiation in a dose-dependent manner and even in the presence of cycloheximide. Yeast MPF exerted its activity in starfish oocytes as well. MPF activity was obtained only from cells in M phase and not from G1, S or G2 phase cells, indicating cyclical changes during the yeast mitotic cell cycle. Preliminary characterization of yeast MPF revealed that its activity was associated with a heat-labile protein having a sedimentation coefficient value of 6S. In contrast to the current assumption that MPF is a Ca-sensitive phospho-protein stabilized by phosphorylated small molecules, such as ATP and Na-β-glycerophosphate, the present study revealed that yeast MPF was still active even after treatment with either Ca2+ or alkaline phosphatase. Furthermore, it was found that yeast MPF and these phosphorylated small molecules were complementary in inducing reinitiation of meiosis, since the meiosis-reinitiating activity was detected only when both were present simultaneously and almost un-detectable when either of them was present alone. These facts suggest that yeast MPF need not necessarily be in a phosphorylated form in order to be active and that the phosphorylated small molecules have another effect, such as preventing the activity of phosphatases that might be present in recipient Xenopus oocytes rather than stabilizing the MPF molecule itself.
Characterization and origin of extrachromosomal DNA granules in Sarcophaga bullata
Authors: Bultmann Hermann , Mezzanotte Roberto
Published: April 2021

We have used endonuclease treatment in situ, followed by Giemsa or ethidium bromide staining, for mapping repetitive sequences on the chromosomes of the flesh fly Sarcophaga bullata and thus for studying extrachromosomal DNA granules in this species. All three restriction enzymes employed (HaeIII, AluI and HindIII) show the same cytological effects, except for a single interstitial band. In both polytene and mitotic chromosomes, chromatin resistant to these endonucleases presumably includes at least three previously unrecognized buoyant density satellites (1·663, 1·670 and 1· 692 g ml−1 in neutral CsCl), and is predominantly localized in the pericentric regions of all five autosomes. Mitotic treated chromosomes show that the entire rodshaped X chromosome, but no part of the dot-like Y chromosome, consists of endonuclease-resistant chromatin. The most unusual heterochromatic component of polytene nuclei in this species, the‘extrachromosomal DNA granules’, are also entirely resistant to digestion with endo-nucleases. We think that these DNA granules represent dispersed X chromatin and not, as previously assumed, extruded autosomal hetero-chromatin.
Observations on rRNA gene replication in Chara vulgaris evaluated by in situ [3H] rRNA-DNA hybridization
Authors: Kwiatkowska Maria , Kononowicz A. K. , Janas Krystyna
Published: April 2021

In situ [3H]rRNA-DNA hybridization showed that: (1) during the cell cycle in antheridial filaments of Chara vulgaris the rDNA gene content doubles during a 2h period in mid-S-phase, which corresponds to about 13% of the duration of this phase; (2) doubling of the number of grains occurs during mid-S-phase irrespective of the developmental stage of the antheridial filaments; (3) the numbers of autoradiographic grains in comparable phases of successive cell cycles preceding spermatid formation are the same. Thus the gradual reduction of total nucleolar volume (estimated per nucleus) occurring in successive cell cycles preceding spermatid formation is not associated with a decrease in the number of rDNA genes.
MHC control of cell position in vitro
Authors: Birkby C. A. , Curtis A. S. G. , McGrath M. , Ripley B. D.
Published: April 2021

Two-dimensional sorting-out behaviour (segregation) in mixtures of pulmonary endothelial cell lines derived from congenic strains of mice was examined using dense confluent cultures in which mitosis is rare. Cell MHC type was detected by autoradiographic labelling or by immunofluorescence techniques. For autoradiography cultures of one type were previously labelled with [3H] thymidine and one component of the mixed cell types was prepared from these cultures. Autoradiographs were prepared from the fixed cultures. Counts of contiguous neighbours (labelled or unlabelled around a randomly chosen central labelled cell) were made: these were analysed statistically using a new model for such a system. The results show that segregation of a clustering type took place if the alleles of the D locus in the H-2 complex were mismatched in the mixed strains, but that matching at the IC locus (or some locus to the right of IJ) overrides the effect of D mismatch. After 2 days culture sorting-out was easily detectable when the cells were stained for their histocompatibility antigens and groups of up to 12 cells of the same type were associated together.
Ultrastructural aspects of encystation and cyst-germination in Phytophthora parasitica
Authors: Hemmes D.E. , Hohl H.R.
Published: April 2021

Encystation in Phytophthora parasitica can be divided into 3 stages. In the first, the zoospores line their peripheries with flattened vesicles and nbrillar vacuoles in preparation for encystation. In the second stage, as the zoospores round up and shed their flagella, an initial wall is produced which takes the form of the mature cyst wall in thickness, but not in density. The participation of the flattened vesicles and fibrillar vacuoles in the formation of this initial wall is suggested by the disappearance of these organelles concomitant with wall formation. The third stage involves the maturation of the cyst wall and occurs only after dictyosomes produce vesicles which move to the cyst periphery and fuse to the plasmalemma.

Germ tubes are formed in direct and indirect germination and involve the evagination of the plasmalemma and cyst wall proximal to an accumulation of dictyosome-derived vesicles. These vesicles remain at the germ-tube tip as it extends. In indirect germination the germ tube stops after having attained an average length of 6 μm and the vesicles appear to fuse at the hyphal apex, thus forming a cap.

Lomasomes do not appear to be cell organelles with a specific function such as wall synthesis, but rather seem to represent aggregations of excess membranous material that have formed as a result of the discharge of vesicles at the cell periphery during wall formation. When dictyosome vesicles are inhibited from forming and moving toward the cell periphery, lomasomes are not formed.
Macromolecular dynamics of the cell surface during the formation of coated pits is revealed by fracture-flip
Authors: Fujimoto Kazushi , Silva Pedro Pinto Da
Published: April 2021

We report here the macromolecular dynamics of the cell surface of rat alveolar macrophages during spreading on a substratum, a process that involves the formation of numerous coated pits. We used ‘fracture-flip’ to prepare high-resolution platinum-shadowed replicas of membrane surfaces. Our observations show the following sequence of events associated with coated pit formation: at 4°C the cell surface of macrophages is covered with a moderate density of particulate components, with most ranging from 10 to 25 nm in diameter. These particles appear to be randomly distributed over the cell surface. Incubation of adherent cells at 37°C for 15 min results in the formation of large loose clusters (area 0·5–4 μm2) of particles on the adherent surfaces. After incubation of macrophages for 30 min at 37°C, these clusters become tighter andeventually form circular depressions (200·300 nm in diameter), which we interpret as part of a process of invagination. After 60 min, the depressions become much steeper. At this time surface particles can be observed on the intervening non-invaginated regions, and the peripheral region of the adherent membrane, as well as the free membrane. Fractureflip reveals the presence of structures undetected in previous electron-microscopic studies and provides ultrastructural evidence for the clustering of surface macromolecules that is involved in the formation of coated pits.
Cell adhesion to hydroxyl groups of a monolayer film
Authors: Owens Norman F. , Gingell David , Trommler Annette
Published: April 2021

We have studied cells on chemically defined mono-molecular films of the long-chain alcohol docosanol. Langmuir-Blodgett films of the alcohol were deposited on glass coverslips, previously made hydrophobic with octadecyl groups. This gives films in which the alcohol headgroups face outwards to the water. Molecular orientation and film integrity were shown by a fluorescence adsorption test. Cell contacts on the films were observed in media without proteins by interference reflection microscopy (IRM) and the mechanics of detachment were examined by hydrodynamic shearing in a flow chamber. Cell contact with docosanol was compared with that on an adjacent area of octadecyl glass without a monolayer.

Dictyostelium amoebae settled and spread on both docosanol and octadecyl glass, but little or no locomotion was seen on docosanol. On octadecyl glass the amoebae moved actively, forming ultrathin cytoplasmic lamellae, which look dark under IRM, and left distinctive trails of membranous debris. Hydrodynamic shearing showed that the amoebae stuck strongly to both surfaces and could not be removed from either at the maximum attainable wall shear stress of 6Nm−2. Red blood cells also adhered to both surfaces and removal from both occurred between 1 and 3Nm−2. IRM and scanning electron microscopy (SEM) studies indicated that this force leads to a minimal measure of red cell adhesion, since removal often involved the breakage of cytoplasmic tethers.

Our results show that alcoholic -OH groups, in a two-dimensional array, provide a surface that is strongly adhesive for cells. No other method has made it possible to demonstrate cell adhesion purely to -OH groups, in a known orientation and density, and in the absence of any other functional groups on the interface.
Differentiated membrane specializations and myofibrillar breakdown and recovery in cultured adult cardiac myocytes treated with TPA and diacylglycerol
Authors: Moses R. L. , Claycomb William C.
Published: April 2021

Cultured adult rat ventricular cardiac muscle cells were treated with either 12-O-tetradecanoyl-phor-bol-13-acetate (TPA) or diacylglycerol (DAG) and observed by in situ transmission electron microscopy. Membrane specializations present in untreated cells (intercalated discs, transverse tubules, plasmalemmal couplings) were also present after TPA and DAG treatment. In the case of the transverse tubular system, there was morphological evidence for active growth. Our studies showed that myofilaments began to become disorganized after 12–24 h of TPA treatment and that after 2 days of exposure to TPA the breakdown of sarcomeres was essentially complete. Myocytes that were treated with TPA for 2 days and then allowed to recover in control medium for 5 days contained sarcomeres in various stages of reassembly. These data indicate that TPA-treated cardiac myocytes retain several membrane specializations, suggesting that there are separate controls for myofilament organization and the maintenance of these differentiated plasmalemmal regions. Furthermore, the ability of the myocytes to recover from TPA treatment may provide investigators with a useful model with which to study myofibrillogenesis.
Subfractionation of the endocytic pathway: Isolation of compartments involved in the processing of internalised epidermal growth factor-receptor complexes
Authors: Futter Clare E. , Hopkins Colin R.
Published: April 2021

The aim of the present study was to isofate different parts of the endocytic pathway in order to examine the rofe of epidermal growth factor (EGF)-receptor internalisation in mediating the biofogical effects of EGF. We have used an antibody to the transferrin receptor complexed with cofloidal gold to modify the density of the endocytic compartments so that they can be purified by sucrose density centrifugation. Using this technique, we have been able to isofate a highly purified preparation of endocytic vesicles from H.Ep.2 cells that contain internalised EGF. By employing pulse-chase protocofs, it is possible to isofate the different parts of the endocytic pathway and show that they are temporally distinct with regard to the processing of EGF. It should now be possible to examine interactions between the EGF receptor and intracellular substrates in different parts of the endocytic pathway.
Extracellular ciliary axonemes associated with the surface of smooth muscle cells of ctenophores
Authors: Tamm Signhild , Tamm Sidney L.
Published: April 2021

We describe the first example of bare ciliary axonemes existing outside eukaryotic cells. The axonemes run in longitudinal invaginations of the surface membrane of giant smooth muscle cells in ctenophores. No motility of the surface-associated axonemes has been detected in living muscles. The axonemes are truly extracellular and in direct contact with the extracellular matrix (mesoglea), as shown by the ultrastructural tracer horseradish peroxidase. The axonemes appear partially degraded and disorganized, and individual doublet microtubules are difficult to distinguish. Nevertheless, immunofluorescence microscopy shows that the axonemes retain antigenic sites reacting with mouse monoclonal anti-beta-tubulin. The origin of the extracellular axonemes is unknown: no attached basal bodies (extracellular or intracellular) have been found. The muscle-associated axonemes may play a unique role in smooth muscle function and/or development, and may be related to the evolution of muscle cells in soft-bodied invertebrates that exploit cilia for a wide variety of functions.
Normal chemotaxis in Dictyostelium discoideum cells with a depolarized plasma membrane potential
Authors: Duijn Bert Van , Vogelzang Sake A. , Ypey Dirk L. , Molen Loek G. Van Der , Haastert Peter J. M. Van
Published: April 2021

We examined a possible role for the plasma membrane potential in signal transduction during cyclic AMP-induced chemotaxis in the cellular slime mold Dictyostelium discoideum. Chemotaxis, cyclic GMP and cyclic AMP responses in cells with a depolarized membrane potential were measured. Cells can be completely depolarized by two different methods: (1) by treatment with azide; this probably causes inhibition of the electrogenic proton pump, which was shown earlier to regulate plasma membrane potential in D. discoideum. (2) By electroporation, which causes the formation of large non-ion-selective pores in the plasma membrane.

It was found that in depolarized cells the cylic AMP-mediated cyclic AMP accumulation was inhibited. In contrast, chemotaxis to a cyclic AMP source was normal; the cyclic AMP-induced accumulation of cyclic GMP, which is known to mediate the chemotactic response, was also not affected. We conclude that membrane-potential-regulated processes, such as voltage-gated ion channels, do not play an essential role in chemotaxis in D. discoideum.
Spreading of trypsinized cells: Cytoskeletal dynamics and energy requirements
Authors: Bereiter-Hahn Jürgen , Lück Michaela , Miebach Toni , Stelzer H. K. , Vöth Monika
Published: April 2021

The spreading of trypsinized XTH-2 cells (a line derived from Xenopus laevis tadpole heart endothélia) on glass was investigated. Three phases can be distinguished: (1) blebbing of rounded cells, first attachment to a solid substratum and formation of a broad smooth contact area; (2) organization of a peripheral zone of actin fibrils and reinforcement of the basal cytoplasm by a stress fibre-like pattern; (3) extension of lamellae.

The first phase seems to be independent of a supply of metabolic energy, while the others clearly depend on it. This is concluded from the close relationship between cellular projection area and energization of mitochondria as revealed by (a) the fluorescence intensity of cells vitally stained with the mitochondria-specific fluorochrome DASPMI (2–4-(dimethyl-amino)-styryl-l-methylpyridinium-iodine); (b) the degree of spreading in the presence of inhibitors of respiration; (c) effective amelioration of spreading (phases (2) and (3)) under conditions of high ATP content.

In phase (2) the extension of the central part of the cells becomes stabilized, the cell body settles on the basal cytoplasmic layer and further expansion of the projection area is achieved by lamella formation (phase (3)); motile and stabile regions of the cells become separated. This sequence of events is interpreted as a self-organizing process based on the development of internal hydraulic pressure, actin polymerization and contraction of the newly developed actomyosin network.

During trypsinization, depolymerization of actin does not occur but rather on addition of Ca2+-containing media. Cellular ATP content drops as well on trypsinization, as on addition of Ca2+. Manganese promotes spreading by decreasing F-actin disassembly and maintaining a high level of cytosolic ATP, most probably because it is not accepted by the calcium pumps. Regarding the association of glycolytic enzymes with F-actin and their influence on actin assembly, lactate dehydrogenase has been inhibited with oxamic acid. This treatment improves the correlation between F-actin content and the degree of spreading; however, the total amount of F-actin remains smaller and the cells spread more.